calpain and calpastatin

Calpain and calpastatin are the key components of the calcium-dependent proteolytic system. Calpains are regulatory, calcium-dependent, cytoplasmic proteinases, and calpastatin is the endogenous inhibitor of calpains. Due to the correlation between changes in the activity of the calpain-calpastatin system in the brain and central nervous system (CNS) pathology states, this proteolytic system is a prime focus of research on CNS pathological processes, generally characterized by calpain activity upregulation. The present review aims to generalize existing data on cerebral calpain distribution and function through mammalian ontogenesis. Special attention is given to the most recent studies on the topic as more information on calpain-calpastatin system involvement in normal CNS development and functioning has become available. We also discuss data on calpain and calpastatin activity and production in different brain regions during ontogenesis as comparative analysis of these results in association with ontogeny processes can reveal brain regions and developmental stages with pronounced function of the calpain system.

Topics: Animals; Brain; Calcium; Calcium-Binding Proteins; Calpain; Mammals; Peptide Hydrolases

Cellular life depends mostly on the creation, modification, interactions and destruction of proteins. This is true for every cell, including human T lymphocytes. One way these cells can ascertain the fidelity and at least partial functionality of their proteomes under constant attack of irreversible modulations (e.g., ROS- or glycation-dependent) is proteostasis. However, with cellular aging proteostasis progressively fails and proteostenosis (decreased amounts and functionalities of remaining proteins) occurs. There are several mechanisms involved in the modulation and protection of the proteome in the T cells which include mainly multiple layers of vesicle-bound and cytoplasmic proteases (e.g., lysosomal and proteasomal ones) acting mostly by degradation of obsolete and age-modified proteins. Recently it was shown that another not yet so widely known system consisting of obligatorily calcium-dependent cysteine proteases, the calpains and their inhibitor, the calpastatin serves in T cells as a dual switch, either activating or inactivating different proteins depending on intracellular conditions. Thus the proteolytic elimination of altered proteins as well as modulation of activity of those remaining leads to dynamic change of proteome composition and function (proteodynamics) in aging lymphocytes, so far in an almost unknown way. Aging T cell proteodynamics requires further comprehensive analysis of the resulting lysoproteomic patterns and their changes.

Topics: Aging; Animals; Calcium-Binding Proteins; Calpain; Humans; Protein Processing, Post-Translational; Proteostasis; T-Lymphocytes

The calpains are a family of intracellular cysteine proteases that function in a wide array of cellular activities, including cytoskeletal remodelling, survival and apoptosis. The ubiquitously expressed micro (µ)-calpain and milli (m)-calpain are archetypal family members that require calcium for function and can be inhibited by their endogenous inhibitor calpastatin. This review describes the role of the calpain system in the prognosis of breast cancer and disease progression, in addition to the role of the calpain system in the response to breast cancer treatments, including chemotherapeutic, endocrine and targeted therapies.

Topics: Breast Neoplasms; Calcium-Binding Proteins; Calpain; Disease Progression; Female; Humans; Prognosis

This article attempts to provide a framework that will help to illustrate the roles of calpains in the process of traumatic brain injury (TBI).. This review provides meaningful points about the essential role of calpains in the neuropathological changes that follow TBI, identifies useful biomarkers of calpain activation and states the important roles of calpain in the treatment of TBI.. Neuronal calpains can be activated within hours or even minutes following contusive or diffuse brain trauma in animals. It has been suggested that they are early mediators of neuronal damage. Trauma can produce sustained calpain activation. In turn, this may result in axonal degeneration and neuronal death in models of TBI. Calpains can cleave cytoskeletal proteins into stable proteolytic fragments that have been widely used as biomarkers of the activation of calpain. The inhibition of calpains can reduce the functional and behavioural deficits by ameliorating axonal pathology and reducing cell deaths in animal models of TBI.. This review concentrates on the current understanding of the role of calpains in neuropathology that has been induced by TBI and the significance of calpains as a therapeutic target for the treatment of primary and secondary injuries that are associated with brain trauma.

Topics: Animals; Biomarkers; Brain Injuries; Calcium-Binding Proteins; Calpain; Cell Death; Cerebral Cortex; Cysteine Proteinase Inhibitors; Female; Humans; Male; Mice; Prognosis; Proteolysis; Treatment Outcome

Calpains are Ca(2 +) -dependent cysteine proteases. Fifteen gene products of calpains are expressed in mammals. Among them, Calpain 1 and Calpain 2 are ubiquitously expressed and have been investigated extensively. Under the physiological conditions, calpain activity is strictly regulated by endogenous inhibitory protein, Calpastatin. Calpains are activated in the various cardiovascular diseases and implicated in their pathogenesis by degrading numerous target proteins. Here we briefly summarize the physiological and pathological role of calpains in the cardiovascular diseases.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Cardiovascular Diseases; Genetic Predisposition to Disease; Humans

Photoreceptor degeneration is the hallmark of several groups of inherited neurodegenerative diseases causing blindness in humans. These diseases are a major cause of visual handicap and to date no satisfactory treatment is available. Here, we briefly review different approaches for the treatment of photoreceptor degeneration, to then focus on neuroprotection. Up to date, translation of experimental neuroprotection into a clinical setting has faced major obstacles, which are in part due to an incomplete understanding of the regulation of pro-survival as well as neurodegenerative mechanisms. Previous approaches were often based on the hypothesis that photoreceptor cell death was governed by a single, apoptotic cell death mechanism. This perception has turned out too simple as recent work has demonstrated that photoreceptor cell death is governed by non-apoptotic mechanisms as well. Moreover, there is evidence, that several different destructive processes are executed in parallel. Briefly reviewing the complexity of degenerative mechanisms, this review discusses relevant pathways, options to target signaling cascades, final common denominators of cell death, and the interplay of events executing cell death. In particular, we focus on cGMP-signaling, epigenetic and proteolytic processes and the corresponding enzymatic activities that were recently shown to be causally related to retinal degeneration. Finally, we illustrate how a better understanding of destructive mechanisms may enable identification and validation of novel targets for neuroprotection, and allow development of next generation neuroprotective treatments as well as combination therapy.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Cyclic GMP; Humans; Nerve Growth Factors; Oxidative Stress; Photoreceptor Cells

Calpains are a family of complex multi-domain intracellular enzymes that share a calcium-dependent cysteine protease core. These are not degradative enzymes, but instead carry out limited cleavage of target proteins in response to calcium signalling. Selective cutting of cytoskeletal proteins to facilitate cell migration is one such function. The two most abundant and extensively studied members of this family in mammals, calpains 1 and 2, are heterodimers of an isoform-specific 80 kDa large subunit and a common 28 kDa small subunit. Structures of calpain-2, both Ca2+-free and bound to calpastatin in the activated Ca2+-bound state, have provided a wealth of information about the enzyme's structure-function relationships and activation. The main association between the subunits is the pairing of their C-terminal penta-EF-hand domains through extensive intimate hydrophobic contacts. A lesser contact is made between the N-terminal anchor helix of the large subunit and the penta-EF-hand domain of the small subunit. Up to ten Ca2+ ions are co-operatively bound during activation. The anchor helix is released and individual domains change their positions relative to each other to properly align the active site. Because calpains 1 and 2 require ~30 and ~350 μM Ca2+ ions for half-maximal activation respectively, it has long been argued that autoproteolysis, subunit dissociation, post-translational modifications or auxiliary proteins are needed to activate the enzymes in the cell, where Ca2+ levels are in the nanomolar range. In the absence of robust support for these mechanisms, it is possible that under normal conditions calpains are transiently activated by high Ca2+ concentrations in the microenvironment of a Ca2+ influx, and then return to an inactive state ready for reactivation.

Topics: Animals; Calcium Signaling; Calcium-Binding Proteins; Calpain; Enzyme Activation; Humans; Protein Conformation; Protein Isoforms; Protein Subunits; Recombinant Proteins; Structure-Activity Relationship; Terminology as Topic

Calpain has long been an enigmatic enzyme, although it is involved in a variety of biological phenomena. Recent progress in calpain genetics has highlighted numerous physiological contexts in which the functions of calpain are of great significance. This review focuses on recent findings in the field of calpain genetics and the importance of calpain function. Calpain is an intracellular Ca(2+)-dependent cysteine protease (EC 3.4.22.17; Clan CA, family C02) found in almost all eukaryotes. It is also present in a few bacteria, but not in archaebacteria. Calpain has limited proteolytic activity; rather, it transforms or modulates the structure and/or activity of its substrates. It is, therefore, referred to as a 'modulator protease'. Within the human genome, 15 genes (CAPN1-3, CAPN5-16) encode a calpain-like protease (CysPc) domain along with several different functional domains. Thus, calpains can be regarded as a distinct family of versatile enzymes that fulfil numerous tasks in vivo. Genetic studies show that a variety of defects in many different organisms, including lethality, muscular dystrophies and gastropathy, actually stem from calpain deficiencies. The cause-effect relationships identified by these studies form the basis for ongoing and future studies regarding the physiological role of calpains.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Eukaryota; Humans; Mice; Muscular Dystrophies; Plants; Protein Structure, Tertiary; Stomach Ulcer

One of the most common causes of unacceptability in meat quality is toughness. Toughness is attributed to a range of factors including the amount of intramuscular connective tissue, intramuscular fat, and the length of the sarcomere. However, it is apparent that the extent of proteolysis of key proteins within muscle fibres is significant determinant of ultimate tenderness. The objective of this manuscript is to describe the main endogenous proteolytic enzyme systems that have the potential to be involved in muscle post-mortem proteolysis and whether the experimental evidence available supports this involvement.

Topics: Animals; Calcium-Binding Proteins; Calpain; Caspases; Cathepsins; Chemical Phenomena; Food Technology; Humans; Meat; Muscle, Skeletal; Peptide Hydrolases; Postmortem Changes; Taste

Calpains, calcium-activated cysteine proteases with a neutral pH optimum, lead to degration of cystoskeletion and structural protein, and delayed neuronal death. The activation of calpains contribute to apoptosis. Calpain inhibitors provide a novel and potential treatment for cerebral ischemia due to improvement of cerebral infarct and ischemia.

Topics: Animals; Apoptosis; Brain Ischemia; Calcium-Binding Proteins; Calpain; Humans

Calpains, particularly conventional dimeric calpains, have claimed to be involved in the cell degeneration processes that characterize numerous disease conditions linked to dysfunctions of cellular Ca2+ homeostasis. The evidence supporting their involvement has traditionally been indirect and circumstantial, but recent work has added more solid evidence supporting the role of ubiquitous dimeric calpains in the process of neurodegeneration. The only disease condition in which a calpain defect has been conclusively involved concerns an atypical monomeric calpain: the muscle specific calpain-3, also known as p94. Inactivating defects in its gene cause a muscular dystrophy termed LGMD-2A. The molecular mechanism by which the absence of the proteolytic activity of calpain-3 causes the dystrophic process is unknown. Another atypical calpain, which has been characterized recently as a Ca2(+)-dependent protease, calpain 10, appears To be involved in the etiology of type 2 diabetes. The involvement has been inferred essentially from genetic evidence. Also in the case of type 2 diabetes the molecular mechanisms that could link the disease to calpain 10 are unknown.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Connectin; Diabetes Mellitus, Type 2; Humans; Muscle Proteins; Muscular Dystrophies, Limb-Girdle; Neurodegenerative Diseases; Protein Kinases

The calpains represent a well-conserved family of calcium-dependent cysteine proteases. They consist of several ubiquitous and tissue specific isoforms and exhibit broad substrate specificity influencing many aspects of cell physiology including migration, proliferation and apoptosis. Calpain activity in vivo is tightly regulated by its natural endogenous inhibitor calpastatin. Calpastatin specifically inhibits calpain and not other cysteine proteases by interaction with several sites on the calpain molecule. Inappropriate regulation of the calpain-calpastatin proteolytic system is associated with several important human pathological disorders including muscular dystrophy, cancer, Alzheimer's disease, neurological injury, ischaemia/reperfusion injury, atherosclerosis, diabetes and cataract formation. Recent advances in elucidating the tertiary structures of calpain 2 and its regulatory domain calpain 4, together with identification of new modes of regulating calpain activity provide new opportunities for the design of novel calpain inhibitors. Several classes of inhibitors, including peptidyl epoxide, aldehyde, and ketoamide inhibitors, targeting the active site have proven effective against the calpains and are in the process of evaluation in animal models of human disease. However, a major limitation to the clinical use of such inhibitors is their lack of specificity among cysteine proteases and other proteolytic enzymes. The development of a new class of calpain inhibitors that interact with domains outside of the catalytic site of calpain may provide greater specificity and therapeutic potential.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Humans

Calpain (Cp) is a calcium (Ca(2+))-dependent cysteine protease. Activation of the major isoforms of Cp, CpI and CpII, are required for a number of important cellular processes including adherence, shape change and migration. The current concept that cytoplasmic Cp locates and associates with its regulatory subunit (Rs) and substrates as well as translocates throughout the cell via random diffusion is not compatible with the spatial and temporal constraints of cellular metabolism. The novel finding that Cp and Rs function relies upon tenacious hydrophobic interactions with organelle membranes offers a unifying explanation for the paradoxical and puzzling features of Cp activation and regulation such as how nM concentrations of intracellular Ca(2+) can activate Cp molecules requiring muM to mM concentrations of Ca(2+) for in vitro activation, and how this protease can spatially and temporally locate specific substrates and translocate throughout the cell. We hypothesize that Cp and its regulatory moieties associate with organelles to facilitate the activation of this protease resulting in the cleavage of substrates and aid in its translocation throughout the cell.

Topics: Animals; Biological Transport, Active; Calcium-Binding Proteins; Calpain; Endoplasmic Reticulum; Golgi Apparatus; Humans; Intracellular Membranes; Models, Biological; Organelles

Amyloid beta peptide (Abeta) is not only a major constituent of extracellular fibrillary pathologies in Alzheimer's disease (AD) brains, but is also physiologically produced and metabolized in neurons. This fact led us to the notion that an age-related decrease in Abeta catabolism may contribute to the molecular pathogenesis of AD, providing a rationale for seeking proteolytic enzymes that degrade Abeta in the brain. Our recent studies have demonstrated that neprilysin is the most potent Abeta-degrading enzyme in vivo. Deficiency of endogenous neprilysin elevates the level of Abeta in brains of neprilysin-knockout mice in a gene dose-dependent manner, and an age-associated decline of neprilysin occurs in several regions of mouse brain. Neuropathological alterations in these same regions have been implicated in cognitive impairments of AD patients at an early stage of the disease. Furthermore, the level of neprilysin mRNA has been found to be significantly and selectively reduced in the hippocampus and temporal cortex of AD patients. A clarification of the role played by decreased neprilysin activity in the pathogenesis of AD has opened up the possibility of neprilysin up-regulation as a novel preventive and therapeutic approach to AD. Since the expression level and activity of neprilysin are likely to be regulated by neuropeptides and their receptors, non-peptidic agonists for these receptors might be effective agents to maintain a sufficient level of Abeta catabolism in brains of the elderly. In addition to Abeta deposits, intraneuronal fibrillary lesions, such as neurofibrillary tangles, are also a pathological hallmark of AD, and the extent of the resultant cytoskeletal disruptions may be dependent upon the activity levels of proteolytic enzymes. Among proteases for which major cytoskeletal components are good substrates, calpains were shown to participate in excitotoxic stress-induced neuritic degeneration in our recent analysis using genetically engineered mice. Moreover, we have found that this pathology can be reduced by controlling the activity of an endogenous calpain inhibitor known as calpastatin, providing a possible approach for the treatment of diverse neurodegenerative disorders, including AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Brain; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Endopeptidases; Humans; Neprilysin; Neurites; tau Proteins; Up-Regulation

The precise mechanism of the progression of advanced heart failure is unknown. We assessed a new scheme in two heart failure models: (I) congenital dilated cardiomyopathy (DCM) in TO-2 strain hamsters lacking delta-sarcoglycan (SG) gene and (II) administration of a high-dose of isoproterenol, as an acute heart failure in normal rats. In TO-2 hamsters, we followed the time course of the histological, physiological and metabolic the progressions of heart failure to the end stage. Dystrophin localization detected by immunostaining age-dependently to the myoplasm and the in situ sarcolemma fragility evaluated by Evans blue entry was increased in the same cardiomyocytes. Western blotting revealed a limited cleavage of the dystrophin protein at the rod domain, strongly suggesting a contribution of endogenous protease(s). We found a remarkable up-regulation of the amount of calpain-1 and -2, and no change of their counterpart, calpastatin. After supplementing TO-2 hearts with the normal delta-SG gene in vivo, these pathological alterations and the animals' survival improved. Furthermore, dystrophin but not delta-SG was disrupted by a high dose of isoproterenol, translocated from the sarcolemma to the myoplasm and fragmented. These results of heart failure, irrespective of the hereditary or acquired origin, indicate a vicious cycle formed by the increased sarcolemma permeability, preferential activation of calpain over calpastatin, and translocation and cleavage of dystrophin would commonly lead to advanced heart failure.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cardiomyopathy, Dilated; Cell Membrane Permeability; Cricetinae; Dependovirus; Disease Models, Animal; Dystrophin; Enzyme Activation; Genetic Therapy; Heart Failure; Isoproterenol; Mesocricetus; Models, Biological; Rats; Sarcoglycans; Sarcolemma

Calpains, the cytoplasmic Ca2+-activated regulatory proteases, have no simple and clearly definable cleavage site specificity, which is in sharp contrast to digestive (e.g., pancreatic) proteases. For calpains, an approximate 10-aa segment having a variety of sequences and spanning the scissile bond, governs proteolytic cleavage. This permissivity is a precondition for calpains to act on several different substrate proteins in the cell. The specificity of calpain action may be ensured by anchoring/targeting proteins. Intriguingly, the established endogenous inhibitor protein, calpastatin, might also serve as a storage site. Furthermore, specificity may be encoded in the 'goodness' of the undecapeptide sequence in substrate proteins. Novel approaches are needed to reveal how calpains find their substrates in cells at the proper time and location.

Topics: Calcium-Binding Proteins; Calpain; Catalysis; Peptide Hydrolases; Substrate Specificity

Calpastatin is a multiheaded inhibitor capable of inhibiting more than one calpain molecule. Each inhibitory domain of calpastatin has three subdomains, A, B, and C; A binds to domain IV and C binds to domain VI of the calpains. Crystallographic evidence shows that binding of C to domain VI involves hydrophobic interactions at a site near the first EF-hand in domain VI. Sequence homology suggests that binding of A to calpain domain IV also involves hydrophobic interactions near the EF1-hand of domain IV. Neither subdomain A nor C have inhibitory activity without subdomain B, but both increase the inhibitory activity of B. Subdomain B peptides have no inhibitory activity unless they contain at least 13 amino acids, and inhibitory activity increases with the number of amino acid residues, suggesting that inhibition requires interaction over a large area of the calpain molecule. Although subdomain B inhibition kinetically is competitive in nature, subdomain B does not seem to interact with the active site of the calpains directly, but may bind to domain III of the calpains and act to block access to the active site. It is possible that subdomain B binds to calpain only after it has been activated by Ca2+.

Topics: Animals; Binding Sites; Calcium-Binding Proteins; Calpain; Humans; Protein Binding; Protein Structure, Tertiary

Spinal cord injury (SCI) evokes an increase in intracellular free Ca(2+) level resulting in activation of calpain, a Ca(2+)-dependent cysteine protease, which cleaves many cytoskeletal and myelin proteins. Calpain is widely expressed in the central nervous system (CNS) and regulated by calpastatin, an endogenous calpain-specific inhibitor. Calpastatin degraded by overactivation of calpain after SCI may lose its regulatory efficiency. Evidence accumulated over the years indicates that uncontrolled calpain activity mediates the degradation of many cytoskeletal and membrane proteins in the course of neuronal death and contributes to the pathophysiology of SCI. Cleavage of the key cytoskeletal and membrane proteins by calpain is an irreversible process that perturbs the integrity and stability of CNS cells leading to cell death. Calpain in conjunction with caspases, most notably caspase-3, can cause apoptosis of the CNS cells following trauma. Aberrant Ca(2+) homeostasis following SCI inevitably activates calpain, which has been shown to play a crucial role in the pathophysiology of SCI. Therefore, calpain appears to be a potential therapeutic target in SCI. Substantial research effort has been focused upon the development of highly specific inhibitors of calpain and caspase-3 for therapeutic applications. Administration of cell permeable and specific inhibitors of calpain and caspase-3 in experimental animal models of SCI has provided significant neuroprotection, raising the hope that humans suffering from SCI may be treated with these inhibitors in the near future.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Glycoproteins; Humans; Spinal Cord Injuries

Calpain is a Ca(2+)-activated proteolytic enzyme involved in neurodegeneration in a variety of injuries and diseases of the central nervous system (CNS). Many calpain homologs have been discovered. Depending on the tissue distribution, calpains are broadly classified as ubiquitous and tissue-specific. Ubiquitous calpain isoforms, -calpain and m-calpain, are abundantly expressed in the CNS. Calpastatin, an endogenous protein inhibitor, regulates the activity of ubiquitous calpain. Overactivation of calpain may degrade calpastatin, limiting its regulatory efficiency. Molecular structures of calpain and calpastatin have been deduced from cDNA cloning. The precise physiological function of calpain remains elusive. However, experimental evidence strongly suggests an important role for calpain in causing neurodegeneration in various injuries and diseases of the CNS. The increase in intracellular free Ca(2+) levels in the course of injuries and diseases in the CNS causes overactivation of calpain, promoting degradation of key cytoskeletal and membrane proteins. Cleavage of these key proteins by calpain is an irreversible process that perturbs the integrity and stability of CNS cells, leading to programmed cell death or apoptosis. Calpain in conjunction with caspases can cause apoptosis of the CNS cells. An aberrant Ca(2+) homeostasis inevitably activates calpain, which plays a crucial role in the pathophysiology of the CNS injuries and diseases. Therefore, calpain is a potential therapeutic target to prevent neurodegeneration. To this end, various cell-permeable calpain inhibitors have been synthesized for pharmacological inhibition of calpain activity. Some calpain inhibitors have shown significant neuroprotection in animal models of the CNS injuries and diseases, indicating their therapeutic potential.

Topics: Brain Injuries; Calcium-Binding Proteins; Calpain; Drug Evaluation, Preclinical; Enzyme Inhibitors; Forecasting; Humans; Isoenzymes; Neurodegenerative Diseases; Spinal Cord Injuries

The calpain system originally comprised three molecules: two Ca2+-dependent proteases, mu-calpain and m-calpain, and a third polypeptide, calpastatin, whose only known function is to inhibit the two calpains. Both mu- and m-calpain are heterodimers containing an identical 28-kDa subunit and an 80-kDa subunit that shares 55-65% sequence homology between the two proteases. The crystallographic structure of m-calpain reveals six "domains" in the 80-kDa subunit: 1). a 19-amino acid NH2-terminal sequence; 2). and 3). two domains that constitute the active site, IIa and IIb; 4). domain III; 5). an 18-amino acid extended sequence linking domain III to domain IV; and 6). domain IV, which resembles the penta EF-hand family of polypeptides. The single calpastatin gene can produce eight or more calpastatin polypeptides ranging from 17 to 85 kDa by use of different promoters and alternative splicing events. The physiological significance of these different calpastatins is unclear, although all bind to three different places on the calpain molecule; binding to at least two of the sites is Ca2+ dependent. Since 1989, cDNA cloning has identified 12 additional mRNAs in mammals that encode polypeptides homologous to domains IIa and IIb of the 80-kDa subunit of mu- and m-calpain, and calpain-like mRNAs have been identified in other organisms. The molecules encoded by these mRNAs have not been isolated, so little is known about their properties. How calpain activity is regulated in cells is still unclear, but the calpains ostensibly participate in a variety of cellular processes including remodeling of cytoskeletal/membrane attachments, different signal transduction pathways, and apoptosis. Deregulated calpain activity following loss of Ca2+ homeostasis results in tissue damage in response to events such as myocardial infarcts, stroke, and brain trauma.

Topics: Animals; Calcium-Binding Proteins; Calpain; Humans; Protein Conformation; Substrate Specificity

Topics: Animals; Calcium-Binding Proteins; Calpain; Glycoproteins; Humans

It is well established that periods of increased contractile activity result in significant changes in muscle structure and function. Such morphological changes as sarcomeric Z-line disruption and sarcoplasmic reticulum vacuolization are characteristic of exercise-induced muscle injury. While the precise mechanism(s) underlying the perturbations to muscle following exercise remains to be elucidated, it is clear that disturbances in Ca2+ homeostasis and changes in the rate of protein degradation occur. The resulting elevation in intracellular [Ca2+] activates the non-lysosomal cysteine protease, calpain. Because calpain cleaves a variety of protein substrates including cytoskeletal and myofibrillar proteins, calpain-mediated degradation is thought to contribute to the changes in muscle structure and function that occur immediately following exercise. In addition, calpain activation may trigger the adaptation response to muscle injury. The purpose of this paper is to: (i) review the chemistry of the calpain-calpastatin system; (ii) provide evidence for the involvement of the non-lysosomal, calcium-activated neutral protease (calpain) in the response of skeletal muscle protein breakdown to exercise (calpain hypothesis); and (iii) describe the possible involvement of calpain in the inflammatory and regeneration response to exercise.

Topics: Calcium; Calcium-Binding Proteins; Calpain; Enzyme Activation; Exercise; Humans; Membrane Fusion; Muscle Proteins; Muscles; Sarcoplasmic Reticulum

Topics: Alzheimer Disease; Amino Acid Sequence; Animals; Apoptosis; Binding Sites; Calcium-Binding Proteins; Calpain; Cataract; Cell Cycle; Cysteine Proteinase Inhibitors; Enzyme Activation; Humans; Long-Term Potentiation; Muscular Dystrophies; Parkinson Disease; Substrate Specificity

Topics: Antithrombin III; Calcium-Binding Proteins; Calpain; DNA; DNA-Binding Proteins; Fluorescence Polarization; Macromolecular Substances; Protein Binding; TATA Box; Temperature; Thrombin

Topics: Amino Acid Sequence; Animals; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Cytosol; Humans; Isoenzymes; Membranes; Models, Biological; Molecular Sequence Data; Molecular Structure; Rats; Terminology as Topic

Selenite overdose cataract, an experimental model of nuclear cataract produced in young rats is reviewed. Topics include procedures for cataract production and assessment, metabolic and molecular changes in the epithelium of the lens, calcium accumulation, activation of calcium-activated protease system, mechanisms for crystallin precipitation, anti-cataract drug testing and relevance to human cataract.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Cataract; Chemical Precipitation; Crystallins; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Disease Models, Animal; Drug Evaluation, Preclinical; Epithelium; Forecasting; Lens, Crystalline; Rats; RNA, Messenger; Sodium Selenite

Extralysosomal degradation of proteins is carried out by proteases complex (proteasomes), calcium dependent calpains, proteases of the rough endoplasmatic reticulum and proteases of the cellular membrane. It depends on a limited proteolysis and includes the main enzymatic proteins, hormones, growth factors and cytoskeletal proteins. Thus it plays an important regulatory role in the metabolism and formation of cellular structures.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Cell Compartmentation; Cysteine Endopeptidases; Endopeptidases; Humans; Multienzyme Complexes; Organelles; Proteasome Endopeptidase Complex; Proteins; Rats

Topics: Biosensing Techniques; Calcium; Calcium-Binding Proteins; Calmodulin; Calpain; Cysteine Proteinase Inhibitors

Topics: Alzheimer Disease; Aminopeptidases; Amyloid beta-Peptides; Brain Ischemia; Calcium-Binding Proteins; Calpain; Caspase 1; Cathepsins; Cysteine Endopeptidases; Humans; Signal Transduction

Calpains are a family of calcium-dependent thiol-proteases which are proposed to be involved in many physiological processes as well as pathological conditions. Calpains are likely to be involved in processing of numerous enzymes and cytoskeletal components, thereby linking their activity to a variety of intracellular events. Although widely studied, the precise mechanism(s) involved in calpain activation and activity in vivo remain poorly understood. Initial studies suggested that calpain exists primarily as an inactive proenzyme that required autolytic cleavage for activation. It was also hypothesized that calpain associated with membrane phospholipids, serving to increase calcium sensitivity, facilitating autolytic conversion and thus activating the enzyme. These hypotheses, however, have not been universally accepted and there is increasing evidence that intact, non-autolyzed calpain is the physiologically active calpain form.

Topics: Calcium; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Enzyme Activation; Hydrolysis; Protein Processing, Post-Translational

Topics: Animals; Arthritis, Rheumatoid; Calcium-Binding Proteins; Calpain; Humans

Calpains (CANPs) are a family of calcium-dependent cysteine proteases under complex cellular regulation. By making selective limited proteolytic cleavages, they activate or alter the regulation of certain enzymes, including key protein kinases and phosphatases, and induce specific cytoskeletal rearrangements, accounting for their suspected involvement in intracellular signaling, vesicular trafficking, and structural stabilization. Calpain activity has been implicated in various aging phenomena, including cataract formation and erythrocyte senescence. Abnormal activation of the large stores of latent calpain in neurons induces cell injury and is believed to underlie neurodegeneration in excitotoxicity, Wallerian degeneration, and certain other neuropathologic states involving abnormal calcium influx. In Alzheimer's disease, we found the ratio of activated calpain I to its latent precursor isoform in neocortex to be threefold higher than that in normal individuals and those with Huntington's or Parkinson's disease. Immunoreactivity toward calpastatin, the endogenous inhibitor of calpain, was also markedly reduced in layers II-V of the neocortex in Alzheimer's disease. The excessive calpain system activation suggested by these findings represents a potential molecular basis for synaptic loss and neuronal cell death in the brain in Alzheimer's disease given the known destructive actions of calpain I and its preferential neuronal and synaptic localization. In surviving cells, persistent calpain activation may also contribute to neurofibrillary pathology and abnormal amyloid precursor protein trafficking/processing through its known actions on protein kinases and the membrane skeleton. The degree of abnormal calpain activation in the brain in Alzheimer's disease strongly correlated with the extent of decline in levels of secreted amyloid precursor protein in brain. Cytoskeletal proteins that are normally good calpain substrates become relatively calpain resistant when they are hyperphosphorylated, which may contribute to their accumulation in neurofibrillary tangles. As a major effector of calcium signals, calpain activity may mirror disturbances in calcium homeostasis and mediate important pathologic consequences of such disturbances.

Topics: Aging; Alzheimer Disease; Amyloid beta-Protein Precursor; Biological Transport; Brain; Calcium-Binding Proteins; Calpain; Humans; Neurofibrils; Protein Processing, Post-Translational

Although the Ca(2+)-dependent proteinase (calpain) system has been found in every vertebrate cell that has been examined for its presence and has been detected in Drosophila and parasites, the physiological function(s) of this system remains unclear. Calpain activity has been associated with cleavages that alter regulation of various enzyme activities, with remodeling or disassembly of the cell cytoskeleton, and with cleavages of hormone receptors. The mechanism regulating activity of the calpain system in vivo also is unknown. It has been proposed that binding of the calpains to phospholipid in a cell membrane lowers the Ca2+ concentration, [Ca2+], required for the calpains to autolyze, and that autolysis converts an inactive proenzyme into an active protease. Recent studies, however, show that the calpains bind to specific proteins and not to phospholipids, and that binding to cell membranes does not affect the [Ca2+] required for autolysis. It seems likely that calpain activity is regulated by binding of Ca2+ to specific sites on the calpain molecule, with binding to each site eliciting a response (proteolytic activity, calpastatin binding, etc.) specific for that site. Regulation must also involve an, as yet, undiscovered mechanism that increases the affinity of the Ca(2+)-binding sites for Ca2+.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Catalysis; Cell Membrane; Cytoskeleton; Invertebrates; Membrane Lipids; Phospholipids; Receptors, Cell Surface; Vertebrates

Topics: Amino Acid Sequence; Animals; Calcium; Calcium-Binding Proteins; Calpain; DNA; Humans; Molecular Sequence Data; Molecular Structure; RNA, Messenger

Topics: Amino Acid Sequence; Animals; Calcium-Binding Proteins; Calpain; Genetic Variation; Humans; Molecular Sequence Data; Structure-Activity Relationship

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Chemical Phenomena; Chemistry; Humans; Proteins

Topics: Animals; Calcium-Binding Proteins; Calpain; Humans; Peptides

Calpain is a Ca2(+)-dependent cysteine endopeptidase and calpastatin is a calpain-specific endogenous inhibitor protein. Both calpain and calpastatin are very widely distributed in various animal tissues and cells. Low (microM) Ca2(+)-requiring calpain I and high (mM) Ca2(+)-requiring calpain II are known to exist. Calpain consists of one heavy (80 kDa) and one light (30 kDa) subunit. The heavy subunits of calpains I and II are different genetic products, whereas the light subunits are the same for both calpains I and II. Molecular cloning as well as protein sequencing revealed that the heavy subunit has four domains, while the light subunit has two domains. The carboxyl terminal domain of each subunit is a calmodulin-like domain, whereas the catalytic site is located in domain 2 of the heavy subunit. Calpastatin has four internally repetitive inhibitory domains. A single domain, or even a truncated 27-mer fragment thereof, possesses inhibitory activity against calpains. Calpain shows a rather broad substrate specificity. It can cleave various enzymes, and cytoskeletal, membrane and receptor proteins. Calpain-catalyzed activation of protein kinase C and transglutaminase may represent a few of the physiological functions of calpain, but a great many other functions can be assigned as well to calpain. Immunohistochemical studies revealed very wide but quite diverse distribution of calpains I and II and calpastatin among various tissues and cells. The expression of the genes for calpain and calpastatin is found to be modulated by retrovirus (HTLV-I) infection to T-lymphocytes. The physiological significance of the calpain and calpastatin system is yet to be elucidated, and accumulating information definitely suggested the role of calpain/calpastatin in health and disease.

Topics: Amino Acid Sequence; Animals; Calcium; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Humans; Molecular Sequence Data; Molecular Structure; Rats; Substrate Specificity; Swine

Seven years have elapsed since the terms calpain and calpastatin were introduced. During these years, significant progress in research has been recorded. Thus, cloning and sequencing of cDNAs for calpains I and II and calpastatin have established amino acid sequences of these molecules. Structure-function relationship of calpastatin has been studied using mutated cDNAs expressed in E. coli. Interleukin 2 receptor-linked expression of calpastatin in HTLV-I-infected T-cells has been reported. Evidence for Ca2+-induced translocation of calpain to the cell membrane, followed by its autolytic activation, has been discussed. A great varieties of proteins such as several kinases, membrane and cytoskeletal proteins, and hormone receptors have been reported to be susceptible to calpains. This paper is to summarize our current knowledge on chemistry and biology of calpain and calpastatin and thereby to speculate the true function of calpains and their regulatory mechanisms.

Topics: Amino Acid Sequence; Animals; Base Sequence; Calcium-Binding Proteins; Calpain; Gene Expression Regulation; Humans; Molecular Sequence Data; Terminology as Topic

The intracellular calcium-dependent proteases (calpains) and their endogenous protein inhibitor (calpastatin) are present in many different mammalian cells. There is emerging evidence for their importance in the turnover of membrane-associated proteins. Accordingly, it is important to understand how these proteinases and their inhibitor interact within cells, in particular at membranes. Bovine myocardial calpastatin appears to be associated in part with intracellular membranes, where it may effectively block the activity of calpain II on membrane-associated proteins. Immuno-electron microscopic studies suggest that canine myocardial calpain and calpastatin are associated with a number of membranous organelles. During canine myocardial autolysis, the amount of calpain at various organelles decreased, but the amount of calpastatin decreased to an even greater extent. Thus there may be a high calpain to calpastatin balance during heart ischemia at these sites. Calpain II aggregation may contribute to localization of the proteinase at sites of high calcium concentration within cells. A model is presented for interaction of calpain II and calpastatin at cellular membranes in the presence of calcium.

Topics: Amino Acid Sequence; Animals; Autolysis; Blotting, Western; Calcium-Binding Proteins; Calpain; Cattle; Cell Membrane; Dogs; Erythrocytes; Molecular Sequence Data; Molecular Weight; Myocardium; Peptide Mapping; Phospholipids; Protein Binding; Sarcolemma; Sarcoplasmic Reticulum

Topics: Amino Acid Sequence; Animals; Calcium; Calcium-Binding Proteins; Calpain; Humans; Ions

There are two forms of Ca2+-dependent cysteine proteinase: low Ca2+ (microM concentration)-requiring calpain I and high Ca2+ (mM concentration)-requiring calpain II. We have recently succeeded in establishing an efficient method for isolating calpains I and II from several tissues and also a method for separating the heavy (80 kDa) and light (30 kDa) subunits from a calpain molecule. These methods enabled us to obtain a variety of calpains I and II and their subunit proteins each in a highly pure state so that these proteins can be compared physically, chemically and immunologically. Calpastatin, a calpain-specific endogenous inhibitor protein, was also purified and characterized. Although the intercellular localization of calpains I and II is being elucidated by use of the respective monospecific antibodies, the true biological functions of calpain and calpastatin still remain rather obscure.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Erythrocytes; Humans; Kidney; Liver; Peptide Fragments; Protease Inhibitors; Rats; Substrate Specificity; Swine

In this study, we characterized the effects of 25-hydroxyvitamin D3 (25-OH D3) and manipulated dietary cation-anion difference (DCAD) on the performance, urine pH, serum constituents, carcass traits, tissue residual vitamin D and its metabolites, beef tenderness, and mRNA and protein concentrations of Ca-dependent proteinases in LM using 24 cull native Korean cows. The cows were divided into 3 groups of 8: control, 25-OH D3 supplemented (25-OH D3), and manipulated DCAD plus 25-OH D3 supplemented (DCAD+25-OH D3). Cows receiving 25-OH D3 or DCAD+25-OH D3 were dosed with 125 mg of 25-OH D3 6 d before slaughter. The manipulated DCAD (-10 mEq/100 g of DM) diet was fed from 20 to 6 d (14 d) before slaughter. The DCAD+25-OH D3 treatment decreased urine pH and increased serum Ca concentrations. Although the vitamin D concentrations in LM, liver, and kidney were not affected by 25-OH D3 or DCAD+25-OH D3, muscle tissue 25-OH D3 concentrations were increased by both regimens. Serum 25-OH D3 concentrations were increased by 25-OH D3 supplementation, and the increase was even greater for DCAD+25-OH D3. The same pattern was observed for serum 1,25- (OH)2 D3. However, the LM concentration of 1,25-(OH)2 D3 was less for DCAD+25-OH D3 than for control. Although Ca concentrations of LM increased numerically in response to 25-OH D3 supplementation, no statistical differences in Warner-Bratzler shear force or sensory traits of LM were detected. The LM of cows receiving 25-OH D3 with or without manipulated DCAD had greater concentrations of mu-calpain and m-calpain mRNA, whereas the reverse was observed for calpastatin mRNA. Expression of mu-calpain protein was increased relative to control by DCAD+25-OH D3. The amount of 25-OH D3 and manipulated DCAD administered to cull native Korean cows was insufficient to improve tenderness of beef by increasing muscle Ca concentration. However, DCAD+25-OH D3 induced greater expressions of mu-calpain protein as well as mRNA.

Topics: Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Body Weight; Calcifediol; Calcitriol; Calcium-Binding Proteins; Calpain; Cattle; Diet; Gene Expression Regulation, Enzymologic; Korea; Male; Meat; Muscle, Skeletal

The objective of this study was to determine whether the improvements in growth and efficiency of gain achieved by recombinant porcine somatotropin (pST) are associated with altered expression of the p94, calpastatin, or alpha-actin genes in porcine longissimus (LD) muscle. Forty-eight barrows (initial 64.2 to 67.4 kg BW) were assigned to four treatments (n = 12) arranged as a 2 x 2 factorial in a randomized complete block design. Factors were duration of treatment (3 or 6 wk) and pST administration (0 or 3 mg x pig(-1) x d(-1)). Plasma samples were obtained 24 h after the first pST injection and at the end of the each treatment period for assays of selected variables. The LD samples were obtained at 3 and 6 wk of pST treatment. Northern blot analysis of calpastatin expression in LD muscle revealed three distinct transcription products of approximately 8.5 (CPST I), 5.5 (CPST II), and 2.5 (CPST III) kb; CPST II was reduced (P < .02) 33 and 61% by pST at 3 and 6 wk, respectively, whereas CPST I and III were not influenced (P > .12). Neither alpha-actin nor p94 was responsive to pST injection. As expected, pST resulted in higher (50%, P < .02) plasma insulin within 24 h and one- and twofold higher (P < .01) concentrations at 3 and 6 wk, respectively. Glucose was increased (P < .01) at 3 (15%) and 6 (10%) wk, whereas urea nitrogen was reduced (32 to 36%, P < .01). The efficacy of pST was evident in that ADG was improved (P < .01) 11 to 13% independent of time. Likewise, feed intake was reduced (P < .01) 10 to 11% and gain: feed improved (P < .01) approximately 26% for pigs receiving pST independent of time. These data indicate that the enhanced muscle growth achieved by pST is not associated with altered expression of p94 or alpha-actin, or an increase in the abundance of any calpastatin transcription product.

Topics: Actins; Animals; Blood Glucose; Blood Urea Nitrogen; Blotting, Northern; Calcium-Binding Proteins; Calpain; Gene Expression; Growth Hormone; Insulin; Male; Recombinant Proteins; RNA, Messenger; Swine; Transcription, Genetic; Weight Gain

Axon degeneration accounts for the poor clinical outcome in Guillain-Barré syndrome (GBS), yet no treatments target this key pathogenic stage. Animal models demonstrate anti-ganglioside antibodies (AGAb) induce axolemmal complement pore formation through which calcium flux activates the intra-axonal calcium-dependent proteases, calpains. We previously showed protection of axonal components using soluble calpain inhibitors in ex vivo GBS mouse models, and herein, we assess the potential of axonally-restricted calpain inhibition as a neuroprotective therapy operating in vivo. Using transgenic mice that over-express the endogenous human calpain inhibitor calpastatin (hCAST) neuronally, we assessed distal motor nerve integrity in our established GBS models. We induced immune-mediated injury with monoclonal AGAb plus a source of human complement. The calpain substrates neurofilament and AnkyrinG, nerve structural proteins, were assessed by immunolabelling and in the case of neurofilament, by single-molecule arrays (Simoa). As the distal intramuscular portion of the phrenic nerve is prominently targeted in our in vivo model, respiratory function was assessed by whole-body plethysmography as the functional output in the acute and extended models. hCAST expression protects distal nerve structural integrity both ex and in vivo, as shown by attenuation of neurofilament breakdown by immunolabelling and Simoa. In an extended in vivo model, while mice still initially undergo respiratory distress owing to acute conduction failure, the recovery phase was accelerated by hCAST expression. Axonal calpain inhibition can protect the axonal integrity of the nerve in an in vivo GBS paradigm and hasten recovery. These studies reinforce the strong justification for developing further animal and human clinical studies using exogenous calpain inhibitors.

Topics: Animals; Axons; Calcium; Calpain; Guillain-Barre Syndrome; Humans; Mice; Mice, Transgenic

Two methods that the beef cattle industry can use to improve efficiency, sustainability, and economic viability are growth promotants and crossbreeding cattle of different breed types. In the United States, over 90% of cattle receive an anabolic implant at some point during production resulting in an overall increase in skeletal muscle growth. Recent research suggests that the two main cattle breed types, Bos indicus and Bos taurus, respond differently to anabolic implants. The objective of this study was to characterize changes that occur in skeletal muscle following implanting in Bos indicus influenced steers or Bos taurus steers. Twenty steers were stratified by initial weight in a 2 × 2 factorial design examining two different breeds: Angus (AN; n = 10) or Santa Gertrudis influenced (SG; n = 10), and two implant strategies: no implant (CON; n = 10) or a combined implant containing 120 mg TBA and 24 mg E2 (IMP; n = 10; Revalor-S, Merck Animal Health). Skeletal muscle biopsies were taken from the longissimus thoracis (LT) 2 and 10 d post-implantation. The mRNA abundance of 24 genes associated with skeletal muscle growth were examined, as well as the protein expression of µ-calpain and calpastatin. Succinate dehydrogenase mRNA abundance was impacted (P = 0.05) by a breed × treatment interaction 2 d post-implanting, with SG-CON having a greater increased abundance than all other steers. A tendency for a breed × treatment interaction was observed for calpain-6 mRNA (P = 0.07), with SG-CON having greater abundance than AN-CON and SG-IMP. Additionally, calpastatin protein expression was altered (P = 0.01) by a breed × treatment interaction, with SG-CON and SG-IMP steers having increased expression (P = 0.01) compared with AN-CON steers. At 2 d post-implanting, a breed × treatment interaction was observed with SG-CON steers having greater (P = 0.05) mRNA abundance of mitogen-activated protein kinase compared with AN-CON steers. Furthermore, breed affected (P = 0.05) calpastatin abundance with AN steers having increased (P = 0.05) abundance 2 d post-implanting compared with SG steers. Meanwhile, implants tended to affect (P = 0.09) muscle RING finger protein-1 mRNA abundance, with CON steers having increased (P = 0.09) abundance compared with that of IMP steers. These findings suggest that cattle breed type and anabolic implants impact calpastatin expression and mRNA abundance associated with protein turnover in the LT of feedlot steers 2 and 10 d post-implant. Two methods that the beef cattle industry can use to potentially improve efficiency, sustainability, and economic viability are growth promotants and crossbreeding cattle of different breed types. In the United States, over 90% of cattle receive at least one anabolic implant during the production cycle resulting in improvements in production and overall economic and environmental sustainability. Research suggests that the two main cattle breed types, Bos indicus and Bos taurus, respond differently to different anabolic implant strategies. The objective of this study was to characterize changes that occur in the skeletal muscle following implanting in Bos indicus influenced animals and Bos taurus animals. This research measured mRNA abundance of 24 genes associated with skeletal muscle growth, and protein expression of calpain-1 and calpastatin. The findings of this research suggest that anabolic implants and cattle breed type interact to cause changes in mRNA abundance in the longissimus thoracis that are related to protein turnover of skeletal muscle. Furthermore, calpastatin protein abundance was also altered by this breed × treatment interaction. This research demonstrates that anabolic implants cause molecular changes in skeletal muscle of feedlot steers, with some of these changes being breed dependent.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Muscle, Skeletal; RNA, Messenger; Trenbolone Acetate

Mitochondrial dysfunction is a common hallmark of neurological disorders, and reducing mitochondrial damage is considered a promising neuroprotective therapeutic strategy. Here, we used high-throughput small molecule screening to identify CHIR99021 as a potent enhancer of mitochondrial function. CHIR99021 improved mitochondrial phenotypes and enhanced cell viability in several models of Huntington's disease (HD), a fatal inherited neurodegenerative disorder. Notably, CHIR99201 treatment reduced HD-associated neuropathology and behavioral defects in HD mice and improved mitochondrial function and cell survival in HD patient-derived neurons. Independent of its known inhibitory activity against glycogen synthase kinase 3 (GSK3), CHIR99021 treatment in HD models suppressed the proteasomal degradation of calpastatin (CAST), and subsequently inhibited calpain activation, a well-established effector of neural death, and Drp1, a driver of mitochondrial fragmentation. Our results established CAST-Drp1 as a druggable signaling axis in HD pathogenesis and highlighted CHIR99021 as a mitochondrial function enhancer and a potential lead for developing HD therapies.

Topics: Animals; Calcium-Binding Proteins; Calpain; Corpus Striatum; Disease Models, Animal; Dynamins; Gene Expression Regulation; Glycogen Synthase Kinase 3 beta; Humans; Huntington Disease; Injections, Intraperitoneal; Male; Mice; Mitochondria; Neurons; Neuroprotective Agents; Primary Cell Culture; Proteasome Endopeptidase Complex; Proteolysis; Pyridines; Pyrimidines; Signal Transduction

Kinases play central roles in signaling cascades, relaying information from the outside to the inside of mammalian cells. De novo designed protein switches capable of interfacing with tyrosine kinase signaling pathways would open new avenues for controlling cellular behavior, but, so far, no such systems have been described. Here we describe the de novo design of two classes of protein switch that link phosphorylation by tyrosine and serine kinases to protein-protein association. In the first class, protein-protein association is required for phosphorylation by the kinase, while in the second class, kinase activity drives protein-protein association. We design systems that couple protein binding to kinase activity on the immunoreceptor tyrosine-based activation motif central to T-cell signaling, and kinase activity to reconstitution of green fluorescent protein fluorescence from fragments and the inhibition of the protease calpain. The designed switches are reversible and function in vitro and in cells with up to 40-fold activation of switching by phosphorylation.

Topics: Amino Acid Motifs; Binding, Competitive; Calcium-Binding Proteins; Calpain; Catalysis; Catalytic Domain; Cell Line; Cyclic AMP-Dependent Protein Kinases; Drug Design; Genes, Synthetic; Green Fluorescent Proteins; Humans; Hydrogen Bonding; Models, Molecular; Phosphorylation; Phosphotyrosine; Protein Binding; Protein Conformation; Protein Domains; Protein Interaction Mapping; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Recombinant Proteins; Signal Transduction; src-Family Kinases; Structure-Activity Relationship

The mechanism of fetal growth restriction (FGR) is not fully understood. In this study, we explored the contribution of the calpain-calpastatin system and the activated states of calpains in human FGR placenta.. The placentas were collected from patients of FGR (n = 17) and controls (n = 23) at elective cesarean sections in Nagoya City University Hospital and used for experiments upon informed consent. The existence and the expression of calpains and calpastatin in human placenta were compared between FGR and controls using immunohistochemistry, SDS-PAGE, and Western blotting.. Staining of calpains (pre-, post-μ-calpain, pre-, post-m-calpain, and calpain-6) and calpastatin was observed in cytoplasm of trophoblast cells, both in FGR and control placenta. Pre-μ-calpain was located in the cytoplasm, and post-μ-calpain was located mainly in proximity to the cytoplasmic membrane. The expression of pre-μ-calpain was significantly higher (P < .001) and calpain-6 was significantly lower (P = .01) in FGR placentas. The inactive μ-calpain (80 kDa) was significantly elevated (P < .01), and active μ-calpain (76 kDa) was significantly decreased (P = .01) in FGR placentas.. The results demonstrate that activation of μ-calpain is suppressed in FGR placentas and that calpain-6 in human placenta is involved in the pathology of FGR.

Topics: Adult; Calcium-Binding Proteins; Calpain; Female; Fetal Growth Retardation; Humans; Placenta; Pregnancy

The protein levels and activities of calpain-1 and calpain-2 are increased in cardiac mitochondria under pathological conditions including ischemia, diabetes, and sepsis, and transgenic overexpression of mitochondrial-targeted calpain-1 induces dilated heart failure, which underscores an important role of increased calpain in mitochondria in mediating myocardial injury. However, it remains to be determined whether selective inhibition of calpain in mitochondria protects the heart under pathological conditions. In this study, we generated transgenic mice overexpressing mitochondrial-targeted calpastatin in cardiomyocytes. Their hearts were isolated and subjected to global ischemia/reperfusion. Hyperglycemia was induced in the transgenic mice by injections of STZ. We showed that transgenic calpastatin was expressed exclusively in mitochondria isolated from their hearts but not from other organs including skeletal muscle and lung tissues. Transgenic overexpression of mitochondrial-targeted calpastatin significantly attenuated mitochondrial oxidative stress and cell death induced by global ischemia/reperfusion in isolated hearts, and ameliorated mitochondrial oxidative stress, cell death, myocardial remodeling and dysfunction in STZ-treated transgenic mice. The protective effects of mitochondrial-targeted calpastatin were correlated with increased ATP5A1 protein expression and ATP synthase activity in isolated hearts subjected to global ischemia/reperfusion and hearts of STZ-treated transgenic mice. In cultured rat myoblast H9c2 cells, overexpression of mitochondrial-targeted calpastatin maintained the protein levels of ATP5A1 and ATP synthase activity, prevented mitochondrial ROS production and decreased cell death following hypoxia/reoxygenation, whereas upregulation of ATP5A1 or scavenging of mitochondrial ROS by mito-TEMPO abrogated mitochondrial ROS production and decreased cell death. These results confirm the role of calpain in myocardial injury, suggesting that selective inhibition of calpain in myocardial mitochondria by mitochondrial-targeted calpastatin is an effective strategy for alleviating myocardial injury and dysfunction in cardiac pathologies.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Cardiomegaly; Diabetes Mellitus, Experimental; Fibrosis; Male; Mice, Transgenic; Mitochondria, Heart; Mitochondrial Proton-Translocating ATPases; Myocytes, Cardiac; Oxidative Stress; Reactive Oxygen Species

Studies that investigate the expression of genes related to the tenderness of meat from entire and immunocastrated male pigs have not yet been performed. The objective of this study was to investigate the association between gender (entire male and immunocastrated) and the meat quality of pigs, as well as to quantify the expression of calpain-1 and the calpastatin gene. Regarding carcass measurements and meat quality, boars presented lower values of muscle depth (P = 0.028), subcutaneous fat thickness (P = 0.046), L* value (P = 0.004) and cook loss (P = 0.008) than the immunocastrated pigs. The boars presented greater calpain-1 gene expression (P = 0.006) and lower calpastatin gene expression (P = 0.003) than immunocastrated pigs. This study shows that combined with other factors the gene expression can contribute to a tender meat from boars due to their higher calpain-1 expression and lower calpastatin expression than those of immunocastrated male pigs.

Topics: Animals; Body Composition; Calcium-Binding Proteins; Calpain; Gene Expression; Male; Muscle, Skeletal; Orchiectomy; Pork Meat; Shear Strength; Sus scrofa

The calpain system is required by many important physiological processes, including the cell cycle, cytoskeleton remodelling, cellular proliferation, migration, cancer cell invasion, metastasis, survival, autophagy, apoptosis and signalling, as well as the pathogenesis of a wide range of disorders, in which it may function to promote tumorigenesis. Calpains are intracellular conserved calcium-activated neutral cysteine proteinases that are involved in mediating cancer progression via catalysing and regulating the proteolysis of their specific substrates, which are important signalling molecules during cancer progression. μ-calpain, m-calpain, and their specific inhibitor calpastatin are the three molecules originally identified as comprising the calpain system and they contain several crucial domains, specific motifs, and functional sites. A large amount of data supports the roles of the calpain-calpastatin system in cancer progression via regulation of cellular adhesion, proliferation, invasion, metastasis, and cellular survival and death, as well as inflammation and angiogenesis during tumorigenesis, implying that the inhibition of calpain activity may be a potential anti-cancer intervention strategy targeting cancer cell survival, invasion and chemotherapy resistance.

Topics: Calcium-Binding Proteins; Calpain; Cell Proliferation; Humans; Neoplasms

Hypertrophic scars, the most common complication of burn injuries, are characterized by excessive deposition of fibroblast-derived extracellular matrix proteins. Calpain, a calcium-dependent protease, is involved in the fibroblast proliferation and extracellular matrix production observed in certain fibrotic diseases. However, its role in the formation of post-burn hypertrophic skin scars remains largely unknown. Here, calpain expression and activity were assessed in skin fibroblasts obtained directly from patients with third-degree burns, who consequently developed post-burn hypertrophic scars. Furthermore, the antifibrotic effect of calpastatin, an endogenous calpain inhibitor, was evaluated in human fibroblasts and a murine burn model. The activity, mRNA levels, and protein levels of calpain were markedly higher in fibroblasts from the burn wounds of patients than in normal cells. Selective calpain inhibition by calpastatin markedly reduced not only the proliferation of burn-wound fibroblasts but also the mRNA and protein expression of calpain, transforming growth factor-beta 1, α-smooth muscle actin, type I and type III collagens, fibronectin, and vimentin in burn-wound fibroblasts. The anti-scarring effects of calpastatin were validated using a murine burn model by molecular, histological, and visual analyses. This study demonstrates the pathological role of calpain and the antifibrotic effect of calpastatin via calpain inhibition in post-burn hypertrophic scar formation.

Topics: Adult; Animals; Burns; Calcium-Binding Proteins; Calpain; Cell Proliferation; Cicatrix, Hypertrophic; Collagen Type III; Extracellular Matrix; Extracellular Matrix Proteins; Female; Fibroblasts; Fibronectins; Humans; Hypertrophy; Male; Mice; Middle Aged; RNA, Messenger; Skin; Transforming Growth Factor beta1; Young Adult

Topics: Aging; Animals; Calcium-Binding Proteins; Calpain; Longevity; Mice; Muscle Weakness; Muscle, Skeletal

Ischemia/reperfusion (I/R) injury unavoidably occurs during hepatic resection and transplantation. Aged livers poorly tolerate I/R during surgical treatment. Although livers have a powerful endogenous inhibitor of calpains, calpastatin (CAST), I/R activates calpains, leading to impaired autophagy, mitochondrial dysfunction, and hepatocyte death. It is unknown how I/R in aged livers affects CAST. Human and mouse liver biopsies at different ages were collected during in vivo I/R. Hepatocytes were isolated from 3-month- (young) and 26-month-old (aged) mice, and challenged with short in vitro simulated I/R. Cell death, protein expression, autophagy, and mitochondrial permeability transition (MPT) between the two age groups were compared. Adenoviral vector was used to overexpress CAST. Significant cell death was observed only in reperfused aged hepatocytes. Before the commencement of ischemia, CAST expression in aged human and mouse livers and mouse hepatocytes was markedly greater than that in young counterparts. However, reperfusion substantially decreased CAST in aged human and mouse livers. In hepatocytes, reperfusion rapidly depleted aged cells of CAST, cleaved autophagy-related protein 5 (ATG5), and induced defective autophagy and MPT onset, all of which were blocked by CAST overexpression. Furthermore, mitochondrial morphology was shifted toward an elongated shape with CAST overexpression. In conclusion, CAST in aged livers is intrinsically short-lived and lost after short I/R. CAST depletion contributes to age-dependent liver injury after I/R.

Topics: Age Factors; Animals; Autophagy; Autophagy-Related Protein 5; Calcium-Binding Proteins; Calpain; Cell Death; Cells, Cultured; Disease Models, Animal; Gene Expression Regulation; Hepatocytes; Humans; Liver; Liver Diseases; Male; Mice, Inbred C57BL; Mitochondria, Liver; Reperfusion Injury; Signal Transduction; Time Factors

Calpains, intracellular proteases specifically inhibited by calpastatin, play a major role in neoangiogenesis involved in tumor invasiveness and metastasis. They are partly exteriorized via the ATP-binding cassette transporter A1(ABCA1) transporter, but the importance of this process in tumor growth is still unknown. The aim of our study was to investigate the role of extracellular calpains in a model of melanoma by blocking their extracellular activity or exteriorization. In the first approach, a B16-F10 model of melanoma was developed in transgenic mice expressing high extracellular levels of calpastatin. In these mice, tumor growth was inhibited by ∼ 3-fold compared with wild-type animals. In vitro cytotoxicity assays and in vivo tumor studies have demonstrated that this protection was associated with a defect in tumor neoangiogenesis. Similarly, in wild-type animals given probenecid to blunt ABCA1 activity, melanoma tumor growth was inhibited by ∼ 3-fold. Again, this response was associated with a defect in neoangiogenesis. In vitro studies confirmed that probenecid limited endothelial cell migration and capillary formation from vascular explants. The observed reduction in fibronectin cleavage under these conditions is potentially involved in the response. Collectively, these studies demonstrate that probenecid, by blunting ABCA1 activity and thereby calpain exteriorization, limits melanoma tumor neoangiogenesis and invasiveness.

Topics: Animals; ATP Binding Cassette Transporter 1; Calcium-Binding Proteins; Calpain; Cell Line, Tumor; Cell Proliferation; Humans; Male; Melanoma, Experimental; Mice; Mice, Transgenic; Neovascularization, Pathologic; Probenecid; Skin Neoplasms

The purpose of this study was to examine the role of mitochondria in postmortem calcium homeostasis and its effect on proteolysis and tenderness. We hypothesized that mitochondria buffer cytosolic calcium levels and delay the activation of calpain-1 and subsequently the development of meat tenderness. To test this hypothesis, pre-rigor bovine longissimus thoracis et lumborum muscle samples were injected with DS16570511 to inhibit mitochondrial calcium uptake. Free calcium, tenderness, texture profile analysis (TPA), calpain-1 activity, and proteolysis were evaluated over a 336 h aging period. Inhibition of mitochondrial calcium uptake increased (P < .0001) cytosolic calcium concentration and calpain-1 autolysis and activity at 24 h compared to control steaks. Further, tenderness and TPA at 168 and 336 h, calpastatin degradation at 24 h, and proteolysis at 168 h were all enhanced (P < .05) in the treated steaks. Collectively, these data indicate that inhibition of mitochondrial calcium uptake can enhance postmortem proteolysis and tenderization through an early activation of calpain-1.

Topics: Animals; Calcium; Calcium Channels; Calcium-Binding Proteins; Calpain; Cattle; Male; Mitochondria, Muscle; Muscle, Skeletal; Proteolysis; Red Meat; Shear Strength

Skeletal muscle atrophy is the net loss of muscle mass that results from an imbalance in protein synthesis and protein degradation. It occurs in response to several stimuli including disease, injury, starvation, and normal aging. Currently, there is no truly effective pharmacological therapy for atrophy; therefore, exploration of the mechanisms contributing to atrophy is essential because it will eventually lead to discovery of an effective therapeutic target. The ether-a-go-go related gene (ERG1A) K. We transduced C. Human ERG1A expression increases both intracellular calcium concentration and combined calpain 1 and 2 activity. The increased calpain activity is likely a result of the increased calcium levels and decreased calpastatin abundance.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Cell Line; ERG1 Potassium Channel; Male; Mice; Muscle Fibers, Skeletal

Myosin heavy chain (MHC) isoforms in goat muscles and their possible relationships with meat quality have not been fully elucidated. This study characterized the MHC isoforms in different caprine muscles using sodium dodecyl sulphate glycerol gel electrophoresis (SDS-GGE). The relationships between MHC isoforms, calpain systems and meat quality characteristics of different muscles in goats were examined. Four muscles, namely infraspinatus (IF), longissimus dorsi (LD), psoas major (PM) and supraspinatus (SS) were obtained from ten Boer crossbred bucks (7-10 months old; 26.5 ± 3.5 kg, BW). The percentages of MHC I, MHC IIa and MHC IIx in SS, IF, PM and LD were 47.2, 38.3, 32.1, 11.9; 28.0, 42.1, 33.0, 36.4; and 24.8, 19.6, 34.9 and 51.7, respectively. IF and SS had higher levels of calpastatin, total collagen and insoluble collagen contents than did PM and LD. PM had longer sarcomere length than did other muscles. LD had higher collagen solubility, troponin-T degradation products and glycogen content than did other muscles. These results infer that variable fiber-type composition could account partially for the differences in the physicochemical properties of goat muscles.

Topics: Animals; Calcium-Binding Proteins; Calpain; Electrophoresis; Electrophoresis, Polyacrylamide Gel; Goats; Meat; Muscle, Skeletal; Myosin Heavy Chains; Protein Isoforms; Troponin T

Pathological cardiac hypertrophy is ultimately accompanied by cardiomyocyte apoptosis. Apoptosis mainly related to calpain-1-mediated apoptotic pathways. Studies had proved that taurine can maintain heart health through antioxidation and antiapoptotic functions, but the effect of taurine on cardiac hypertrophy is still unclear. This study aimed to determine whether taurine could inhibit calpain-1-mediated mitochondria-dependent apoptotic pathways in isoproterenol (ISO)-induced hypertrophic cardiomyocytes. We found that taurine could inhibit the increase in cell surface area and reduce the protein expression levels of the hypertrophic markers atrial natriuretic peptide, brain natriuretic polypeptide, and β-myosin heavy chain. Taurine also reduced ROS, intracellular Ca

Topics: Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Atrial Natriuretic Factor; bcl-2-Associated X Protein; Calcium; Calcium-Binding Proteins; Calpain; Cardiomegaly; Caspase 3; Caspase 9; Cell Line; Cytochromes c; Isoproterenol; Membrane Potential, Mitochondrial; Mitochondria; Myocytes, Cardiac; Natriuretic Peptide, Brain; Natriuretic Peptides; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Rats; Taurine; Ventricular Myosins

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by loss of the survival motor neuron 1 (SMN1) gene. SMA is characterized by the degeneration of spinal cord motoneurons (MNs), progressive skeletal muscle atrophy, and weakness. The cellular and molecular mechanisms causing MN loss of function are only partially known. Recent advances in SMA research postulate the role of calpain protease regulating survival motor neuron (SMN) protein and the positive effect on SMA phenotype of treatment with calpain inhibitors. We analyzed the level of calpain pathway members in mice and human cellular SMA models. Results indicate an increase of calpain activity in SMN-reduced MNs. Spinal cord analysis of SMA mice treated with calpeptin, a calpain inhibitor, showed an increase of SMN, calpain, and its endogenous inhibitor calpastatin in MNs. Finally, in vitro calpeptin treatment prevented microtubule-associated protein 1A/1B-light chain 3 (LC3) increase in MNs neurites, indicating that calpain inhibition may reduce autophagosome accumulation in neuron prolongations, but not in soma. Thus, our results show that calpain activity is increased in SMA MNs and its inhibition may have a beneficial effect on SMA phenotype through the increase of SMN in spinal cord MNs.

Topics: Animals; Calcium-Binding Proteins; Calpain; Carrier Proteins; Cell Differentiation; Cell Line; Cell Survival; Cells, Cultured; Dipeptides; Disease Models, Animal; Fibroblasts; Humans; Induced Pluripotent Stem Cells; Mice; Mice, Mutant Strains; Microfilament Proteins; Microtubule-Associated Proteins; Motor Neurons; Muscular Atrophy, Spinal; Proteolysis; Spinal Cord; Survival of Motor Neuron 1 Protein

Intrinsically disordered proteins (IDPs) play important roles in the regulation of cellular function and in disease, and thus they represent an important group of therapeutic targets. Yet, members of this "disorderome" have not yet been successfully targeted by drugs, primarily because traditional design principles cannot be applied to their highly dynamic, heterogeneous structural states. Binders developed against IDPs so far suffer from very weak binding and inability to act in a cellular context. Here, we describe a possible generic method for the targeting of IDPs via covalent modification, which could entail specific and strong binding and inhibitory potential, making such "warheads" reasonable starting points of drug-development efforts. We demonstrate this principle by addressing the cysteine-specific covalent modification of calpastatin, the IDP inhibitor of the calcium-dependent cysteine protease calpain. We describe the protocol for monitoring the covalent modification of the inhibitor, measuring the K

Topics: Calcium-Binding Proteins; Calpain; Circular Dichroism; Cysteine; Dithionitrobenzoic Acid; Drug Design; Electrophoresis, Polyacrylamide Gel; Humans; Interferometry; Intrinsically Disordered Proteins; Kinetics; Molecular Structure; Molecular Targeted Therapy; Protein Binding; Structure-Activity Relationship; Tandem Mass Spectrometry

Mitochondrial dynamic disorder is involved in myocardial ischemia/reperfusion (I/R) injury. To explore the effect of mitochondrial calcium uniporter (MCU) on mitochondrial dynamic imbalance under I/R and its related signal pathways, a mouse myocardial I/R model and hypoxia/reoxygenation model of mouse cardiomyocytes were established. The expression of MCU during I/R increased and related to myocardial injury, enhancement of mitochondrial fission, inhibition of mitochondrial fusion and mitophagy. Suppressing MCU functions by Ru360 during I/R could reduce myocardial infarction area and cardiomyocyte apoptosis, alleviate mitochondrial fission and restore mitochondrial fusion and mitophagy. However, spermine administration, which could enhance MCU function, deteriorated the above-mentioned myocardial cell injury and mitochondrial dynamic imbalanced. In addition, up-regulation of MCU promoted the expression and activation of calpain-1/2 and down-regulated the expression of Optic atrophy type 1 (OPA1). Meantime, in transgenic mice (overexpression calpastatin, the endogenous inhibitor of calpain) I/R model and OPA1 knock-down cultured cell. In I/R models of transgenic mice over-expressing calpastatin, which is the endogenous inhibitor of calpain, and in H/R models with siOPA1 transfection, inhibition of calpains could enhance mitochondrial fusion and mitophagy, and inhibit excessive mitochondrion fission and apoptosis through OPA1. Therefore, we conclude that during I/R, MCU up-regulation induces calpain activation, which down-regulates OPA1, consequently leading to mitochondrial dynamic imbalance.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Apoptosis; Calcium Channels; Calcium-Binding Proteins; Calpain; Enzyme Inhibitors; GTP Phosphohydrolases; Homeostasis; Male; Mice, Inbred C57BL; Mice, Transgenic; Mitochondria, Heart; Mitochondrial Dynamics; Mitophagy; Myocardial Infarction; Myocardial Reperfusion Injury; Protective Agents; Rats; Up-Regulation

Despite the high and preferential expression of p38γ MAPK in the myocardium, little is known about its function in the heart. The aim of the current study was to elucidate the physiologic and biochemical roles of p38γ in the heart. Expression and subcellular localization of p38 isoforms was determined in mouse hearts. Comparisons of the cardiac function and structure of wild-type and p38γ knockout (KO) mice at baseline and after abdominal aortic banding demonstrated that KO mice developed less ventricular hypertrophy and that contractile function is better preserved. To identify potential substrates of p38γ, we generated an analog-sensitive mutant to affinity tag endogenous myocardial proteins. Among other proteins, this technique identified calpastatin as a direct p38γ substrate. Moreover, phosphorylation of calpastatin by p38γ impaired its ability to inhibit the protease, calpain. We have identified p38γ as an important determinant of the progression of pathologic cardiac hypertrophy after aortic banding in mice. In addition, we have identified calpastatin, among other substrates, as a novel direct target of p38γ that may contribute to the protection observed in p38γKO mice.-Loonat, A. A., Martin, E. D., Sarafraz-Shekary, N., Tilgner, K., Hertz, N. T., Levin, R., Shokat, K. M., Burlingame, A. L., Arabacilar, P., Uddin, S., Thomas, M., Marber, M. S., Clark, J. E. p38γ MAPK contributes to left ventricular remodeling after pathologic stress and disinhibits calpain through phosphorylation of calpastatin.

Topics: Animals; Calcium-Binding Proteins; Calpain; Echocardiography; Electrophoresis, Polyacrylamide Gel; HEK293 Cells; Humans; Immunohistochemistry; Male; Mice; Mitogen-Activated Protein Kinase 12; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Isoforms; Tandem Mass Spectrometry; Ventricular Remodeling

The transformation of fibroblasts to myofibroblasts plays a major role in fibrogenic responses during dermal wound healing. We show a contribution of calpain systems (intracellular regulatory protease systems) in vascular endothelial cells (ECs) to myofibroblast differentiation in wound sites. Dermal wound healing experiments in mice found that calpastatin (an endogenous inhibitor of calpains) is enriched in preexisting vessels but not in newly formed capillaries. Transgenic overexpression of calpastatin in ECs delayed wound healing in mice as well as reducing the keratinocyte layer, extracellular matrix deposition, and myofibroblast accumulation in wound sites. EC and leukocyte markers, however, remain unchanged. Calpastatin overexpression reduced the expression of genes encoding platelet-derived growth factor-B and PDGF receptor-β (PDGFR-β). Topical application of platelet-derived growth factor-BB-containing ointment to wounds accelerated healing in control mice, but calpastatin overexpression prevented this acceleration. In cultured human dermal fibroblasts, α-smooth muscle actin and PDGFR-β were up-regulated by coculturing with ECs, but this action was inhibited by suppression of EC calpain activity. EC-driven transformation of mouse dermal fibroblasts was also suppressed by calpastatin overexpression in ECs. These results suggest that endothelial calpain systems influence PDGFR-β signaling in fibroblasts, EC-driven myofibroblast differentiation, and subsequent fibrogenic responses in wounds.-Miyazaki, T., Haraguchi, S., Kim-Kaneyama, J.-R., Miyazaki, A. Endothelial calpain systems orchestrate myofibroblast differentiation during wound healing.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Differentiation; Coculture Techniques; Dermis; Endothelial Cells; Extracellular Matrix; Keratinocytes; Mice; Mice, Transgenic; Myofibroblasts; Receptor, Platelet-Derived Growth Factor beta; Wound Healing

The objective of this study was to investigate the effect of phosphorylation on the sensitivity of μ-calpain to the inhibition induced by calpastatin. Purified μ-calpain was incubated with alkaline phosphatase (AP) or protein kinase A (PKA) to modulate the phosphorylation level of μ-calpain. Accurately 25, 50, 100 and 150 units of AP/PKA-treated μ-calpain were mixed with the same amounts of heat stable proteins and incubated at 4 °C. In the calpastatin-free system, AP and PKA-treated μ-calpain had higher proteolytic activity compared to the control. Intact AP-treated μ-calpain degraded fastest in the 50, 100 and 150 unit μ-calpain incubation systems. However, the degradation rate of μ-calpain in control and PKA group was non-significant in 100 and 150 unit μ-calpain systems. Our results demonstrated that, compared to dephosphorylated and control μ-calpain, calpastatin presents greater inhibition to PKA phosphorylated μ-calpain. This study increases understanding of the mechanism of μ-calpain activity regulated by phosphorylation.

Topics: Alkaline Phosphatase; Animals; Calcium-Binding Proteins; Calpain; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Phosphorylation; Proteolysis

Expression of members of the calpain system are associated with clinical outcome of patients with, amongst others, breast and ovarian cancers, with calpain-2 expression in ovarian cancer being implicated in chemo-resistance and survival. This study aimed, using a large patient cohort and in vitro models, to verify its importance and further investigate the role in ovarian cancer chemoresponse.. Calpain-1, calpain-2, calpain-4 and calpastatin expression were evaluated in primary ovarian carcinomas (n = 575) by immunohistochemistry. Protein expression was assessed, via western blotting, in five ovarian cancer cell lines with various sensitivities towards cisplatin/carboplatin. In vitro calpain activity was inhibited by calpeptin treatment to assess changes in platinum sensitivity by proliferation assay, with expression of genes associated with epithelial-mesenchymal transition being examined by RT. The current study confirmed previous data that high calpain-2 expression is associated with poor overall survival (P = 0.026) and that calpain-1 was not associated with overall survival or progression-free survival. Low expression of calpastatin (P = 0.010) and calpain-4 (P = 0.003) were also associated with adverse survival. Such prognostic associations do not seem to be linked with altered tumour sensitivity towards platinum-based chemotherapy. Interestingly, low calpain-1 expression was more frequent in patients with confined tumours (stage 1) (χ. The conventional calpains and calpastatin have been confirmed to play an important role in ovarian cancer; however, the precise mechanisms whereby they exert effects remain to be elucidated.

Topics: Adenocarcinoma, Clear Cell; Adenocarcinoma, Mucinous; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Calcium-Binding Proteins; Calpain; Cell Proliferation; Cohort Studies; Cystadenocarcinoma, Serous; Endometrial Neoplasms; Epithelial-Mesenchymal Transition; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Ovarian Neoplasms; Prognosis; Survival Rate; Tumor Cells, Cultured

Purification and separation of calpains and calpastatin are used to determine the individual activities of calpain-1 and calpain-2 and their inhibitor calpastatin. We discuss here a method to purify these enzymes using dialysis followed by separation using anion-exchange chromatography coupled with gradient elution. Swollen DEAE Sephacel is used as the column matrix in this method. Calpastatin and both domains of calpain are weakly basic molecules that effectively bind with the DEAE Sephacel and separate well using a stepwise, increasing gradient of NaCl to elute the proteins. Calpastatin binds most weakly with the column matrix, so it elutes first, followed by calpain-1 and, finally, calpain-2.

Topics: Animals; Anions; Calcium-Binding Proteins; Calpain; Chickens; Chromatography, Ion Exchange; DEAE-Dextran; Molecular Biology

The production of recombinant calpastatin in E. coli has become an efficient tool to obtain discrete amounts of a specific calpastatin species that can be present concomitantly with other calpastatin fragments/forms in the same tissue or cell type in a given condition. Indeed, at present, it is still difficult to distinguish the various calpastatin species for several reasons among which: calpastatins differ only at the N-terminus, can undergo calpain-dependent cleavage generating discrete fragments, and show anomalous electrophoretic mobility. Another benefit of using recombinant calpastatin is that, as the wild-type forms, it is heat resistant and thus can be efficiently isolated taking advantage of a simple quick purification step. Finally, the lack of posttranslational modifications makes recombinant calpastatin species particularly suitable for studying in vitro the biochemical features of specific parts of the inhibitor that following controlled posttranslational modifications change their functional interaction with calpain. In this chapter, we describe, starting from the mRNA sequence, how to produce rat calpastatin Type I in E. coli. We use routinely the same method, with minor modifications, for the production of other calpastatin species deriving from different tissues or organisms and calpastatin constructs having only specific domains. The possibility to obtain large amounts of a single calpain inhibitor form is a great advantage for studying the calpain/calpastatin system in vitro.

Topics: Animals; Calcium-Binding Proteins; Calpain; Escherichia coli; Molecular Biology; Protein Processing, Post-Translational; Rats; Recombinant Proteins

We here describe the purification of calpastatin from human erythrocytes. When calpastatin is purified from tissues, it is necessary to measure its inhibitory activity against calpain in the presence of Ca

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Erythrocytes; Humans; Molecular Biology

Immunoblotting is a procedure routinely used to analyze calpastatin expression. However, immunoblotting alone may not be adequate for this task, since calpastatin isoforms can vary by tissue, can be modified by partial digestion, and can undergo posttranslational modifications. Here we describe a method for more precise evaluation of calpastatin expression by combining immunoblot analysis with an assay for the inhibitory activity of a single calpastatin species isolated by SDS-PAGE and protein elution from the gel.

Topics: Calcium-Binding Proteins; Calpain; Electrophoresis, Polyacrylamide Gel; Gene Expression; Immunoblotting; Muscles

This chapter describes the strategy and procedures for the calcium-mediated affinity purification of calpain. The affinity capture method exploits the reversible binding properties of calpain's intrinsically disordered protein (IDP) inhibitor, calpastatin. IDPs are easily produced in heterologous expression hosts and purified to homogeneity. Combining these properties with in vivo biotinylation leads to a simplified purification strategy whereby biotinylated human calpastatin domain 1 (hCSD1) can capture calpain efficiently from a complex biological mixture with only a single chromatographic step and in a considerably reduced time. Our approach is generally applicable through the in vivo biotinylation of any IDP of interest in order to capture its binding partner in a calcium- and chelator-based protocol.

Topics: Biotinylation; Calcium; Calcium-Binding Proteins; Calpain; Chromatography, Affinity; Escherichia coli; Humans

The aim of this study was to investigate the dual effect of the nitric oxide donor NOR-3 and calpastatin on µ-calpain activity, autolysis, and proteolytic ability. µ-Calpain and calpastatin were purified and allocated to the following five treatments: µ-calpain, µ-calpain + calpastatin, µ-calpain + NOR-3, µ-calpain + calpastatin + NOR-3, and µ-calpain + NOR-3 + calpastatin. µ-Calpain autolysis and the activity against purified myofibrils was initiated by addition of calcium. Results showed that NOR-3 could induce µ-calpain S-nitrosylation and effectively block the activity via the inhibition of µ-calpain autolysis. Calpastatin inhibited µ-calpain activity in a dose-dependent manner. The combined treatment of NOR-3 and calpastatin exerted a further inhibitory effect on µ-calpain activity, autolysis and proteolysis which was affected by the addition order of NOR-3 and calpastatin. Our data suggest that S-nitrosylation may play a regulatory role in mediating µ-calpain activity in the presence of calpastatin.

Topics: Animals; Autolysis; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Myofibrils; Nitric Oxide; Nitro Compounds; Proteolysis; Swine

Goat meat is considered healthy because of its low fat content, but it is often rather tough. Tenderness is the most important attribute of quality during meat consumption and there is scarce information about the expression of genes involved in the meat tenderization process in goats. The aim of this trial was to assess certain meat quality traits and the expression, at the messenger RNA (mRNA) and protein levels, of specific genes involved in the tenderization process of the longissimus lumborum (LL) in young male goats (Capra hircus) at different ages. Samples of LL were collected at slaughter from 32 Alpine goats that were divided into three categories: 9 suckling kids (Sk) at 5.4±0.15 weeks of age, 16 chevons (Ch) at 17.1±0.55 weeks of age and 7 post-puberal goats (Pu) at 34.3±2.5 weeks of age. Animal and carcass variables (live weight gain, live weight, carcass weight and fat deposits) and quality traits of meat (lipid content, ultimate pH, color parameters, cooking loss and shear force) were determined. The mRNA abundances of calpain-1 (Capn1), calpain-2 (Capn2), calpastatin (Cast), caspase 3 (Casp3), caspase 9 (Casp9), αB-crystallin (Cryab), heat shock protein 27 (Hsp27), heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) were detected by quantitative PCR. Capn1, Cast, Cryab and Hsp27 protein expression was investigated by ELISA. The Sk group had the leanest carcasses. The meat of the Pu group was the darkest (P<0.05) and the toughest (P<0.05). The redness of meat increased with the age of the goats. The Sk group showed lower mRNA abundances for the Capn2/Cast ratio, Casp3, Cryab, Hsp27, Hsp40 and Hsp70 than the Pu group (P<0.05). Intermediate values were found for the Ch group. Similar results were highlighted for the protein expression of Cryab and Hsp27. The experiment acknowledged a differentiation of the experimental groups based on performance, carcass and meat characteristics, and the genes considered. Moreover, Sk and Pu groups, characterized by a different tenderness of their meat, were clearly discriminated by a different expression of the Hsp.

Topics: Age Factors; Animals; Body Composition; Calcium-Binding Proteins; Calpain; Caspases; Goats; Heat-Shock Proteins; Male; Muscle, Skeletal; Phenotype; Red Meat

Aged people have a high chance to develop two prevalent diseases, diabetes and Alzheimer's disease (AD), which are characterized with hyperglycemia and neurodegeneration, respectively. Interestingly, recent evidence suggest that diabetes is a predisposing factor for AD. Nevertheless, the mechanisms underlying the association of diabetes with AD remain poorly defined. Here, we studied the effects of diabetes on AD in mice. The APP-PS1 mouse, an AD-prone strain, was administrated with streptozotocin (STZ) to destroy 75% beta cell mass to induce sustained hyperglycemia. We found that STZ-treated APP-PS1 mice exhibited poorer performance in the social recognition test, Morris water maze, and plus-maze discriminative avoidance task, compared to saline-treated normoglycemic APP-PS1 mice, likely resulting from increases in brain deposition of amyloid-β peptide aggregates (Aβ). Since formation of Aβ is known to be induced by protein hyperphosphorylation mediated by calpain (CAPN)-induced cleavage of p35 into p25, we examined levels of these proteins in mouse brain. We detected not only increased p35-to-p25 conversion, but also enhanced CAPN1 activity via increased protein but not mRNA levels. The internal CAPN1 inhibitor, calpastatin (CAST), was downregulated in STZ-treated APP-PS1 mouse brain, as a basis for the increase in CAPN1. In vitro, a human neuronal cell line, HCN-2, increased CAPN1 activity and downregulated CAST levels when incubated for 8 days in high glucose level, resulting in increased cell apoptosis. Together, these data suggest that chronic hyperglycemia may promote AD development through downregulating CAST.

Topics: Alzheimer Disease; Animals; Brain; Calcium-Binding Proteins; Calpain; Cell Line; Diabetes Mellitus, Experimental; Down-Regulation; Female; Humans; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurons

The aim of this study was to determine the extent to which calpastatin (CASN) variants (based on two chromatographic peaks; CASN-P1 and CASN-P2) explain variation in μ-calpain autolysis, protein degradation, and changes in the sarcoplasmic proteome observed during postmortem aging of beef. The Longissimus lumborum (LL) and Triceps brachii (TB) muscles were obtained from six crossbred steers and samples prepared from day 0, 1 and 7 postmortem (pm). The decline of CASN activity during aging was due to decrease of CASN-P2 in both muscles. The CASN-P2:μ-calpain ratio at day 0 was greater for TB, which presented lesser calpain autolysis, myofibrillar protein degradation, and fewer sarcoplasmic proteome changes during aging. Changes in abundance of Heat shock protein 70 family in the sarcoplasmic fraction were positively associated to proteolysis during aging, with greater differences in LL.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; HSP70 Heat-Shock Proteins; Male; Muscle, Skeletal; Myofibrils; Postmortem Changes; Proteolysis; Proteome; Red Meat

Alzheimer's disease (AD) and other tauopathies are characterized by intracellular accumulation of microtubule-associated tau protein leading to neurodegeneration. Calpastatin is the endogenous inhibitor of calpain, a calcium-dependent cysteine protease that has been increasingly implicated in tauopathies. In this study, we generated a neuron specific calpastatin overexpressing knock-in transgenic mouse model and crossed it with the PS19 tauopathy mouse model expressing human P301S mutant tau protein. The forced expression of calpastatin in neurons significantly alleviated tau hyperphosphorylation measured by immunocytochemistry and immunoblot. The genetic inhibition of calpain by calpastatin also greatly suppressed characteristic hippocampal neuron loss and widespread astrogliosis and microgliosis in PS19 mice. Consistently, PS19 mice with neuronal calpastatin overexpression exhibited remarkably alleviated cognitive deficits, muscle weakness, skeletal muscle atrophy, and neuromuscular denervation, together implying the neuroprotective effects of neuronal calpastatin in PS19 mice of tauopathy. In sum, this study provides additional evidence supporting the pathological role of calpain in neurodegenerative diseases associated with tau pathology, and suggests that targeting calpain is likely a promising therapeutic approach for these devastating diseases.

Topics: Animals; Calcium-Binding Proteins; Calpain; Female; Gene Knock-In Techniques; Gliosis; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurons; tau Proteins; Tauopathies

We examined the lateral gastrocnemius (LG), plantaris (PL), and extensor digitorum longus (EDL) muscles to determine whether differential activation of the calpain system is related to the degree of atrophy in these fast-twitch skeletal muscles during hibernation in Daurian ground squirrels (

Topics: Adaptation, Physiological; Animals; Calcium; Calcium-Binding Proteins; Calpain; Cytosol; Female; Hibernation; Male; Muscle Fibers, Fast-Twitch; Muscle Fibers, Slow-Twitch; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Sciuridae; Troponin T

Fibrosis is a common pathologic outcome of chronic disease resulting in the replacement of normal tissue parenchyma with a collagen-rich extracellular matrix produced by myofibroblasts. Although the progenitor cell types and cellular programs giving rise to myofibroblasts through mesenchymal transition can vary between tissues and diseases, their contribution to fibrosis initiation, maintenance, and progression is thought to be pervasive. Here, we showed that the ability of transforming growth factor-β (TGFβ) to efficiently induce myofibroblast differentiation of cultured epithelial cells, endothelial cells, or quiescent fibroblasts is dependent on the induced expression and activity of dimeric calpains, a family of non-lysosomal cysteine proteases that regulate a variety of cellular events through posttranslational modification of diverse substrates. siRNA-based gene silencing demonstrated that TGFβ-induced mesenchymal transition of a murine breast epithelial cell line was dependent on induction of expression of calpain 9 (CAPN9), an isoform previously thought to be restricted to the gastrointestinal tract. Mice lacking functional CAPN9 owing to biallelic targeting of

Topics: Angiotensin II; Animals; Bleomycin; Calcium-Binding Proteins; Calpain; Carbon Tetrachloride; Cell Line; Dogs; Epithelial-Mesenchymal Transition; Fibrosis; Humans; Isoenzymes; Liver Cirrhosis; Male; Mice, Inbred C57BL; Molecular Targeted Therapy; Myocardium; Protein Biosynthesis; Protein Multimerization; RNA Stability; Signal Transduction; Transforming Growth Factor beta

The effects of beef carcass weight on muscle pH/temperature profile and selected meat quality attributes were evaluated. Twenty-six carcasses from light (≤260kg, n=15) and heavy (≥290kg, n=11) feedlot steers were randomly allocated and stimulated with low voltage electrical stimulation (LVES) for 30s at 7min post-mortem (pm). Quality evaluations were carried out on samples from the Longissimus et lumborum (LL) muscle from the left side of each carcass. Heavier carcasses showed faster pH decline and slower (P<0.05) temperature decline at 45min, 3, 6, 12 and 24h pm. Heavier carcasses passed through the heat shortening window (i.e. at pH6, temperature was >35°C) but there was no sign of sarcomere shortening in any carcass. Significantly lower (P<0.05) shear force values were recorded in the heavier carcasses at 3days pm but at 14days pm, heavier carcasses had numerically lower but not significantly different shear force. Heavier carcasses produced numerically higher but not significant (P>0.05) drip loss at 3 and 14days pm as well as higher L* (meat lightness) (P<0.05) and C* (chroma) (P<0.05) values early (2days) pm. However, at 14days pm, there were no significant differences between the light and heavy carcasses in terms of L* and C*. No significant difference was observed between heavy and light carcasses in terms of H* at 2 and 14days pm. The study showed that heavier carcasses which favor slaughter house pricing can be produced and processed alongside lighter carcasses without significant detrimental effects on meat quality by using low voltage electrical stimulation (LVES).

Topics: Abattoirs; Animals; Body Composition; Body Weight; Calcium-Binding Proteins; Calpain; Cattle; Color; Electric Stimulation; Hydrogen-Ion Concentration; Meat-Packing Industry; Muscle, Skeletal; Postmortem Changes; Red Meat; Temperature; Time Factors

Genetic marker effects and type of inheritance are estimated with poor precision when minor marker allele frequencies are low. An Angus population was subjected to marker assisted selection for multiple years to equalize CAPN1 haplotypes, CAST, and GHR genetic marker frequencies. The objective was to estimate the pleiotropic effects of these carcass quality oriented markers for body weight, reproduction, and first calf performance traits in 174 replacement beef females which were managed under 2 post-weaning development protocols. Heifers were weighed at 11-, 12-, and 13-mo, at first breeding season pregnancy evaluation, and prior to first calving season. Pubertal status was determined at 11-, 12-, and 13-mo of age. Antral follicles were counted, reproductive tracts were scored, and tract dimensions were measured at 13-mo. Body condition and hip height were scored and measured at pregnancy evaluation and prior to calving season. Heifer pregnancy and weaning rates and ordinal birth date were recorded. Calf body weights at birth and weaning were analyzed. Single df linear contrasts for recessive effects of the GHR heterozygous genotype showed significant decreases of 2.5-3.6% in 11-, 12-, and 13-mo heifer body weights and heifer weight prior to calving. The additive differences between GHR homozygotes were small and not significant for all body weights measured but a 1 wk difference in calf birth date was significant. For all 13-mo uterine measurements, scores, and antral follicle counts, only the CAST dominance contrast for medium antral follicle count was significant. The CAPN1 haplotype with a strong additive effect for increased beef tenderness also had a significant additive effect on calving date. Heifers homozygous for the tender haplotype calved 7.9 days later than heifers homozygous for the tough haplotype. Most heifer reproductive traits were not significantly affected by CAST and CAPN1 markers that are widely used to improve beef tenderness by selection and breeders should not be concerned with how these markers affect reproduction and other heifer traits with the possible exception of CAPN1 effects on calving date.

Topics: Animals; Body Composition; Calcium-Binding Proteins; Calpain; Cattle; Female; Gene Expression Regulation; Genetic Markers; Genotype; Haplotypes; Ovarian Follicle; Parturition; Pregnancy; Receptors, Somatotropin; Reproduction

The objective of this study was to determine associations of small heat shock proteins (sHSPs) in tenderness development of loins from callipyge and normal genotype lambs. Loins (M. longissimus lumborum) from sixteen lambs across four genotypes were collected throughout 9 days of postmortem aging. The loins from callipyge lambs had more intact desmin and troponin T throughout aging periods, as well as less μ-calpain autolysis and more calpastatin compared to loins from other genotypes (P < 0.05). Delayed onset of apoptosis was found in the callipyge loins indicated by less cytochrome c and more inactive procaspase-3 compared to normal lamb loins (P < 0.05). Less degraded HSP27 was also consistently found in the callipyge loins compared with loins from normal lambs (P < 0.001). The results found up-regulation of anti-apoptotic activities coincided with toughness in callipyge loins, which suggest apoptosis is likely involved in postmortem proteolysis and subsequent meat tenderization.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Caspase 3; Cytochromes c; Desmin; Heat-Shock Proteins, Small; Muscle, Skeletal; Proteolysis; Red Meat; Sheep, Domestic; Troponin T

To determine whether the regulation of calpain system is involved in non-hibernators and hibernators in disused condition, the soleus (SOL) and extensor digitorum longus (EDL) muscles were used for investigating the muscle mass, the ratio of muscle wet weight/body weight (MWW/BW), fiber-type distribution, fiber cross-sectional area (CSA), and the protein expression of MuRF1, calpain-1, calpain-2, calpastatin, desmin, troponin T, and troponin C in hindlimb unloading rats and hibernating Daurian ground squirrels. The muscle mass, MWW/BW, and fiber CSA were found significantly decreased in SOL and EDL of hindlimb unloading rats, but unchanged in hibernating ground squirrels. The MuRF1 expression was increased in both SOL and EDL of unloading rats, while it was only increased in SOL, but maintained in EDL of hibernating ground squirrels. The expression levels of calpain-1 and calpain-2 were increased in different degrees in unloaded SOL and EDL in rats, while they were maintained in EDL and even reduced in SOL of hibernating ground squirrels. Besides, the expression of calpastatin was decreased in unloaded rats, but increased in hibernating ground squirrels. The desmin expression was decreased in unloaded rats, but maintained in hibernating squirrels. Interestingly, the levels of troponin T and troponin C were decreased in both SOL and EDL of unloaded rats, but increased in hibernating ground squirrels with muscle-type specificity. In conclusion, differential calpain activation and substrate-selective degradation in slow and fast muscles are involved in the mechanisms of muscle atrophy of unloaded rats and remarkable ability of muscle maintenance of hibernating ground squirrels.

Topics: Animals; Calcium-Binding Proteins; Calpain; Desmin; Female; Hibernation; Hindlimb; Muscle Proteins; Muscle, Skeletal; Rats, Sprague-Dawley; Sciuridae; Tripartite Motif Proteins; Troponin C; Troponin T; Ubiquitin-Protein Ligases

This study was conducted to determine whether the application of high pressure processing (HPP) could prevent the process of rigor in fresh pork and to identify the physical and chemical changes occurring in the meat that are responsible for this effect. Pressure (0-400 MPa) and time (10 min) were applied to the pork loin and enzyme samples. The effects of treatment on shear force, myofibrillar fragmentation, enzyme activity and meat texture were measured. The results showed that, compared with untreated samples, HPP treatment inhibited the occurrence of rigor in pork. The myofibrillar fragmentation indices of HPP treated samples were higher than those of control samples and increased linearly with increasing pressure. Application of HPP prevented the process of rigor at 0-300 MPa, reduced calpain activity and markedly decreased calpastatin activity, resulting in an increase of total calpains. Above 300 MPa, the mechanical action of pressure led to continuous prevention of rigor.

Topics: Animals; Calcium-Binding Proteins; Calpain; Food Handling; Humans; Muscle, Skeletal; Myofibrils; Pressure; Red Meat; Rigor Mortis; Stress, Mechanical; Swine

Calpains are intracellular, calcium-activated cysteine proteases. Calpain-3 is abundant in skeletal muscle, where its mutation-induced loss of function causes limb-girdle muscular dystrophy type 2A. Unlike the small subunit-containing calpain-1 and -2, the calpain-3 isoform homodimerizes through pairing of its C-terminal penta-EF-hand domain. It also has two unique insertion sequences (ISs) not found in the other calpains: IS1 within calpain-3's protease core and IS2 just prior to the penta-EF-hand domain. Production of either native or recombinant full-length calpain-3 to characterize the function of these ISs is challenging. Therefore, here we used recombinant rat calpain-2 as a stable surrogate and inserted IS1 into its equivalent position in the protease core. As it does in calpain-3, IS1 occupied the catalytic cleft and restricted the enzyme's access to substrate and inhibitors. Following activation by Ca

Topics: Calcium; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; DNA Transposable Elements; Isoenzymes; Leucine; Leupeptins; Muscle Proteins; Protein Conformation; Proteolysis; Recombinant Proteins

Calpains are calcium-activated proteases that have biomedical and biotechnological potential. Their activity is tightly regulated by their endogenous inhibitor, calpastatin that binds to the enzyme only in the presence of calcium. Conventional approaches to purify calpain comprise multiple chromatographic steps, and are labor-intensive, leading to low yields. Here we report a new purification procedure for the human m-calpain based on its reversible calcium-mediated interaction with the intrinsically disordered calpastatin. We exploit the specific binding properties of human calpastatin domain 1 (hCSD1) to physically capture human m-calpain from a complex biological mixture. The dissociation of the complex is mediated by chelating calcium, upon which heterodimeric calpain elutes while hCSD1 remains immobilized onto the stationary phase. This novel affinity-based purification was compared to the conventional multistep purification strategy and we find that it is robust, it yields a homogeneous preparation, it can be scaled up easily and it rests on a non-disruptive step that maintains close to physiological conditions that allow further biophysical and functional studies.

Topics: Biotinylation; Calcium Chelating Agents; Calcium-Binding Proteins; Calpain; Chromatography, Affinity; Chromatography, Gel; Chromatography, Ion Exchange; Circular Dichroism; Computational Biology; Escherichia coli; Humans; Interferometry; Kinetics; Mutation; Protein Binding; Protein Domains; Proteolysis; Recombinant Proteins; Sequence Analysis, Protein; Sequence Homology, Amino Acid

Calpains activate during myocardial ischemia-reperfusion and contribute to reperfusion injury. Studies in transgenic animals with altered calpain/calpastatin system subjected to permanent ischemia suggest that calpains are also involved in post-infarction remodelling and heart failure.. To determine whether delayed oral administration of the calpain inhibitor SNJ-1945 reduces adverse myocardial remodelling and dysfunction following transient coronary occlusion.. Male Sprague-Dawley rats were subjected to 30 min of ischemia followed by 21 days of reperfusion and received the calpain inhibitor SNJ-1945 intraperitoneally at the onset of reperfusion (Acute group), orally starting after 24 h of reperfusion and for 14 days (Chronic group), or the combination of both treatments. Calpain-1 and calpain-2 protein content increased and correlated with higher calpain activity in control hearts. Administration of SNJ-1945 attenuated calpain activation, and reduced scar expansion, ventricular dilation and dysfunction in both acute and chronic groups. Acute treatment reduced infarct size in hearts reperfused for 24 h and inflammation measured after 3 days. Delayed, chronic oral administration of SNJ-1945 attenuated inflammation, cardiomyocyte hypertrophy and collagen infiltration in the non-infarcted myocardium at 21 days in correlation with increased levels of IĸB and reduced NF-ĸB activation. In cultured fibroblasts, SNJ-1945 attenuated TGF-β1-induced fibroblast activation.. Our data demonstrate for the first time that long-term calpain inhibition is possible with delayed oral treatment, attenuates adverse post-infarction remodelling, likely through prevention of NF-ĸB activation, and may be a promising therapeutic intervention to prevent adverse remodelling and heart failure in patients with acute myocardial infarction.

Topics: Animals; Calcium-Binding Proteins; Calpain; Carbamates; Glycoproteins; Heart; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Rats, Sprague-Dawley

Single nucleotide polymorphisms (SNPs) carried in calpain (CAPN1), calpastatin (CAST), and leptin (LEP) genes are associated with meat tenderness. Due to the economic importance of this meat quality attribute, the development of fast, reliable, and affordable methods to identify bovine carriers of favorable alleles is of great importance for genetic improvement. Currently, PCR-RFLP is accepted as the standard gold method for genotyping SNPs associated with meat tenderness. But these SNPs can be detected by other techniques as high-resolution melting (HRM) analysis - a post-PCR method - that offers several advantages and has great application potential in the meat industry. In this study, we standardized, validated, and compared the performance of PCR-HRM to that of PCR-RFLP in genotyping bovine SNPs associated with meat tenderness: CAPN4751, CAPN316, CAST2959, CAST282, LEPE2FB, and LEPE2JW. We analyzed genotypes of a total of 380 bovines, 110 Bos taurus and 270 Bos indicus. Results obtained with PCR-HRM were consistent with those found by PCR-RLFP. Furthermore, HRM was found to be highly sensitive, and our results confirmed the repeatability (intra-assay precision) and reproducibility (inter-assay precision) of this assay. An internal control for endonuclease activity was created using site-directed mutagenesis to generate an additional enzymatic restriction point useful to discriminate SNP alleles. Our results show that PCR-HRM is an efficient method that produces reliable and rapid results. However, should be had in account that the method of DNA extraction, the quality and quantity of DNA, analyst-related variations, and primer design may generate challenges for allele discrimination.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Genotyping Techniques; Leptin; Meat; Polymerase Chain Reaction; Polymorphism, Single Nucleotide

Calpains are ubiquitous pro-inflammatory proteases, whose activity is controlled by calpastatin, their specific inhibitor. Transgenic mice over-expressing rabbit calpastatin (CalpTG) are protected against vascular remodelling and angiotensin II-dependent inflammation. We hypothesized that specific calpain inhibition would protect against aging-related lesions in arteries and kidneys. We analysed tissues from 2-months and 2-years-old CalpTG and wild-type mice and performed high throughput RNA-Sequencing of kidney tissue in aged mice. In addition, we analysed inflammatory response in the kidney of aged CalpTG and wild-type mice, and in both in vivo (monosodium urate peritonitis) and in vitro models of inflammation. At two years, CalpTG mice had preserved kidney tissue, less vascular remodelling and less markers of senescence than wild-type mice. Nevertheless, CalpTG mice lifespan was not extended, due to the development of lethal spleen tumors. Inflammatory pathways were less expressed in aged CalpTG mice, especially cytokines related to NF-κB and NLRP3 inflammasome activation. CalpTG mice had reduced macrophage infiltration with aging and CalpTG mice produced less IL-1α and IL-1β in vivo in response to inflammasome activators. In vitro, macrophages from CalpTG mice produced less IL-1α in response to particulate activators of inflammasome. Calpains inhibition protects against inflammaging, limiting kidney and vascular lesions related to aging.

Topics: Aging; Animals; Arteries; Calcium-Binding Proteins; Calpain; Cells, Cultured; Cysteine Proteinase Inhibitors; Cytokines; Inflammasomes; Kidney; Mice; Mice, Inbred C57BL; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Peritonitis; Rabbits

Adipose tissue macrophages have been proposed as a link between obesity and insulin resistance. However, the mechanisms underlying these processes are not completely defined. Calpains are calcium-dependent neutral cysteine proteases that modulate cellular function and have been implicated in various inflammatory diseases. To define whether activated calpains influence diet-induced obesity and adipose tissue macrophage accumulation, mice that were either wild type (WT) or overexpressing calpastatin (CAST Tg), the endogenous inhibitor of calpains were fed with high (60% kcal) fat diet for 16 weeks. CAST overexpression did not influence high fat diet-induced body weight and fat mass gain throughout the study. Calpain inhibition showed a transient improvement in glucose tolerance at 5 weeks of HFD whereas it lost this effect on glucose and insulin tolerance at 16 weeks HFD in obese mice. However, CAST overexpression significantly reduced adipocyte apoptosis, adipose tissue collagen and macrophage accumulation as detected by TUNEL, Picro Sirius and F4/80 immunostaining, respectively. CAST overexpression significantly attenuated obesity-induced inflammatory responses in adipose tissue. Furthermore, calpain inhibition suppressed macrophage migration to adipose tissue in vitro. The present study demonstrates a pivotal role for calpains in mediating HFD-induced adipose tissue remodeling by influencing multiple functions including apoptosis, fibrosis and inflammation.

Topics: 3T3 Cells; Adipocytes; Adipose Tissue; Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Collagen; Diet, High-Fat; Disease Models, Animal; Fibrosis; Inflammation; Liver; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myocardium; Obesity; Weight Gain

Previous studies have shown that catecholamines in vivo and in vitro inhibit the activity of Ca

Topics: Adrenal Medulla; Animals; Calcium; Calcium-Binding Proteins; Calpain; Carrier Proteins; Catecholamines; Cold Temperature; Epinephrine; Male; Microfilament Proteins; Muscle Proteins; Muscle, Skeletal; Proteolysis; Rats; Rats, Wistar; RNA, Messenger; Signal Transduction

Impaired apoptosis is one of the hallmarks of cancer. Caspase-3 and -8 are key regulators of the apoptotic response and have been shown to interact with the calpain family, a group of cysteine proteases, during tumorigenesis. The current study sought to investigate the prognostic potential of caspase-3 and -8 in breast cancer, as well as the prognostic value of combinatorial caspase and calpain expression. A large cohort (n = 1902) of early stage invasive breast cancer patients was used to explore the expression of caspase-3 and -8. Protein expression was examined using standard immunohistochemistry on tissue microarrays. High caspase-3 expression, but not caspase-8, is significantly associated with adverse breast cancer-specific survival (P = 0.008 and P = 0.056, respectively). Multivariate analysis showed that caspase-3 remained an independent factor when confounding factors were included (hazard ratio (HR) 1.347, 95% confidence interval (CI) 1.086-1.670; P = 0.007). The analyses in individual subgroups demonstrated the significance of caspase-3 expression in clinical outcomes in receptor positive (ER, PR or HER2) subgroups (P = 0.001) and in non-basal like subgroup (P = 0.029). Calpain expression had been previously assessed. Significant association was also found between high caspase-3/high calpain-1 and breast cancer-specific survival in the total patient cohort (P = 0.005) and basal-like subgroup (P = 0.034), as indicated by Kaplan-Meier analysis. Caspase-3 expression is associated with adverse breast cancer-specific survival in breast cancer patients, and provides additional prognostic values in distinct phenotypes. Combinatorial caspase and calpain expression can predict worse prognosis, especially in basal-like phenotypes. The findings warrant further validation studies in independent multi-centre patient cohorts.

Topics: Adolescent; Adult; Aged; Breast Neoplasms; Calcium-Binding Proteins; Calpain; Carcinoma; Caspase 3; Caspase 8; Cell Line, Tumor; Estrogens; Female; Genes, erbB-2; Humans; Kaplan-Meier Estimate; Middle Aged; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Progesterone; Prognosis; Tissue Array Analysis; Young Adult

The objectives of this study were to characterize the allelic and genotypic frequencies of polymorphisms in the µ-calpain and calpastatin genes, and to assess their association with meat tenderness and animal growth in Nellore cattle. We evaluated 605 Nellore animals at 24 months of age, on average, at slaughter. The polymorphisms were determined for the molecular markers CAPN316, CAPN530, CAPN4751, CAPN4753, and UOGACAST1. Analyses of meat tenderness at 7, 14, and 21 days of maturation were performed in samples of longissimus thoracis obtained between the 12th and 13th rib and sheared using a Warner Bratzler Shear Force. Significant effects were observed for meat tenderness at days 7, 14, and 21 of maturation for the marker CAPN4751, at day 21 for the marker CAPN4753, and at days 14 and 21 for the marker UOGCAST1. For genotypic combinations of markers, the results were significant for the combination CAPN4751/UOGCAST1 in the three maturation periods and CAPN4753/UOGCAST1 at days 14 and 21 of maturation.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Gene Frequency; Genetic Markers; Genotype; Polymorphism, Single Nucleotide; Quantitative Trait Loci; Red Meat

Baseline research on the toughness of Egyptian goose meat is required. This study therefore investigates the post mortem pH and temperature decline (15 min-4 h 15 min post mortem) in the pectoralis muscle (breast portion) of this gamebird species. It also explores the enzyme activity of the Ca(2+)-dependent protease (calpain system) and the lysosomal cathepsins during the rigor mortis period.. No differences were found for any of the variables between genders. The pH decline in the pectoralis muscle occurs quite rapidly (c = -0.806; ultimate pH ∼ 5.86) compared with other species and it is speculated that the high rigor temperature (>20 °C) may contribute to the increased toughness. No calpain I was found in Egyptian goose meat and the µ/m-calpain activity remained constant during the rigor period, while a decrease in calpastatin activity was observed. The cathepsin B, B & L and H activity increased over the rigor period.. Further research into the connective tissue content and myofibrillar breakdown during aging is required in order to know if the proteolytic enzymes do in actual fact contribute to tenderisation.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cathepsins; Egypt; Female; Geese; Humans; Hydrogen-Ion Concentration; Male; Meat; Pectoralis Muscles; Rigor Mortis; Stress, Mechanical; Temperature

Calpain mediates collagen synthesis and cell proliferation and plays an important role in pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). In the present study, we investigated whether and how calpain is activated by PAH mediators in pulmonary artery smooth muscle cells (PASMCs). These data show that smooth muscle-specific knockout of calpain attenuated and knockout of calpastatin potentiated pulmonary vascular remodeling and pulmonary hypertension. Treatment of PASMCs with the PAH mediators platelet-derived growth factor (PDGF), serotonin, H2O2, endothelin-1, and IL-6 caused significant increases in calpain activity, cell proliferation, and collagen-I protein level without changes in protein levels of calpain-1 and -2. The calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA/AM) did not affect calpain activation, but the extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 and knocking down of calpain-2 prevented calpain activation in PAH mediator-treated PASMCs. Mass spectrometry data showed that the phosphorylation of calpain-2 at serine (Ser) 50 was increased and the phosphorylation of calpain-2 at Ser369 was decreased in PDGF-treated PASMCs. The PDGF-induced increase in Ser50 phosphorylation of calpain-2 was prevented by PD98059, whereas dephosphorylation of calpain-2 at Ser369 was blocked by the protein phosphatase 2A inhibitor fostriecin. Furthermore, smooth muscle of pulmonary arteries in PAH animal models and patients with PAH showed higher levels of phospho-Ser50-calpain-2 (P-Ser50) and lower levels of phospho-Ser369-calpain-2 (P-Ser369). These data support that calpain modulates pulmonary vascular remodeling in PAH. PAH mediator-induced activation of calpain is caused by ERK1/2-dependent phosphorylation of calpain-2 at Ser50 and protein phosphatase 2A-dependent dephosphorylation of calpain-2 at Ser369 in pulmonary vascular remodeling of PAH.

Topics: Animals; Calcium-Binding Proteins; Calpain; Disease Models, Animal; Enzyme Activation; Enzyme Activators; Extracellular Signal-Regulated MAP Kinases; HEK293 Cells; Humans; Hypertension, Pulmonary; Hypoxia; Mice, Knockout; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphorylation; Protein Kinase Inhibitors; Protein Phosphatase 2; Pulmonary Artery; RNA Interference; Signal Transduction; Transfection; Vascular Remodeling

Calpain plays a critical role in cardiomyopathic changes in type 1 diabetes (T1D). This study investigated how calpain regulates mitochondrial reactive oxygen species (ROS) generation in the development of diabetic cardiomyopathy. T1D was induced in transgenic mice overexpressing calpastatin, in mice with cardiomyocyte-specific capn4 deletion, or in their wild-type littermates by injection of streptozotocin. Calpain-1 protein and activity in mitochondria were elevated in diabetic mouse hearts. The increased mitochondrial calpain-1 was associated with an increase in mitochondrial ROS generation and oxidative damage and a reduction in ATP synthase-α (ATP5A1) protein and ATP synthase activity. Genetic inhibition of calpain or upregulation of ATP5A1 increased ATP5A1 and ATP synthase activity, prevented mitochondrial ROS generation and oxidative damage, and reduced cardiomyopathic changes in diabetic mice. High glucose concentration induced ATP synthase disruption, mitochondrial superoxide generation, and cell death in cardiomyocytes, all of which were prevented by overexpression of mitochondria-targeted calpastatin or ATP5A1. Moreover, upregulation of calpain-1 specifically in mitochondria induced the cleavage of ATP5A1, superoxide generation, and apoptosis in cardiomyocytes. In summary, calpain-1 accumulation in mitochondria disrupts ATP synthase and induces ROS generation, which promotes diabetic cardiomyopathy. These findings suggest a novel mechanism for and may have significant implications in diabetic cardiac complications.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Cardiomyopathies; Disease Models, Animal; Mice; Mice, Transgenic; Mitochondria, Heart; Mitochondrial Proton-Translocating ATPases; Myocardium; Myocytes, Cardiac; Reactive Oxygen Species; Superoxides

This study describes the changes taking place during rigour in springbok (Antidorcas marsupialis) Longissimus thoracis et lumborum (LTL) and Biceps femoris (BF) muscles. Samples from six male and six female springbok were snap-frozen at 2, 3, 5, 8, 12, 18, 24 and 30h post-mortem (PM) and the pH, calpains I, II and calpastatin activities and cathepsins B, BL and H activities were determined. The temperature was also recorded. Significant third-order interactions were found for the pH and temperature, with the female LTL cooling more rapidly and acidifying slower than the other samples. Female muscles were at risk of developing cold-shortening and all the samples cooled more rapidly than recommended for cattle or sheep. Cathepsin BL activity increased PM, likely due to the degradation of the lysosomes. Calpains I, II and calpastatin activity declined during rigour, indicating that the calpains were activated early PM. Gender and muscle had a significant effect on calpain and cathepsin activity.

Topics: Animals; Antelopes; Calcium-Binding Proteins; Calpain; Cathepsins; Chemical Phenomena; Female; Food Quality; Food Storage; Hydrogen-Ion Concentration; Isoenzymes; Male; Meat; Mechanical Phenomena; Muscle, Skeletal; Proteolysis; Refrigeration; Rigor Mortis; Sex Characteristics; South Africa; Stress, Physiological; Stress, Psychological

Preterm infants suffer central nervous system (CNS) injury from hypoxia-ischemia and inflammation - termed encephalopathy of prematurity. Mature CNS injury activates caspase and calpain proteases. Erythropoietin (EPO) limits apoptosis mediated by activated caspases, but its role in modulating calpain activation has not yet been investigated extensively following injury to the developing CNS. We hypothesized that excess calpain activation degrades developmentally regulated molecules essential for CNS circuit formation, myelination and axon integrity, including neuronal potassium-chloride co-transporter (KCC2), myelin basic protein (MBP) and phosphorylated neurofilament (pNF), respectively. Further, we predicted that post-injury EPO treatment could mitigate CNS calpain-mediated degradation. Using prenatal transient systemic hypoxia-ischemia (TSHI) in rats to mimic CNS injury from extreme preterm birth, and postnatal EPO treatment with a clinically relevant dosing regimen, we found sustained postnatal excess cortical calpain activation following prenatal TSHI, as shown by the cleavage of alpha II-spectrin (αII-spectrin) into 145-kDa αII-spectrin degradation products (αII-SDPs) and p35 into p25. Postnatal expression of the endogenous calpain inhibitor calpastatin was also reduced following prenatal TSHI. Calpain substrate expression following TSHI, including cortical KCC2, MBP and NF, was modulated by postnatal EPO treatment. Calpain activation was reflected in serum levels of αII-SDPs and KCC2 fragments, and notably, EPO treatment also modulated KCC2 fragment levels. Together, these data indicate that excess calpain activity contributes to the pathogenesis of encephalopathy of prematurity. Serum biomarkers of calpain activation may detect ongoing cerebral injury and responsiveness to EPO or similar neuroprotective strategies.

Topics: Animals; Animals, Newborn; Apoptosis; Axons; Brain Injuries; Calcium-Binding Proteins; Calpain; Caspases; Enzyme Activation; Erythropoietin; Female; Hypoxia-Ischemia, Brain; Membrane Proteins; Myelin Basic Protein; Rats, Sprague-Dawley

Aneurysms of the ascending aorta are an outstanding challenge to clinicians as they may persist asymptomatic until they present with dissection or rupture. Intensive research is performed to reveal the molecular mechanisms causing aneurysm formation. Calpains are ubiquitous non-lysosomal cysteine proteases which are classically activated by calcium signaling. The two major forms of the calpain-family are calpain-I and calpain-II. Calpastatin specifically inhibits the proteolytic activity of calpain-I and -II. Recently it has been demonstrated in aneurysm tissues from ascending aortas obtained from Marfan syndrome patients that calpain-II expression is increased and calpastatin expression is decreased. Thus, we were interested in the probable role of calpains in aneurysms of ascending aorta in non-Marfan patients. Therefore, ascending aortic samples of dilated and non-dilated aortas were analyzed according to their calpain-I, -II and calpastatin content as well as the expression levels of MMPs and elastin as well as the infiltration of inflammatory cells. We have found significant differences in calpain-I and calpastatin protein expression and serum levels in patients with aneurysm of the ascending aorta. Furthermore, MMP-1 and MMP-3 expression levels correlate with calpain-I protein levels. Due to our findings we conclude that calpain-1 seems to be related to fibrotic alteration in aortic aneurysm tissue in our experimental group. The change in calpain-1 modulates the structure of aortic tissue causing alteration in elastin structure, thus enabling macrophage infiltration and elevation of MMP levels. Circulating levels of calpain-1 may be used as a prognostic marker in the future if further correlation analyses are done.

Topics: Aged; Aorta; Aortic Aneurysm; Calcium-Binding Proteins; Calpain; Elastin; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunohistochemistry; Male; Metalloproteases; Middle Aged; Polymerase Chain Reaction

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease with a poorly understood cause and no effective treatment. Given that calpains mediate neurodegeneration in other pathological states and are abnormally activated in ALS, we investigated the possible ameliorative effects of inhibiting calpain over-activation in hSOD1(G93A) transgenic (Tg) mice in vivo by neuron-specific over-expression of calpastatin (CAST), the highly selective endogenous inhibitor of calpains. Our data indicate that over-expression of CAST in hSOD1(G93A) mice, which lowered calpain activation to levels comparable to wild-type mice, inhibited the abnormal breakdown of cytoskeletal proteins (spectrin, MAP2 and neurofilaments), and ameliorated motor axon loss. Disease onset in hSOD1(G93A) /CAST mice compared to littermate hSOD1(G93A) mice is delayed, which accounts for their longer time of survival. We also find that neuronal over-expression of CAST in hSOD1(G93A) transgenic mice inhibited production of putative neurotoxic caspase-cleaved tau and activation of Cdk5, which have been implicated in neurodegeneration in ALS models, and also reduced the formation of SOD1 oligomers. Our data indicate that inhibition of calpain with CAST is neuroprotective in an ALS mouse model. CAST (encoding calpastatin) inhibits hyperactivated calpain to prevent motor neuron disease operating through a cascade of events as indicated in the schematic, with relevance to amyotrophic lateral sclerosis (ALS). We propose that over-expression of CAST in motor neurons of hSOD1(G93A) mice inhibits activation of CDK5, breakdown of cytoskeletal proteins (NFs, MAP2 and Tau) and regulatory molecules (Cam Kinase IV, Calcineurin A), and disease-causing proteins (TDP-43, α-Synuclein and Huntingtin) to prevent neuronal loss and delay neurological deficits. In our experiments, CAST could also inhibit cleavage of Bid, Bax, AIF to prevent mitochondrial, ER and lysosome-mediated cell death mechanisms. Similarly, CAST over-expression in neurons attenuated pathological effects of TDP-43, α-synuclein and Huntingtin. These results suggest a potential value of specific small molecule inhibitors of calpains in delaying the development of ALS. Read the Editorial Highlight for this article on page 140.

Topics: Age Factors; Amyotrophic Lateral Sclerosis; Animals; Axons; Calcium-Binding Proteins; Calpain; Cell Death; Cyclin-Dependent Kinase 5; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Disease Models, Animal; Disease Progression; Gene Expression Regulation; Humans; Mice; Mice, Transgenic; Motor Activity; Motor Neurons; Nerve Degeneration; Spinal Cord; Superoxide Dismutase

The aim of this study was to develop a simple, specific and rapid analytical method for accurate identification of calpain and calpastatin from chicken blood and muscle samples. The method is based on liquid-liquid extraction technique followed by casein Zymography detection. The target compounds were extracted from blood and meat samples by tris buffer, and purified and separated on anion exchange chromatography. It has been observed that buffer (pH 6.7) containing 50 mM tris-base appears to be excellent extractant as activity of analytes was maximum for all samples. The concentrations of μ-, m-calpain and calpastatin detected in the extracts of blood, breast and thigh samples were 0.28-0.55, 1.91-2.05 and 1.38-1.52 Unit/g, respectively. For robustness, the analytical method was applied to determine the activity of calpains (μ and m) in eighty postmortem muscle samples. It has been observed that μ-calpain activity in breast and thigh muscles declined very rapidly at 48 h and 24 h, respectively while activity of m-calpain remained stable. Shear force values were also declined with the increase of post-mortem aging showing the presence of ample tenderness of breast and thigh muscles. Finally, it is concluded that the method standardized for the detection of calpain and calpastatin has the potential to be applied to identify post-mortem aging of chicken meat samples.

Topics: Animals; Calcium-Binding Proteins; Calpain; Chickens; Hydrogen-Ion Concentration; Meat; Postmortem Changes; Refrigeration

Angiotensin II (AngII) infusion profoundly increases activity of calpains, calcium-dependent neutral cysteine proteases, in mice. Pharmacological inhibition of calpains attenuates AngII-induced aortic medial macrophage accumulation, atherosclerosis, and abdominal aortic aneurysm in mice. However, the precise functional contribution of leukocyte-derived calpains in AngII-induced vascular pathologies has not been determined. The purpose of this study was to determine whether calpains expressed in bone marrow (BM)-derived cells contribute to AngII-induced atherosclerosis and aortic aneurysms in hypercholesterolemic mice.. To study whether leukocyte calpains contributed to AngII-induced aortic pathologies, irradiated male low-density lipoprotein receptor(-/-) mice were repopulated with BM-derived cells that were either wild-type or overexpressed calpastatin, the endogenous inhibitor of calpains. Mice were fed a fat-enriched diet and infused with AngII (1000 ng/kg per minute) for 4 weeks. Overexpression of calpastatin in BM-derived cells significantly attenuated AngII-induced atherosclerotic lesion formation in aortic arches, but had no effect on aneurysm formation. Using either BM-derived cells from calpain-1-deficient mice or mice with leukocyte-specific calpain-2 deficiency generated using cre-loxP recombination technology, further studies demonstrated that independent deficiency of either calpain-1 or -2 in leukocytes modestly attenuated AngII-induced atherosclerosis. Calpastatin overexpression significantly attenuated AngII-induced inflammatory responses in macrophages and spleen. Furthermore, calpain inhibition suppressed migration and adhesion of macrophages to endothelial cells in vitro. Calpain inhibition also significantly decreased hypercholesterolemia-induced atherosclerosis in the absence of AngII.. The present study demonstrates a pivotal role for BM-derived calpains in mediating AngII-induced atherosclerosis by influencing macrophage function.

Topics: Angiotensin II; Animals; Aortic Aneurysm, Abdominal; Atherosclerosis; Bone Marrow Transplantation; Calcium-Binding Proteins; Calpain; Cell Adhesion; Cell Movement; Cells, Cultured; Coculture Techniques; Cysteine Proteinase Inhibitors; Diet, High-Fat; Disease Models, Animal; Endothelial Cells; Genetic Predisposition to Disease; Inflammation; Leukocytes; Macrophages; Male; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Phenotype; Receptors, LDL; Whole-Body Irradiation

Excessive growth of pulmonary arterial (PA) smooth muscle cells (SMCs) is a major component of PA hypertension (PAH). The calcium-activated neutral cysteine proteases calpains 1 and 2, expressed by PASMCs, contribute to PH but are tightly controlled by a single specific inhibitor, calpastatin. Our objective was to investigate calpastatin during pulmonary hypertension (PH) progression and its potential role as an intracellular and/or extracellular effector. We assessed calpains and calpastatin in patients with idiopathic PAH and mice with hypoxic or spontaneous (SM22-5HTT(+) strain) PH. To assess intracellular and extracellular roles for calpastatin, we studied effects of the calpain inhibitor PD150606 on hypoxic PH in mice with calpastatin overexpression driven by the cytomegalovirus promoter (CMV-Cast) or C-reactive protein (CRP) promoter (CRP-Cast), inducing increased calpastatin production ubiquitously and in the liver, respectively. Chronically hypoxic and SM22-5HTT(+) mice exhibited increased lung calpastatin and calpain 1 and 2 protein levels and activity, both intracellularly and extracellularly. Prominent calpastatin and calpain immunostaining was found in PASMCs of remodeled vessels in mice and patients with PAH, who also exhibited increased plasma calpastatin levels. CMV-Cast and CRP-Cast mice showed similarly decreased PH severity compared with wild-type mice, with no additional effect of PD150606 treatment. In cultured PASMCs from wild-type and CMV-Cast mice, exogenous calpastatin decreased cell proliferation and migration with similar potency as PD150606 and suppressed fibronectin-induced potentiation. These results indicate that calpastatin limits PH severity via extracellular mechanisms. They suggest a new approach to the development of treatments for PH.

Topics: Acrylates; Animals; Calcium-Binding Proteins; Calpain; Cell Movement; Cell Proliferation; Cytomegalovirus; Disease Progression; Extracellular Space; Heart Function Tests; Humans; Hypertension, Pulmonary; Hypoxia; Intracellular Space; Male; Mice, Inbred C57BL; Mice, Transgenic; Myocytes, Smooth Muscle; Promoter Regions, Genetic; Pulmonary Artery

Mutations in rhodopsin (RHO) are a common cause of retinal dystrophy and can be transmitted by dominant or recessive inheritance. Clinical symptoms caused by dominant and recessive mutations in patients and animal models are very similar but the molecular mechanisms leading to retinal degeneration may differ. We characterized three murine models of retina degeneration caused by either Rho loss of function or expression of the P23H dominant mutation in Rho. Rho loss of function is characterized by activation of calpains and apoptosis-inducing factor (Aif) in dying photoreceptors. Retinas bearing the P23H dominant mutations activate both the calpain-Aif cell death pathway and ER-stress responses that together contribute to photoreceptor cell demise. In vivo treatment with the calpastatin peptide, a calpain inhibitor, was strongly neuroprotective in mice lacking Rho while photoreceptor survival in retinas expressing the P23H dominant mutation was more affected by treatment with salubrinal, an inhibitor of the ER-stress pathway. The further reduction of photoreceptor cell demise by co-treatment with calpastatin and salubrinal suggests co-activation of the calpain and ER-stress death pathways in mice bearing dominant mutations in the Rho gene.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Calcium-Binding Proteins; Calpain; Disease Models, Animal; Mice; Mutation; Photoreceptor Cells, Vertebrate; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Despite the high response rates of individuals with myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)) to treatment with lenalidomide (LEN) and the recent identification of cereblon (CRBN) as the molecular target of LEN, the cellular mechanism by which LEN eliminates MDS clones remains elusive. Here we performed an RNA interference screen to delineate gene regulatory networks that mediate LEN responsiveness in an MDS cell line, MDSL. We identified GPR68, which encodes a G-protein-coupled receptor that has been implicated in calcium metabolism, as the top candidate gene for modulating sensitivity to LEN. LEN induced GPR68 expression via IKAROS family zinc finger 1 (IKZF1), resulting in increased cytosolic calcium levels and activation of a calcium-dependent calpain, CAPN1, which were requisite steps for induction of apoptosis in MDS cells and in acute myeloid leukemia (AML) cells. In contrast, deletion of GPR68 or inhibition of calcium and calpain activation suppressed LEN-induced cytotoxicity. Moreover, expression of calpastatin (CAST), an endogenous CAPN1 inhibitor that is encoded by a gene (CAST) deleted in del(5q) MDS, correlated with LEN responsiveness in patients with del(5q) MDS. Depletion of CAST restored responsiveness of LEN-resistant non-del(5q) MDS cells and AML cells, providing an explanation for the superior responses of patients with del(5q) MDS to LEN treatment. Our study describes a cellular mechanism by which LEN, acting through CRBN and IKZF1, has cytotoxic effects in MDS and AML that depend on a calcium- and calpain-dependent pathway.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Calcium; Calcium-Binding Proteins; Calpain; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Ikaros Transcription Factor; Immunologic Factors; Lenalidomide; Leukemia, Myeloid, Acute; Myelodysplastic Syndromes; Peptide Hydrolases; Receptors, G-Protein-Coupled; RNA Interference; Thalidomide; Ubiquitin-Protein Ligases

The calpains are a family of intracellular cysteine proteases that function in a variety of important cellular functions, including cell signalling, motility, apoptosis and survival. In early invasive breast cancer expression of calpain-1, calpain-2 and their inhibitor, calpastatin, have been associated with clinical outcome and clinicopathological factors.The expression of calpain-1, calpain-2 and calpastatin was determined using immunohistochemistry on core biopsy samples, in a cohort of large but operable inflammatory and non-inflammatory primary breast cancer patients treated with neoadjuvant chemotherapy. Information on treatment and prognostic variables together with long-term clinical follow-up was available for these patients. Diagnostic pre-chemotherapy core biopsy samples and surgically excised specimens were available for analysis.Expression of calpastatin, calpain-1 or calpain-2 in the core biopsies was not associated with breast cancer specific survival in the total patient cohort; however, in patients with non-inflammatory breast cancer, high calpastatin expression was significantly associated with adverse breast cancer-specific survival (P=0.035), as was low calpain-2 expression (P=0.031). Low calpastatin expression was significantly associated with adverse breast cancer-specific survival of the inflammatory breast cancer patients (P=0.020), as was low calpain-1 expression (P=0.003).In conclusion, high calpain-2 and low calpastatin expression is associated with improved breast cancer-specific survival in non-inflammatory large but operable primary breast cancer treated with neoadjuvant chemotherapy. In inflammatory cases, high calpain-1 and high calpastatin expression is associated with improved breast cancer-specific survival. Determining the expression of these proteins may be of clinical relevance. Further validation, in multi-centre cohorts of breast cancer patients treated with neoadjuvant chemotherapy, is warranted.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Calcium-Binding Proteins; Calpain; Female; Humans; Immunohistochemistry; Inflammatory Breast Neoplasms; Middle Aged; Neoadjuvant Therapy; Prognosis; Survival Analysis; Young Adult

Brahman cattle are important in tropical regions due to their ability to tolerate excessive heat and parasites. However, Brahman cattle exhibit lower carcass quality characteristics when compared to Bos taurus breeds. The objective of this study was to evaluate potential associations between single nucleotide polymorphisms (SNPs) in six candidate genes for carcass quality and composition traits in a population of Brahman and Brahman-influenced steers. Steers were evaluated through the American Brahman Breeders Association carcass evaluation project in Gonzales, Texas. Carcass traits measured included hot carcass weight, ribeye area, marbling score, yield grade, quality grade, dressing percent, and Warner-Bratzler shear force score. Six previously described candidate genes were chosen for SNP analysis based on their previous association with growth and carcass traits. Candidate genes utilized in the current study included calpastatin (CAST), calpain (CAPN3), thyroglobulin (TG), growth hormone, insulin growth factor 1, and adiponectin. Six unique SNPs from three candidate genes (TG, CAST, and CAPN3) were significantly associated (P < 0.001) with carcass quality traits (marbling score and quality grade). A genotypic effect was observed for all significant SNPs, with differing levels of performance observed for animals inheriting different genotypes. Although multiple SNPs in the current study were significantly (P < 0.001) associated with growth and carcass traits, they should be validated in larger populations prior to implementation in selection strategies.

Topics: Adiponectin; Animals; Calcium-Binding Proteins; Calpain; Cattle; Genotype; Growth Hormone; Insulin-Like Growth Factor I; Male; Meat; Polymorphism, Single Nucleotide; Quantitative Trait, Heritable; Thyroglobulin

This research aimed to evaluate the effects of the beta-agonist zilpaterol hydrochloride (ZH) on carcass traits, subprimal yield, meat quality, palatability traits, and gene expression in Nellore heifers. Zilpaterol increased Longissimus lumborum area and did not change back fat thickness, meat color, and cooking loss. Heifers fed ZH had greater hindquarter weight and carcass percentage. Muscles from hindquarter were heavier for animals fed ZH. Forequarter (% of carcass) decreased and brisket did not change with ZH supplementation. There were no differences between treatments for steak aroma, beef flavor, and off-flavor. However, tenderness and juiciness were reduced by ZH, depending on postmortem aging. Zilpaterol increased Calpain-1, Calpain-2, and calpastatin mRNA expression, with no effect of day of slaughter or ZH×Day interaction. In conclusion, ZH supplementation improved hypertrophy, meat production, and debone yield in Nellore heifers, which led to decreased tenderness and to increased mRNA expression in the calpain-calpastatin system.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Color; Consumer Behavior; Cooking; Food Additives; Food Quality; Gene Expression; Humans; Hydrogen-Ion Concentration; Red Meat; RNA, Messenger; Taste; Trimethylsilyl Compounds

Calpain is an intracellular Ca(2+)-dependent protease, and the activation of calpain has been implicated in neurodegenerative diseases. Calpain activity can be regulated by calpastatin, an endogenous specific calpain inhibitor. Several lines of evidence have demonstrated a potential role of calpastatin in preventing calpain-mediated pathogenesis. Additionally, several studies have revealed that calpain activation and mitochondrial damage are involved in the cell death process; however, recent evidence has not clearly indicated a neuroprotective mechanism of calpastatin against calpain-dependent mitochondrial impairment in the process of neuronal cell death. Therefore, the purpose of this study was to investigate the potential ability of calpastatin to inhibit calpain activation and mitochondrial impairment in oxidative stress-induced neuron degeneration. Calpastatin was stably overexpressed in human neuroblastoma SH-SY5Y cells. In non-calpastatin overexpressing SH-SY5Y cells, hydrogen peroxide significantly decreased cell viability, superoxide dismutase activity, mitochondrial membrane potential, ATP production and mitochondrial fusion protein (Opa1) levels in the mitochondrial fraction but increased reactive oxygen species formation, calpain and calcineurin activation, mitochondrial fission protein (Fis1 and Drp1) levels in the mitochondrial fraction and apoptotic cells. Nevertheless, these toxic effects were abolished in hydrogen peroxide-treated calpastatin-overexpressing SH-SY5Y cells. The results of the present study demonstrate the potential ability of calpastatin to diminish calpain and calcineurin activation and mitochondrial impairment in neurons that are affected by oxidative damage.

Topics: Calcineurin; Calcium-Binding Proteins; Calpain; Cell Death; Cell Line, Tumor; Gene Expression; Humans; Hydrogen Peroxide; Mitochondrial Dynamics; Neurons; Oxidative Stress

Restenosis limits the efficacy of vascular percutaneous intervention, in which vascular smooth muscle cell (VSMC) proliferation and activation of inflammation are two primary causal factors. Calpains influence VSMC proliferation and collagen synthesis. However, the roles of calpastatin and calpains in vascular restenosis remain unclear. Here, restenosis was induced by ligating the left carotid artery, and VSMCs were pretreated with platelet-derived growth factor (PDGF)-BB. Adenovirus vector carrying MMP2 sequence and specific small interfering RNA against calpain-1/2 were introduced. Finally, restenosis enhanced the expression of calpain-1/2, but reduced calpastatin content. In calpastatin transgenic mice, lumen narrowing was attenuated gradually and peaked on days 14-21. Cell proliferation and migration as well as collagen synthesis were inhibited in transgenic mice, and expression of calpain-1/2 and MMP2/transforming growth factor-β1 (TGF-β1). Consistently, in VSMCs pretreated with PDGF-BB, calpastatin induction and calpains inhibition suppressed the proliferation and migration of VSMCs and collagen synthesis, and reduced expression of calpain-1/2 and MMP2/TGF-β1. Moreover, simvastatin improved restenosis indicators by suppressing the HIF-1α/calpains/MMP2/TGF-β1 pathway. However, MMP2 supplementation eliminated the vascular protection of calpastatin induction and simvastatin. Collectively, calpains inhibition plays crucial roles in vascular restenosis by preventing neointimal hyperplasia at the early stage via suppression of the MMP2/TGF-β1 pathway.

Topics: Animals; Calcium-Binding Proteins; Calpain; Carotid Arteries; Cell Movement; Cell Proliferation; Collagen; Coronary Restenosis; Hypoxia-Inducible Factor 1, alpha Subunit; Matrix Metalloproteinase 2; Mice, Inbred C57BL; Mice, Transgenic; Models, Biological; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Platelet-Derived Growth Factor; RNA, Small Interfering; Signal Transduction; Simvastatin; Transforming Growth Factor beta1

Recent evidence indicates the involvement of calpains (CAPNs), a family of cysteine proteases, in cancer development and progression, as well as the insufficient response to cancer therapies. The contribution of CAPNs and regulatory calpastatin (CAST) and ERK1/2 kinases to aggressiveness, disease course, and outcome in laryngeal cancer remains elusive. This study was aimed to evaluate the CAPN1/2-CAST-ERK1/2 enzyme system mRNA/protein level and to investigate whether they can promote the dynamic of tumor growth and prognosis. The mRNA expression of marker genes was determined in 106 laryngeal cancer (SCLC) cases and 73 non-cancerous adjacent mucosa (NCLM) controls using quantitative real-time PCR. The level of corresponding proteins was analyzed by Western Blot. SLUG expression, as indicator of pathological advancement was determined using IHC staining. Significant increases of CAPN1/2-CAST-ERK1/2 levels of mRNA/protein were noted in SCLC compared to NCLM (p < 0.05). As a result, a higher level of CAPN1 and ERK1 genes was related to larger tumor size, more aggressive and deeper growth according to TFG scale and SLUG level (p < 0.05). There were also relationships of CAPN1/2 and ERK1 with incidences of local/nodal recurrences (p < 0.05). An inverse association for CAPN1/2, CAST, and ERK1/2 transcripts was determined with regard to overall survival (p < 0.05). In addition, a higher CAPN1 and phospho-ERK1 protein level was related to higher grade and stage (p < 0.05) and was found to promote worse prognosis. This is the first study to show that activity of CAPN1/2- CAST-ERK1/2 axis may be an indicator of tumor phenotype and unfavorable outcome in SCLC.

Topics: Aged; Aged, 80 and over; Calcium-Binding Proteins; Calpain; Disease Progression; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Laryngeal Mucosa; Laryngeal Neoplasms; Male; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Grading; Neoplasm Recurrence, Local; Phenotype; Prognosis; Risk Factors; RNA, Messenger; Tumor Burden

Viral myocarditis (VMC) treatment has long been lacking of effective methods. Our former studies indicated roles of calpain in VMC pathogenesis. This study aimed at verifying the potential of calpain in Coxsackievirus B3 (CVB3)-induced myocarditis treatment.. A transgenic mouse overexpressing the endogenous calpain inhibitor, calpastatin, was introduced in the study. VMC mouse model was established via intraperitoneal injection of CVB3 in transgenic and wild mouse respectively. Myocardial injury was assayed histologically (HE staining and pathology grading) and serologically (myocardial damage markers of CK-MB and cTnI). CVB3 replication was observed in vivo and in vitro via the capsid protein VP1 detection or virus titration. Inflammation/fibrotic factors of MPO, perforin, IFNγ, IL17, Smad3 and MMP2 were evaluated using western blot or immunohistology stain. Role of calpain in regulating fibroblast migration was studied in scratch assays.. Calpastatin overexpression ameliorated myocardial injury induced by CVB3 infection significantly in transgenic mouse indicated by reduced peripheral CK-MB and cTnI levels and improved histology injury. Comparing with CVB3-infected wild type mouse, the transgenic mouse heart tissue carried lower virus load. The inflammation factors of MPO, perforin, IFNγ and IL17 were down-regulated accompanied with fibrotic agents of Smad3 and MMP2 inhibition. And calpain participated in the migration of fibroblasts in vitro, which further proves its role in regulating fibrosis.. Calpain plays dual roles of facilitating CVB3 replication and inflammation promotion. Calpain inhibition in CVB3-induced myocarditis showed significant treatment effect. Calpain might be a novel target for VMC treatment in clinical practices.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Migration Inhibition; Coxsackievirus Infections; Disease Models, Animal; Enterovirus B, Human; Fibroblasts; Fibrosis; Inflammation; Mice; Myocarditis; Virus Replication

The islet in type 2 diabetes (T2D) shares many features of the brain in protein misfolding diseases. There is a deficit of β cells with islet amyloid derived from islet amyloid polypeptide (IAPP), a protein coexpressed with insulin. Small intracellular membrane-permeant oligomers, the most toxic form of IAPP, are more frequent in β cells of patients with T2D and rodents expressing human IAPP. β Cells in T2D, and affected cells in neurodegenerative diseases, share a comparable pattern of molecular pathology, including endoplasmic reticulum stress, mitochondrial dysfunction, attenuation of autophagy, and calpain hyperactivation. While this adverse functional cascade in response to toxic oligomers is well described, the sequence of events and how best to intervene is unknown. We hypothesized that calpain hyperactivation is a proximal event and tested this in vivo by β cell-specific suppression of calpain hyperactivation with calpastatin overexpression in human IAPP transgenic mice. β Cell-specific calpastatin overexpression was remarkably protective against β cell dysfunction and loss and diabetes onset. The critical autophagy/lysosomal pathway for β cell viability was protected with calpain suppression, consistent with findings in models of neurodegenerative diseases. We conclude that suppression of calpain hyperactivation is a potentially beneficial disease-modifying strategy for protein misfolding diseases, including T2D.

Topics: Animals; Calcium-Binding Proteins; Calpain; Diabetes Mellitus, Type 2; Female; Humans; Insulin-Secreting Cells; Islet Amyloid Polypeptide; Male; Mice; Mice, Transgenic

The immune response is determined by the speed of the T cell reaction to antigens assured by a state of readiness for proliferation and cytokine secretion. Proliferation, apoptosis and motion of many cell types are controlled by cytoplasmic proteases - µ- and m-calpain - and their inhibitor calpastatin, together forming the "calpain-calpastatin system" (CCS), assumed to modify their targets only upon activation-dependent cytoplasmic Ca2+ increase. Contrastingly to this notion, using quantitative real time PCR and semiquantitative flow cytometry respectively, we show here that the CCS genes are constitutively expressed, and that both calpains are constitutively active in resting, circulating human CD4+ and CD8+ lymphocytes. Furthermore, we demonstrate that calpain inhibition in the resting T cells prevents them from proliferation in vitro and greatly reduces secretion of multiple cytokines. The mechanistic reason for these effects of calpain inhibition on T cell functions might be the demonstrated significant reduction of the expression of active (phosphorylated) upstream signalling molecules, including the phospholipase C gamma, p56Lck and NFκB, in the inhibitor-treated cells. Thus, we propose that the constitutive, self-regulatory calpain-calpastatin system activity in resting human T cells is a necessary, controlling element of their readiness for complex and effective response to antigenic challenge.

Topics: Antigens, CD; Apoptosis; Calcium-Binding Proteins; Calpain; Cell Survival; Cytokines; Gene Expression Profiling; Humans; Lymphocyte Activation; Phosphorylation; Resting Phase, Cell Cycle; Signal Transduction; T-Lymphocyte Subsets; T-Lymphocytes

The present study investigated the effects of the nutritional levels of diets on meat quality and related gene expression in Hainan black goat. Twenty-four goats were divided into six dietary treatments and were fed a concentrate-based diet with two levels of crude protein (CP) (15% or 17%) and three levels of digestive energy (DE) (11.72, 12.55 or 13.39 MJ/kg DM) for 90 days. Goats fed the concentrate-based diet with 17% CP had significantly (P < 0.05) higher average daily gains (ADG) and better feed conversion rates (FCR). The pH 24h value tended to decrease (P < 0.05) with increasing DE levels. The tenderness of Longissimus dorsi muscle (LD) and Semimembranosus muscle (SM) reduced with increasing CP levels (P < 0.05). With increasing DE levels, tenderness was increased (P < 0.05). The heart fatty acid-binding protein (H-FABP) mRNA expression levels in LD and SM increased with increasing DE levels (P < 0.05), but decreased with increasing CP levels (P < 0.05). The calpastatin (CAST) and μ-calpain mRNA expressions levels in LD and SM were affected significantly (P < 0.05) by CP and DE levels in the diet. Therefore, the nutritional levels of diets affect meat quality and expression levels of genes associated with meat quality in Hainan black goats.

Topics: Animal Feed; Animals; Body Composition; Calcium-Binding Proteins; Calpain; Dietary Proteins; Digestion; Energy Metabolism; Fatty Acid-Binding Proteins; Food Quality; Gene Expression; Goats; Meat; Molecular Sequence Data; Muscle, Skeletal; Nutritive Value; RNA, Messenger

Cardiotrophin-1 (CT1) has been used to prevent cell death in different models of liver injury in rats. D-galactosamine induces cell death in culture rat and human hepatocytes. The present study evaluated the cytoprotective effects of CT1 in an experimental model of apoptosis induced by D-galactosamine in hepatocytes.. DNA fragmentation, calpain activity and Western blots of caspase-3, calpastatin and Stat3, and Akt phosphorylation were measured. Stat3 and Akt inhibitors were used to analyze the mechanisms of action of CT1.. CT1 caused an increase in Stat3 and Akt phosphorylation and a decrease of DNA fragmentation, calpain activity, and caspase-3 induced by D-galactosamine. The reduction of calpain activity by CT1 was associated with an increase of calpastatin (its endogenous inhibitor). The effects of CT1 were also dependent on the activation of Sta3 or Akt.. CT1 decreases cell death through a mechanism related to Stat3 and Akt phosphorylation and activation of calpastatin in D-galactosamine-treated hepatocytes.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Caspase 3; Cytokines; Cytoprotection; Disease Models, Animal; DNA Fragmentation; Galactosamine; Hepatocytes; Male; Phosphorylation; Primary Cell Culture; Proto-Oncogene Proteins c-akt; STAT3 Transcription Factor; Swine

Over recent years, accumulated evidence suggests that autophagy induction is protective in animal models of a number of neurodegenerative diseases. Intense research in the field has elucidated different pathways through which autophagy can be upregulated and it is important to establish how modulation of these pathways impacts upon disease progression in vivo and therefore which, if any, may have further therapeutic relevance. In addition, it is important to understand how alterations in these target pathways may affect normal physiology when constitutively modulated over a long time period, as would be required for treatment of neurodegenerative diseases. Here we evaluate the potential protective effect of downregulation of calpains. We demonstrate, in Drosophila, that calpain knockdown protects against the aggregation and toxicity of proteins, like mutant huntingtin, in an autophagy-dependent fashion. Furthermore, we demonstrate that, overexpression of the calpain inhibitor, calpastatin, increases autophagosome levels and is protective in a mouse model of Huntington's disease, improving motor signs and delaying the onset of tremors. Importantly, long-term inhibition of calpains did not result in any overt deleterious phenotypes in mice. Thus, calpain inhibition, or activation of autophagy pathways downstream of calpains, may be suitable therapeutic targets for diseases like Huntington's disease.

Topics: Animals; Autophagy; Calcium-Binding Proteins; Calpain; Disease Models, Animal; Drosophila; Drosophila Proteins; Female; Gene Knockdown Techniques; Huntington Disease; Inbreeding; Male; Mice; Mice, Inbred C57BL; Peptides; Signal Transduction

The protective effects of taurine against closed head injury (CHI) have been reported. This study was designed to investigate whether taurine reduced white matter damage and hippocampal neuronal death through suppressing calpain activation after CHI in rats. Taurine (50 mg/kg) was administered intravenously 30 min and 4 h again after CHI. It was found that taurine lessened the corpus callosum damage, attenuated the neuronal cell death in hippocampal CA1 and CA3 subfields and improved the neurological functions 7 days after CHI. Moreover, it suppressed the over-activation of calpain, enhanced the levels of calpastatin, and reduced the degradation of neurofilament heavy protein, myelin basic protein and αII-spectrin in traumatic tissue 24 h after CHI. These data confirm the protective effects of taurine against gray and white matter damage due to CHI, and suggest that down-regulating calpain activation could be one of the protective mechanisms of taurine against CHI.

Topics: Animals; CA1 Region, Hippocampal; CA3 Region, Hippocampal; Calcium-Binding Proteins; Calpain; Cell Death; Corpus Callosum; Disease Models, Animal; Gray Matter; Head Injuries, Closed; Male; Myelin Basic Protein; Neurofilament Proteins; Neurons; Neuroprotective Agents; Random Allocation; Rats, Sprague-Dawley; Taurine; White Matter

Calpastatin participates in apoptotic cell death and cell signaling, but its role in skeletal myoblast development and molecular involvements in cell growth still remains unknown. The current study aimed to investigate the role of calpastatin on the expression patterns of calpains, caspases, and heat shock proteins (HSPs). In addition, the cell viability during myoblast growth under calpastatin silence condition was also investigated. Three small interference RNA sequences (siRNAs) were used to silence calpastatin gene and ligated into pSilencer plasmid vector to construct short hairpin RNA (shRNA) expression. The all three siRNAs significantly silence the calpastatin gene. Moreover, suppression of calpastatin significantly reduced the viability of myoblasts during growth phase when compared to control cells. Additionally, knockdown of calpastatin significantly increased the mRNA expression of μ-calpain, caspase-3, caspase-7, and caspase-9, as well as HSP-27, -70, and -90. The present study results suggested that the suppression of calpastatin resulted in the increased expression of μ-calpain, caspases, and HSPs which in turn regulate the apoptotic cell death. The present study throws light on the central role of calpastatin in the control of calpain activity, cell proliferation, cell survival, and apoptotic pathways.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Caspases; Cattle; DNA Primers; Gene Expression Regulation; Gene Knockdown Techniques; Heat-Shock Proteins; Muscle Development; Plasmids; Real-Time Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Satellite Cells, Skeletal Muscle; Transfection

Here we demonstrate that heat shock protein 90 (HSP90) interacts with calpain-1, but not with calpain-2, and forms a discrete complex in which the protease maintains its catalytic activity, although with a lower affinity for Ca2+. Equilibrium gel distribution experiments show that this complex is composed by an equal number of molecules of each protein partner. Moreover, in resting cells, cytosolic calpain-1 is completely associated with HSP90. Since calpain-1, in association with HSP90, retains its proteolytic activity, and the chaperone is displaced by calpastatin also in the absence of Ca2+, the catalytic cleft of the protease is not involved in this association. Thus, calpain-1 can form two distinct complexes depending on the availability of calpastatin in the cytosol. The occurrence of a complex between HSP90 and calpain-1, in which the protease is still activable, can prevent the complete inhibition of the protease even in the presence of high calpastatin levels. We also demonstrate that in basal cell conditions HSP90 and calpain-1, but not calpain-2, are inserted in the multi-protein N-Methyl-D-Aspartate receptor (NMDAR) complex. The amount of calpain-1 at the NMDAR cluster is not modified in conditions of increased [Ca2+]i, and this resident protease is involved in the processing of NMDAR components. Finally, the amount of calpain-1 associated with NMDAR cluster is independent from Ca2+-mediated translocation. Our findings show that HSP90 plays an important role in maintaining a given and proper amount of calpain-1 at the functional sites.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Cell Line; HSP90 Heat-Shock Proteins; Humans; Immunoprecipitation; Ions; Male; Mice; Microscopy, Confocal; Peptide Hydrolases; Protein Binding; Rats; Receptors, N-Methyl-D-Aspartate; Recombinant Proteins

Janus kinase/signal transducer and activator of transcription (JAK/STAT) signals and their endogenous inhibitor, suppressor of cytokine signaling 3 (SOCS3), in vascular endothelial cells (ECs) reportedly dominate the pathological angiogenesis. However, how these inflammatory signals are potentiated during pathological angiogenesis has not been fully elucidated. We suspected that an intracellular protease calpain, which composes the multifunctional proteolytic systems together with its endogenous inhibitor calpastatin (CAST), contributes to the JAK/STAT regulations.. To specify the effect of EC calpain/CAST systems on JAK/STAT signals and their relationship with pathological angiogenesis.. The loss of CAST, which is ensured by several growth factor classes, was detectable in neovessels in murine allograft tumors, some human malignant tissues, and oxygen-induced retinopathy lesions in mice. EC-specific transgenic introduction of CAST caused downregulation of JAK/STAT signals, upregulation of SOCS3 expression, and depletion of vascular endothelial growth factor (VEGF)-C, thereby counteracting unstable pathological neovessels and disease progression in tumors and oxygen-induced retinopathy lesions in mice. Neutralizing antibody against VEGF-C ameliorated pathological angiogenesis in oxygen-induced retinopathy lesions. Small interfering RNA-based silencing of endogenous CAST in cultured ECs facilitated μ-calpain-induced proteolytic degradation of SOCS3, leading to VEGF-C production through amplified interleukin-6-driven STAT3 signals. Interleukin-6-induced angiogenic tube formation in cultured ECs was accelerated by CAST silencing, which is suppressible by pharmacological inhibition of JAK/STAT signals, antibody-based blockage of VEGF-C, and transfection of calpain-resistant SOCS3, whereas transfection of wild-type SOCS3 exhibited modest angiostatic effects.. Loss of CAST in angiogenic ECs facilitates μ-calpain-induced SOCS3 degradation, which amplifies pathological angiogenesis through interleukin-6/STAT3/VEGF-C axis.

Topics: Adenocarcinoma; Amino Acid Sequence; Animals; Aorta; Calcium-Binding Proteins; Calpain; Carcinoma, Lewis Lung; Cells, Cultured; Cytokines; Endothelial Cells; Female; Glioblastoma; Humans; Janus Kinases; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Molecular Sequence Data; Mutagenesis, Site-Directed; Neoplasms; Neovascularization, Pathologic; Recombinant Fusion Proteins; Retinopathy of Prematurity; Signal Transduction; STAT Transcription Factors; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Vascular Endothelial Growth Factor C

Both calpain activation and endoplasmic reticulum (ER) stress are implicated in ischemic heart injury. However, the role of calpain in ER stress remains largely elusive. This study investigated whether calpain activation causes ER stress, thereby mediating cardiomyocyte apoptosis in an in vitro model of hypoxia/re-oxygenation (H/R). In neonatal mouse cardiomyocytes and rat cardiomyocyte-like H9c2 cells, up-regulation of calpain-1 sufficiently induced ER stress, c-Jun N-terminal protein kinase1/2 (JNK1/2) activation and apoptosis. Inhibition of ER stress or JNK1/2 prevented apoptosis induced by calpain-1. In an in vitro model of H/R-induced injury in cardiomyocytes, H/R was induced by a 24-hour hypoxia followed by a 24-hour re-oxygenation. H/R activated calpain-1, induced ER stress and JNK1/2 activation, and triggered apoptosis. Inhibition of calpain and ER stress blocked JNK1/2 activation and prevented H/R-induced apoptosis. Furthermore, blockade of JNK1/2 signaling inhibited apoptosis following H/R. The role of calpain in ER stress was also demonstrated in an in vivo model of ischemia/reperfusion using transgenic mice over-expressing calpastatin. In summary, calpain-1 induces ER stress and JNK1/2 activation, thereby mediating apoptosis in cardiomyocytes. Accordingly, inhibition of calpain prevents ER stress, JNK1/2 activation and apoptosis in H/R-induced cardiomyocytes. Thus, ER stress/JNK1/2 activation may represent an important mechanism linking calpain-1 to ischemic injury.

Topics: Animals; Animals, Newborn; Apoptosis; Blotting, Western; Calcium-Binding Proteins; Calpain; Cell Hypoxia; Cell Line; Cells, Cultured; Endoplasmic Reticulum Stress; Enzyme Activation; Humans; Mice, Inbred C57BL; Mice, Transgenic; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinase 9; Myocardial Reperfusion Injury; Myocytes, Cardiac; Oxygen; Rats; RNA Interference

Sumoylation is a posttranslational modification that regulates a wide spectrum of cellular activities. Cardiomyopathy is the leading cause of heart failure. Whether sumoylation, particularly SUMO-2/3 conjugation, is involved in cardiomyopathy has not been investigated. We report here that SUMO-2/3 conjugation was elevated in the human failing hearts, and we investigated the impact of increased SUMO-2 conjugation on heart function by using the gain-of-function approach in mice, in which cardiac specific expression of constitutively active SUMO-2 was governed by alpha myosin heavy chain promoter (MHC-SUMO-2 transgenic, SUMO-2-Tg). Four of five independent SUMO-2-Tg mouse lines exhibited cardiomyopathy with various severities, ranging from acute heart failure leading to early death to the development of chronic cardiomyopathy with aging. We further revealed that SUMO-2 directly regulated apoptotic process by at least partially targeting calpain 2 and its natural inhibitor calpastatin. SUMO conjugation to calpain 2 promoted its enzymatic activity, and SUMO attachment to calpastatin mainly promoted its turnover and altered its subcellular distribution. Thus, enhanced SUMO-2 conjugation led to increased apoptosis and played a pathogenic role in the development of cardiomyopathy and heart failure.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Cardiomyopathies; Heart Failure; HeLa Cells; Humans; Mice; Protein Binding; Protein Transport; Small Ubiquitin-Related Modifier Proteins; Ubiquitins

Previous studies have shown that inflammation is associated with excessive activation of calpains. Acetylcholine (ACh) has been reported to inhibit pro-inflammatory cytokine release and protect against cardiomyocyte injury. However, there is no direct evidence regarding whether ACh can regulate calpains to exert cardioprotection. To this end, we investigated the effect of ACh on tumour necrosis factor alpha (TNF-α)-induced cardiomyocyte injury and further explored the underlying mechanism.. Flow cytometry and transmission electron microscopy were performed to evaluate apoptosis and cellular ultrastructure. Western blotting was performed to assess changes in protein expression. siRNA was employed to silence specific proteins.. TNF-α treatment increased the expression of cleaved caspase-3, calpain-1 and p38-mitogen-activated protein kinase (p38- MAPK). The calpain inhibitor PD150606 and the p38-MAPK inhibitor SB203580 inhibited apoptosis induced by TNF-α. Moreover, SB203580 decreased the expression and activity of calpain-1, possibly related to the up-regulation of calpastatin. ACh significantly inhibited TNF-α-induced cell apoptosis, as evidenced by decreases in caspase-3 cleavage, p38-MAPK phosphorylation, and calpain-1 expression and activity as well as increases in calpastatin expression. These beneficial effects of ACh were abolished by atropine or M2AChR siRNA.. Our results suggest that ACh ameliorated TNF-α-induced calpain activation by decreasing p38-MAPK phosphorylation and enhancing calpastatin expression, indicating that calpain may be an important link between inflammatory factors and myocardial cell apoptosis.

Topics: Acetylcholine; Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Cell Line; MAP Kinase Signaling System; Myocytes, Cardiac; Proto-Oncogene Proteins c-akt; Rats; Tumor Necrosis Factor-alpha

This study aimed to determine the optimum ageing period for vacuum-packed springbok (Antidorcas marsupialis) Longissimus thoracis et lumborum (LTL) and Biceps femoris (BF) muscle stored at 5.4 ± 1.0°C. Portions of muscle from seven male and six female springbok were aged 1, 2, 5, 8, 14 or 21 days. The Warner Bratzler shear force declined most during the first five days post-mortem (PM), while purge and cooking losses increased significantly with ageing. Calpains I and II and calpastatin activity declined significantly up to five days PM, suggesting that they may be responsible for tenderization. Cathepsins B, BL and H activity increased significantly during ageing. The BF muscle had significantly higher pH, lower purge loss, higher cooking loss, higher WBSF and higher calpain and calpastatin activity than the LTL. No significant differences between the genders or muscles were found for the collagen content or collagen solubility. Springbok LTL and BF muscles should not be aged for longer than five days.

Topics: Animals; Antelopes; Calcium-Binding Proteins; Calpain; Cathepsins; Cold Temperature; Collagen; Female; Food Handling; Humans; Hydrogen-Ion Concentration; Male; Meat; Muscle, Skeletal; Stress, Mechanical

The activation of the calpain system is involved in the repair process following myocardial infarction (MI). However, the impact of the inhibition of calpain by calpastatin, its natural inhibitor, on scar healing and left ventricular (LV) remodeling is elusive. Male mice ubiquitously overexpressing calpastatin (TG) and wild-type (WT) controls were subjected to an anterior coronary artery ligation. Mortality at 6 wk was higher in TG mice (24% in WT vs. 44% in TG, P < 0.05) driven by a significantly higher incidence of cardiac rupture during the first week post-MI, despite comparable infarct size and LV dysfunction and dilatation. Calpain activation post-MI was blunted in TG myocardium. In TG mice, inflammatory cell infiltration and activation were reduced in the infarct zone (IZ), particularly affecting M2 macrophages and CD4(+) T cells, which are crucial for scar healing. To elucidate the role of calpastatin overexpression in macrophages, we stimulated peritoneal macrophages obtained from TG and WT mice in vitro with IL-4, yielding an abrogated M2 polarization in TG but not in WT cells. Lymphopenic Rag1(-/-) mice receiving TG splenocytes before MI demonstrated decreased T-cell recruitment and M2 macrophage activation in the IZ day 5 after MI compared with those receiving WT splenocytes. Calpastatin overexpression prevented the activation of the calpain system after MI. It also impaired scar healing, promoted LV rupture, and increased mortality. Defective scar formation was associated with blunted CD4(+) T-cell and M2-macrophage recruitment.

Topics: Animals; Calcium-Binding Proteins; Calpain; CD4-Positive T-Lymphocytes; Chemotaxis, Leukocyte; Disease Models, Animal; Enzyme Activation; Genotype; Heart Rupture, Post-Infarction; Homeodomain Proteins; Lymphocyte Activation; Macrophage Activation; Macrophages; Male; Mice, Inbred C57BL; Mice, Knockout; Myocardial Infarction; Myocardium; Phenotype; Time Factors; Up-Regulation; Ventricular Dysfunction, Left; Ventricular Function, Left; Ventricular Remodeling; Wound Healing

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by pathological deposits of β-amyloid (Aβ) in senile plaques, intracellular neurofibrillary tangles (NFTs) comprising hyperphosphorylated aggregated tau, synaptic dysfunction and neuronal death. Substantial evidence indicates that disrupted neuronal calcium homeostasis is an early event in AD that could mediate synaptic dysfunction and neuronal toxicity. Sodium calcium exchangers (NCXs) play important roles in regulating intracellular calcium, and accumulating data suggests that reduced NCX function, following aberrant proteolytic cleavage of these exchangers, may contribute to neurodegeneration. Here, we show that elevated calpain, but not caspase-3, activity is a prominent feature of AD brain. In addition, we observe increased calpain-mediated cleavage of NCX3, but not a related family member NCX1, in AD brain relative to unaffected tissue and that from other neurodegenerative conditions. Moreover, the extent of NCX3 proteolysis correlated significantly with amounts of Aβ1-42. We also show that exposure of primary cortical neurons to oligomeric Aβ1-42 results in calpain-dependent cleavage of NCX3, and we demonstrate that loss of NCX3 function is associated with Aβ toxicity. Our findings suggest that Aβ mediates calpain cleavage of NCX3 in AD brain and therefore that reduced NCX3 activity could contribute to the sustained increases in intraneuronal calcium concentrations that are associated with synaptic and neuronal dysfunction in AD.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Amyloid beta-Peptides; Animals; Brain; Calcium-Binding Proteins; Calpain; Caspase 3; Cells, Cultured; Female; Gene Knockdown Techniques; Humans; Male; Middle Aged; Oligonucleotides, Antisense; Postmortem Changes; Protein Subunits; Rats; Sodium-Calcium Exchanger; Spectrin; Substrate Specificity; Tauopathies

Calcium (Ca) and potassium (K) are essential nutrients in animal nutrition. Furthermore, the Ca content can influence meat tenderness because it is needed by the proteolytic system of calpains and calpastatins, major factors in postmortem tenderization of skeletal muscles. K content, which is needed for muscle contraction, can also affect meat tenderness. This study showed that K positively affects the Warner-Bratzler shear force (WBSF), measured at 14days of meat aging, which means that higher levels of K are related to lower meat tenderness. Additionally, a significant effect (P≤0.015) of a SNP in the calcium-activated neutral protease 1 (CAPN1) gene on Ca content was observed. Metal content in beef can affect not only nutritional values but also meat quality traits. Part of this effect may be related to variation in specific genes.

Topics: Animal Nutritional Physiological Phenomena; Animals; Biomarkers; Calcium; Calcium-Binding Proteins; Calpain; Cattle; Food Handling; Genetic Variation; Genotype; Meat; Muscle Proteins; Muscle, Skeletal; Phenotype; Polymorphism, Single Nucleotide; Potassium

Eukaryotic genes have intervening sequences, introns, in their coding regions. Since introns are spliced out from m-RNA before translation, they are considered to have no effect on the protein structure. Here, we report a novel relationship between introns and the tertiary structures of retinol binding protein and calpain domain VI. We identified "intron-positions" as amino acid residues on which or just after which introns are found in their corresponding nucleotide sequences, and then found that four intron-positions form a plane. We also found that the four intron-positions of retinol-binding protein encloses its ligand retinol. The tertiary structure of calpain domain VI changes after Ca(2+) binding, and the four intron-positions form a plane that includes its ligand calpastatin. To evaluate the statistical significance of the planarity, we calculated the mean distance of each intron-position from the plane defined by the other three intron-positions, and showed that it is significantly smaller than the one calculated for randomly generated locations based on exon size distribution. On the basis of this finding, we discuss the evolution of retinol binding protein and the origin of introns.

Topics: Amino Acids; Animals; Calcium; Calcium-Binding Proteins; Calpain; Cattle; Exons; Gene Deletion; Humans; Introns; Ligands; Probability; Protein Binding; Protein Structure, Tertiary; Retinol-Binding Proteins; RNA Splicing; Swine; Vitamin A

First we looked for putative causative mutations in the CAST and CAPN1 genes associated with meat tenderness and found a total of 31 and 7 polymorphisms, respectively, in the Parda de Montaña and Pirenaica breeds. Tenderness was not affected by mutations in CAPN1. However, three SNPs located at intron 5 (BTA7: g.98533962C>G on UMD 3.0), exon 7 (g.98535683A>G) and intron 12 (g.98545188T>A) of the CAST gene were significantly associated with meat tenderness at 7 days post-mortem in the Parda de Montaña breed. The haplotypes h2 and h5 showed significant associations with meat toughness being consistent with the SNP association results, which showed that the g.98535683A>G SNP in CAST might be the causative mutation of the effect found in this study. This mutation changes the amino acid sequence at position p.Thr182Ala (NM_174003). This amino acid substitution could affect the interacting regions between the calpastatin L-domain and calpain, and then could generate a more stable union between calpain and calpastatin.

Topics: Amino Acid Substitution; Animals; Breeding; Calcium-Binding Proteins; Calpain; Cattle; Exons; Gene Frequency; Haplotypes; Introns; Meat; Mutation; Polymorphism, Single Nucleotide

The objective of this study was to determine calpain system activity and postmortem protein degradation in three muscles from growing (n=6, 7.3 ± 0.5 months) and mature (n=6, 106.7 ± 43.1 months) beef cattle. The ratio of μ-calpain:total calpastatin activity tended to be lower in mature animals (P=0.08), suggesting reduced potential for proteolysis. Additionally, muscles from the mature group had greater calpastatin activity compared to calves at 6 days postmortem and had less μ-calpain autolysis and troponin-T and titin degradation during the aging period (P<0.01). Between the longissimus, semimembranosus, and triceps brachii muscles, the triceps brachii had the least postmortem proteolysis, with greater calpastatin activity and less troponin-T and titin degradation compared to other muscles (P<0.01). These data suggest that calpastatin activity in muscle from older animals is more persistent postmortem. This difference may contribute to the decreased protein degradation and increased toughness of beef from mature cattle, even after aging.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Electrophoresis, Polyacrylamide Gel; Meat; Muscle, Skeletal; Postmortem Changes; Proteolysis; Troponin T

The objective of this study was to investigate the contribution of caspase and calpain, on the proteolysis of calpastatin in postmortem beef muscle, by examining the influences of calpain inhibitor MDL-28170 and caspase-3 inhibitor DEVD-CHO on calpastatin degradation and the in vitro proteolysis of calpastatin by caspase-3, -6 and μ-calpain. In this study, both calpain- and caspase-3-inhibitors suppressed postmortem degradation of calpastatin. In vitro treatment of calpastatin with μ-calpain resulted in degradation products similar in size to those occurring naturally in aged beef muscle. With addition of caspase-3, only the 100 kDa degradation fragment was present during the early phase of ageing, and subsequently, was likely to have been inactivated by calpain or other factors. Therefore, calpain was the major contributor to the proteolysis of calpastatin in postmortem beef muscle. While caspase-3 was involved in calpastatin degradation during the early postmortem period, calpastatin maybe plays an important role in bridging the gap between caspase and calpain systems.

Topics: Animals; Calcium-Binding Proteins; Calpain; Caspase 3; Cattle; Food Handling; Glycoproteins; Meat; Muscle, Skeletal; Postmortem Changes; Proteolysis

Endoplasmic reticulum (ER) stress, implicated in various neurodegenerative processes, increases the level of intracellular Ca(2+) and leads to activation of calpain, a Ca(2+)-dependent cysteine protease. We have shown previously that S-allyl-L-cysteine (SAC) in aged garlic extracts significantly protects cultured rat hippocampal neurons (HPNs) against ER stress-induced neurotoxicity. The neuroprotective effect of SAC was compared with those of the related antioxidant compounds, L-cysteine (CYS) and N-acetylcysteine (NAC), on calpain activity in HPNs and also in vitro. SAC, but not CYS or NAC, reversibly restored the survival of HPNs and increased the degradation of α-spectrin, a substrate for calpain, induced by tunicamycin, a typical ER stress inducer. Activities of μ- and m-calpains in vitro were also concentration dependently suppressed by SAC, but not by CYS or NAC. At submaximal concentration, although ALLN (5 pM), which blocks the active site of calpain, and calpastatin (100 pM), an endogenous calpain-inhibitor protein, additively inhibited μ-calpain activity in vitro in combination with SAC, the effect of PD150606 (25 μM), which prevents interaction of Ca(2+) with the Ca(2+)-binding site of calpain, was unaffected by SAC. In contrast, SAC (1 mM) significantly reversed the effect of PD150606 at a concentration that elicited supramaximal inhibition (100 μM), but did not affect ALLN (1 nM)- and calpastatin (100 nM)-induced inhibition of μ-calpain activity. These results suggest that the protective effects of SAC against ER stress-induced neuronal cell death are not attributable to antioxidant activity, but to suppression of calpain through interaction with its Ca(2+)-binding site.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Cell Survival; Cells, Cultured; Cysteine; Dipeptides; Endoplasmic Reticulum Stress; Hippocampus; Leupeptins; Neurons; Neuroprotective Agents; Oxidative Stress; Rats; Rats, Wistar; Spectrin

The objective of this study was to assess the association of markers in the calpastatin and mu-calpain loci with iron in beef cattle muscle. The population consisted of 259 cross-bred steers from Beefmaster, Brangus, Bonsmara, Romosinuano, Hereford and Angus sires. Total iron and heme iron concentrations were measured. Markers in the calpastatin (referred to as CAST) and mu-calpain (referred to as CAPN4751) genes were used to assess their association with iron levels. The mean and standard error for iron and heme iron content in the population was 35.6 ± 1.3 μg and 27.1 ± 1.4 μg respectively. Significant associations (P < 0.01) of markers were observed for both iron and heme iron content. For CAST, animals with the CC genotype had higher levels of iron and heme iron in longissimus dorsi muscle. For CAPN4751, individuals with the TT genotype had higher concentrations of iron and heme iron than did animals with the CC and CT genotypes. Genotypes known to be associated with tougher meat were associated with higher levels of iron concentration.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Genotype; Iron; Meat; Muscles; Polymorphism, Genetic

A rise in intracellular myocardial Ca(2+) during cardiac ischemia activates calpain (Calpn) thereby causing damage to myocardial proteins, which leads to myocyte death and consequently to loss of myocardial structure and function. Calcineurin (CaN) interacts with Calpn and causes cellular damage eventually leading to cell death. Calpastatin (Calp) and high molecular weight calmodulin-binding protein (HMWCaMBP) (homolog of Calp), inhibit Calpn activity and thus prevent cell death. CaN stimulation can also result in self-repair of damaged cardiomyocytes. The present study attempts to elucidate the expression of these proteins in cells under pre-ischemic condition (control), following ischemia induction and also reperfusion subsequent to ischemia. For the first time, flow cytometric analysis (FACS) has been used for analyzing protein expression concurrently with viability. We induced ischemia and subsequently reperfusion in 80% confluent cultures of neonatal murine cardiomyocytes (NMCC). Viability following induction was assessed with 7-AAD staining and the cells were simultaneously checked for protein expression by FACS. We observed that ischemia induction results in increased expression of CaN, Calp and Calpn. HMWCaMBP expression was reduced in live cells following ischemia which suggests that there is a poor survival outcome of cells expressing HMWCaMBP thereby making it a potential biomarker for such cells. Most live cells following ischemia expressed CaN pointing towards self-repair and favorable survival outcomes.

Topics: Animals; Animals, Newborn; Calcineurin; Calcium-Binding Proteins; Calmodulin-Binding Proteins; Calpain; Cells, Cultured; Gene Expression Regulation; Mice; Myocardium; Myocytes, Cardiac; Reperfusion Injury

Genetic marker effects and interactions are estimated with poor precision when minor marker allele frequencies are low. An Angus population was subjected to marker assisted selection for multiple years to increase divergent haplotype and minor marker allele frequencies to 1) estimate effect size and mode of inheritance for previously reported SNP on targeted beef carcass quality traits; 2) estimate effects of previously reported SNP on nontarget performance traits; and 3) evaluate tenderness SNP specific residual variance models compared to a single residual variance model for tenderness. Divergent haplotypes within µ-calpain (CAPN1), and SNP within calpastatin (CAST) and growth hormone receptor (GHR) were successfully selected to increase their frequencies. Traits evaluated were birth BW, weaning BW, final BW, fat thickness, LM area, USDA marbling score, yield grade, slice shear force (SSF), and visible and near infrared predicted slice shear force. Both CAPN1 and CAST exhibited additive (P < 0.001) modes of inheritance for SSF and neither exhibited dominance (P ≥ 0.19). Furthermore, the interaction between CAPN1 and CAST for SSF was not significant (P = 0.55). Estimated additive effects of CAPN1 (1.049 kg) and CAST (1.257 kg) on SSF were large in this study. Animals homozygous for tender alleles at both CAPN1 and CAST would have 4.61 kg lower SSF (38.6% of the mean) than animals homozygous tough for both markers. There was also an effect of CAST on yield grade (P < 0.02). The tender CAST allele was associated with more red meat yield and less trimmable fat. There were no significant effects (P ≥ 0.23) for GHR on any of the traits evaluated in this study. Furthermore, CAST specific residual variance models were found to fit significantly better (P < 0.001) than single residual variance models for SSF, with the tougher genotypes having larger residual variance. Thus, the risk of a tough steak from the undesired CAST genotype is increased through both an increase in mean and an increase in variation. This work confirms the importance of CAPN1 and CAST for tenderness in beef, provides a new effect of CAST on beef tenderness, and questions the utility of GHR as a selection marker for beef quality.

Topics: Alleles; Animals; Body Composition; Calcium-Binding Proteins; Calpain; Cattle; Genetic Markers; Haplotypes; Muscle, Skeletal; Polymorphism, Single Nucleotide; Receptors, Somatotropin; Selection, Genetic

Recent studies in septic models have shown that myocardial calpain activity and TNF-α expression increase during sepsis and that inhibition of calpain activation downregulates myocardial TNF-α expression and improves cardiac dysfunction. However, the mechanism underlying this pathological process is unclear. Thus, in the present study, we aimed to explore whether IκBα/NF-κB signaling linked myocardial calpain activity and TNF-α expression in septic mice. Adult male mice were injected with LPS (4 mg/kg ip) to induce sepsis. Myocardial calpain activity, IκBα/NF-κB signaling activity, and TNF-α expression were assessed, and myocardial function was evaluated using the Langendorff system. In septic mice, myocardial calpain activity and TNF-α expression were increased and IκBα protein was degraded. Furthermore, NF-κB was activated, as indicated by increased NF-κB p65 phosphorylation, cleavage of p105 into p50, and its nuclear translocation. Administration of the calpain inhibitors calpain inhibitor Ш and PD-150606 prevented the LPS-induced degradation of myocardial IκBα, NF-κB activation, and TNF-α expression and ultimately improved myocardial function. In calpastatin transgenic mice, an endogenous calpain inhibitor and cultured neonatal mouse cardiomyocytes overexpressing calpastatin also inhibited calpain activity, IκBα protein degradation, and NF-κB activation after LPS treatment. In conclusion, myocardial calpain activity was increased in septic mice. Calpain induced myocardial NF-κB activation, TNF-α expression, and myocardial dysfunction in septic mice through IκBα protein cleavage.

Topics: Acrylates; Animals; Calcium-Binding Proteins; Calpain; Dipeptides; Disease Models, Animal; Heart; Heart Diseases; I-kappa B Proteins; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Myocardium; NF-kappa B; NF-KappaB Inhibitor alpha; Sepsis; Signal Transduction; Tumor Necrosis Factor-alpha

The objective of this study was to evaluate the effects of seven single nucleotide polymorphisms (SNPs) in Calpain 1 and Calpastatin genes previously associated with meat tenderness attributes in other cattle breeds in Korean Hanwoo cattle. The Hanwoo resource population was used to study association of 7 SNPs with beef tenderness, flavor, juiciness, intramuscular fat and shear force. In this association study, CAST:c.182A>G (+0.14, P=0.04) and CAST:c.1985G>C (-0.12, P=0.02) had significant effects on juiciness, but no effects on other traits. In contrast, CAPN1:c.1589G>A was associated with meat tenderness (P=0.01) and juiciness (P=0.04). The CAPN1:c.1589G>A (Val530Ile) SNP marker displayed significant effect on the meat tenderness score which is strongly supported by molecular modeling of the CAPN1:c.1589G>A (Val530Ile) variant that inhibits CAST protein from binding more strongly than the wild-type protein, which may explain its effect on meat tenderness.

Topics: Animals; Breeding; Calcium-Binding Proteins; Calpain; Cattle; Genotype; Humans; Meat; Models, Molecular; Mutation; Phenotype; Polymorphism, Single Nucleotide; Stress, Mechanical; Taste; Water

The present study was carried out to understand the effect of cortisol on calpain system in the C2C12 and 3T3-L1 adipocyte cells under co-culture system. Cells were co-cultured by using transwell inserts with a 0.4 μm porous membrane to separate C2C12 and 3T3-L1 preadipocyte cells. Each cell type was grown independently on the transwell plates. Following cell differentiation, inserts containing 3T3-L1 cells were transferred to C2C12 plates. Ten microgram per milliliter of cortisol was added to the medium. Following treatment for 3 days, the cells in the lower well were harvested for analysis. Calpains such as μ-calpain, m-calpain, and calpastatin were selected for the analysis. RT-PCR results indicated the significant increase in the mRNA expression of μ-calpain, m-calpain, and calpastatin. In addition, the confocal microscopical investigation indicated the cortisol treatment increases calpain expression in the C2C12 and 3T3-L1 cells. Taking all these together, cortisol treatment with co-culture system shows most reliable status of calpains expression in the cells, which is quite distinct from one-dimensional monocultured cells.

Topics: 3T3-L1 Cells; Adipocytes; Animals; Anti-Inflammatory Agents; Calcium-Binding Proteins; Calpain; Cell Differentiation; Coculture Techniques; Diffusion Chambers, Culture; Gene Expression; Hydrocortisone; Mice; Myoblasts; RNA, Messenger

Lewy bodies, a pathological hallmark of Parkinson's disease (PD), contain aggregated alpha-synuclein (αSyn), which is found in several modified forms and can be discovered phosphorylated, ubiquitinated and truncated. Aggregation-prone truncated species of αSyn caused by aberrant cleavage of this fibrillogenic protein are hypothesized to participate in its sequestration into inclusions subsequently leading to synaptic dysfunction and neuronal death. Here, we investigated the role of calpain cleavage of αSyn in vivo by generating two opposing mouse models. We crossed into human [A30P]αSyn transgenic (i) mice deficient for calpastatin, a calpain-specific inhibitor, thus enhancing calpain activity (SynCAST(-)) and (ii) mice overexpressing human calpastatin leading to reduced calpain activity (SynCAST(+)). As anticipated, a reduced calpain activity led to a decreased number of αSyn-positive aggregates, whereas loss of calpastatin led to increased truncation of αSyn in SynCAST(-). Furthermore, overexpression of calpastatin decreased astrogliosis and the calpain-dependent degradation of synaptic proteins, potentially ameliorating the observed neuropathology in [A30P]αSyn and SynCAST(+) mice. Overall, our data further support a crucial role of calpains, particularly of calpain 1, in the pathogenesis of PD and in disease-associated aggregation of αSyn, indicating a therapeutic potential of calpain inhibition in PD.

Topics: alpha-Synuclein; Animals; Calcium-Binding Proteins; Calpain; Disease Models, Animal; Gene Expression Regulation; Humans; Lewy Bodies; Mice; Mice, Transgenic; Neurons; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Proteolysis; Signal Transduction; Synapses

The objective of this study was to investigate whether single nucleotide polymorphisms (SNP) in the calpain 1 (CAPN1), calpain 3 (CAPN3) and calpastatin (CAST) genes, which have been shown to be associated with shear force and tenderness differences in the skeletal muscle of cattle, contribute to phenotypic variation in muscle tenderness by modulating the transcriptional activity of their respective gene. The mRNA expression of the calpain and CAST genes was assessed in the longissimus lumborum muscle (LLM) of cattle from two herds located in distinct production zones on the east (New South Wales, NSW) and west (Western Australia, WA) of Australia. The cattle in the herds were mainly Brahman cattle (Bos indicus) with smaller populations of Angus cattle (Bos taurus). There were 191 steers in the WA herd and 107 steers and 106 heifers in the NSW herd. These herds were established by choosing cattle from the diverse population which had different single nucleotide polymorphism (SNP) genotypes at the CAPN1, CAPN3 and CAST loci. Using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), the transcriptional activities of the CAPN1 and the CAST genes, but not the CAPN3 gene, were found to differ between favorable, positively associated with tenderness, and unfavorable, negatively associated with tenderness, allelic variants of these genes. These findings suggest that the muscle shear force and consumer taste panel differences in tenderness explained by the CAPN1 and CAST gene markers are a consequence of alterations in their mRNA levels, which may ultimately influence the protein activity of these genes, thereby altering the rate and(or) the extent of postmortem proteolysis in skeletal muscle. Of particular importance were the significantly lower type II and type III CAST 5' splice variant mRNA levels that were detected in the LLM muscle of Brahman and Angus cattle with 2 favourable alleles of the CAST:c.2832A > G polymorphism. Moreover, a reduction in the abundance of an alternative polyadenylated variant of the CAST transcript, terminated at the proximal polyadenylation site, provides a unique insight into the potential involvement of a post-transcriptional regulatory mechanism which may influence protein expression levels in bovine skeletal muscle.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Genetic Markers; Genotype; Muscle, Skeletal; Polymorphism, Single Nucleotide; RNA Processing, Post-Transcriptional; RNA, Messenger

The process of photoreceptor cell death in retinitis pigmentosa is still not well characterized, and identification of common mechanisms will be instrumental for development of therapeutic strategies. Here we investigated activation of Bax in rd1, P23H transgenic, and Rho knockout retinas.. Bax activation was evaluated by immunofluorescence using anti-activated Bax-specific antibodies and by Western blotting on mitochondrial protein extracts. Knockdown of cathepsin D, calpain 1, and calpain 2 was achieved by short hairpin RNA (shRNA) delivery in rd1 mutant photoreceptors cells differentiated from retinal neurospheres. The mechanism of Bax activation through calpains was evaluated in vivo by intravitreal injection of calpastatin.. We defined activation and mitochondrial localization of Bax as well as activation of calpains and cathepsin D in the three models of retinitis pigmentosa. Taking advantage of an in vitro culture system for rd1 mutant photoreceptors, we unraveled the mechanism of Bax activation. We demonstrated that calpain 1 and cathepsin D contributed to activation of Bax and to apoptosis-inducing factor (Aif) nuclear translocation. In vivo interference with calpain activity blocks Bax activation in the rd1 and Rho knockout retinas and reduces activation in the P23H transgenic retina.. Activation of Bax was observed in all three models of retinitis pigmentosa and leads to neurodamage by localization at the mitochondrion. Our data suggest that Bax can be envisaged as one of the promising target molecules for restraining photoreceptor degeneration.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Calcium-Binding Proteins; Calpain; Cells, Cultured; Cysteine Proteinase Inhibitors; Disease Models, Animal; In Situ Nick-End Labeling; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mutation; Photoreceptor Cells, Vertebrate; Real-Time Polymerase Chain Reaction; Retinitis Pigmentosa; RNA; Transcriptional Activation

Enzymatic proteolysis by calpains, Ca(2+)-dependent intracellular cysteine proteases, has been implicated in pathological processes such as cellular degeneration or death. Here, we investigated the role of calpain activation in the hearts subjected to myocardial infarction. We produced myocardial infarction in Cast(-/-) mice deficient for calpastatin, the specific endogenous inhibitory protein for calpains, and Cast(+/+) mice. The activity of cardiac calpains in Cast(+/+) mice was not elevated within 1 day but showed a gradual elevation after 7 days following myocardial infarction, which was further pronounced in Cast(-/-) mice. Although the prevalence of cardiomyocyte death was indistinguishable between Cast(-/-) and Cast(+/+) mice, Cast(-/-) mice exhibited profound contractile dysfunction and chamber dilatation and showed a significant reduction in survival rate after myocardial infarction as compared with Cast(+/+) mice. Notably, immunofluorescence revealed that at 28 days after myocardial infarction, calpains were activated in cardiomyocytes exclusively at the border zone and that Cast(-/-) mice showed higher intensity and a broader extent of calpain activation at the border zone than Cast(+/+) mice. In the border zone of Cast(-/-) mice, pronounced activation of calpains was associated with a decrease in N-cadherin expression and up-regulation of molecular markers for cardiac hypertrophy and fibrosis. In cultured rat neonatal cardiomyocytes, calpain activation by treatment with ionomycin induced cleavage of N-cadherin and decreased expression levels of β-catenin and connexin 43, which was attenuated by calpain inhibitor. These results thus demonstrate that activation of calpains disassembles cell-cell adhesion at intercalated discs by degrading N-cadherin and thereby promotes left ventricular remodeling after myocardial infarction.

Topics: Animals; beta Catenin; Cadherins; Calcium-Binding Proteins; Calpain; Connexin 43; Enzyme Activation; Mice; Mice, Knockout; Muscle Proteins; Myocardial Infarction; Myocytes, Cardiac; Nerve Tissue Proteins; Rats; Rats, Wistar; Ventricular Remodeling

We investigated changes in muscle mass, calpains, calpastatin and Z-disk ultrastructure in the soleus muscle (SOL) of Daurian ground squirrels (Spermophilus dauricus) after hibernation or hindlimb suspension to determine possible mechanisms by which muscle atrophy is prevented in hibernators. Squirrels (n=30) were divided into five groups: no hibernation group (PRE, n=6); hindlimb suspension group (HLS, n=6); two month hibernation group (HIB, n=6); two day group after 90±12 days of hibernation (POST, n=6); and forced exercise group (one time forced, moderate-intensity treadmill exercise) after arousal (FE, n=6). Activity and protein expression of calpains were determined by casein zymography and western blotting, and Z-disk ultrastructure was observed by transmission electron microscopy. The following results were found. Lower body mass and higher SOL muscle mass (mg) to total body mass (g) ratio were observed in HIB and POST; calpain-1 activity increased significantly by 176% (P=0.034) in HLS compared to the PRE group; no significant changes were observed in calpain-2 activity. Protein expression of calpain-1 and calpain-2 increased by 83% (P=0.041) and 208% (P=0.029) in HLS compared to the PRE group, respectively; calpastatin expression increased significantly by 180% (P<0.001) and 153% (P=0.007) in HIB and POST, respectively; the myofilaments were well-organized, and the width of the sarcomere and the Z-disk both appeared visually similar among the pre-hibernation, hibernating and post-hibernation animals. Inhibition of calpain activity and consequently calpain-mediated protein degradation by highly elevated calpastatin protein expression levels may be an important mechanism for preventing muscle protein loss during hibernation and ensuring that Z-lines remained ultrastructurally intact.

Topics: Animals; Calcium-Binding Proteins; Calpain; Female; Hibernation; Hindlimb; Male; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Sciuridae

Tau pathogenicity in Alzheimer's disease and other tauopathies is thought to involve the generation of hyperphosphorylated, truncated, and oligomeric tau species with enhanced neurotoxicity, although the generative mechanisms and the implications for disease therapy are not well understood. Here, we report a striking rescue from mutant tau toxicity in the JNPL3 mouse model of tauopathy. We show that pathological activation of calpains gives rise to a range of potentially toxic forms of tau, directly, and by activating cdk5. Calpain overactivation in brains of these mice is accelerated as a result of the marked depletion of the endogenous calpain inhibitor, calpastatin. When levels of this inhibitor are restored in neurons of JNPL3 mice by overexpressing calpastatin, tauopathy is prevented, including calpain-mediated breakdown of cytoskeletal proteins, cdk5 activation, tau hyperphosphorylation, formation of potentially neurotoxic tau fragments by either calpain or caspase-3, and tau oligomerization. Calpastatin overexpression also prevents loss of motor axons, delays disease onset, and extends survival of JNPL3 mice by 3 months to within the range of normal lifespan. Our findings support the therapeutic promise of highly specific calpain inhibition in the treatment of tauopathies and other neurodegenerative states.

Topics: Animals; Behavior, Animal; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Female; Longevity; Male; Mice; Mice, Transgenic; Nerve Degeneration; Survival Rate; tau Proteins; Tauopathies; Treatment Outcome

Activation of calpains (calpain 2 and Lp82) in rodent lenses readily causes proteolysis and cataract formation. In contrast, primate lenses are quite resistant to activation of calpains. The hypothesis is that high levels of human endogenous calpain inhibitor, calpastatin (CS), prevent calpain activation in human lenses. The purpose of the present study was to directly test if CS is a major inhibitory factor in a human lens epithelial cell line, HLE B-3.. Small interfering RNAs (siRNAs) were used to knock down expression of CS in HLE B-3. The cells then were cultured with the calcium ionophore, ionomycin, with or without a calpain inhibitor SNJ-1945. Transcripts for calpain 2 and CS were measured by quantitative PCR (qPCR). Calpain 2 activity was detected by immunoblotting for the calpain-specific, α-spectrin breakdown product and for activation-associated, fragments of calpain 2.. Expression of CS in HLE B-3 was remarkably higher than in α-TN4 (mouse comparator cell line). Proteolysis of α-spectrin was observed in the soluble proteins from α-TN4 incubated with Ca(2+), but not in the human HLE B-3. When CS-reduced HLE B-3 cells (transfected with CS siRNA) were cultured with ionomycin, calpain 2 was activated, specific proteolysis of α-spectrin occurred, and cell death ensued; SNJ-1945 inhibited these changes.. Our data demonstrated that the high levels of endogenous CS do, indeed, inhibit calpain activity in normal human lens epithelial cells. We speculate that age-related oxidation might cause loss of CS activity in human lens epithelial cells, allowing activation of long-dormant calpain 2, proteolysis of critical cytoskeletal proteins, and cataract formation.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Cell Line; Epithelial Cells; Glycoproteins; Humans; Lens, Crystalline; Mice; Proteolysis; RNA, Small Interfering; Spectrin

Improving the traits that underlie meat quality is a major challenge in the beef industry. The objective of this paper was to detect QTL linked to sensory meat quality traits in 3 French beef cattle breeds. We genotyped 1,059, 1,219, and 947 young bulls and their sires belonging to the Charolais, Limousin, and Blonde d'Aquitaine breeds, respectively, using the Illumina BovineSNP50 BeadChip (Illumina Inc., San Diego, CA). After estimating relevant genetic parameters using VCE software, we performed a linkage disequilibrium and linkage analysis on 4 meat traits: intramuscular fat content, muscle lightness, shear force, and tenderness score. Heritability coefficients largely ranged between 0.10 and 0.24; however, they reached a maximum of 0.44 and 0.50 for intramuscular fat content and tenderness score, respectively, in the Charolais breed. The 2 meat texture traits, shear force and tenderness score, were strongly genetically correlated (-0.91 in the Charolais and Limousin breed and -0.86 in the Blonde d'Aquitaine breed), indicating that they are 2 different measures of approximately the same trait. The genetic correlation between tenderness and intramuscular fat content differed across breeds. Using a significance threshold of 5 × 10(-4) for QTL detection, we found more than 200 significant positions across the 29 autosomal chromosomes for the 4 traits in the Charolais and Blonde d'Aquitaine breeds; in contrast, there were only 78 significant positions in the Limousin breed. Few QTL were common across breeds. We detected QTL for intramuscular fat content located near the myostatin gene in the Charolais and Blonde d'Aquitaine breeds. No mutation in this gene has been reported for the Blonde d'Aquitaine breed; therefore, it suggests that an unknown mutation could be segregating in this breed. We confirmed that, in certain breeds, markers in the calpastatin and calpain 1 gene regions affect tenderness. We also found new QTL as several QTL on chromosome 3 that are significantly associated with meat tenderness in the Blonde d'Aquitaine breed. Overall, these results greatly contribute to the goal of building a panel of markers that can be used to select animals of high meat quality.

Topics: Animals; Breeding; Calcium-Binding Proteins; Calpain; Cattle; Chromosome Mapping; Female; Food Quality; France; Linkage Disequilibrium; Male; Meat; Muscle, Skeletal; Mutation; Myostatin; Quantitative Trait Loci; Taste

Recent work indicates that infections are a major contributor to diaphragm weakness in patients who are critically ill and mechanically ventilated, and that diaphragm weakness is a risk factor for death and prolonged mechanical ventilation. Infections activate muscle calpain, but many believe this is an epiphenomenon and that other proteolytic processes are responsible for infection-induced muscle weakness. We tested the hypothesis that muscle-specific overexpression of calpastatin (CalpOX; an endogenous calpain inhibitor) would attenuate diaphragm dysfunction in cecal ligation puncture (CLP)-induced sepsis. We studied 1) wild-type (WT) sham-operated mice, 2) WT CLP-operated mice, 3) CalpOX sham-operated mice, and 4) CalpOX CLP-operated mice (n = 9-10/group). Twenty-four hours after surgery, we assessed the diaphragm force-frequency relationship, diaphragm mass, and total protein content and diaphragm levels of talin and myosin heavy chain (MHC). CLP markedly reduced diaphragm-specific force generation (force/cross-sectional area), which was prevented by calpastatin overexpression (force averaged 21.4 ± 0.5, 6.9 ± 0.8, 22.4 ± 1.0, and 18.3 ± 1.3 N/cm(2), respectively, for WT sham, WT CLP, CalpOX sham, and CalpOX CLP groups, P < 0.001). Diaphragm mass and total protein content were similar in all groups. CLP induced talin cleavage and reduced MHC levels; CalpOX prevented these alterations. CLP-induced sepsis rapidly reduces diaphragm-specific force generation and is associated with cleavage and/or depletion of key muscle proteins (talin, MHC), effects prevented by muscle-specific calpastatin overexpression. These data indicate that calpain activation is a major cause of diaphragm weakness in response to CLP-induced sepsis.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cecum; Diaphragm; Ligation; Mice; Muscle Proteins; Muscle Weakness; Muscles; Myosin Heavy Chains; Sepsis; Talin

HCV-induced CAPN activation and its effects on virus-infected cells in a host-immune system have been studied recently. It has been shown that the HCV-nonstructural 5A protein acts as both an inducer and a substrate for host CAPN protease; it participates in suppressing the TNF-α-induced apoptosis response and downstream IFN-induced antiviral processes. However, little is known regarding the disturbance of antiviral responses generated by bovine CAPN activation by BVDV, which is a surrogate model of HCV and is one of the most destructive diseases leading to great economic losses in cattle herds worldwide. This is also thought to be associated with the effects of either small CAPN inhibitors or the natural inhibitor CAST. They mainly bind to the binding site of CAPN substrate proteins and competitively inhibit the binding of the enzyme substrates to possibly defend against the two viruses (HCV and BVDV) for anti-viral immunity. To devise a new stratagem to discover lead candidates for an anti-BVDV drug, we first attempted to understand the bovine CAPN-CAST interaction sites and the interaction constraints of local binding architectures, were well reflected in the geometry between the pharmacophore features and its shape constraints identified using our modeled bovine CAPN1/CAST4 complex structures. We propose a computer-aided molecular design of an anti-BVDV drug as a mimetic CAST inhibitor to develop a rule-based screening function for adjusting the puzzle of relationship between bovine CAPN1 and the BVDV nonstructural proteins from all of the data obtained in the study.

Topics: Animals; Binding Sites; Bovine Virus Diarrhea-Mucosal Disease; Calcium-Binding Proteins; Calpain; Cattle; Diarrhea Viruses, Bovine Viral; Humans; Models, Molecular; Multiprotein Complexes; Viral Nonstructural Proteins; Virus Replication

Entamoeba histolytica is a tissue-invasive protozoan parasite causing dysentery in humans. During infection of colonic tissues, amoebic trophozoites are able to kill host cells via apoptosis or necrosis, both of which trigger IL-8-mediated acute inflammatory responses. However, the signaling pathways involved in host cell death induced by E. histolytica have not yet been fully defined. In this study, we examined whether calpain plays a role in the cleavage of pro-survival transcription factors during cell death of colonic epithelial cells, induced by live E. histolytica trophozoites. Incubation with amoebic trophozoites induced activation of m-calpain in a time- and dose-dependent manner. Moreover, incubation with amoebae resulted in marked degradation of STAT proteins (STAT3 and STAT5) and NF-κB (p65) in Caco-2 cells. However, IκB, an inhibitor of NF-κB, was not cleaved in Caco-2 cells following adherence of E. histolytica. Entamoeba-induced cleavage of STAT proteins and NF-κB was partially inhibited by pretreatment of cells with a cell-permeable calpain inhibitor, calpeptin. In contrast, E. histolytica did not induce cleavage of caspase-3 in Caco-2 cells. Furthermore, pretreatment of Caco-2 cells with a calpain inhibitor, calpeptin (but not the pan-caspase inhibitor, z-VAD-fmk) or m-calpain siRNA partially reduced Entamoeba-induced DNA fragmentation in Caco-2 cells. These results suggest that calpain plays an important role in E. histolytica-induced degradation of NF-κB and STATs in colonic epithelial cells, which ultimately accelerates cell death.

Topics: Caco-2 Cells; Calcium-Binding Proteins; Calpain; Caspase 3; Caspases; Cell Death; Colon; Entamoeba histolytica; Epithelial Cells; Humans; I-kappa B Proteins; Intestinal Mucosa; NF-kappa B; RNA Interference; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; STAT5 Transcription Factor

Genetic marker effects and type of inheritance are estimated with poor precision when minor marker allele frequencies are low. A stable composite population (MARC III) was subjected to marker-assisted selection for multiple years to equalize specific marker frequencies to 1) estimate effect size and mode of inheritance for previously reported SNP on targeted beef carcass quality traits (n=254), 2) estimate pleiotropic effects of previously reported SNP on nontarget performance traits (n=542 or 254), and 3) evaluate tenderness SNP specific residual variance for LM tenderness. Three haplotypes within μ-calpain (CAPN1), a SNP in calpastatin (CAST), and a dinucleotide substitution in diacylglycerol O-acyltransferase 1 (DGAT1) were successfully selected to equalize their frequencies. Traits evaluated were birth BW, weaning BW, yearling BW, final BW, dressing percent, HCW, fat thickness, LM area, USDA marbling score, yield grade, LM slice shear force (SSF), and visible and near-infrared (VISNIR)-predicted SSF. While the CAPN1 genotype effect on SSF was not significant (P=0.12), the direction and size of CAPN1 contrasts were consistent with previous research. Effects on SSF between divergent CAPN1 haplotypes (1.153 kg) and the additive effect of CAST (0.902 kg) were large, and animals homozygous for tender alleles at both CAPN1 and CAST would have 4.11 kg lower SSF (27.5% of the mean) than animals homozygous tough for both markers. Furthermore, the interaction between CAPN1 and CAST for SSF was not significant (P=0.40). There were significant effects for DGAT1 on adjusted fat thickness (P=0.02) and VISNIR-predicted SSF (P<0.001) with additive and dominance modes of inheritance (P<0.05) for both traits. Furthermore, CAST genotype specific residual variance models fit significantly better (P<0.001) than single residual variance models for SSF, with the tougher genotypes having progressively larger residual (and hence phenotypic) variances. Therefore, risk of a tough steak from the undesired CAST genotype is increased through both an increase in mean and an increase in variation. This work is supportive of the importance of CAPN1 and CAST for mean tenderness in beef, confirms an effect of CAST on beef LM tenderness variation, and identifies an effect of DGAT1 on subcutaneous fat thickness.

Topics: Animals; Body Composition; Breeding; Calcium-Binding Proteins; Calpain; Cattle; Diacylglycerol O-Acyltransferase; Gene Frequency; Genetic Markers; Genotype; Haplotypes; Homozygote; Inheritance Patterns; Meat; Phenotype

In the heart, calpastatin (Calp) and its homologue high molecular weight calmodulin-binding protein (HMWCaMBP) regulate calpains (Calpn) by inhibition. A rise in intracellular myocardial Ca2+ during cardiac ischemia activates Calpn thereby causing damage to myocardial proteins, which leads to myocyte death and consequently to loss of myocardial structure and function. The present study aims to elucidate expression of Calp and HMWCaMBP with respect to Calpn during induced ischemia and reperfusion in primary murine cardiomyocyte cultures. Ischemia and subsequently reperfusion was induced in ∼ 80% confluent cultures of neonatal murine cardiomyocytes (NMCC). Flow cytometric analysis (FACS) has been used for analyzing protein expression concurrently with viability. Confocal fluorescent microscopy was used to observe protein localization. We observed that ischemia induces increased expression of Calp, HMWCaMBP and Calpn. Calpn expressing NMCC on co-expressing Calp survived ischemic induction compared to NMCC co-expressing HMWCaMBP. Similarly, living cells expressed Calp in contrast to dead cells which expressed HMWCaMBP following reperfusion. A significant difference in the expression of Calp and its homologue HMWCaMBP was observed in localization studies during ischemia. The current study adds to the existing knowledge that HMWCaMBP could be a putative isoform of Calp. NMCC on co-expressing Calp and Calpn-1 survived ischemic and reperfusion inductions compared to NMCC co-expressing HMWCaMBP and Calpn-1. A significant difference in expression of Calp and HMWCaMBP was observed in localization studies during ischemia.

Topics: Animals; Animals, Newborn; Blotting, Western; Calcium-Binding Proteins; Calmodulin-Binding Proteins; Calpain; Cells, Cultured; Female; Flow Cytometry; Mice; Microscopy, Confocal; Molecular Weight; Myocardial Ischemia; Myocytes, Cardiac; Protein Binding; Protein Isoforms; Reperfusion

Doxorubicin causes damage to the heart, which may present as cardiomyopathy. However, the mechanisms by which doxorubicin induces cardiotoxicity remain not fully understood and no effective prevention for doxorubicin cardiomyopathy is available. Calpains, a family of calcium-dependent thiol-proteases, have been implicated in cardiovascular diseases. Their activities are tightly controlled by calpastatin. This study employed transgenic mice over-expressing calpastatin to investigate the role of calpain in doxorubicin-induced cardiotoxicity.. Doxorubicin treatment decreased calpain activities in cultured neonatal mouse cardiomyocytes and in vivo mouse hearts, which correlated with down-regulation of calpain-1 and calpain-2 proteins. Over-expression of calpastatin or incubation with pharmacological calpain inhibitors enhanced apoptosis in neonatal and adult cardiomyocytes induced by doxorubicin. In contrast, over-expression of calpain-2 but not calpain-1 attenuated doxorubicin-induced apoptosis in cardiomyocytes. The pro-apoptotic effects of calpain inhibition were associated with down-regulation of protein kinase B (AKT) protein and mRNA expression, and a concomitant reduction in glycogen synthase kinase-3beta (GSK-3β) phosphorylation (Ser9) in doxorubicin-treated cardiomyocytes. Blocking AKT further increased doxorubicin-induced cardiac injuries, suggesting the effects of calpain inhibition may be mediated by inactivating the AKT signalling. In an in vivo model of doxorubicin-induced cardiotoxicity, over-expression of calpastatin exacerbated myocardial dysfunction as assessed by echocardiography and haemodynamic measurement in transgenic mice 5 days after doxorubicin injection. The 5-day mortality was higher in transgenic mice (29.16%) compared with their wild-type littermates (8%) after doxorubicin treatment.. Over-expression of calpastatin enhances doxorubicin-induced cardiac injuries through calpain inhibition and thus, calpains may protect cardiomyocytes against doxorubicin-induced cardiotoxicity.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Calcium-Binding Proteins; Calpain; Cells, Cultured; Cysteine Proteinase Inhibitors; Disease Models, Animal; Dose-Response Relationship, Drug; Doxorubicin; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Heart Diseases; Hemodynamics; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myocytes, Cardiac; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; RNA, Messenger; Time Factors; Transfection; Ultrasonography; Up-Regulation; Ventricular Function, Left

In both the central nervous system (CNS) and peripheral nervous system (PNS), transected axons undergo Wallerian degeneration. Even though Augustus Waller first described this process after transection of axons in 1850, the molecular mechanisms may be shared, at least in part, by many human diseases. Early pathology includes failure of synaptic transmission, target denervation, and granular disintegration of the axonal cytoskeleton (GDC). The Ca(2+)-dependent protease calpains have been implicated in GDC but causality has not been established. To test the hypothesis that calpains play a causal role in axonal and synaptic degeneration in vivo, we studied transgenic mice that express human calpastatin (hCAST), the endogenous calpain inhibitor, in optic and sciatic nerve axons. Five days after optic nerve transection and 48 h after sciatic nerve transection, robust neurofilament proteolysis observed in wild-type controls was reduced in hCAST transgenic mice. Protection of the axonal cytoskeleton in sciatic nerves of hCAST mice was nearly complete 48 h post-transection. In addition, hCAST expression preserved the morphological integrity of neuromuscular junctions. However, compound muscle action potential amplitudes after nerve transection were similar in wild-type and hCAST mice. These results, in total, provide direct evidence that calpains are responsible for the morphological degeneration of the axon and synapse during Wallerian degeneration.

Topics: Animals; Axons; Calcium-Binding Proteins; Calpain; Cerebral Cortex; Cytoskeleton; Electromyography; Electrophysiological Phenomena; Female; Humans; Immunohistochemistry; Mice; Mice, Transgenic; Neurofilament Proteins; Neuromuscular Junction; Optic Nerve; Retina; Sciatic Nerve; Wallerian Degeneration

Conventional calpains are ubiquitous cysteine proteases whose activity is promoted by calcium signaling and specifically limited by calpastatin. Calpain expression has been shown to be increased in human malignant cells, but the contribution of the calpain/calpastatin system in tumorigenesis remains unclear. It may play an important role in tumor cells themselves (cell growth, migration, and a contrario cell death) and/or in tumor niche (tissue infiltration by immune cells, neo-angiogenesis). In this study, we have used a mouse model of melanoma as a tool to gain further understanding of the role of calpains in tumor progression. To determine the respective importance of each target, we overexpressed calpastatin in tumor and/or host in isolation. Our data demonstrate that calpain inhibition in both tumor and host blunts tumor growth, while paradoxically increasing metastatic dissemination to regional lymph nodes. Specifically, calpain inhibition in melanoma cells limits tumor growth in vitro and in vivo but increases dissemination by amplifying cell resistance to apoptosis and accelerating migration process. Meanwhile, calpain inhibition restricted to host cells blunts tumor infiltration by immune cells and angiogenesis required for antitumor immunity, allowing tumor cells to escape tumor niche and disseminate. The development of highly specific calpain inhibitors with potential medical applications in cancer should take into account the opposing roles of the calpain/calpastatin system in initial tumor growth and subsequent metastatic dissemination.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Line, Tumor; Disease Models, Animal; Male; Melanoma; Mice; Mice, Inbred C57BL; Mice, Transgenic

The objective was to investigate if the association between working chute behavior and beef tenderness found in our previous study is related to protein degradation and calpain system activity. Crossbred steers (n=183) allotted to 16 pens were weighed every 28 d. Temperament was evaluated as exit velocity (EV), chute score (CS), and catch score (CAPS). Between 14 and 16 mo of age (606±52 kg), steers were harvested. Strip steaks were collected and aged for 14 d. Subsamples were collected at 36 h and 7d postmortem and analyzed for calpastatin activity, μ-calpain autolysis, and troponin-T degradation. Shear force (WBSF) was correlated (P<0.05) with calpastatin activity and measurements of troponin-T. Calpastatin activity, μ-calpain autolysis, and troponin-T measurements did not correlate with the measurements of EV, CS, and CAPS. Therefore, activation of the calpain system or differences in protein degradation did not appear to influence the differences in tenderness that are correlated with working chute behavior.

Topics: Abattoirs; Animals; Behavior, Animal; Calcium-Binding Proteins; Calpain; Cattle; Hybridization, Genetic; Male; Meat; Muscle, Skeletal; Postmortem Changes; Proteolysis; Temperament; Troponin T

Syk is a 72kDa non-receptor tyrosine kinase that is best characterized in hematopoietic cells. While Syk is pro-tumorigenic in some cancer cell types, it also has been reported as a negative regulator of metastatic cell growth in others. An examination of the RelA (p65) subunit of NF-κB expressed in MCF7 breast cancer cells indicated that either treatment with pervanadate or stable expression of Syk protected RelA from calpain-mediated proteolysis. Similar results were observed with the tyrosine phosphatase, PTP1B, another sensitive calpain substrate. The activity of calpain in MCF7 cell lysates was inhibited by both treatment with hydrogen peroxide and expression of Syk, the former due to oxidative inactivation of calpain and the latter to enhanced expression of calpastatin (CAST), the endogenous calpain inhibitor. The level of CAST was elevated in the cytosolic fraction of Syk-positive breast cancer cells resulting in more CAST present in complex with calpain in cell lysates. The high levels of CAST coincided with elevated basal levels of calcium-and of intracellular calpain activity-in Syk-expressing cells resulting from decreased levels of Bcl-2, an inhibitor of IP3-receptor-mediated calcium release. The inhibition of cellular calpain stimulated the Syk-mediated enhancement of NF-κB induced by TNF-α, enhanced tyrosine phosphorylation resulting from integrin crosslinking, and increased the localization of Syk to the plasma membrane.

Topics: Blotting, Western; Breast Neoplasms; Calcium; Calcium-Binding Proteins; Calpain; Cell Adhesion; Cell Movement; Cell Proliferation; Female; Fluorescent Antibody Technique; Humans; Hydrogen Peroxide; Immunoprecipitation; Intracellular Signaling Peptides and Proteins; NF-kappa B; Oxidants; Phosphorylation; Protein-Tyrosine Kinases; Proteolysis; Proto-Oncogene Proteins c-bcl-2; Subcellular Fractions; Syk Kinase; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Breast tumor kinase (Brk), also known as protein kinase-6 (PTK6), is a nonreceptor protein-tyrosine kinase that has a close functional relationship with the human epidermal growth factor receptor 2 (HER2). High levels of Brk were found in HER2-positive tumor specimens from patients with invasive ductal breast cancer; however, the underlying mechanism of the co-overexpression of Brk and HER2 remains elusive. In the current study, we explored the mechanism of HER2 and Brk co-overexpression in breast cancer cells by investigating the effect of overexpression and knockdown of HER2 on the level of Brk in breast cancer cells. We found that Brk was more stable in HER2-elevated cells than in control vector-transfected cells and was less stable in HER2 siRNA-treated cells than in control siRNA-treated cells, suggesting that HER2 regulates Brk protein stability. Further studies indicated that degradation of Brk involved a calpain-1-mediated proteolytic pathway and indicated an inverse relationship between the level of HER2 expression and calpain-1 activity. We found that HER2 inhibited calpain-1 activity through upregulating calpastatin, an endogenous calpain inhibitor. Silencing of HER2 downregulated calpastatin, and the downregulation could be rescued by overexpression of constitutively active MEK. Together, these data offer novel mechanistic insights into the functional relationship between Brk and HER2.

Topics: Breast; Breast Neoplasms; Calcium-Binding Proteins; Calpain; Enzyme Stability; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Protein-Tyrosine Kinases; Proteolysis; Receptor, ErbB-2; RNA Interference; RNA, Small Interfering; Up-Regulation

Calpain (intracellular Ca(2+)-dependent protease) and calpastatin (calpain specific endogenous inhibitor) are widely distributed in biological systems, and have been implicated in many cellular physiological and pathological processes. Calpastatin level is of central importance to the control of calpain activity. We demonstrated for the first time that calpastatin is overexpressed in mycoplasma-contaminated cultured cells (SH-SY5Y cells that are infected by a strain of Mycoplasma hyorhinis (NDMh)). We have found that the calpastatin-upregulating activity resides in the mycoplasmal membrane lipoproteins, and is associated with NF-κB activation. Calpain-promoted proteolysis is attenuated in the NDMh lipoprotein-treated cells. Here we show that the NDMh lipoproteins promoted an increase in calpastatin in SH-SY5Y cells via the TLR2/TAK1/NF-κB pathway. The synthetic mycoplasmal lipopeptide MALP-2 and the bacterial lipopeptide PAM3CSK4 (TLR2 agonists) also promoted calpastatin upregulation. LPS (TLR4 agonist) activated NF-κB without calpastatin increase in the cell. In contrast, lipoteichoic acid (TLR2 agonist) upregulated calpastatin not via NF-κB activation, but via the MEK1/ELK1 pathway. Zymosan and peptidoglycan, TLR2 agonists that lack lipids, did not induce calpastatin upregulation. Cell treatment with a calpastatin-upregulating agonist (lipoteichoic acid) led to the attenuation of Ca(2+)-promoted calpain activity, whereas agonists that do not upregulate calpastatin (LPS, Zymosan) were ineffective. Overall, the results indicate that in these non-immune cells, calpastatin is upregulated by TLR2-agonists containing lipids, with more than one downstream pathway involved. Such agonists may be useful for studying mechanisms and factors involved in calpastatin regulation. In addition, suitable TLR2 agonists may be of interest in devising treatments for pathological processes involving excessive calpain activation.

Topics: Calcium-Binding Proteins; Calpain; ets-Domain Protein Elk-1; Humans; Immunoblotting; Lipopeptides; Lipopolysaccharides; Lipoproteins; MAP Kinase Kinase 1; Mycoplasma hyorhinis; Neuroblastoma; NF-kappa B; Proteolysis; Teichoic Acids; Toll-Like Receptor 2; Tumor Cells, Cultured; Up-Regulation

While multiple molecular mechanisms contribute to midbrain nigrostriatal dopaminergic degeneration in Parkinson's disease (PD), the mechanism of damage in non-dopaminergic sites within the central nervous system, including the spinal cord, is not well-understood. Thus, to understand the comprehensive pathophysiology underlying this devastating disease, postmortem spinal cord tissue samples (cervical, thoracic, and lumbar segments) from patients with PD were analyzed compared to age-matched normal subjects or Alzheimer's disease for selective molecular markers of neurodegeneration and inflammation. Distal axonal degeneration, relative abundance of both sensory and motor neuron death, selective loss of ChAT(+) motoneurons, reactive astrogliosis, microgliosis, increased cycloxygenase-2 (Cox-2) expression, and infiltration of T cells were observed in spinal cord of PD patients compared to normal subjects. Biochemical analyses of spinal cord tissues revealed associated inflammatory and proteolytic events (elevated levels of Cox-2, expression and activity of μ- and m-calpain, degradation of axonal neurofilament protein, and concomitantly low levels of endogenous inhibitor - calpastatin) in spinal cord of PD patients. Thus, pathologically upregulated calpain activity in spinal cords of patients with PD may contribute to inflammatory response-mediated neuronal death, leading to motor dysfunction. We proposed calpain over-activation and calpain-calpastatin dysregulation driving in a cascade of inflammatory responses (microglial activation and T cell infiltration) and degenerative pathways culminating in axonal degeneration and neuronal death in spinal cord of Parkinson's disease patients. This may be one of the crucial mechanisms in the degenerative process.

Topics: Alzheimer Disease; Axons; Calcium-Binding Proteins; Calpain; Case-Control Studies; Cell Death; Cytoskeletal Proteins; Gliosis; Humans; Huntington Disease; Inflammation; Multiple Sclerosis; Nerve Degeneration; Neurons; Parkinson Disease; Spinal Cord; T-Lymphocytes

Although it is well established that carbohydrate and lipid metabolism are profoundly altered by cold stress, the effects of short-term cold exposure on protein metabolism in skeletal muscle are still poorly understood. Because cold acclimation requires that an organism adjust its metabolic flux, and muscle amino acids may be an important energy source for heat production, we hypothesize that muscle proteolysis is increased and protein synthesis is decreased under such a stress condition. Herein, cold exposure for 24 h decreased rates of protein synthesis and increased overall proteolysis in both soleus and extensor digitorum longus (EDL) muscles, but it did not affect muscle weight. An increase in proteolysis was accompanied by hyperactivity of the ubiquitin-proteasome system (UPS) in both soleus and EDL, and Ca(2+)-dependent proteolysis in EDL. Furthermore, muscles of rats exposed to cold showed increased mRNA and protein levels of atrogin-1 and muscle RING finger enzyme-1 (MuRF1). Additionally, cold stress reduced phosphorylation of Akt and Forkhead box class O1 (FoxO1), a well-known effect that increases FoxO translocation to the nucleus and leads to activation of proteolysis. Plasma insulin levels were lower, whereas catecholamines, corticosterone, and thyroid hormones were higher in cold-exposed rats compared with control rats. The present data provide the first direct evidence that short-term cold exposure for 24 h decreases rates of protein synthesis and increases the UPS and Ca(2+)-dependent proteolytic processes, and increases expression of atrogin-1 and MuRF1 in skeletal muscles of young rats. The activation of atrophy induced by acute cold stress seems to be mediated at least in part through the inactivation of Akt/FoxO signaling and activation of AMP-activated protein kinase.

Topics: Acclimatization; AMP-Activated Protein Kinases; Animals; Calcium-Binding Proteins; Calpain; Carrier Proteins; Cold Temperature; Cold-Shock Response; Forkhead Transcription Factors; Hormones; Kinetics; Lysosomes; Male; Microfilament Proteins; Muscle Proteins; Muscle, Skeletal; Nerve Tissue Proteins; Phosphorylation; Proteasome Endopeptidase Complex; Proteolysis; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; RNA, Messenger; Signal Transduction; SKP Cullin F-Box Protein Ligases; Tripartite Motif Proteins; Ubiquitin-Protein Ligase Complexes; Ubiquitin-Protein Ligases

Tenderness is a key component of palatability, which influences consumers' perception of meat quality. There are a variety of factors that contribute to the tenderness of beef carcasses, including postmortem proteolysis. A more complete understanding of this biological mechanism regulating tenderness is needed to ensure consistently tender beef. Numerous reports indicate µ-calpain is primarily responsible for the degradation of proteins postmortem. Meanwhile, it has been shown that caspase-3 can cleave calpastatin, the inhibitor of µ-calpain. Therefore, the objective of this study was to determine if in vitro degradation of calpastatin by caspase-3 can enhance the postmortem breakdown of myofibrillar proteins by µ-calpain. Bovine semitendinosus muscles were excised from two carcasses 20 min postmortem. Muscle strips were dissected from the semitendinosus, restrained to maintain length, and placed in a neutral buffer containing protease inhibitors. Upon rigor completion, myofibrils were isolated from each strip, and sarcomere length was determined. Samples with similar sarcomere lengths were selected to minimize the effect of sarcomere length on proteolysis. Myofibrils were then incubated at 22°C with either µ-calpain, µ-calpain+calpastatin, µ-calpain+caspase-3+calpastatin, or caspase-3+calpastatin for 0.25, 1, 3, 24, 48, or 72 h at a pH of 6.8. Proteolysis of troponin T (TnT) and calpastatin was evaluated using SDS-PAGE and Western blotting techniques. Analysis of Western blots confirmed significant degradation of calpastatin by caspase-3 (P<0.05). Additionally, Western blots revealed intact calpastatin disappeared rapidly as a result of digestion by µ-calpain. Although caspase-3 did not significantly degrade TnT (P>0.05), all µ-calpain digestion treatments resulted in substantial TnT breakdown (P<0.05). Degradation of TnT did not differ between the µ-calpain+calpastatin and µ-calpain+caspase-3+calpastatin digestions (P>0.05). Results of this study indicate caspase-3 cleavage of calpastatin does not enhance in vitro degradation of TnT by µ-calpain.

Topics: Animals; Blotting, Western; Calcium-Binding Proteins; Calpain; Caspase 3; Cattle; Electrophoresis, Polyacrylamide Gel; Myofibrils; Troponin T

Calpains are involved in calcium-induced neuronal cell toxicity, which is associated with the pathophysiology of Alzheimer's disease (AD). The activity of calpains is regulated by the inhibitor calpastatin, and increased activity of calpains and decreased calpastastin are often found in AD. Histone deacetylase (HDAC) inhibitors are implicated in AD treatment through the improvement of learning and memory but the underlying mechanism is yet to be understood. Here, using SH-SY5Y neuroblastoma cells and a calcium ionophore ionomycin, we examined whether and how HDAC inhibitor trichostatin A (TSA) inhibits calcium-induced neuronal cell death. TSA increased both the mRNA and protein levels of calpastatin, with no alterations in those of calpain 1 and calpain 2. Furthermore, TSA-stimulated increase of calpastatin was accompanied by a significant attenuation of ionomycin-induced autolysis of calpain 1, but not of calpain 2, and calpain-dependent 150 kDa αII spectrin cleavage. Under these conditions, however, caspase activity was unaltered. Moreover, ectopic expression of small interfering RNA of calpastatin reversed the inhibitory effect of TSA on ionomycin-induced calpain 1 autolysis and αII spectrin cleavage. Chromatin immunoprecipitation assay revealed the increased levels of acetylation at lysine 5 of histone H4 (H4K5-Ac), H3K9-Ac and H3K14-Ac within the calpastatin promoter region in TSA-treated cells relative to control cells. Finally, TSA significantly decreased ionomycin-induced cell toxicity. This study demonstrates that TSA attenuates calcium-induced neuronal cell death by the inhibition of calpain activity which is mediated in part by increased calpastatin expression via histone hyperacetylation within the calpastatin promoter region. Our study provides a novel mechanism for the neuroprotective effect of HDAC inhibitors on AD.

Topics: Acetylation; Apoptosis; Blotting, Western; Calcium; Calcium-Binding Proteins; Calpain; Cell Proliferation; Chromatin Immunoprecipitation; Epigenomics; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Ionomycin; Luciferases; Neuroblastoma; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Tumor Cells, Cultured

Axon degeneration is widespread both in neurodegenerative disease and in normal neural development, but the molecular pathways regulating these degenerative processes and the extent to which they are distinct or overlapping remain incompletely understood. We report that calpastatin, an inhibitor of calcium-activated proteases of the calpain family, functions as a key endogenous regulator of axon degeneration. Calpastatin depletion was observed in degenerating axons after physical injury, and maintaining calpastatin inhibited degeneration of transected axons in vitro and in the optic nerve in vivo. Calpastatin depletion also occurred in a caspase-dependent manner in trophic factor-deprived sensory axons and was required for this in vitro model of developmental degeneration. In vivo, calpastatin regulated the normal pruning of retinal ganglion cell axons in their target field. These findings identify calpastatin as a key checkpoint for axonal survival after injury and during development, and demonstrate downstream convergence of these distinct pathways of axon degeneration.

Topics: Animals; Animals, Newborn; Armadillo Domain Proteins; Axotomy; Brain; Calcium-Binding Proteins; Calpain; Cell Survival; Cells, Cultured; Cytoskeletal Proteins; Disease Models, Animal; Embryo, Mammalian; Enzyme Inhibitors; Ganglia, Spinal; Gene Expression Regulation; Green Fluorescent Proteins; HEK293 Cells; Humans; In Vitro Techniques; Mice; Microscopy, Electron, Transmission; Nerve Degeneration; Nerve Growth Factor; Nerve Tissue Proteins; Neurons; Nicotinamide-Nucleotide Adenylyltransferase; RNA, Messenger; RNA, Small Interfering; Sciatic Neuropathy; Time Factors; Transduction, Genetic; Wallerian Degeneration

Five single-nucleotide polymorphisms (SNPs) located in the calpain 1, (mu/I) large subunit (CAPN1), calpastatin (CAST), and cathepsin D (CTSD) genes were analyzed in a large sample of Piemontese cattle. The aim of this study was to evaluate allele and genotype frequencies of these SNPs and to investigate associations of CAPN1, CAST, and CTSD gene variants with meat quality traits. Minor allele frequencies ranged from 30 to 48%. The presence of the A allele at CAPN530 increased yellowness and drip loss. The CAST282 G allele was associated with an increased drip loss compared to the C allele, and the CAST2959 A allele decreased redness compared to the G allele.

Topics: Analysis of Variance; Animals; Bayes Theorem; Calcium-Binding Proteins; Calpain; Cathepsin D; Cattle; Gene Frequency; Genotype; Italy; Meat; Muscle, Skeletal; Polymorphism, Single Nucleotide

Sickle cell disease (SCD) is a globally distributed hereditary red blood cell (RBC) disorder. One of the hallmarks of SCD is the presence of circulating dense RBCs, which are important in SCD-related clinical manifestations. In human dense sickle cells, we found reduced calpastatin activity and protein expression compared to either healthy RBCs or unfractionated sickle cells, suggesting an imbalance between activator and inhibitor of calpain-1 in favor of activator in dense sickle cells. Calpain-1 is a nonlysosomal cysteine proteinase that modulates multiple cell functions through the selective cleavage of proteins. To investigate the relevance of this observation in vivo, we evaluated the effects of the orally active inhibitor of calpain-1, BDA-410 (30 mg/kg/d), on RBCs from SAD mice, a mouse model for SCD. In SAD mice, BDA-410 improved RBC morphology, reduced RBC density (D(20); from 1106 ± 0.001 to 1100 ± 0.001 g/ml; P<0.05) and increased RBC-K(+) content (from 364 ± 10 to 429 ± 12.3 mmol/kg Hb; P<0.05), markedly reduced the activity of the Ca(2+)-activated K(+)channel (Gardos channel), and decreased membrane association of peroxiredoxin-2. The inhibitory effect of calphostin C, a specific inhibitor of protein kinase C (PKC), on the Gardos channel was eliminated after BDA-410 treatment, which suggests that calpain-1 inhibition affects the PKC-dependent fraction of the Gardos channel. BDA-410 prevented hypoxia-induced RBC dehydration and K(+) loss in SAD mice. These data suggest a potential role of BDA-410 as a novel therapeutic agent for treatment of SCD.

Topics: Anemia, Sickle Cell; Animals; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Dehydration; Disease Models, Animal; Erythrocytes, Abnormal; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Mice; Mice, Mutant Strains; Mice, Transgenic; Sulfonamides

Spinocerebellar ataxia type 3 (SCA3) is pathologically characterized by the formation of intranuclear aggregates which contain ataxin-3, the mutated protein in SCA3, in a specific subtype of neurons. It has been proposed that ataxin-3 is cleaved by proteolytic enzymes, in particular by calpains and caspases, eventually leading to the formation of aggregates. In our study, we examined the ability of calpains to cleave ataxin-3 in vitro and in vivo. We demonstrated in cell culture and mouse brain homogenates that cleavage of overexpressed ataxin-3 by calpains and in particular by calpain-2 occur and that polyglutamine expanded ataxin-3 is more sensitive to calpain degradation. Based on these results, we investigated the influence of calpains on the pathogenesis of SCA3 in vivo. For this purpose, we enhanced calpain activity in a SCA3 transgenic mouse model by knocking out the endogenous calpain inhibitor calpastatin. Double-mutant mice demonstrated an aggravated neurological phenotype with an increased number of nuclear aggregates and accelerated neurodegeneration in the cerebellum. This study confirms the critical importance of calcium-dependent calpain-type proteases in the pathogenesis of SCA3 and suggests that the manipulation of the ataxin-3 cleavage pathway and the regulation of intracellular calcium homeostasis may represent novel targets for therapeutic intervention in SCA3.

Topics: Animals; Ataxin-3; Calcium; Calcium-Binding Proteins; Calpain; Cerebellum; Disease Models, Animal; Gene Deletion; Gene Expression Regulation; Gene Knockout Techniques; Genotype; Glycoproteins; HEK293 Cells; Homeostasis; Humans; Immunohistochemistry; Machado-Joseph Disease; Mice; Mice, Knockout; Mutation; Nerve Tissue Proteins; Nuclear Proteins; Peptides; Phenotype; Repressor Proteins; Transcription Factors

Surgery is critical in the management of gastro-oesophageal cancer, and the addition of neo-adjuvant chemotherapy has proved to be of benefit. The calpain system has been implicated in tumour progression and response to various anti-cancer therapies, and therefore expression of the system was determined in this tumour type.. Two cohorts of gastro-oesophageal adenocarcinomas were investigated for calpain-1, calpain-2, calpain-9 and calpastatin expression using conventional immunohistochemistry. 88 patients who received neo-adjuvant chemotherapy and 140 patients who received surgery alone were investigated using a tissue microarray approach.. Calpain-1, calpain-2 and calpastatin expression was associated with adverse cancer-specific survival in the neo-adjuvant cohort (P = 0.004, P = 0.001 and P = 0.012 respectively); which remained significant in multivariate analysis (Hazard ratio (HR) = 0.337; 95% confidence interval (CI) = 0.140-0.81; P = 0.015, HR = 0.375; 95% CI = 0.165-0.858; P = 0.020 and HR = 0.481; 95% CI = 0.257-0.900; P = 0.022 respectively). Calpain-1 and calpastatin expression was also associated with adverse cancer specific survival in the primary surgery cohort (P = 0.001 and P = 0.013 respectively); which remained significant in multivariate analysis (HR = 0.309; 95% CI = 0.159-0.601; P = 0.001 and HR = 0.418; 95% CI = 0.205-0.850; P = 0.016 respectively). Calpain-9 expression was not associated with cancer-specific survival in the neo-adjuvant and primary surgery cohorts.. Determining the expression levels of calpain-1, calpain-2 and calpastatin may provide clinically relevant prognostic information for gastro-oesophageal adenocarcinomas; these findings warrant further studies in larger cohorts of patients.

Topics: Adenocarcinoma; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Calcium-Binding Proteins; Calpain; Chemotherapy, Adjuvant; Esophageal Neoplasms; Humans; Kaplan-Meier Estimate; Neoplasm Invasiveness; Neoplasm Staging; Prognosis; Tissue Array Analysis; Treatment Outcome

Proteolytic enzymes have been implicated in the pathogenesis of Multiple Sclerosis (MS) for both their ability to degrade myelin proteins and for their presence in MS plaques.In this study we investigated whether interferon-beta (IFN-β) could differently modulate the activity and the expression of proteolytic activities against myelin basic protein (MBP) present in lipopolysaccharide (LPS)-activated astrocytes.. Rat astrocyte cultures were activated with LPS and simultaneously treated with different doses of IFN-β. To assess the presence of MBP-cleaving proteolytic activity, culture supernatants and cellular extracts collected from astrocytes were incubated with exogenous MBP. A MBP-degrading activity was found in both lysates and supernatants from LPS-activated astrocytes and was dose-dependently inhibited by IFN-β. The use of protease inhibitors as well as the zymographic analysis indicated the presence of calpain II (CANP-2) in cell lysates and gelatinases A (MMP-2) and B (MMP-9) in cell supernatants. RT-PCR revealed that the expression of CANP-2 as well as of MMP-2 and MMP-9 was increased in LPS-activated astrocytes and was dose-dependently inhibited by IFN-β treatment. The expression of calpastatin, the natural inhibitor of CANPs, was not affected by IFN-β treatment. By contrast, decreased expression of TIMP-1 and TIMP-2, the natural inhibitors of MMP-9 and MMP-2, respectively, was observed in IFN-β-treated astrocytes compared to LPS-treated cells. The ratio enzyme/inhibitor indicated that the effect of IFN-β treatment is more relevant to CANP-2 than on MMPs.. These results suggest that the neuroinflammatory damage during MS involves altered balance between multiple proteases and their inhibitors and indicate that IFN-β is effective in regulating different enzymatic systems involved in MS pathogenesis.

Topics: Animals; Astrocytes; Calcium-Binding Proteins; Calpain; Cytosol; Dose-Response Relationship, Drug; Extracellular Space; Gene Expression; Interferon-beta; Lipopolysaccharides; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Myelin Basic Protein; Myelin Sheath; Primary Cell Culture; Protease Inhibitors; Rats; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2

The aim was to analyze the allelic and genotypic frequencies for two genes associated with tenderness of meat (CAPN1 and CAST) and one with fat deposits (DGAT1) in three endangered Spanish cattle breeds: Berrenda en Colorado (BC), Berrenda en Negro (BN), and Cardena Andaluza (CA) to utility of their involvement in the selection of them and to help the adoption of conservation measurement. Seventy-five males and 298 females of those breeds were genotyped. Genotypic and allelic frequencies for each polymorphic locus were estimated. There were significant differences in the genotypic frequencies among breeds in CAPN1 and DGTA1 genes and in the case of the genic frequencies in CAPN1, CAST, and DGAT1 genes. The three breeds analyzed (BC, BN, and CA) presented high allelic frequencies for the favorable allele of the three markers (from 0.41 to 0.75). The association between the favorable allele and meat quality must be confirmed. In cases of association with differences in quality meat, the absence of differences in the genotypic and genic frequency distributions between the sexes is advantageous in mating planning because it implies that there is no handicap to be overcome for the conservation program and it would allow the use of sires to promote the increase in improvements within a short period of time.

Topics: Animals; Breeding; Calcium-Binding Proteins; Calpain; Cattle; Diacylglycerol O-Acyltransferase; Endangered Species; Female; Gene Frequency; Genetic Markers; Genotype; Heterozygote; Livestock; Male; Meat

We conducted an in vivo experiment to determine whether vitamin D₃ acts as a fat synthesizer and/or meat tenderizer in mice. At 6 weeks of age, 20 male C57BL/6 wild-type mice were randomly divided into two groups (10 mice per group) and fed a modified AIN93G diet with (vitamin D₃ diet) or without (basal diet) 10 IU 25-OH-cholecalciferol kg⁻³ for 3 weeks.. When vitamin D₃ was fed to mice for 3 weeks, body fat was significantly increased compared to mice fed a basal diet. There was, however, no difference in body weight between the two groups. Vitamin D₃ increased the gene expressions of pro-inflammatory cytokines and peroxisome proliferator-activated receptor gamma, but decreased interleukin-15 in adipose tissue through nuclear vitamin D receptor and uncoupling protein-2 signals. The muscle inducible nitrate oxide synthase content of mice fed vitamin D₃ was higher than those fed a basal diet, while muscle arginase l showed a reverse phenomenon. longissimuss dorsi muscle of vitamin D₃-fed mice showed more severe fat deposition than those fed a basal diet. Vitamin D₃ amplified muscle u- and m-calpain protein content and suppressed muscle calpastatin protein content.. These findings suggest that vitamin D3 can be used as a fat synthesizer and meat tenderizer in meat-producing animals.

Topics: Adiposity; Animals; Arginase; Calcifediol; Calcium-Binding Proteins; Calpain; Cytokines; Dietary Supplements; Gene Expression Regulation; Ion Channels; Lipid Metabolism; Male; Mice, Inbred C57BL; Mitochondrial Proteins; Muscle, Skeletal; Nitric Oxide Synthase Type II; PPAR gamma; Random Allocation; Receptors, Calcitriol; Signal Transduction; Uncoupling Protein 2

The aim of the present experiment was to measure the protease activities in ice-stored and super-chilled Atlantic salmon (Salmo salar) fillets, and the effect on texture. Pre-rigour fillets of Atlantic salmon were either super-chilled to a core temperature of -1.5°C or directly chilled on ice prior to 144h of ice storage. A significantly higher calpain activity was detected in the super-chilled fillets at 6h post-treatment compared to the ice-stored fillets and followed by a significant decrease below its initial level, while the calpastatin activity was significantly lower for the super-chilled fillets at all time points. The cathepsin B+L and B activities increased significantly with time post-treatment; however, no significant differences were observed at any time points between the two treatments. For the ice stored fillets, the cathepsin L activity decreased significantly from 6 to 24h post-treatment and thereafter increased significantly to 144h post-treatment. There was also a significantly lower cathepsin L activity in the super-chilled fillets at 0h post-treatment. No significant difference in breaking force was detected; however, a significant difference in maximum compression (Fmax) was detected at 24h post-treatment with lower Fmax in the super-chilled fillets. This experiment showed that super-chilling had a significant effect on the protease activities and the ATP degradation in salmon fillets. The observed difference in Fmax may be a result of these observed differences, and may indicate a softening of the super-chilled salmon muscle at 24h post-treatment.

Topics: Animals; Calcium-Binding Proteins; Calpain; Food Handling; Food Preservation; Ice; Salmo salar; Seafood; Temperature

Calpain, an intracellular cysteine protease, has been widely reported to be involved in neuronal cell death. The purpose of this study is to investigate the role of calpain activation in axonal damage-induced retinal ganglion cell (RGC) death. Twelve-week-old male calpstatin (an endogenous calpain inhibitor) knockout mice (CAST KO) and wild-type (WT) mice were used in this study. Axonal damage was induced by optic nerve crush (NC) or tubulin destruction induced by leaving a gelatin sponge soaked with vinblastine (VB), a microtubule disassembly chemical, around the optic nerve. Calpain activation was assessed by immunoblot analysis, which indirectly quantified the cleaved α-fodrin, a substrate of calpain. RGCs were retrogradely labeled by injecting a fluorescent tracer, Fluoro-Gold (FG), and the retinas were harvested and flat-mounted retinas prepared. The densities of FG-labeled RGCs harvested from the WT and CAST KO groups were assessed and compared. Additionally, a calpain inhibitor (SNJ-1945, 100 mg/kg/day) was administered orally, and the density of surviving RGCs was compared with that of the vehicle control group. The mean density of surviving RGCs in the CAST KO group was significantly lower than that observed in the WT group, both in NC and in VB. The mean density of surviving RGCs in the SNJ-1945-treated group was significantly higher than that of the control group. The calpain inhibitor SNJ-1945 has a neuroprotective effect against axonal damage-induced RGC death. This pathway may be an important therapeutic target for preventing this axonal damage-induced RGC death, including glaucoma and diabetic optic neuropathy and other CNS diseases that share a common etiology.

Topics: Amino Acids; Animals; Axons; Brain-Derived Neurotrophic Factor; Calcium-Binding Proteins; Calpain; Carbamates; Carrier Proteins; Cell Count; Cell Death; Cells, Cultured; Disease Models, Animal; Enzyme Inhibitors; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microfilament Proteins; Neurofilament Proteins; Optic Nerve Injuries; Retina; Retinal Ganglion Cells; Stilbamidines; Time Factors; Tubulin

This study was designed to investigate the effects of calpastatin genotypes determined by PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) on calpastatin activity (CAC) and Warner-Bratzler Shear Force (WBS). Longissimus muscles were prepared from 379 Hanwoo bulls aged approximately 20months. The selection of PCR primers was based on exons (27 and 28) of the bovine calpastatin cDNA sequences, and genetic variants were detected by SSCP analysis using Taq I restriction enzymes. Sequencing analysis confirmed 4 restriction sites (nucleotide positions 52, 67, 796, and 1369), and a genetic variant was verified at a nucleotide position 641 (C/T substitutions) based on sequences (AF281256). The CAST28 genotypes showing allele frequencies of C (0.429) and T (0.571) were significantly associated with CAC and WBS. A significant positive residual correlation (r=0.121, P=0.02) between CAC and WBS was obtained.

Topics: Animals; Body Composition; Calcium-Binding Proteins; Calpain; Cattle; Exons; Gene Frequency; Genetic Markers; Genetic Variation; Genotype; Male; Meat; Muscle, Skeletal; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Postmortem Changes; Sequence Analysis, DNA

The mechanism by which amyloid-β peptide (Aβ) accumulation causes neurodegeneration in Alzheimer's disease (AD) remains unresolved. Given that Aβ perturbs calcium homeostasis in neurons, we investigated the possible involvement of calpain, a calcium-activated neutral protease. We first demonstrated close postsynaptic association of calpain activation with Aβ plaque formation in brains from both patients with AD and transgenic (Tg) mice overexpressing amyloid precursor protein (APP). Using a viral vector-based tracer, we then showed that axonal termini were dynamically misdirected to calpain activation-positive Aβ plaques. Consistently, cerebrospinal fluid from patients with AD contained a higher level of calpain-cleaved spectrin than that of controls. Genetic deficiency of calpastatin (CS), a calpain-specific inhibitor protein, augmented Aβ amyloidosis, tau phosphorylation, microgliosis, and somatodendritic dystrophy, and increased mortality in APP-Tg mice. In contrast, brain-specific CS overexpression had the opposite effect. These findings implicate that calpain activation plays a pivotal role in the Aβ-triggered pathological cascade, highlighting a target for pharmacological intervention in the treatment of AD.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Brain; Calcium-Binding Proteins; Calpain; Caspases; Enzyme Activation; Female; Fluorescent Antibody Technique; Humans; Immunoblotting; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Middle Aged; Neurodegenerative Diseases; Phosphorylation; Plaque, Amyloid; Survival Analysis; tau Proteins

The experiment was conducted to determine the effect of temperature during post-mortem muscle storage on the activity of the calpain system, the myofibril fragmentation and the free calcium concentration. Porcine longissimus muscle were incubated from 2h post-mortem at temperatures of 2, 15, 25 and 30 °C and sampling times were at 2, 6, 24, 48 and 120 h post-mortem. After 120 h at 30 °C the free calcium concentration increased to 530 μM from 440 μM at 2 °C. Incubation at temperatures higher than 2 °C resulted in the appearance of autolyzed m-calpain activity and a decrease of native m-calpain activity. Native m-calpain decreased more slowly than native μ-calpain, and the autolysis process started later. Myofibril fragmentation increased with storage time and incubation temperature, while calpastatin activity decreased. The study showed that high temperature incubation not only rapidly activated μ-calpain but at higher temperatures and later time points also m-calpain.

Topics: Animals; Autolysis; Calcium; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Food Handling; Meat; Muscle, Skeletal; Myofibrils; Particle Size; Proteolysis; Sarcomeres; Sus scrofa; Temperature; Time Factors

Sepsis, a leading cause of death worldwide, involves widespread activation of inflammation, massive activation of coagulation, and lymphocyte apoptosis. Calpains, calcium-activated cysteine proteases, have been shown to increase inflammatory reactions and lymphocyte apoptosis. Moreover, calpain plays an essential role in microparticle release.. We investigated the contribution of calpain in eliciting tissue damage during sepsis.. To test our hypothesis, we induced polymicrobial sepsis by cecal ligation and puncture in wild-type (WT) mice and transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor.. In WT mice, calpain activity increased transiently peaking at 6 hours after cecal ligation and puncture surgery. Calpastatin overexpression improved survival, organ dysfunction (including lung, kidney, and liver damage), and lymphocyte apoptosis. It decreased the sepsis-induced systemic proinflammatory response and disseminated intravascular coagulation, by reducing the number of procoagulant circulating microparticles and therefore delaying thrombin generation. The deleterious effect of microparticles in this model was confirmed by transferring microparticles from septic WT to septic transgenic mice, worsening their survival and coagulopathy.. These results demonstrate an important role of the calpain/calpastatin system in coagulation/inflammation pathways during sepsis, because calpain inhibition is associated with less severe disseminated intravascular coagulation and better overall outcomes in sepsis.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Cell-Derived Microparticles; Cytokines; Disease Models, Animal; Disseminated Intravascular Coagulation; Lymphocytes; Mice; Mice, Inbred C57BL; Mice, Transgenic; Multiple Organ Failure; NF-kappa B; Sepsis; Thromboplastin

Mycoplasma hyorhinis frequently contaminates cultured cells, with effects on synthetic and metabolic pathways. We demonstrated for the first time that contamination of cells by a strain of M. hyorhinis (NDMh) results in increased levels of calpastatin (the endogenous inhibitor of the ubiquitous Ca(2+) -dependent protease calpain). We now show that the calpastatin upregulation by NDMh in neuroblastoma SH-SY5Y cells resides in the NDMh lipoprotein fraction (LPP), via the NF-κB transcription pathway. NF-κB activation requires dissociation of the cytoplasmic NF-κB/IκB complex followed by NF-κB translocation to the nucleus. NDMh-LPP induced translocation of the NF-κB RelA subunit to the nucleus and upregulated calpastatin. RelA translocation and calpastatin elevation were prevented when dissociation of the NF-κB/IκB complex was inhibited either by transfection with the non-phosphorylatable IκB mutant ΔNIκBα, or by using PS1145, an inhibitor of the IκB kinase (IKK complex). Increased calpastatin levels attenuate calpain-related amyloid-β-peptide and Ca(2+) -toxicity (these are central to the pathogenesis of Alzheimer's Disease). LPP-induced elevation of calpastatin provides an example of effects on non-inflammatory intracellular proteins, the outcome being significant alterations in host cell functions. Since calpastatin level is important in the control of calpain activity, mycoplasmal LPP may be of interest in treating some pathological processes involving excessive calpain activation.

Topics: Amyloid beta-Peptides; Bacterial Outer Membrane Proteins; Calcium Ionophores; Calcium-Binding Proteins; Calpain; Carrier Proteins; Cell Line, Tumor; Host-Pathogen Interactions; Humans; Ionomycin; Lipoproteins; Microfilament Proteins; Mycoplasma hyorhinis; Mycoplasma Infections; NF-kappa B; Signal Transduction; Up-Regulation

Accumulating evidence suggests that the calpain/calpastatin system is involved in skeletal muscle remodelling induced by β(2) -adrenoceptor agonist treatment. In addition to other pathways, the Akt/mammalian target of rapamycin (mTOR) pathway, controlling protein synthesis, and the calcium/calmodulin-dependent protein kinase 2 (CamK2) and AMP-activated protein kinase (AMPK) pathways, recently identified as calpain substrates, could be relevant in β(2) -adrenoceptor agonist-induced skeletal muscle remodelling. In the present study we investigated muscle hypertrophy and phenotypic shifts, as well as the molecular response of components of the Akt/mTOR pathway (i.e. Akt, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), ribosomal protein S6 (rpS6), CamK2 and AMPK), in response to calpastatin overexpression in the skeletal muscle of mice treated with 1 mg/kg per day clenbuterol for 21 days. Using gene electrotransfer of a calpastatin expression vector into the tibialis anterior of adult mice, we found that calpastatin overexpression attenuates muscle hypertrophy and phenotypic shifts induced by clenbuterol treatment. At the molecular level, calpastatin overexpression markedly decreased calpain activity, but was ineffective in altering the phosphorylation of Akt, 4E-BP1 and rpS6. In contrast, calpastatin overexpression increased the protein expression of both total AMPK and total CamK2. In conclusion, the results support the contention that the calpain/calpastatin system plays a crucial role in skeletal muscle hypertrophy and phenotypic shifts under chronic clenbuterol treatment, with AMPK and CamK2 probably playing a minor role. Moreover, the calpastatin-induced inhibition of hypertrophy under clenbuterol treatment was not related to a decreased mTOR-dependent initiation of protein translation.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Clenbuterol; Gene Expression Regulation; Hypertrophy; Male; Mice; Mice, Inbred CBA; Muscle, Skeletal; Phenotype

Calpains are cysteine proteases that have been implicated as both effectors and suppressors of apoptosis. Previously, we showed that c-myc transformation regulated calpain activity and sensitized cells to apoptosis induced by calpain inhibition. The objective of this study was to investigate the role of calpain in the Eμ-myc transgenic model of B-cell lymphoma. Calpain activity assays, apoptosis, cell cycle assays, and expression measurements were used to determine the activity and role of calpain in vitro and in vivo. We found that Eμ-myc transgenic cells have highly elevated calpain activity. Calpastatin, the negative calpain regulator, was expressed at much lower levels in Eμ-myc lymphoma cells compared to normal splenic B cells. The primary isoform in Eμ-myc lymphoma is calpain 1. Treatment of Eμ-myc lymphoma cells with the calpain inhibitors PD150606 or calpain inhibitor III induced caspase-3-dependent apoptosis in vitro. General caspase inhibitors or caspase-3/7 inhibitor protected cells from death induced by calpain inhibitor, whereas caspase-9 inhibitors failed to rescue cells. Human Burkitt's lymphoma (BL2) cells display a pattern of sensitivity and caspase-3 dependence similar to calpain inhibition. Treatment of Eμ-myc lymphoma-bearing mice with PD150606 inhibited calpain activity in vivo and induced cell death in these cells as determined by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining. Multiple daily treatments resulted in reduced tumor load, particularly in combination with etoposide. In conclusion, calpain is highly elevated in the Eμ-myc lymphoma and calpain inhibition has therapeutic potential.

Topics: Acrylates; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; B-Lymphocytes; Calcium-Binding Proteins; Calpain; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Etoposide; Humans; Lymphoma; Mice; Mice, Transgenic

We investigated the effects of calpain-system genetic markers on consumer beef quality ratings, including interactions of marker effects with hormonal growth promotant (HGP) use and tenderstretch hanging. Brahman cattle in New South Wales (NSW; n = 164) and Western Australia (WA; n = 141) were selected at weaning from commercial and research herds to achieve balance and divergence in calpastatin (CAST) and calpain 3 (CAPN3) gene marker status. Genotypes for μ-calpain (CAPN1-4751 and CAPN1-316) were also determined. Angus cattle (49 in NSW, 17 in WA) with favorable CAST and CAPN3 alleles, balanced for CAPN1-316 status, were also studied. Half the cattle at each site had HGP (Revalor-H, containing 200 mg trenbolone acetate and 20 mg 17β-estradiol) implants during grain finishing. One side of each carcass was suspended from the Achilles tendon (AT) and the other from the pelvis [tenderstretch (TS)]. Meat Standards Australia consumer panels scored 7-d aged striploin steaks from both AT and TS sides, and 7-d aged rump and oyster blade steaks from the AT side of each carcass. Two favorable CAST alleles increased tenderness ratings of AT-striploin, TS-striploin, rump, and oyster blade steaks by, respectively, 6.1, 4.2, 4.2, and 3.1 units, and overall liking by 4.7, 2.8, 2.9, 3.7 (all P < 0.04). Two favorable CAPN1-4751 alleles increased tenderness of AT-striploin, TS-striploin, and rump steaks by 6.5, 4.3, and 3.9 units, and overall liking by 5.6, 3.1, and 4.1 units. Two favorable CAPN3 alleles improved rump steaks by 3.7, 3.3, 3.7, and 3.5 units, for tenderness, juiciness, liking the flavor, and overall liking. There were no significant CAPN1-316 effects. The effect of HGP was greatest for the AT-striploin (reducing tenderness and overall liking by 8.2 units, P < 0.001), then TS-striploin (-5.6 for tenderness, -5.0 for overall liking, P < 0.001), and then rump (-4.4 for tenderness, -3.3 for overall liking, P < 0.007). Processing conditions differed considerably between NSW and WA. Rump steaks from NSW scored about 10 units greater than those from WA, but Angus and Brahman steaks from the same location with the same marker alleles had similar scores. In contrast, NSW Angus striploin steaks scored about 15 units greater for tenderness and overall liking (P < 0.001) than cattle with the same marker alleles at the other 3 location × breed combinations, which had generally similar scores. Therefore, calpain-system gene markers have beneficial effects on eating quali

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Female; Food Preferences; Gene Expression Regulation; Genetic Markers; Genotype; Humans; Male; Meat

The enteropathogen Shigella flexneri invades epithelial cells, leading to inflammation and tissue destruction. We report that Shigella infection of epithelial cells induces an early genotoxic stress, but the resulting p53 response and cell death are impaired due to the bacterium's ability to promote p53 degradation, mainly through calpain protease activation. Calpain activation is promoted by the Shigella virulence effector VirA and dependent on calcium flux and the depletion of the endogenous calpain inhibitor calpastatin. Further, although VirA-induced calpain activity is critical for regulating cytoskeletal events driving bacterial uptake, calpain activation ultimately leads to necrotic cell death, thereby restricting Shigella intracellular growth. Therefore, calpains work at multiple steps in regulating Shigella pathogenesis by disrupting the p53-dependent DNA repair response early during infection and regulating both formation and ultimate death of the Shigella epithelial replicative niche.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Ataxia Telangiectasia Mutated Proteins; Calcium-Binding Proteins; Calpain; Cell Cycle Proteins; DNA Damage; DNA-Binding Proteins; Enzyme Activation; Epithelial Cells; Fibroblasts; HeLa Cells; Host-Pathogen Interactions; Humans; L-Lactate Dehydrogenase; Mice; Necrosis; NF-kappa B; Phosphorylation; Protein Serine-Threonine Kinases; Protein Stability; Proteolysis; Proto-Oncogene Proteins c-mdm2; Shigella flexneri; Signal Transduction; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Virulence Factors

Noncanonical amino acids (NCAAs) can be used in a variety of protein design contexts. For example, they can be used in place of the canonical amino acids (CAAs) to improve the biophysical properties of peptides that target protein interfaces. We describe the incorporation of 114 NCAAs into the protein-modeling suite Rosetta. We describe our methods for building backbone dependent rotamer libraries and the parameterization and construction of a scoring function that can be used to score NCAA containing peptides and proteins. We validate these additions to Rosetta and our NCAA-rotamer libraries by showing that we can improve the binding of a calpastatin derived peptides to calpain-1 by substituting NCAAs for native amino acids using Rosetta. Rosetta (executables and source), auxiliary scripts and code, and documentation can be found at (http://www.rosettacommons.org/).

Topics: Amino Acids; Calcium-Binding Proteins; Calpain; Computational Biology; Fluorescence Polarization; Models, Molecular; Peptide Library; Peptides; Protein Binding; Proteins; Software; Thermodynamics

Calpains are calcium regulated cysteine proteases that have been described in a wide range of cellular processes, including apoptosis, migration and cell cycle regulation. In addition, calpains have been implicated in differentiation, but their impact on neural differentiation requires further investigation. Here, we addressed the role of calpain 1 and calpain 2 in neural stem cell (NSC) self-renewal and differentiation. We found that calpain inhibition using either the chemical inhibitor calpeptin or the endogenous calpain inhibitor calpastatin favored differentiation of NSCs. This effect was associated with significant changes in cell cycle-related proteins and may be regulated by calcium. Interestingly, calpain 1 and calpain 2 were found to play distinct roles in NSC fate decision. Calpain 1 expression levels were higher in self-renewing NSC and decreased with differentiation, while calpain 2 increased throughout differentiation. In addition, calpain 1 silencing resulted in increased levels of both neuronal and glial markers, β-III Tubulin and glial fibrillary acidic protein (GFAP). Calpain 2 silencing elicited decreased levels of GFAP. These results support a role for calpain 1 in repressing differentiation, thus maintaining a proliferative NSC pool, and suggest that calpain 2 is involved in glial differentiation.

Topics: Animals; Astrocytes; Biomarkers; Calcium; Calcium-Binding Proteins; Calpain; Cell Count; Cell Cycle Proteins; Cell Death; Cell Differentiation; Cell Proliferation; Dipeptides; Gene Silencing; Intermediate Filament Proteins; Mice; Nerve Tissue Proteins; Nestin; Neural Stem Cells; Neurogenesis; Neurons; Phenotype; Tubulin

Messenger RNA (mRNA) expression of calpain-1 (µ-calpain), -2 (m-calpain), -3 (p94), small subunit (calpain-4; 28 kDa), and three types of calpastatin (CSTN) isoform were investigated for 10 skeletal muscles of Holstein cattle by real-time and/or semi-quantitative reverse transcription polymerase chain reaction. Noticeably, effect of muscle type was observed on 28 kDa expression (P < 0.001) with a tendency of higher 28 kDa expression in myosin heavy chain (MyHC)-2x-rich muscles compared to MyHC-slow-rich muscles. The CSTN-I and -III expression in Longissimus thoracis (LT) showed the lowest value among the muscles tested. Moreover, 28 kDa/CSTN-I ratio was higher in the diaphragm (DP), psoas major (PM), and LT than those in the lingual muscles (TN), masseter (MS) and pectoralis (PP) (P < 0.05). Calpain-1/CSTN I, calpain-2/CSTN I in LT and PM were higher than that in TN (P < 0.05). Calpain-3/CSTN-I and -III in LT and/or PM showed higher values than that in TN (P < 0.05). These results indicated that the calpain and CSTN expressions are regulated by muscle type, suggesting especially by muscle fiber type. Calpains/CSTN-I ratios, especially 28 kDa/CSTN-I, may account for higher extent of post mortem proteolysis previously observed in LT and PM muscles.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Female; Isoenzymes; Muscle, Skeletal; Polymerase Chain Reaction; RNA, Messenger

Ovarian cancer is routinely treated with surgery and platinum-based chemotherapy. Resistance is a major obstacle in the efficacy of this chemotherapy regimen and the ability to identify those patients at risk of developing resistance is of considerable clinical importance. The expression of calpain-1, calpain-2 and calpastatin were determined using standard immunohistochemistry on a tissue microarray of 154 primary ovarian carcinomas from patients subsequently treated with platinum-based adjuvant chemotherapy. High levels of calpain-2 expression was significantly associated with platinum resistant tumours (P = 0.031). Furthermore, high expression of calpain-2 was significantly associated with progression-free (P = 0.049) and overall survival (P = 0.006) in this cohort. The association between calpain-2 expression and overall survival remained significant in multivariate analysis accounting for tumour grade, stage, optimal debulking and platinum sensitivity (hazard ratio = 2.174; 95% confidence interval = 1.144-4.130; P = 0.018). The results suggest that determining calpain-2 expression in ovarian carcinomas may allow prognostic stratification of patients treated with surgery and platinum-based chemotherapy. The findings of this study warrant validation in a larger clinical cohort.

Topics: Adult; Aged; Aged, 80 and over; Calcium-Binding Proteins; Calpain; Chemotherapy, Adjuvant; Disease Progression; Female; Follow-Up Studies; Gene Expression Regulation; Humans; Immunohistochemistry; Middle Aged; Multivariate Analysis; Ovarian Neoplasms; Platinum Compounds; Prognosis; Proportional Hazards Models; Retrospective Studies

The USA300 strains of Staphylococcus aureus are the major cause of skin and soft tissue infection in the United States. Invasive USA300 infection has been attributed to several virulence factors, including protein A and the α-hemolysin (Hla), which cause pathology by activating host signaling cascades. Here we show that S. aureus exploits the proinflammatory bias of human keratinocytes to activate pyroptosis, a caspase 1-dependent form of inflammatory cell death, which was required for staphylococci to penetrate across a keratinocyte barrier. Keratinocyte necrosis was mediated by calpains, Ca(2+)-dependent intracellular proteases whose endogenous inhibitor, calpastatin, is targeted by Hla-induced caspase 1. Neither Panton-Valentine leukocidin nor protein A expression was essential, but inhibition of either calpain or caspase 1 activity was sufficient to prevent staphylococcal invasion across the keratinocytes. These studies suggest that pharmacological interruption of specific keratinocyte signaling cascades as well as targeting the Hla might prevent invasive skin infection by staphylococci.

Topics: Apoptosis; Calcium-Binding Proteins; Calpain; Caspase 1; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Dipeptides; Enzyme Activation; Humans; Keratinocytes; Methicillin-Resistant Staphylococcus aureus; Mutation; Signal Transduction; Soft Tissue Infections; Staphylococcal Infections; Staphylococcal Skin Infections; Virulence Factors

We performed a genome-wide association study for Warner-Bratzler shear force (WBSF), a measure of meat tenderness, by genotyping 3360 animals from five breeds with 54 790 BovineSNP50 and 96 putative single-nucleotide polymorphisms (SNPs) within μ-calpain [HUGO nomenclature calpain 1, (mu/I) large subunit; CAPN1] and calpastatin (CAST). Within- and across-breed analyses estimated SNP allele substitution effects (ASEs) by genomic best linear unbiased prediction (GBLUP) and variance components by restricted maximum likelihood under an animal model incorporating a genomic relationship matrix. GBLUP estimates of ASEs from the across-breed analysis were moderately correlated (0.31-0.66) with those from the individual within-breed analyses, indicating that prediction equations for molecular estimates of breeding value developed from across-breed analyses should be effective for genomic selection within breeds. We identified 79 genomic regions associated with WBSF in at least three breeds, but only eight were detected in all five breeds, suggesting that the within-breed analyses were underpowered, that different quantitative trait loci (QTL) underlie variation between breeds or that the BovineSNP50 SNP density is insufficient to detect common QTL among breeds. In the across-breed analysis, CAPN1 was followed by CAST as the most strongly associated WBSF QTL genome-wide, and associations with both were detected in all five breeds. We show that none of the four commercialized CAST and CAPN1 SNP diagnostics are causal for associations with WBSF, and we putatively fine-map the CAPN1 causal mutation to a 4581-bp region. We estimate that variation in CAST and CAPN1 explains 1.02 and 1.85% of the phenotypic variation in WBSF respectively.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Genetic Variation; Genome-Wide Association Study; Genotype; Meat; Polymorphism, Single Nucleotide; Quantitative Trait Loci

Traumatic brain injury (TBI) results in abrupt, initial cell damage leading to delayed neuronal death. The calcium-activated proteases, calpains, are known to contribute to this secondary neurodegenerative cascade. Although the specific inhibitor of calpains, calpastatin, is present within neurons, normal levels of calpastatin are unable to fully prevent the damaging proteolytic activity of calpains after injury. In this study, increased calpastatin expression was achieved using transgenic mice that overexpress the human calpastatin (hCAST) construct under control of a calcium-calmodulin-dependent kinase II α promoter. Naïve hCAST transgenic mice exhibited enhanced neuronal calpastatin expression and significantly reduced protease activity. Acute calpain-mediated spectrin proteolysis in the cortex and hippocampus induced by controlled cortical impact brain injury was significantly attenuated in calpastatin overexpressing mice. Aspects of posttraumatic motor and cognitive behavioral deficits were also lessened in hCAST transgenic mice compared to their wildtype littermates. However, volumetric analyses of neocortical contusion revealed no histological neuroprotection at either acute or long-term time points. Partial hippocampal neuroprotection observed at a moderate injury severity was lost after severe TBI. This study underscores the effectiveness of calpastatin overexpression in reducing calpain-mediated proteolysis and behavioral impairment after TBI, supporting the therapeutic potential for calpain inhibition. In addition, the reduction in spectrin proteolysis without accompanied neocortical neuroprotection suggests the involvement of other factors that are critical for neuronal survival after contusion brain injury.

Topics: Animals; Brain Injuries; Calcium-Binding Proteins; Calpain; Gene Expression Regulation; Hippocampus; Humans; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neocortex; Proteolysis

Wound healing is a multistep phenomenon that relies on complex interactions between various cell types. Calpains are ubiquitously expressed proteases regulating several processes including cellular adhesion and motility as well as inflammation and angiogenesis. Calpains can be targeted by inhibitors, and their inhibition was shown to reduce organ damage in various disease models. We aimed to assess the role of calpains in skin healing and the potential benefit of calpain inhibition on scar formation. We used a pertinent model where calpain activity is inhibited only in lesional organs, namely transgenic mice overexpressing calpastatin (CPST), a specific natural calpain inhibitor. CPST mice showed a striking delay in wound healing particularly in the initial steps compared to wild types (WT). CPST wounds displayed reduced proliferation in the epidermis and delayed re-epithelization. Granulation tissue formation was impaired in CPST mice, with a reduction in CD45+ leukocyte infiltrate and in CD31+ blood vessel density. Interestingly, wounds on WT skin grafted on CPST mice (WT/CPST) showed a similar delayed healing with reduced angiogenesis and inflammation compared to wounds on WT/WT mice demonstrating the implication of calpain activity in distant extra-cutaneous cells during wound healing. CPST wounds showed a reduction in alpha-smooth muscle actin (αSMA) expressing myofibroblasts as well as αSMA RNA expression suggesting a defect in granulation tissue contraction. At later stages of skin healing, calpain inhibition proved beneficial by reducing collagen production and wound fibrosis. In vitro, human fibroblasts exposed to calpeptin, a pan-calpain inhibitor, showed reduced collagen synthesis, impaired TGFβ-induced differentiation into αSMA-expressing myofibroblasts, and were less efficient in a collagen gel contraction assay. In conclusion, calpains are major players in granulation tissue formation. In view of their specific effects on fibroblasts a late inhibition of calpains should be considered for scar reduction.

Topics: Actins; Animals; Blood Vessels; Calcium-Binding Proteins; Calpain; Cell Adhesion; Cell Differentiation; Cell Movement; Cells, Cultured; Cicatrix; Collagen; Female; Fibroblasts; Fibrosis; Granulation Tissue; Humans; Inflammation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myofibroblasts; Platelet Endothelial Cell Adhesion Molecule-1; Skin; Wound Healing

Earlier observations in the literature suggest that proteolytic degradation of excess unmatched α-globin chains reduces their accumulation and precipitation in β-thalassaemia erythroid precursor cells and have linked this proteolytic degradation to the activity of calpain protease. The aim of this study was to correlate the activity of calpain and its inhibitor, calpastatin, with different degrees of disease severity in β-thalassaemia. CD34(+) cells were enriched from peripheral blood of healthy individuals (control group) and patients with mild and severe clinical presentations of β(0)-thalassaemia/Hb E disease. By ex vivo cultivation promoting erythroid cell differentiation for 7 days, proerythroblasts, were employed for the functional characterization of the calpain-calpastatin proteolytic system. In comparison to the control group, enzymatic activity and protein amounts of μ-calpain were found to be more than 3-fold increased in proerythroblasts from patients with mild clinical symptoms, whereas no significant difference was observed in patients with severe clinical symptoms. Furthermore, a 1.6-fold decrease of calpastatin activity and 3.2-fold accumulation of a 34 kDa calpain-mediated degradation product of calpastatin were observed in patients with mild clinical symptoms. The increased activity of calpain may be involved in the removal of excess α-globin chains contributing to a lower degree of disease severity in patients with mild clinical symptoms.

Topics: alpha-Globins; Animals; Antigens, CD34; beta-Thalassemia; Calcium-Binding Proteins; Calpain; Case-Control Studies; Cell Differentiation; Cells, Cultured; Erythroid Precursor Cells; Humans; Male; Proteolysis; Rats; Recombinant Proteins; Severity of Illness Index

The current study was conducted to evaluate the functions of μ-calpain (CAPN1), calpastatin, HSPs (heat shock proteins), and caspases during myogenesis and cell death induced by sodium azide (NaN(3)) hypoxia. The cell samples were divided into three groups: satellite cells formed at confluent monolayer (stage 1), stage 1 cells fusion into myotubes on d eight post-differentiation (stage 2), and stage 2 cells treated with 1 mM NaN(3) for 24 h (stage 3). Real-time RT-PCR showed that stage 2 cells had increased CAPN1, calpastatin, caspase 7, and CARD9 (Caspase activation and recruitment domain 9) mRNA expressions compared to stage 1 cells (*p < 0.05). By Western blotting caspase 3, caspase 7, caspase 8, and caspase 9 protein levels increased in cells at stage 2 compared to cells at stage 1 (*p < 0.05). Real-time RT-PCR showed that stage 3 cells had increased CAPN1, calpastatin, caspase 7, HSP70 (70 kDA heat shock proteins), and HSP90 (90 kDA heat shock proteins-alpha) and decreased CARD9 mRNA expression compared to stage 2 cells (*p < 0.05). Stage 3 samples had increase caspase 7 and caspase 12 activities compared to stage 2 samples, and by Western blotting protein levels of both HSP70 and HSP90 expressions, increased significantly under hypoxia condition (*p < 0.05). Here, we conclude that CAPN1, calpastatin, caspase 3, caspase 7, caspase 8, and CARD9 have important roles for satellite cell myogenesis; and that caspase 7, 12, HSP70, and HSP90 are involved in the process of apoptotic cell death under hypoxia conditions and we speculate that these proteins may be involved in early postmortem proteolysis and meat tenderization.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Calpain; CARD Signaling Adaptor Proteins; Caspases; Cattle; Cell Hypoxia; Cells, Cultured; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Meat; Muscle Development; Muscle Fibers, Skeletal; Proteolysis; RNA, Messenger; Satellite Cells, Skeletal Muscle; Sodium Azide

Calpain has been considered to be the most important protease involved in tenderization during the conversion of muscle into meat. However, recent evidence suggests the possible involvement of the key apoptosis protease, caspase, on post-mortem tenderization. This study used inhibitors of calpain and caspase-3 to treat chicken muscle immediately after slaughter and followed the changes in caspase-3 and calpain activities together with their expression during 5 days of aging. Addition of calpain inhibitors to the system resulted in significantly higher caspase-3 activities (p < 0.01) during storage. Western blot analysis of pro-caspase-3 and α-spectrin cleavage of the 120 kDa peptide (SBDP 120) showed that the addition of calpain inhibitors resulted in the formation of higher amounts of the active form of caspase-3 compared with the control (p < 0.01). Inclusion of inhibitors of caspase-3 led to lower calpain activities (p < 0.01) and dramatically reduced the expression of calpain-1 and calpain-2 (p < 0.01). Concomitantly, this inhibition resulted in greater calpastatin expression compared with the control (p < 0.01). The findings of this investigation show that calpain prevented the activation of caspase-3, whereas caspase-3 appeared to enhance the calpain activity during post-mortem aging through inhibition of calpastatin. It is therefore suggested that there is a relationship between caspase-3 and calpain which contributes to the tenderizing process during the conversion of muscle tissue into meat.

Topics: Animals; Calcium-Binding Proteins; Calpain; Caspase 3; Caspase Inhibitors; Chickens; Dipeptides; Muscle, Skeletal; Oligopeptides; Poultry; Protease Inhibitors

Basal-like and triple-negative breast tumours encompass an important clinical subgroup and biomarkers that can prognostically stratify these patients are required.. We investigated two breast cancer tissue microarrays for the expression of calpain-1, calpain-2 and calpastatin using immunohistochemistry. The first microarray was comprised of invasive tumours from 1371 unselected patients, and the verification microarray was comprised of invasive tumours from 387 oestrogen receptor (ER)-negative patients.. The calpain system contains a number of proteases and an endogenous inhibitor, calpastatin. Calpain activity is implicated in important cellular processes including cytoskeletal remodelling, apoptosis and survival. Our results show that the expression of calpastatin and calpain-1 are significantly associated with various clinicopathological criteria including tumour grade and ER expression. High expression of calpain-2 in basal-like or triple-negative disease was associated with adverse breast cancer-specific survival (P = 0.003 and <0.001, respectively) and was verified in an independent cohort of patients. Interestingly, those patients with basal-like or triple-negative disease with a low level of calpain-2 expression had similar breast cancer-specific survival to non-basal- or receptor- (oestrogen, progesterone or human epidermal growth factor receptor 2 (HER2)) positive disease.. Expression of the large catalytic subunit of m-calpain (calpain-2) is significantly associated with clinical outcome of patients with triple-negative and basal-like disease.

Topics: Adolescent; Adult; Aged; Breast Neoplasms; Calcium-Binding Proteins; Calpain; Carcinoma, Ductal, Breast; Disease-Free Survival; Female; Humans; Kaplan-Meier Estimate; Middle Aged; Multivariate Analysis; Neoplasm Invasiveness; Neoplasms, Basal Cell; Proportional Hazards Models; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Retrospective Studies; Young Adult

Machado-Joseph disease is the most frequently found dominantly-inherited cerebellar ataxia. Over-repetition of a CAG trinucleotide in the MJD1 gene translates into a polyglutamine tract within the ataxin 3 protein, which upon proteolysis may trigger Machado-Joseph disease. We investigated the role of calpains in the generation of toxic ataxin 3 fragments and pathogenesis of Machado-Joseph disease. For this purpose, we inhibited calpain activity in mouse models of Machado-Joseph disease by overexpressing the endogenous calpain-inhibitor calpastatin. Calpain blockage reduced the size and number of mutant ataxin 3 inclusions, neuronal dysfunction and neurodegeneration. By reducing fragmentation of ataxin 3, calpastatin overexpression modified the subcellular localization of mutant ataxin 3 restraining the protein in the cytoplasm, reducing aggregation and nuclear toxicity and overcoming calpastatin depletion observed upon mutant ataxin 3 expression. Our findings are the first in vivo proof that mutant ataxin 3 proteolysis by calpains mediates its translocation to the nucleus, aggregation and toxicity and that inhibition of calpains may provide an effective therapy for Machado-Joseph disease.

Topics: Active Transport, Cell Nucleus; Adult; Animals; Ataxin-3; Brain Chemistry; Calcium-Binding Proteins; Calpain; Female; Glycoproteins; Humans; Machado-Joseph Disease; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Middle Aged; Mutation; Neuroprotective Agents; Nuclear Proteins; Proteolysis; Transcription Factors

Methamphetamine (METH) is an abused psychostimulant drug that can cause neurotoxicity to dopaminergic cells. It has been demonstrated that METH can induce caspase- and calpain-dependent death cascades. The purpose of the present study was to investigate the functional role of calpastatin, a specific endogenous calpain inhibitor protein, on caspase and calpain activation in METH-induced degeneration in neuroblastoma SH-SY5Y cell cultures. In this study, we found that METH significantly decreased cell viability, tyrosine hydroxylase phosphorylation and calpastatin levels. Supplementation of cells with exogenous calpastatin was able to reverse the toxic effect of METH on reduction in cell viability and tyrosine hydroxylase phosphorylation. METH also significantly increased calpain levels, the formation of calpain-specific breakdown products and cleaved caspase-3 levels; once again, these effects were diminished by pretreating the cells with calpastatin. These data suggest the contribution of calpastatin as a potential regulatory factor for calpain- and caspase-dependent death processes.

Topics: Calcium-Binding Proteins; Calpain; Caspase 3; Caspases; Cell Line, Tumor; Cell Survival; Central Nervous System Stimulants; Enzyme Activation; Humans; Methamphetamine; Neuroblastoma; Neuroprotective Agents; Phosphorylation; Tyrosine 3-Monooxygenase

Survival of hepatic stellate cells (HSCs) is a hallmark of liver fibrosis, while the induction of HSC apoptosis may induce recovery. Activated HSC are resistant to many pro-apoptotic stimuli. To this issue, the role of Endoplasmic Reticulum (ER) stress in promoting apoptosis of HSCs and consequently fibrosis resolution is still debated.. To evaluate the potential ER stress-mediated apoptosis of HSCs and fibrosis resolution. HSCs were incubated with the ER stress agonists, tunicamycin or thapsigargin. In vivo, HSC were isolated from normal, bile duct-ligated (BDL) and bile duct-diverted (BDD) rats.. In activated HSC, the specific inhibitor of ER stress-induced apoptosis, calpastatin, is significantly increased vs. quiescent HSCs. Calpain is conversely reduced in activated HSCs. This pattern of protein expression provides HSCs resistance to the ER stress signals of apoptosis (apoptosis-resistant phenotype). However, both tunicamycin and thapsigargin are able to induce apoptosis in HSCs in vitro, completely reversing the calpain/calpastatin pattern expression. Furthermore, in vivo, the fibrosis resolution observed in rat livers subjected to bile duct ligation (BDL) and subsequent bile duct diversion (BDD), leads to fibrosis resolution through a mechanism of HSCs apoptosis, potentially associated with ER stress: in fact, BDD rat liver shows an increased number of apoptotic HSCs associated with reduced calapstatin and increased calpain protein expression, leading to an apoptosis-sensible phenotype.. ER stress sensitizes HSC to apoptosis both in vitro and in vivo. Thus, ER stress represents a key target to trigger cell death in activated HSC and promotes fibrosis resolution.

Topics: Animals; Apoptosis; Bile Ducts; Blotting, Western; Calcium-Binding Proteins; Calpain; Caspase 8; Endoplasmic Reticulum Stress; Fibrosis; Hepatic Stellate Cells; Immunohistochemistry; In Situ Nick-End Labeling; Ligation; Liver; Rats; RNA, Small Interfering; Thapsigargin; Tunicamycin

Sarcopenia, the age-related loss of muscle mass, is a highly-debilitating consequence of aging. In this investigation, we show sarcopenia is greatly reduced by muscle-specific overexpression of calpastatin, the endogenous inhibitor of calcium-dependent proteases (calpains). Further, we show that calpain cleavage of specific structural and regulatory proteins in myofibrils is prevented by covalent modification of calpain by nitric oxide (NO) through S-nitrosylation. We find that calpain in adult, non-sarcopenic muscles is S-nitrosylated but that aging leads to loss of S-nitrosylation, suggesting that reduced S-nitrosylation during aging leads to increased calpain-mediated proteolysis of myofibrils. Further, our data show that muscle aging is accompanied by loss of neuronal nitric oxide synthase (nNOS), the primary source of muscle NO, and that expression of a muscle-specific nNOS transgene restores calpain S-nitrosylation in aging muscle and prevents sarcopenia. Together, the findings show that in vivo reduction of calpain S-nitrosylation in muscle may be an important component of sarcopenia, indicating that modulation of NO can provide a therapeutic strategy to slow muscle loss during old age.

Topics: Aging; Animals; Calcium-Binding Proteins; Calpain; Gene Expression; Humans; Mice; Muscle, Skeletal; Myofibrils; Nitric Oxide; Nitric Oxide Synthase Type I; Protein Isoforms; Proteolysis; Sarcopenia

We have designed a highly specific inhibitor of calpain by mimicking a natural protein-protein interaction between calpain and its endogenous inhibitor calpastatin. To enable this goal we established a new method of stabilizing an α-helix in a small peptide by screening 24 commercially available cross-linkers for successful cysteine alkylation in a model peptide sequence. The effects of cross-linking on the α-helicity of selected peptides were examined by CD and NMR spectroscopy, and revealed structurally rigid cross-linkers to be the best at stabilizing α-helices. We applied this strategy to the design of inhibitors of calpain that are based on calpastatin, an intrinsically unstable polypeptide that becomes structured upon binding to the enzyme. A two-turn α-helix that binds proximal to the active site cleft was stabilized, resulting in a potent and selective inhibitor for calpain. We further expanded the utility of this inhibitor by developing irreversible calpain family activity-based probes (ABPs), which retained the specificity of the stabilized helical inhibitor. We believe the inhibitor and ABPs will be useful for future investigation of calpains, while the cross-linking technique will enable exploration of other protein-protein interactions.

Topics: Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Models, Molecular; Molecular Structure; Protein Binding; Protein Structure, Secondary; Structure-Activity Relationship

Pancreatic cancer, including cancer of the ampulla of Vater and bile duct, is very aggressive and has a poor five year survival rate; improved methods of patient stratification are required.. We assessed the expression of calpain-1, calpain-2 and calpastatin in two patient cohorts using immunohistochemistry on tissue microarrays. The first cohort was composed of 68 pancreatic adenocarcinomas and the second cohort was composed of 120 cancers of the bile duct and ampulla.. In bile duct and ampullary carcinomas an association was observed between cytoplasmic calpastatin expression and patient age (P = 0.036), and between nuclear calpastatin expression and increased tumour stage (P = 0.026) and the presence of vascular invasion (P = 0.043). In pancreatic cancer, high calpain-2 expression was significantly associated with improved overall survival (P = 0.036), which remained significant in multivariate Cox-regression analysis (hazard ratio = 0.342; 95% confidence interva l = 0.157-0.741; P = 0.007). In cancers of the bile duct and ampulla, low cytoplasmic expression of calpastatin was significantly associated with poor overall survival (P = 0.012), which remained significant in multivariate Cox-regression analysis (hazard ratio = 0.595; 95% confidence interval = 0.365-0.968; P = 0.037).. The results suggest that calpain-2 and calpastatin expression is important in pancreatic cancers, influencing disease progression. The findings of this study warrant a larger follow-up study.

Topics: Adult; Aged; Aged, 80 and over; Ampulla of Vater; Bile Duct Neoplasms; Biomarkers, Tumor; Calcium-Binding Proteins; Calpain; Carcinoma; Cohort Studies; Disease Progression; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Male; Middle Aged; Pancreatic Neoplasms; Proportional Hazards Models; Tissue Array Analysis

The nicotinic acetylcholine receptor α1 (nAChRα1) was investigated as a potential proinflammatory molecule in the kidney, given a recent report that it is an alternative urokinase plasminogen activator (uPA) receptor, in addition to the classical receptor uPAR. Two animal models and in vitro monocyte studies were involved: (1) In an ApoE(-/-) mouse model of chronic kidney disease, glomerular-resident cells and monocytes/macrophages were identified as the primary cell types that express nAChRα1 during hypercholesterolemia/uninephrectomy-induced nephropathy. Silencing of the nAChRα1 gene for 4 months (6 months on Western diet) prevented the increases in renal monocyte chemoattractant protein-1 and osteopontin expression levels and F4/80+ macrophage infiltration compared with the nonsilenced mice. These changes were associated with significantly reduced transforming growth factor-β1 mRNA (50% decrease) and α smooth muscle actin-positive (αSMA+) myofibroblasts (90% decrease), better glomerular and tubular basement membranes (GBM/TBM) preservation (threefold less disintegration), and better renal function preservation (serum creatinine 40% lower) in the nAChRα1-silenced mice. The nAChRα1 silencing was also associated with significantly reduced renal tissue calcium deposition (78% decrease) and calpain-1 (but not calpain-2) activation (70% decrease). (2) The nAChRα1 was expressed in vitro by mouse monocyte cell line WEHI-274.1. The silencing of nAChRα1 significantly reduced both calpain-1 and -2 activities, and reduced the degradation of the calpain substrate talin. (3) To further explore the role of calpain-1 activity in hypercholesterolemic nephropathy, disease severities were compared in CAST(-/-)ApoE(-/-) (calpain overactive) mice and ApoE(-/-) mice fed with Western diet for 10 months (n=12). Macrophages were the main cell type of renal calpain-1 production in the model. The number of renal F4/80+ macrophages was 10-fold higher in the CAST(-/-)ApoE(-/-) mice (P<0.05), and was associated with a significantly higher level of αSMA+ cells, increased GBM/TBM destruction, and higher serum creatinine levels. Our studies suggest that the receptor nAChRα1 is an important regulator of calpain-1 activation and inflammation in the chronic hypercholesterolemic nephropathy. This new proinflammatory pathway may also be relevant to other disorders beyond hyperlipidemic nephropathy.

Topics: Actins; Animals; Antigens, Differentiation; Apolipoproteins E; Blotting, Northern; Blotting, Western; Calcium-Binding Proteins; Calpain; Cell Line; Female; Hypercholesterolemia; Inflammation; Kidney; Kidney Diseases; Macrophages; Male; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Monocytes; Nephrectomy; Receptors, Nicotinic; RNA Interference; Transforming Growth Factor beta1

Important to the function of calpains is temporal and spatial regulation of their proteolytic activity. Here, we demonstrate that cytoplasm-resident calpain 2 cleaves human nuclear topoisomerase I (hTOP1) via Ca(2+)-activated proteolysis and nucleoplasmic shuttling of proteases. This proteolysis of hTOP1 was induced by either ionomycin-caused Ca(2+) influx or addition of Ca(2+) in cellular extracts. Ca(2+) failed to induce hTOP1 proteolysis in calpain 2-knockdown cells. Moreover, calpain 2 cleaved hTOP1 in vitro. Furthermore, calpain 2 entered the nucleus upon Ca(2+) influx, and calpastatin interfered with this process. Calpain 2 cleavage sites were mapped at K(158) and K(183) of hTOP1. Calpain 2-truncated hTOP1 exhibited greater relaxation activity but remained able to interact with nucleolin and to form cleavable complexes. Interestingly, calpain 2 appears to be involved in ionomycin-induced protection from camptothecin-induced cytotoxicity. Thus, our data suggest that nucleocytoplasmic shuttling may serve as a novel type of regulation for calpain 2-mediated nuclear proteolysis.

Topics: Active Transport, Cell Nucleus; Amino Acid Sequence; Calcium; Calcium-Binding Proteins; Calpain; Camptothecin; Cell Line, Tumor; Cell Nucleus; Cytoplasm; DNA Topoisomerases, Type I; Gene Knockdown Techniques; Humans; Ionomycin; Molecular Sequence Data; Nucleolin; Peptide Hydrolases; Phosphoproteins; RNA-Binding Proteins

Relationships of the calpain system with meat tenderness and carcass traits were examined for 94 purebred Angus bulls with genotypes of the calpain classified by RFLP (restriction fragment length polymorphism) and SSCP (Single strand conformation polymorphism) analysis. Designing of primers based on the calpain regulatory subunit (CAPNS) and u-calpian (CAPN1) genes. Bulls from 15 months of age were slaughtered, and carcass traits, including fat thickness (FAT); longissimus muscle area (LMA); percentage of kidney, pelvic, and heart fat (KPH); hot carcass weight (HCW); marbling score (MAR); and quality grade (QUL), were analyzed. Measurements regarding meat tenderness involved activities of calpastatin (CAC), u-calpain (UAC), m-calpain (MAC), Warner-Bratzler Shear Force (WBS) and myofibril fragmentation index (MFI). Statistical significances of the calpain genotypes accounted for variations in MAR and QUL at CAPNS locus, and both loci explained variations of UAC and MAC. Significant mean differences in genotypes of CAPNS locus were found for MAR (BB > AB > AA) and QUL (AB > BB > AA). UAC showed significant correlations with MAC, CAC, MFI, FAT, and MAR, and we found that MAC correlated with WBS, FAT, HCW, MAR, and QUL. Strong positive correlation detected between LMA and HCW, and MAR and QUL, and a negative correlation between MFI and MAR was estimated. From the result it may be possible to use the calpain genotypes classified by RFLP and SSCP analysis in marker assisted selection programs to estimate UAC and MAC precisely regardless meat tenderness and to improve MAR and QUL of beef cattle.

Topics: Animals; Biomechanical Phenomena; Calcium-Binding Proteins; Calpain; Cattle; Exons; Genotype; Introns; Least-Squares Analysis; Male; Meat; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Polymorphism, Single-Stranded Conformational; Quantitative Trait, Heritable

The calpain family, and their endogenous inhibitor calpastatin, has been implicated in cancer progression, and recent in vitro data have indicated a role in trastuzumab resistance. The aims of our study were to examine expression levels of calpastatin, calpain-1 and calpain-2 in breast tumours from patients treated with trastuzumab following adjuvant chemotherapy to determine their potential as biomarkers to predict therapeutic response. The expression of calpastatin, calpain-1 and calpain-2 was determined, using immunohistochemistry (IHC), in tumours from a series of 93 patients with primary breast cancer treated with surgery and adjuvant chemotherapy with or without trastuzumab followed by trastuzumab to complete 1 year of therapy. IHC was performed using tissue microarrays constructed from cores taken from intratumour and peripheral tumour areas. Expression was correlated with clinicopathologic variables and patient outcome. Calpastatin expression was correlated with Nottingham prognostic index (p = 0.003) and lymph node status (p = 0.007). Trastuzumab resistance was defined as disease relapse during therapy. Calpain-1 expression is associated with relapse-free survival (p = 0.001) and remained significant in multivariate analysis accounting for confounding pathological and treatment variables (hazard ratio 4.60, 95% confidence interval 1.05-20.25; p = 0.043). Calpain-1 may be a useful biomarker to predict relapse-free survival in breast cancer patients treated with adjuvant trastuzumab and chemotherapy. A larger verification study is warranted.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Biomarkers, Tumor; Breast Neoplasms; Calcium-Binding Proteins; Calpain; Chemotherapy, Adjuvant; Disease-Free Survival; Female; Humans; Middle Aged; Prognosis; Trastuzumab

The objectives of the study were to evaluate allelic frequencies and to test the association of polymorphisms in the calpastatin (CAST) and µ-calpain (CAPN1) genes with meat tenderness in 3 French beef breeds. A total of 1,114 Charolais, 1,254 Limousin, and 981 Blonde d'Aquitaine purebred young bulls were genotyped for 3 SNP in the CAST gene and 4 SNP in the CAPN1 gene. Two of these markers, 1 in each gene, can be found in Australian or American commercial genetic tests. Others have previously been reported in American studies or are newly evidenced SNP. The quantitative traits studied were Warner-Bratzler shear force and a tenderness score evaluated by trained sensory panels. All the SNP were informative in the 3 breeds. Associations of individual markers or haplotypes with traits were analyzed. The results differed in the 3 breeds. The G allele of a CAST marker (position 97574679 on Btau4.0) was found to exert a significant effect on the shear force (+0.18 phenotypic SD; RSD) and tenderness score (-0.22 RSD) in the Blonde d'Aquitaine breed. In the same breed, this marker was associated with another CAST SNP (position 97576054 on Btau4.0) such that the GA haplotype appeared to be associated with tougher meat. Two CAPN1 markers (positions 45221250 and 45241089 on Btau4.0) had a significant effect on both traits in the Charolais breed (from |0.11| to |0.25| RSD). In the same breed, these markers were associated with another CAPN1 SNP (position 45219395 on Btau4.0) such that the ACA and AGG haplotypes appeared to be associated with a tender meat and a tougher meat, respectively. Consequently, the present results indicate that the effects of the markers studied are breed-specific and cannot be extended to all Bos taurus breeds. Further studies are also required to identify other more appropriate markers for French beef breeds.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; France; Gene Expression Regulation; Genetic Markers; Genetic Variation; Genotype; Male; Meat; Polymorphism, Genetic

Effects of genotype (GEN) and implant program (IMP) on LM and gluteus muscle (GM) tenderization were investigated using crossbred steer (n = 185) and heifer (n = 158) calves. The 3-marker GeneSTAR Tenderness panel [CAST (calpastatin), CAPN1 316 (µ-calpain), and CAPN1 4751 (µ-calpain)] was used to determine the GEN of each animal (reported as total number of favorable alleles, 0 through 6). Calves were randomly assigned to 1 of 2 IMP, conventional (CNV) or delayed. Cattle in the CNV group were implanted at the beginning of the finishing period with Revalor-IS or Revalor-IH (Intervet Inc., Millsboro, DE), and then reimplanted 59 d later with Revalor-S or Revalor-H (Intervet Inc.). Calves in the delayed group received a single terminal implant (Revalor-S or Revalor-H) administered 45 d after initiation of the finishing period. Warner-Bratzler shear force (WBSF) was measured on LM and GM steaks at 3, 7, 14, 21, and 28 d postmortem. No interactions between the main effects of sex, IMP, or GEN were detected (P > 0.05) for WBSF. An IMP × postmortem aging (age) interaction was detected (P < 0.05) for LM and GM WBSF. For both muscles, steaks from CNV cattle had WBSF values that were approximately 0.2 kg greater (P < 0.05) than the values for steaks from delayed animals, but only during the early postmortem period (3 to 7 d). A linear effect of GEN on WBSF was detected (P < 0.05) for LM and GM steaks. Within each muscle, steaks from cattle with 6 favorable alleles had WBSF values 0.33 kg less than the values for steaks from cattle with 1 favorable allele. The GEN × age interaction was not significant for LM (P = 0.14) or GM (P = 0.20), but a numerical trend was observed for the effect of GEN on WBSF to diminish as age increased. To investigate how genetic markers could be interfaced with current beef carcass quality grading, cattle were sorted into 2 gene marker groups (GMG), ≤3 vs. ≥4 favorable alleles. For both muscles, GMG was effective only at identifying tenderness differences within the Select grade. When aged ≤14 d, Select LM steaks from cattle with ≥4 alleles had smaller (P < 0.05) WBSF values than did LM steaks from animals with ≤3 alleles. Preslaughter factors (sex, IMP, and GMG) controlled in the present study each accounted for less than 7% of the explained variation in tenderness of the test population. Results from this study suggest that the 3 GeneSTAR Tenderness markers were associated with small differences (0.33 kg) in WBSF and may be useful for i

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Drug Combinations; Drug Implants; Estradiol; Female; Genetic Markers; Genotype; Least-Squares Analysis; Male; Meat; Muscle, Skeletal; Random Allocation; Trenbolone Acetate

Time-lapse imaging analysis was previously used to show that spontaneous proteolysis of PrP(C), which is fluorescence-labeled at both NH(2)- and COOH-termini, occurred in mouse neuroblastoma neuro2a (N2a) cells susceptible to PrP(Sc). We demonstrated that, unlike other protease inhibitors, a calpain inhibitor, calpastatin, drastically inhibited endoproteolysis of PrP(C), as observed with time-lapse imaging in living cells, suggesting calpain-like activity. Calpastatin also inhibited cleavage of endogenous PrP(C), and unprocessed molecules and the double-labeled PrP(C) accumulated around the perinuclear region. The molecular weight of PrP(C) fragments generated by spontaneous proteolysis was identical to those produced when PrP(C) synthesized in vitro was exposed to exogenous calpain. These results suggest that a calpain-like activity mediates normal processing of PrP(C) in N2a cells.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Line, Tumor; Enzyme Activation; Mice; Neuroblastoma; Peptide Hydrolases; Protein Processing, Post-Translational; PrPC Proteins

Rejection of solid organ allograft involves alloreactive T-cell expansion. The importance of NF-κB and NFAT in this process is underscored by the therapeutic efficacy of immunosuppressive agents, which target the two transcription factors. Since calpains, calcium-activated proteases, are involved in the activation of NF-κB and NFAT, we investigated the role of calpains in allograft rejection. In human transplant kidneys undergoing acute or chronic rejection, we show an increased expression of CAPN 1 gene encoding μ-calpain, associated with a marked expression of μ-calpain, mainly in infiltrating T cells. To address the role of calpain in rejection, we used a skin transplant model in transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor. We show that calpain inhibition extended skin allograft survival, from 11 to 20 days. This delay was associated with a limitation in allograft infiltration by T cells. In vitro, calpain inhibition by calpastatin transgene expression limited dramatically T-cell migration but, unexpectedly, increased slightly T-cell proliferation. Amplification of IL-2 signaling via the stabilization of IL-2R common γ-chain provided an explanation for the proliferation response. This is the first study establishing that calpain inhibition delays allograft rejection by slowing down T-cell migration rather than proliferation.

Topics: Acrylates; Adoptive Transfer; Animals; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cell Movement; Cell Proliferation; Gene Expression; Graft Rejection; Homeodomain Proteins; Humans; Interleukin-2; Kidney; Kidney Transplantation; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; NF-kappa B; NFATC Transcription Factors; Skin Transplantation; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Th17 Cells; Transplantation, Homologous

Substrate identification is the key to defining molecular pathways or cellular processes regulated by proteases. Although phage display with random peptide libraries has been used to analyze substrate specificity of proteases, it is difficult to deduce endogenous substrates from mapped peptide motifs. Phage display with conventional cDNA libraries identifies high percentage of non-open reading frame (non-ORF) clones, which encode short unnatural peptides, owing to uncontrollable reading frames of cellular proteins. We recently developed ORF phage display to identify endogenous proteins with specific binding or functional activity with minimal reading frame problem. Here we used calpain 2 as a protease to demonstrate that ORF phage display is capable of identifying endogenous substrates and showed its advantage to re-verify and characterize the identified substrates without requiring pure substrate proteins. An ORF phage display cDNA library with C-terminal biotin was bound to immobilized streptavidin and released by cleavage with calpain 2. After three rounds of phage selection, eleven substrates were identified, including calpastatin of endogenous calpain inhibitor. These results suggest that ORF phage display is a valuable technology to identify endogenous substrates for proteases.

Topics: Animals; Calcium-Binding Proteins; Calpain; Isoenzymes; Open Reading Frames; Peptide Library; Peptides; Rats

This study aimed to detect variability in CAST, CAPN1 and CAPN3 porcine genes and to investigate the effect of CAST and CAPN1 polymorphisms on the activity of native and autolyzed μ-calpain and m-calpain, measured from 1 to 72 h post-mortem in Longissimus dorsi (LD) muscle of 30 pigs. Effects of polymorphisms on meat quality parameter such as pH, color and drip loss were also evaluated. Samples carrying CAST EU137105:g.76,872AA genotype showed higher autolyzed μ-calpain activity 24 and 72 h post-mortem, as well as lower drip loss values. Expression of CAST, CAPN1 and CAPN3 was assessed in LD muscles divergent for shear force. Higher CAST and CAPN3 expression was found in LD with high shear force (P<0.2), confirming a direct role for calpastatin but not for calpain 3 in meat tenderization. In conclusion, CAST gene affected post-mortem activation time of calpain and drip loss.

Topics: Animals; Calcium-Binding Proteins; Calpain; Gene Expression; Genotype; Meat; Muscle Proteins; Muscle, Skeletal; Polymorphism, Single Nucleotide; Swine

The aim of this study was to determine whether or not over-activation of calpains during running exercise or tetanic contractions was a major factor to induce sarcomere lesions in atrophic soleus muscle. Relationship between the degrees of desmin degradation and sarcomere lesions was also elucidated. We observed ultrastructural changes in soleus muscle fibers after 4-week unloading with or without running exercise. Calpain activity and desmin degradation were measured in atrophic soleus muscles before or after repeated tetani in vitro. Calpain-1 activity was progressively increased and desmin degradation was correspondingly elevated in 1-, 2-, and 4-week of unloaded soleus muscles. Calpain-1 activity and desmin degradation had an additional increase in unloaded soleus muscles after repeated tetani in vitro. PD150606, an inhibitor of calpains, reduced calpain activity and desmin degradation during tetanic contractions in unloaded soleus muscles. The 4-week unloading decreased the width of myofibrils and Z-disk in soleus fibers. After running exercise in unloaded group, Z-disks of adjacent myofibrils were not well in register but instead were longitudinally displaced. Calpain inhibition compromised exercise-induced misalignment of the Z-disks in atrophic soleus muscle. These results suggest that tetanic contractions induce an over-activation of calpains which lead to higher degrees of desmin degradation in unloaded soleus muscle. Desmin degradation may loose connections between adjacent myofibrils, whereas running exercise results in sarcomere injury in unloaded soleus muscle.

Topics: Acrylates; Animals; Calcium-Binding Proteins; Calpain; Desmin; Enzyme Assays; In Vitro Techniques; Male; Motor Activity; Muscle Contraction; Muscle, Skeletal; Muscular Disorders, Atrophic; Rats; Rats, Sprague-Dawley; Sarcomeres; Signal Transduction; Troponin; Weightlessness Simulation

Metastasis of breast cancer is a major contributor to mortality. Histological assessment of vascular invasion (VI) provides important prognostic information and demonstrates that VI occurs predominantly via lymphatics in breast cancer. We sought to examine genes and proteins involved in lymphovascular invasion (LVI) to understand the mechanisms of this key disease process. A gene expression array of 91 breast cancer patients was analysed by an Artificial Neural Network (ANN) approach using LVI to supervise the analysis. 89 transcripts were significantly associated (p<0.001) with the presence of LVI. Calpastatin, a specific calpain inhibitor, had the second lowest selection error and was investigated in breast cancer specimens using real-time PCR (n=56) and immunohistochemistry (n=53). Both calpastatin mRNA and protein levels were significantly associated with the presence of LVI (p=0.014 and p=0.025 respectively). The data supports the hypothesis that calpastatin may play a role in regulating the initial metastatic dissemination of breast cancer.

Topics: Adult; Breast Neoplasms; Calcium-Binding Proteins; Calpain; Female; Humans; Immunohistochemistry; Lymphatic Metastasis; Lymphatic Vessels; Middle Aged; Neoplasm Invasiveness; Predictive Value of Tests; Real-Time Polymerase Chain Reaction

Molecular markers for beef tenderness are classic examples of the contribution of genome technology to animal breeding through marker-assisted selection (MAS). Markers on the μ-calpain (CAPN1) and calpastatin (CAST) genes have been extensively evaluated for their association with tenderness. However, little is known about their potential effect on other economically important traits. In this work, the association of molecular markers for beef tenderness with growth traits was evaluated in Angus cattle of Argentina. Expected progeny differences were extracted from the 2008 Angus Sire Summary of Argentina. Information corresponding to 268 influential bulls that had been genotyped for two markers in CAPN1 and two markers in CAST was provided by the Argentine Angus Association. Genotype probabilities were assigned, by segregation analysis, to those bulls in the Sire Summary that had no marker information. Expected progeny differences of 1365 sires were regressed on the number of alleles favouring tenderness at each locus. There was a significant effect of markers on expected progeny differences of birth weight, weaning weight (direct), weight at 18 months and rib eye area. In general, there was a negative effect of alleles favouring tenderness on growth traits. These correlated responses should be taken into account when molecular markers are used in selection schemes that aim to improve beef tenderness.

Topics: Alleles; Animals; Biomarkers; Body Composition; Breeding; Calcium-Binding Proteins; Calpain; Cattle; Genetic Markers; Genotype; Meat

Calpain, an intracellular Ca²⁺-dependent cysteine protease, is known to play a role in a wide range of metabolic pathways through limited proteolysis of its substrates. However, only a limited number of these substrates are currently known, with the exact mechanism of substrate recognition and cleavage by calpain still largely unknown. While previous research has successfully applied standard machine-learning algorithms to accurately predict substrate cleavage by other similar types of proteases, their approach does not extend well to calpain, possibly due to its particular mode of proteolytic action and limited amount of experimental data. Through the use of Multiple Kernel Learning, a recent extension to the classic Support Vector Machine framework, we were able to train complex models based on rich, heterogeneous feature sets, leading to significantly improved prediction quality (6% over highest AUC score produced by state-of-the-art methods). In addition to producing a stronger machine-learning model for the prediction of calpain cleavage, we were able to highlight the importance and role of each feature of substrate sequences in defining specificity: primary sequence, secondary structure and solvent accessibility. Most notably, we showed there existed significant specificity differences across calpain sub-types, despite previous assumption to the contrary. Prediction accuracy was further successfully validated using, as an unbiased test set, mutated sequences of calpastatin (endogenous inhibitor of calpain) modified to no longer block calpain's proteolytic action. An online implementation of our prediction tool is available at http://calpain.org.

Topics: Algorithms; Area Under Curve; Artificial Intelligence; Binding Sites; Biochemistry; Calcium; Calcium-Binding Proteins; Calpain; Computational Biology; Humans; Models, Statistical; Normal Distribution; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Reproducibility of Results; Software

In this study, 20 young steers received no beta-agonist (C), 100 animals all received zilpaterol hydrochloride (Z), with 1 group only receiving Z while the other 4 groups received zilpaterol and vitamin D3 at the following levels (IU/animal/day) and durations before slaughter: 7 million for 3 days (3D7M); 7 million for 6 days (6D7M); 7 million for 6 days with 7 days no supplementation (6D7M7N) and 1 million for 9 days (9D1M). Left carcass sides were electrically stimulated (ES) and the right side not stimulated (NES). Samples were aged for 3 or 14 days post mortem. Parameters included Warner-Bratzler shear force (WBSF), myofibril filament length, sarcomere length and calpastatin and calpain enzyme activity. Both ES and prolonged aging reduced WBSF (P<0.001). 6D7M, 6D7M7N and Z remained significantly tougher than C (P<0.001), while 3D7M and 9D1M improved WBSF under NES conditions. ES is more effective to alleviate beta-agonist induced toughness than high vitamin D3 supplements.

Topics: Animal Feed; Animals; Calcium; Calcium-Binding Proteins; Calpain; Cattle; Cholecalciferol; Dietary Supplements; Electric Stimulation; Male; Meat; Myofibrils; Parathyroid Hormone; Proteolysis; Sarcomeres; Trimethylsilyl Compounds

Retinitis pigmentosa (RP) is a heterogeneous group of inherited neurodegenerative diseases affecting photoreceptors and causing blindness. Many human cases are caused by mutations in the rhodopsin gene. An important question regarding RP pathology is whether different genetic defects trigger the same or different cell death mechanisms. To answer this question, we analysed photoreceptor degeneration in P23H and S334ter transgenic rats carrying rhodopsin mutations that affect protein folding and sorting respectively. We found strong activation of calpain and poly(ADP-ribose) polymerase (PARP) in both mutants, concomitant with calpastatin down-regulation, increased oxidative DNA damage and accumulation of PAR polymers. These parameters were strictly correlated with the temporal progression of photoreceptor degeneration, mirroring earlier findings in the phosphodiesterase-6 mutant rd1 mouse, and suggesting execution of non-apoptotic cell death mechanisms. Interestingly, activation of caspases-3 and -9 and cytochrome c leakage-key events in apoptotic cell death--were observed only in the S334ter mutant, which also showed increased expression of PARP-1. The identification of the same metabolic markers triggered by different mutations in two different species suggests the existence of common cell death mechanisms, which is a major consideration for any mutation independent treatment.

Topics: Animals; Animals, Newborn; Biomarkers; Calcium-Binding Proteins; Calpain; Caspase 3; Caspase 9; Cell Death; Cell Shape; Cytochromes c; DNA Damage; Enzyme Activation; Humans; In Situ Nick-End Labeling; Mutation; Oxidative Stress; Photoreceptor Cells, Vertebrate; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Protein Transport; Rats; Rats, Mutant Strains; Rats, Transgenic; Rhodopsin; Staining and Labeling

Although the calpain-calpastatin system has been implicated in a number of pathological conditions, its normal physiological role remains largely unknown. To investigate the functions of this system, we generated conventional and conditional calpain-2 knockout mice. The conventional calpain-2 knockout embryos died around embryonic day 15, preceded by cell death associated with caspase activation and DNA fragmentation in placental trophoblasts. In contrast, conditional knockout mice in which calpain-2 is expressed in the placenta but not in the fetus were spared. These results suggest that calpain-2 contributes to trophoblast survival via suppression of caspase activation. Double-knockout mice also deficient in calpain-1 and calpastatin resulted in accelerated and rescued embryonic lethality, respectively, suggesting that calpain-1 and -2 at least in part share similar in vivo functions under the control of calpastatin. Triple-knockout mice exhibited early embryonic lethality, a finding consistent with the notion that this protease system is vital for embryonic survival.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Death; Embryo Loss; Embryo, Mammalian; Female; Mice; Mice, Knockout; Placenta; Pregnancy; Protein Isoforms

To assess the expression of calpains and calpain-induced aggrecan fragmentation in early and advanced stages of degeneration of human intervertebral discs (IVDs).. Disc tissue samples of 55 patients (mean age, 51.2 ± 22.3 years) who underwent intervertebral fusion were divided into groups with early and advanced degeneration based on the Thompson magnetic resonance imaging (MRI) scale. In advanced degeneration group, five patients (mean age, 35.5 ± 11.4 years) of lumbar disc herniation (LDH) were included. Protein levels of m- and μ-calpains and their inhibitor calpastatin were assayed, and immunohistochemical techniques were used to localize and quantify the production of the enzymes. To investigate calpain activity, we assayed purified aggrecan fragmentation in disc tissue by Western blotting and immunohistochemistry with VPGVA antibody, which recognizes the m-calpain generated neo-epitope GVA.. Discs at early stages of degeneration expressed low levels of m- and μ-calpains and calpastatin, and few cells expressed degenerative enzymes. At more advanced stages of degeneration, the expression and number of cells immunopositive for m-calpain, μ-calpain and calpastatin were significantly higher. Further finding showed that anti-GVA-reactive aggrecan fragments were significantly higher in discs at advanced compared with early stages of degeneration. Herniated disc samples showed stronger expression and more cells immunopositive for calpains, calpastatin and GVA in the nucleus pulposus than in the annulus fibrous.. The expression of calpains, together with m-calpain-induced degradation products of extracellular matrix, was correlated with the degree of disc degeneration in human IVD tissue. These findings suggest that calpains may be involved in IVD degeneration via proteoglycan (PG) cleavage.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Aggrecans; Blotting, Western; Calcium-Binding Proteins; Calpain; Child; Extracellular Matrix; Extracellular Matrix Proteins; Female; Humans; Intervertebral Disc Degeneration; Lumbar Vertebrae; Magnetic Resonance Imaging; Male; Middle Aged; Sacrum

Calpains are proteolytic enzymes that modulate cellular function through cleavage of targets, thereby modifying their actions. An important role is emerging for calpains in regulating inflammation and immune responses, although specific mechanisms by which this occurs have not been clearly defined. In this study, we identify a novel target of calpain, selenoprotein K (SelK), which is an endoplasmic reticulum transmembrane protein important for Ca(2+) flux in immune cells. Calpain-mediated cleavage of SelK was detected in myeloid cells (macrophages, neutrophils, and dendritic cells) but not in lymphoid cells (B and T cells). Both m- and μ-calpain were capable of cleaving immunoprecipitated SelK, but m-calpain was the predominant isoform expressed in mouse immune cells. Consistent with these results, specific inhibitors were used to show that only m-calpain cleaved SelK in macrophages. The cleavage site in SelK was identified between Arg(81) and Gly(82) and the resulting truncated SelK was shown to lack selenocysteine, the amino acid that defines selenoproteins. Resting macrophages predominantly expressed cleaved SelK and, when activated through different Toll-like receptors (TLRs), SelK cleavage was inhibited. We found that decreased calpain cleavage was due to TLR-induced up-regulation of the endogenous inhibitor, calpastatin. TLR-induced calpastatin expression not only inhibited SelK cleavage, but cleavage of another calpain target, talin. Moreover, the expression of the calpain isoforms and calpastatin in macrophages were different from T and B cells. Overall, our findings identify SelK as a novel calpain target and reveal dynamic changes in the calpain/calpastatin system during TLR-induced activation of macrophages.

Topics: Amino Acid Sequence; Animals; Bone Marrow Cells; Calcium; Calcium-Binding Proteins; Calpain; Cell Movement; Endoplasmic Reticulum; Humans; Ligands; Macrophages; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Selenoproteins; Toll-Like Receptors

Recently we have shown that calpain-1 activation contributes to cardiomyocyte apoptosis induced by hyperglycemia. This study was undertaken to investigate whether targeted disruption of calpain would reduce myocardial hypertrophy and fibrosis in mouse models of type 1 diabetes.. Diabetes in mice was induced by injection of streptozotocin (STZ), and OVE26 mice were also used as a type 1 diabetic model. The function of calpain was genetically manipulated by cardiomyocyte-specific knockout Capn4 in mice and the use of calpastatin transgenic mice. Myocardial hypertrophy and fibrosis were investigated 2 and 5 months after STZ injection or in OVE26 diabetic mice at the age of 5 months. Cultured isolated adult mouse cardiac fibroblast cells were also investigated under high glucose conditions.. Calpain activity, cardiomyocyte cross-sectional areas, and myocardial collagen deposition were significantly increased in both STZ-induced and OVE26 diabetic hearts, and these were accompanied by elevated expression of hypertrophic and fibrotic collagen genes. Deficiency of Capn4 or overexpression of calpastatin reduced myocardial hypertrophy and fibrosis in both diabetic models, leading to the improvement of myocardial function. These effects were associated with a normalization of the nuclear factor of activated T-cell nuclear factor-κB and matrix metalloproteinase (MMP) activities in diabetic hearts. In cultured cardiac fibroblasts, high glucose-induced proliferation and MMP activities were prevented by calpain inhibition.. Myocardial hypertrophy and fibrosis in diabetic mice are attenuated by reduction of calpain function. Thus targeted inhibition of calpain represents a potential novel therapeutic strategy for reversing diabetic cardiomyopathy.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cardiomyopathy, Hypertrophic; Cell Proliferation; Cells, Cultured; Diabetes Mellitus, Type 1; Diabetic Cardiomyopathies; Disease Models, Animal; Fibrosis; Gene Expression Regulation; Heart; Hyperglycemia; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Molecular Targeted Therapy; Myocardium; Streptozocin

The peptidyl-proline isomerase, protein never in mitosis gene A interacting-1 (PIN1) binds and isomerizes proteins phosphorylated on serine/threonine before a proline. It was previously found that depletion of PIN1 greatly increased induction of cyclooxygenase-2 and inducible nitric oxide synthase by lowering calpain activity in murine aortic endothelial cells (MAEC). Here we investigated the effect of PIN1 on the endogenous inhibitor of heterodimeric μ- and m-calpains, calpastatin. MAEC were transduced with small hairpin (sh) RNA to knock down PIN1 (KD) or an inactive Control shRNA. Cells were also treated with non-targeted double stranded small inhibitory RNA (siRNA) or siRNA designed to deplete calpastatin. Despite reducing calpain activity, PIN1 KD did not significantly affect the expression of μ- and m-calpains, or calpastatin, compared to Control shRNA. Instead, depletion of PIN1 increased the inhibitory activity of calpastatin. Calpastatin co-immunoprecipitated with endogenous PIN1 and was pulled down with glutathione-S-transferase (GST)-PIN1 fusion protein. Adding GST-PIN1 to KD cell extracts lacking PIN1 reduced calpastatin inhibitory activity. Substrate binding and catalytic domain mutants of PIN1 failed to do so. These results suggest that protein interaction and the proline isomerase functions of PIN1 are required for it to inhibit calpastatin. Furthermore, depletion of calpastatin raised calpain activity and reduced calpain inhibitory activity to similar levels in KD and Control MAEC, indicating that calpastatin is required for PIN1 depletion to lower calpain activity. Thus, PIN1 apparently restrains the ability of calpastatin to inhibit calpain, maintaining calpain activity in endothelial cells. PIN1 may act directly via phosphorylated serine/threonine-proline motifs in calpastatin, or indirectly via other PIN1 substrates that control calpastatin.

Topics: Animals; Calcium-Binding Proteins; Calpain; Endothelium, Vascular; Gene Knockdown Techniques; Mice; Mitosis; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phosphorylation; RNA, Small Interfering

We are here reporting that in peripheral blood mononuclear cells (PBMC) of patients homozygous for F508del-CFTR the calpain-calpastatin system undergoes a profound alteration. In fact, calpain basal activity, almost undetectable in control PBMC, becomes measurable at a significant extent in cells from cystic fibrosis (CF) patients, also due to a 40-60% decrease in both calpastatin protein and inhibitory activity. Constitutive protease activation in CF patients' cells induces a large accumulation of the mutated cystic fibrosis transmembrane conductance regulator (CFTR) in the 100kD+70kD split forms as well as a degradation of proteins associated to the CFTR complex. Specifically, the scaffolding protein Na(+)/H(+) exchanger 3 regulatory factor-1 (NHERF-1) is converted in two distinct fragments showing masses of 35kD and 20kD, being however the latter form the most represented one, thereby indicating that in CF-PBMC the CFTR complex undergoes a large disorganization. In conclusion, our observations are providing new information on the role of calpain in the regulation of plasma membrane ion conductance and provide additional evidence on the transition of this protease activity from a physiological to a pathological function.

Topics: Adolescent; Adult; Calcium-Binding Proteins; Calpain; Case-Control Studies; Child; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytoskeletal Proteins; Enzyme Activation; Enzyme Assays; Female; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Mutation, Missense; Phosphoproteins; Protein Isoforms; Protein Transport; Proteolysis; Sodium-Hydrogen Exchangers; Young Adult

Subchronic gestational stress leads to permanent modifications in the hippocampus-hypothalamus-pituitary-adrenal axis of offspring probably due to the increase in circulating glucocorticoids known to affect prenatal programming. The aim of this study was to investigate whether cell turnover is affected in the hippocampus-hypothalamus-pituitary axis by subchronic prenatal stress and the intracellular mechanisms involved. Restraint stress was performed in pregnant rats during the last week of gestation (45 minutes; 3 times/day). Only male offspring were used for this study and were sacrificed at 6 months of age. In prenatally stressed adults a decrease in markers of cell death and proliferation was observed in the hippocampus, hypothalamus and pituitary. This was associated with an increase in insulin-like growth factor-I mRNA levels, phosphorylation of CREB and calpastatin levels and inhibition of calpain -2 and caspase -8 activation. Levels of the anti-apoptotic protein Bcl-2 were increased and levels of the pro-apoptotic factor p53 were reduced. In conclusion, prenatal restraint stress induces a long-term decrease in cell turnover in the hippocampus-hypothalamus-pituitary axis that might be at least partly mediated by an autocrine-paracrine IGF-I effect. These changes could condition the response of this axis to future physiological and pathophysiological situations.

Topics: Animals; Calcium-Binding Proteins; Calpain; Caspase 8; Cyclic AMP Response Element-Binding Protein; Female; Hypothalamo-Hypophyseal System; Insulin-Like Growth Factor I; Male; Phosphorylation; Pituitary-Adrenal System; Pregnancy; Prenatal Exposure Delayed Effects; Proto-Oncogene Proteins c-bcl-2; Rats; RNA, Messenger; Stress, Physiological; Time; Tumor Suppressor Protein p53

Calpains, also called calcium activated neutral proteases (CANP), are expressed ubiquitously. They are intracellular, non-lysosomal cytoplasmic cysteine endopeptidases. Calcium is required for their activation. Their endogenous specific inhibitor is calpastatin, which is expressed ubiquitously and coexists within cells besides calpain. When calcium is present, calpastatin and calpain attach to each other inhibiting the protease. The calpain system plays an important role in many processes including apoptosis, necrosis, ischemia formation and exocytosis. So far, many reports exist on studies about the influence of calpains in different tumors (skin, breast, renal cell and prostate cancers). The role of calpains in pathogenesis or further tumor progression has always been proved in related studies, but their exact function could not be demonstrated. So far, no studies on calpains being involved in the pathogenesis of ovarian cancer have been published. In our study we focused on the expression of the enzymes calpain 1, calpain 2 and their inhibitor calpastatin in normal and malign ovarian tissue. Therefore, we performed immunohistochemical stainings of paraffin slices and evaluated staining intensity (SI), percentage of positive cells (PP) and immunoreactive score (IRS). We evaluated the correlation between enzyme expression in malign and benign ovarian tissues. In malignant ovarian tissue, we found decreased expression, staining intensity and immunoreactive score of calpastatin. With higher grading of the ovarian carcinoma, staining intensity and immunoreactive score of calpain 1 decreased. Staining intensity of calpain 2 in ovarian carcinoma decreased with increasing lymph node status. We clearly demonstrated differences between enzyme expressions in malign and benign tissue. This study could not find any specific function of calpains. Only few studies in the literature have been found that deal with calpain evaluation of ovarian cancer. Additional studies including more patients are required to elucidate the functional role and impact of calpain in tumors in detail.

Topics: Calcium-Binding Proteins; Calpain; Female; Humans; Immunohistochemistry; Ovarian Neoplasms

The objective of this study is to investigate the expression of calpain-1, calpain-2, and calpastatin in the human periurethral vaginal tissues and to show the potential link between calpain system and stress urinary incontinence (SUI).. The periurethral vaginal tissues of 39 women with SUI and 31 women without SUI were collected to detect the expressions of calpains and calpastatin by using semi-quantitative competitive reverse transcription-polymerase chain reaction and Western blotting.. There were no significant differences on the expressions of calpain-1 at the levels of messenger RNA (mRNA) and protein in both groups (P > 0.05), but the patients with SUI had significantly higher levels of calpain-2 mRNA and protein than the control (P < 0.05); and the mRNA expressions of calpastatin in women with SUI were significantly higher than the control (P < 0.05), while the protein expressions were significantly lower when compared to the control (P < 0.01).. Overexpression of calpain-2 and low expression of calpastatin may involve in the pathological development of SUI.

Topics: Calcium-Binding Proteins; Calpain; Case-Control Studies; Female; Humans; Middle Aged; RNA, Messenger; Urinary Incontinence, Stress; Vagina

Calcium homeostasis is disturbed in many ways in the course of chronic kidney disease (CKD). The concentration of free cytoplasmic calcium in erythrocytes is increased. Maintenance of a high concentration gradient (between the cystoplasmic and extracellular space) is possible only due to a finely tuned cooperation between many regulating systems in the cytoplasmic membranes and cell organelles. The aim of our study was to evaluate the activity of Ca(2+)-Mg(2+)-dependent ATPase (PMCA), calmodulin and calpain-calpastatin (CANP-CAST) system in erythrocytes of CKD children treated conservatively in the stages II-IV.. A total of 36 patients with CKD were enrolled in the study. Group A contained patients with CKD stage II; group B with CKD stage III; and group C with CKD stage IV. The control group D consisted of 30 healthy subjects. In the serum, we determined the following: intact parathormon, total calcium, creatinine; in the red blood cells: free cytosolic calcium concentration (Ca(i)(2+)), activity of Ca(2+)-Mg(2+)-transporting ATPase (PMCA), basal PMCA (bPMCA), calmodulin (CALM), CANP, CAST.. In all groups, Ca(i)(2+) concentrations were significantly higher, whereas PMCA and bPMCA activity were lower than in the controls. CANP concentrations in group A were elevated compared to the controls, whereas in groups B and C they were significantly lower. In group C, the mean CAST activity reached the highest values. CALM concentrations were decreased versus controls in all groups of patients.. The intracellular Ca(i)(2+) homeostasis is disturbed in children with CKD and aggravates the deterioration of renal function as well. The reasons for the progressing increase of erythrocyte calcium concentration are multifactorial. Undoubtedly, the decreased PMCA activity, the calmodulin deficiency and the dysregulated CANP-CAST system are responsible for that phenomenon. The impact of many other biological modulators, creating a network defending the cell against the calcium accumulation, cannot be excluded.

Topics: Adolescent; Ca(2+) Mg(2+)-ATPase; Calcium-Binding Proteins; Calmodulin; Calpain; Child; Chronic Disease; Erythrocytes; Humans; Kidney Diseases

Persistent dysregulation in Ca(2+) homeostasis is a pervasive pathogenic mechanism in most neurodegenerative diseases, and accordingly, calpain activation has been implicated in neuronal cells dysfunction and death. In this study we examined the intracellular functional state of the calpain-calpastatin system in -G93A(+) SOD1 transgenic mice to establish if and how uncontrolled activation of calpain can be prevented in vivo during the course of prolonged [Ca(2+)](i) elevation. The presented data indicate that 1) calpain activation is more extensive in motor cortex, in lumbar, and sacral spinal cord segments compared with the lower or almost undetectable activation of the protease in other brain areas, 2) direct measurements of the variations of Ca(2+) levels established that the degree of the protease activation is correlated to the extent of elevation of [Ca(2+)](i), 3) intracellular activation of calpain is always associated with diffusion of calpastatin from perinuclear aggregated forms into the cytosol and the formation of a calpain-calpastatin complex, and 4) a conservative fragmentation of calpastatin is accompanied by its increased expression and inhibitory capacity in conditions of prolonged increase in [Ca(2+)](i). Thus, calpastatin diffusion and formation of the calpain-calpastatin complex together with an increased synthesis of the inhibitor protein represent a cellular defense response to conditions of prolonged dysregulation in intracellular Ca(2+) homeostasis. Altogether these findings provide a new understanding of the in vivo molecular mechanisms governing calpain activation that can be extended to many neurodegenerative diseases, potentially useful for the development of new therapeutic approaches.

Topics: Animals; Brain; Calcium; Calcium-Binding Proteins; Calpain; Enzyme Activation; Gene Expression Regulation, Enzymologic; Homeostasis; Humans; Intracellular Space; Isoenzymes; Mice; Mice, Transgenic; Muscles; Neurons; Rats; RNA, Messenger; Spinal Cord; Substrate Specificity; Tissue Extracts

We have recently demonstrated the localization of associated m-calpain and calpastatin in the endoplasmic reticulum (ER) of bovine pulmonary artery smooth muscle. Herein, we sought to determine the role of m-calpain on calcium-dependent proteolytic cleavage of Na(+)/Ca(2+) exchanger (NCX) in the ER. Treatment of the ER with Ca(2+) (5 mM) dissociates m-calpain-calpastatin association leading to the activation of m-calpain, which subsequently cleaves the ER integral transmembrane protein NCX1 (116 kDa) to an 82 kDa fragment. Pre-treatment of the ER with calpain inhibitors, calpeptin (10 microM) or MDL28170 (10 microM), or Ca(2+) chelator, EGTA (10 mM) does not cleave NCX1. In vitro cleavage of the ER purified NCX1 by the ER purified m-calpain also supports our finding. Cleavage of NCX1 by m-calpain in the ER may be interpreted as the main cause of intracellular Ca(2+) overload in the smooth muscle, which could be important for the manifestation of pulmonary hypertension.

Topics: Animals; Blotting, Western; Calcium; Calcium-Binding Proteins; Calpain; Cattle; Dipeptides; Egtazic Acid; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum; Immunoprecipitation; In Vitro Techniques; Muscle, Smooth; Protein Binding; Pulmonary Artery; Sodium-Calcium Exchanger

Androgen ablation therapy is effective in treating androgen-dependent prostate tumors; however, tumors that can proliferate in castrate levels of androgen eventually arise. We previously reported that in CWR22Rv1 (Rv1) cells, the protease calpain 2 can cleave the androgen receptor (AR) into a constitutively active approximately 80,000 low molecular weight (LMW) form. In this study, we further dissect the mechanisms that produce the AR LMW forms using Rv1 cells and the related CWR22-R1 (R1) cells. The 39-amino acid insertional mutation in the Rv1-AR (E3DM-AR) sensitizes this AR to calpain 2 proteolysis. R1 cells encode the same AR molecule as the parental CWR22 xenograft. Using calpain 2 small interfering RNA and calpeptin, we find that calpain 2 plays a role in the generation of the LMW-AR in R1 cells. Furthermore, LMW-AR expression is regulated by the activation of calpain 2 by ERK 1 and 2. Inhibition of ERK phosphorylation or small interfering RNA-mediated decrease of ERK expression reduces LMW-AR levels in R1 cells. Conversely, activation of the MAPK pathway results in increased ERK phosphorylation and increased levels of LMW-AR. Finally, analyses of human tumor samples found that LMW-AR levels are higher in tumors that have an increased calpain/calpastatin ratio and/or increased levels of phospho-ERK (pERK). This suggests that a higher calpain/calpastatin ratio collaborates with activated ERK to promote the generation of the LMW-AR.

Topics: Calcium-Binding Proteins; Calpain; Cell Line, Transformed; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Molecular Weight; Phosphorylation; Prostatic Neoplasms; Receptors, Androgen; Recurrence; RNA, Small Interfering

The klotho mouse shows multiple phenotypes resembling human aging caused by the mutation of a single gene. This mutation is caused by the insertion of ectopic DNA into the regulatory region of the alpha-klotho gene. The alpha-klotho gene encodes a type I membrane protein that is expressed predominantly in the kidney and brain. As a result of a defect in alpha-klotho gene expression, the klotho mouse exhibits multiple age-associated disorders, such as arteriosclerosis, osteoporosis, pulmonary emphysema and short life span. However, the mechanism by which the alpha-klotho gene product suppresses the aging phenomena has not been identified. Analysis of the pathophysiology of klotho mice is expected to give clues not only to understanding the mechanisms of individual diseases associated with aging but also the molecular mechanisms during human aging. We previously reported that the aberrant activation of mu-calpain is caused by the alpha-klotho mutation, and such change leads to degradation of cytoskeletal elements. Similar phenomena were observed in normal aged mice. Such deterioration may trigger tissue abnormalities in klotho mice and aged mice, but klotho protein may suppress these processes. We will summarize the function of alpha-klotho protein based on our research on the relationship between proteolysis and age-related disorders and the recent advanced researches.

Topics: Aging; Animals; Calcium-Binding Proteins; Calpain; DNA; Glucuronidase; Humans; Klotho Proteins; Membrane Proteins; Mice; Mutagenesis, Insertional

Methamphetamine (METH) is a potent psychostimulant drug that may cause neuronal cell degeneration. The underlying mechanisms of METH-induced neuronal toxicity remains poorly understood. In this study, we investigated an important role of calpain-dependent cascades in methamphetamine-induced toxicity in human dopaminergic neuroblastoma SH-SY5Y cultured cell lines. In addition, the protective effect of melatonin against METH-induced calpain-dependent death pathway was also investigated. The results of this study show that METH significantly decreased cell viability and tyrosine hydroxylase phosphorylation in SH-SY5Y cultured cells. Melatonin reversed the toxic effect of METH by inducing cell viability. In addition, melatonin was able to restore the reduction in mitochondrial function and phosphorylation of tyrosine hydroxylase in SH-SY5Y treated cells. An induction of calpain expression and activity but a reduction of calpain inhibitor (calpastatin) protein levels were observed in SH-SY5Y cells treated with METH but these effects were diminished by melatonin. These results implicated calpain-dependent death pathways in the processes of METH-induced toxicity and also indicated that melatonin has the capacity to reverse this toxic effect in SH-SY5Y cultured cells.

Topics: Calcium-Binding Proteins; Calpain; Cell Line, Tumor; Cell Survival; Humans; Melatonin; Methamphetamine; Neuroblastoma

Mycoplasmas often contaminate cultured cells, leading to alterations in cellular gene expression, protein synthesis, signal transduction and metabolic pathways. Mycoplasmal contamination is often unnoticed, so that mycoplasma-induced alterations in cell functions may not be appreciated, unless specifically studied. Here, we show for the first time that contamination of SH-SY5Y cells by Mycoplasma hyorhinis leads to increased levels of calpastatin (the endogenous inhibitor of the Ca(2+)-dependent protease calpain), resulting in inhibition of Ca(2+)-induced calpain activation and inhibition of calpain-promoted proteolysis in the mycoplasmal-infected cells. Calpain activity is recovered upon calpastatin removal from extracts of contaminated cells. The calpain-calpastatin system has been implicated in a variety of physiological and pathological processes (signal transduction, motility, cell cycle, cell differentiation, membrane damage and apoptosis). Because the ratio of calpastatin to calpain is an important factor in the control of calpain activity within the cell, the elevated calpastatin may protect the mycoplasma-infected cells against certain types of damage (e.g. caused by high Ca(2+)). Thus, our results are important for studies on the modulation of host cells by mycoplasmas, and relevant to the pathobiology of processes involving mycoplasmal infections. The mycoplasma-infected cells provide a system for identifying factors that participate in the regulation of cellular calpastatin.

Topics: Calcium; Calcium-Binding Proteins; Calpain; Cell Differentiation; Equipment Contamination; Host-Pathogen Interactions; Mycoplasma hyorhinis; Neuroblastoma; Neurons; Proteins; Tumor Cells, Cultured; Up-Regulation

Two intracellular cysteine proteases (calpains and caspases) and inducible nitric oxide synthase (iNOS) participate in the ischemic brain injury. In vitro nitric oxide (NO) regulates calpain and caspase-3 activation. The present study investigated whether aminoguanidine (AG), an iNOS inhibitor, protected brain against experimental stroke through inhibiting calpain and caspase-3 activation. Rats received 1h ischemia by intraluminal filament, then, reperfused for 23 h (R 23 h). AG (100 mg/kg) was administered intraperitoneally 5 min before ischemia. Our data showed that treatment with AG markedly improved neurological deficit, reduced brain swelling, decreased infarct volume, and attenuated the necrotic cell death in ischemic penumbra and core, and apoptotic cell death in penumbra at R 23 h. Enzymatic studies demonstrated the significant inhibition of the activities of mu- and m-calpain and caspase-3, and Western blot analysis revealed marked increases in the levels of MAP-2 and spectrin in penumbra and core in AG-treated rats versus vehicle-treated rats. AG also significantly enhanced the calpastatin levels in core, although it had no significant effects on that in penumbra. These data demonstrate that inhibiting calpain and caspase-3 activation is one mechanism of AG against experimental stroke, suggesting that NO produced by iNOS may be involved in calpain- and caspase-3-mediated ischemic cell death, at least in part.

Topics: Animals; Apoptosis; Biotransformation; Blotting, Western; Brain; Brain Edema; Calcium-Binding Proteins; Calpain; Caspase 3; Cerebral Infarction; Cytosol; Enzyme Activation; Guanidines; Male; Microtubule-Associated Proteins; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Recovery of Function; Spectrin; Stroke

Previously, we reported that bovine pulmonary smooth muscle endoplasmic reticulum (ER) membrane possesses associated m-calpain and calpastatin and ER lumen contains only m-calpain. Herein, we report characteristic properties of ER membrane m-calpain (MCp), calpastatins and lumen m-calpain (LCp) and a brief comparative study between MCp and LCp. MCp containing 80 kDa large and 28 kDa small subunit is non-phosphorylated, whereas LCp containing only 80 kDa large subunit is phosphorylated. Optimum pH, Ca(2+) concentration and pI value of both MCp and LCp are 7.5, 5 mM and 4.5, respectively. MCp and LCp have similar kinetic parameters and circular dichroism (CD) spectra. Autolysis of MCp and LCp are different. Coimmunoprecipitation studies revealed that LCp is associated with ERp57 in the ER lumen, which suggests that the regulation of LCp differs from the regulation of MCp. In presence of Ca(2+), the activated LCp cleaves inositol 1,4,5-trisphosphate receptor-1 (IP(3)R1) in the ER lumen, whereas the activated MCp cleaves Na(+)/Ca(2+) exchanger-1 (NCX1) in the ER membrane. We have determined pI (4.6 and 4.7, respectively) and IC(50) (0.52 and 0.8 nM, respectively) values of 110 and 70 kDa calpastatins. For first time, we have determined the characteristic properties, regulation and functional activity of LCp in the ER lumen.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Cattle; Cell Membrane; Circular Dichroism; Endoplasmic Reticulum; Hydrogen-Ion Concentration; Kinetics; Muscle, Smooth, Vascular

Rhabdomyosarcoma (RMS) are soft-tissue sarcoma commonly encountered in childhood. RMS cells can acquire invasive behavior and form metastases. The metastatic dissemination implicates many proteases among which are mu-calpain and m-calpain. Study of calpain expression and activity underline the deregulation of calpain activity in RMS. Analysis of kinetic characteristics of RMS cells, compared to human myoblasts LHCN-M2 cells, shows an important migration velocity in RMS cells. One of the major results of this study is the positive linear correlation between calpain activity and migration velocity presenting calpains as a marker of tumor aggressiveness. The RMS cytoskeleton is disorganized. Specifying the role of mu- and m-calpain using antisense oligonucleotides led to show that both calpains up-regulate alpha- and beta-actin in ARMS cells. Moreover, the invasive behavior of these cells is higher than that of LHCN-M2 cells. However, it is similar to that of non-treated LHCN-M2 cells, when calpains are inhibited. In summary, calpains may be involved in the anarchic adhesion, migration and invasion of RMS. The direct relationship between calpain activity and migration velocities or invasive behavior indicates that calpains could be considered as markers of tumor aggressiveness and as potential targets for limiting development of RMS tumor as well as their metastatic behavior.

Topics: Actins; Blotting, Western; Calcium-Binding Proteins; Calpain; Cell Adhesion; Cell Movement; Cell Proliferation; Cells, Cultured; Cytoskeleton; Humans; Myoblasts; Neoplasm Invasiveness; Reverse Transcriptase Polymerase Chain Reaction; Rhabdomyosarcoma, Alveolar; RNA, Messenger

Calpains contribute to reperfusion-induced myocardial cell death. However, it remains controversial whether its activation occurs during ischemia or reperfusion. We investigated the regulation and time-course of calpain activation secondary to transient ischemia and the efficacy of its inhibition at reperfusion as a therapeutic strategy to limit infarct size. In isolated rat hearts (Sprague-Dawley), ischemia induced a time-dependent translocation of m-calpain to the membrane that was not associated with calpain activation as assessed by proteolysis of its substrate alpha-fodrin. Translocation of calpain was dependent on Ca(2+) entry through reverse mode Na(+)/Ca(2+)-exchange and was independent of acidosis. Calpain activation occurred during reperfusion, but only after intracellular pH (pHi) normalization, and was not prevented by inhibiting its translocation during ischemia with methyl-beta-cyclodextrin. The intravenous infusion of MDL-28170 in an in vivo rat model with transient coronary occlusion during the first minutes of reperfusion resulted in a reduction of infarct size (43.9+/-3.9% vs. 60.2+/-4.7, P=0.046, n=18) and alpha-fodrin degradation. These results suggest that (1) Ca(2+)-induced calpain translocation to the membrane during ischemia is independent of its activation, (2) intracellular acidosis inhibits calpain activation during ischemia and pHi normalization allows activation upon reperfusion, and (3) calpain inhibition at the time of reperfusion appears as a potentially useful strategy to limit infarct size.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Death; Enzyme Activation; Hemodynamics; Hydrogen-Ion Concentration; In Vitro Techniques; Intracellular Space; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Protease Inhibitors; Protein Transport; Rats; Rats, Sprague-Dawley

Accumulation of abnormal proteins and endoplasmic reticulum stress accompany neurodegenerative diseases including Huntington's disease. We show that the expression of mutant huntingtin proteins with extended polyglutamine repeats differentially affected endoplasmic reticulum signaling cascades linked to the inositol-requiring enzyme-1 (IRE1) pathway. Thus, the p38 and c-Jun N-terminal kinase pathways were activated, while the levels of the nuclear factor-kappaB-p65 (NF-kappaB-p65) protein decreased. Downregulation of NF-kappaB signaling was linked to decreased antioxidant levels, increased oxidative stress, and enhanced cell death. Concomitantly, calpain was activated, and treatment with calpain inhibitors restored NF-kappaB-p65 levels and increased cell viability. The calpain regulator, calpastatin, was low in cells expressing mutant huntingtin, and overexpression of calpastatin counteracted the deleterious effects caused by N-terminal mutant huntingtin proteins. These results show that calpastatin and an altered NF-kappaB-p65 signaling are crucial factors involved in oxidative stress and cell death mediated by mutant huntingtin proteins.

Topics: Animals; Base Sequence; Calcium Signaling; Calcium-Binding Proteins; Calpain; Cell Death; Cell Line; DNA Primers; Down-Regulation; Humans; Huntingtin Protein; Mitochondrial Proteins; Mutation; Nerve Tissue Proteins; NF-kappa B; Nuclear Proteins; Oxidative Stress; PC12 Cells; Rats; Recombinant Fusion Proteins; Signal Transduction; Superoxide Dismutase; Thioredoxins; Transcription Factor RelA

The calcium-activated neutral proteases, mu- and m-calpain, along with their inhibitor, calpastatin, have been demonstrated to mediate a variety of Ca(2+)-dependent processes including signal transduction, cell proliferation, cell cycle progression, differentiation, apoptosis, membrane fusion, platelet activation and skeletal muscle protein degradation. The cDNA coding for yak calpastatin was amplified and cloned by RT-PCR to investigate and characterize the nucleotide/amino-acid sequence and to predict structure and function of the calpastatin. The present study suggests that the yak calpastatin gene encodes a protein of 786 amino acids that shares 99 % sequence identity with the amino-acid sequence of cattle calpastatin, and that the yak protein is composed of an N-terminal region (domains L and XL) and four repetitive homologous C-terminal domains (d1-d4), in which several prosite motifs are present including short peptide L54-64 (EVKPKEHTEPK in domain L) and GXXE/ DXTIPPXYR (in subdomain B), where X is a variable amino acid. Our results suggest the existence of other functional sites including potential phosphorylation sites for protein kinase C, cAMP- and cGMP-dependent protein kinase, casein kinase II, as well as N-myristoylation and amidation sites that play an important role in molecular regulation of the calpain/calpastatin system. The regulation of the calpain/calpastatin system is determined by the interaction between dIV and dVI in calpains and subdomains A, B, and C in calpastatin.

Topics: Amino Acid Sequence; Animals; Base Sequence; Calcium-Binding Proteins; Calpain; Cattle; Cloning, Molecular; Computational Biology; Conserved Sequence; Humans; Models, Molecular; Molecular Sequence Data; Open Reading Frames; Protein Structure, Quaternary; Sequence Alignment

The mechanisms underlying neuronal pathology and death in the spinal cord (SC) during inflammation remain elusive. We previously showed the important role of plasma membrane calcium ATPases (PMCAs) in the survival of SC neurons, in vitro. We also postulated that a decrease in PMCA2 expression could cause neuronal death during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. The current studies were undertaken to define the specific contribution of PMCA2 to degeneration of SC neurons, the effectors downstream to PMCA2 mediating neuronal death and the triggers that reduce PMCA2 expression. We report that knockdown of PMCA2 in SC neurons decreases collapsin response mediator protein 1 (CRMP1) levels. This is followed by cell death. Silencing of CRMP1 expression also leads to neuronal loss. Kainic acid reduces both PMCA2 and CRMP1 levels and induces neuronal death. Administration of an alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)/kainate receptor antagonist, at onset or peak of EAE, restores the decreased PMCA2 and CRMP1 levels to control values and ameliorates clinical deficits. Thus, our data link the reduction in PMCA2 expression with perturbations in the expression of CRMP1 and the ensuing death of SC neurons. This represents an additional mechanism underlying AMPA/kainate receptor-mediated excitotoxicity with relevance to neurodegeneration in EAE.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Death; Cell Survival; Cells, Cultured; Cysteine Proteinase Inhibitors; Embryo, Mammalian; Encephalomyelitis, Autoimmune, Experimental; Gene Expression; Kainic Acid; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Neurons; Phosphoproteins; Plasma Membrane Calcium-Transporting ATPases; Proteome; Proteomics; Quinoxalines; Rats; Rats, Inbred Strains; Receptors, AMPA; Receptors, Kainic Acid; RNA, Small Interfering; Spinal Cord

The study was designed to investigate the effects of nutritional level (control diet (CD), 14.19% crude protein, 13.81MJ of DE/kg; low nutritional level diet (LND), 11.08% crude protein, 12.55MJ of DE/kg) on pork quality and gene expression of mu-calpain and calpastatin in muscle of finishing pigs. The LND treatment increased drip loss (P<0.05), had a trend to increase intramuscular fat (IMF) content (P=0.09), decreased Warner-Bratzler shear force (WBSF) of pork (P<0.05), improved mRNA level of mu-calpain (P<0.05) in skeletal muscle, but had no effect on gene expression of calpastatin, compared with the CD treatment. These data suggest that a moderately reduced energy and protein diet increased pork tenderness and intramuscular fat. The increase in tenderness by LND treatment may be partly due to increased gene expression of mu-calpain in muscle.

Topics: Animal Feed; Animals; Calcium-Binding Proteins; Calpain; Diet; Energy Intake; Fats; Food Technology; Gene Expression; Meat; Muscle Proteins; Muscle, Skeletal; Nutritive Value; RNA, Messenger; Stress, Mechanical; Swine

Effects and interactions of calpain-system tenderness gene markers on objective meat quality traits of Brahman (Bos indicus) cattle were quantified within 2 concurrent experiments at different locations. Cattle were selected for study from commercial and research herds at weaning based on their genotype for calpastatin (CAST) and calpain 3 (CAPN3) gene markers for beef tenderness. Gene marker status for mu-calpain (CAPN1-4751 and CAPN1-316) was also determined for inclusion in statistical analyses. Eighty-two heifer and 82 castrated male cattle with 0 or 2 favorable alleles for CAST and CAPN3 were studied in New South Wales (NSW), and 143 castrated male cattle with 0, 1, or 2 favorable alleles for CAST and CAPN3 were studied in Western Australia (WA). The cattle were backgrounded for 6 to 8 mo and grain-fed for 117 d (NSW) or 80 d (WA) before slaughter. One-half the cattle in each experiment were implanted with a hormonal growth promotant during feedlotting. One side of each carcass was suspended from the Achilles tendon (AT) and the other from the pelvis (tenderstretch). The M. longissimus lumborum from both sides and the M. semitendinosus from the AT side were collected; then samples of each were aged at 1 degrees C for 1 or 7 d. Favorable alleles for one or more markers reduced shear force, with little effect on other meat quality traits. The size of effects of individual markers varied with site, muscle, method of carcass suspension, and aging period. Individual marker effects were additive as evident in cattle with 4 favorable alleles for CAST and CAPN3 markers, which had shear force reductions of 12.2 N (P < 0.001, NSW) and 9.3 N (P = 0.002, WA) in AT 7 d aged M. longissimus lumborum compared with those with no favorable alleles. There was no evidence (all P > 0.05) of interactions between the gene markers, or between the hormonal growth promotant and gene markers for any meat quality traits. This study provides further evidence that selection based on the CAST or CAPN3 gene markers improves meat tenderness in Brahman cattle, with little if any detrimental effects on other meat quality traits. The CAPN1-4751 gene marker also improved beef tenderness without affecting other objective meat quality traits in heterozygous cattle compared with homozygotes for the unfavorable allele.

Topics: Alleles; Animals; Body Composition; Calcium-Binding Proteins; Calpain; Cattle; Female; Gene Expression Regulation; Genetic Markers; Genotype; Heterozygote; Homozygote; Male; Meat; Shear Strength; Temperament

The overexpression of the cysteine protease calpain is associated with many diseases, including brain trauma, spinal cord injury, Alzheimer's disease, Parkinson's disease, muscular dystrophy, arthritis, and cataract. Calpastatin is the naturally occurring specific regulator of calpain activity. It has previously been reported that a 20-mer peptide truncated from region B of calpastatin inhibitory domain 1 (named CP1B) retains both the affinity and selectivity of calpastatin toward calpain, exhibiting a K(i) of 26 nM against mu-calpain, and is 1000-fold more selective for mu-calpain than cathepsin L. Both the wild-type and beta-Ala mutant CP1B peptides exhibit a propensity to adopt a looplike conformation between Glu10 and Lys13. A computational study of human wild-type CP1B and the beta-Ala mutants of this peptide was conducted. The resulting structural predictions were compared with the crystal structure of the calpain-calpastatin complex and were correlated with experimental IC(50) values. These findings suggest that the conformational preference of the loop region between Glu10 and Lys13 of CP1B in the absence of calpain may contribute to the inhibitory activity of this series of peptides against calpain.

Topics: Calcium-Binding Proteins; Calpain; Crystallography, X-Ray; Cysteine Proteinase Inhibitors; Humans; Models, Molecular; Mutation; Peptide Fragments; Peptides; Protein Conformation

Experiments were conducted concurrently at 2 locations to quantify effects and interactions of calpain-system tenderness gene markers on growth, efficiency, temperament, and carcass traits of Brahman cattle. Cattle were selected at weaning from commercial and research herds based on their genotype for commercially available calpastatin (CAST) and calpain 3 (CAPN3) gene markers for beef tenderness. Genotypes for mu-calpain gene markers (CAPN1-4751 and CAPN1-316) were also determined and included in statistical analyses. The New South Wales (NSW) herd was composed of 82 heifers and 82 castrated male cattle with 0 or 2 favorable alleles for CAST and CAPN3. The Western Australia (WA) herd was composed of 173 castrated male cattle with 0, 1, or 2 favorable alleles for CAST and CAPN3. One-half of the cattle at each site were implanted with a hormonal growth promotant (HGP: Revalor-H) during grain finishing. Cattle were backgrounded at pasture for 6 to 8 mo and grain-fed for 117 d (NSW) or 80 d (WA) before slaughter. Individually, or in combination with each other and with CAPN1-4751 status, CAST and CAPN3 status had no significant (all P > 0.05) effects on BW, growth, feed efficiency, or temperament traits. The only significant effect of CAST or CAPN3 on carcass characteristics was a small increase in rib fat with increasing number of favorable CAST alleles (P = 0.042) in the WA herd. There were no significant interactions (all P > 0.05) between the markers, or between the markers and sex or HGP treatment apart from CAST x HGP for area of the M. longissimus lumborum (P = 0.024) in the NSW experiment. Favorable CAST or CAPN3 alleles appear unlikely to have detrimental effects on growth, efficiency, temperament, or carcass characteristics of Brahman cattle; however, some effects evident for CAPN1 status indicate the need for further production studies on effects of these markers. Overall, the findings of the present study indicate that calpain-system gene markers are suitable for use in marker-assisted selection to improve meat tenderness in Brahman cattle without negative effects on other production and carcass characteristics.

Topics: Animals; Body Composition; Calcium-Binding Proteins; Calpain; Cattle; Female; Gene Expression Regulation; Genetic Markers; Genotype; Male; Meat; Shear Strength; Temperament

The present study was performed to investigate changes of cytosolic and mitochondrial calpain activities, and effects of intravitreously injected calpain inhibitor on photoreceptor apoptosis in Royal College of Surgeon's (RCS) rats. Time courses of activities for both cytosolic and mitochondrial calpains and amount of calpastatin in RCS rat retina were analyzed by subcellular fractionation, calpain assay and western blotting. Calpain assay was colorimetrically performed using Suc-LLVY-Glo as substrate. Effects of intravitreously injected calpain inhibitor (ALLN and PD150606) on RCS rat retinal degeneration were analyzed by TUNEL staining. Effects of mitochondrial calpain activity on activation and translocation of apoptosis-inducing factor (AIF) were analyzed by western blotting. Mitochondrial calpain started to be significantly activated at postnatal (p) 28 days in RCS rat retina, whereas cytosolic micro-calpain was activated at p 35 days, although specific activity of mitochondrial calpain was 13% compared to cytosolic micro-calpain. Intravitreously injected ALLN and PD150606 effectively inhibited photoreceptor apoptosis only when injected at p 25 days, but did not inhibit photoreceptor apoptosis when injected at p 32 days. Parts of AIF were truncated/activated by mitochondrial calpains and translocated to the nucleus. These results suggest that 1), calpain presents not only in the cytosolic fraction but also in the mitochondrial fraction in RCS rat retina; 2), mitochondrial calpain is activated earlier than cytosolic calpain during retinal degeneration in RCS rats; 3), photoreceptor apoptosis may be regulated by not only calpain systems but also other mechanisms; 4), mitochondrial calpain may activate AIF to induce apoptosis; and 5), calpain inhibitors may be partially effective to inhibit photoreceptor apoptosis in RCS rats. The present study provides new insights into the molecular basis for photoreceptor apoptosis in RCS rats and the future possibility of new pharmaceutical treatments for retinitis pigmentosa.

Topics: Acrylates; Animals; Apoptosis; Apoptosis Inducing Factor; Blotting, Western; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Cytosol; Electrophoresis, Polyacrylamide Gel; Immunoenzyme Techniques; In Situ Nick-End Labeling; Leupeptins; Mitochondria; Photoreceptor Cells, Vertebrate; Rats; Rats, Mutant Strains; Retinal Degeneration

Abnormal activation of calpain is implicated in synaptic dysfunction and participates in neuronal death in Alzheimer disease (AD) and other neurological disorders. Pharmacological inhibition of calpain has been shown to improve memory and synaptic transmission in the mouse model of AD. However, the role and mechanism of calpain in AD progression remain elusive. Here we demonstrate a role of calpain in the neuropathology in amyloid precursor protein (APP) and presenilin 1 (PS1) double-transgenic mice, an established mouse model of AD. We found that overexpression of endogenous calpain inhibitor calpastatin (CAST) under the control of the calcium/calmodulin-dependent protein kinase II promoter in APP/PS1 mice caused a remarkable decrease of amyloid plaque burdens and prevented Tau phosphorylation and the loss of synapses. Furthermore, CAST overexpression prevented the decrease in the phosphorylation of the memory-related molecules CREB and ERK in the brain of APP/PS1 mice and improved spatial learning and memory. Interestingly, treatment of cultured primary neurons with amyloid-beta (Abeta) peptides caused an increase in the level of beta-site APP-cleaving enzyme 1 (BACE1), the key enzyme responsible for APP processing and Abeta production. This effect was inhibited by CAST overexpression. Consistently, overexpression of calpain in heterologous APP expressing cells up-regulated the level of BACE1 and increased Abeta production. Finally, CAST transgene prevented the increase of BACE1 in APP/PS1 mice. Thus, calpain activation plays an important role in APP processing and plaque formation, probably by regulating the expression of BACE1.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Calcium-Binding Proteins; Calpain; Cell Death; Cell Line; Disease Models, Animal; Disease Progression; Enzyme Activation; Female; Gene Expression Regulation, Enzymologic; Humans; Male; Memory; Mice; Mice, Transgenic; Phosphorylation; Plaque, Amyloid; Presenilin-1; Synapses; tau Proteins; Up-Regulation

Sixty four young red deer (Cervus elaphus) stags (<2 years old) were slaughtered at four different times (December (Group 1); n=17, March (Group 2); n=8, July (Group 3); n=20 and September (Group 4); n=19) to evaluate seasonal effects on venison quality. M. longissimus dorsi samples for calpain analysis were collected on the slaughter line and the rest of these muscles were collected at 1 day post-slaughter. Loins were divided into four parts and randomly allocated to storage for 1 day, 3, 9 or 14weeks at -1.5°C and then vacuum packaged. Seasonal variation was demonstrated in venison pH. Highly significant positive regressions were found for shear force (P<0.001) and colour display life (P<0.001) on pH, where higher pH values were associated with tougher venison and longer colour display life. A clear trend of increasing fluid loss during storage, calculated as amount of purge at 14 weeks of storage minus the amount of drip loss at 1 day post-slaughter, was evident, averaging 2.5% (SEM 0.17) over the four groups. The relative activities of the calpastatin-bound calpain, μ-calpain and m-calpain all exhibited a seasonal pattern although there was no evidence (P>0.05) that this affected tenderness. There was a highly significant (P<0.001) negative regression for the average over the four storage times of drip and purge on calpastatin-bound calpain activity.

Topics: Animals; Calcium-Binding Proteins; Calpain; Color; Deer; Food Technology; Hydrogen-Ion Concentration; Meat; Muscle Proteins; Muscle, Skeletal; Seasons; Stress, Mechanical; Water

Beta agonists used as growth enhancers are known to affect the aging potential of beef muscle negatively. On the other hand, procedures like electrical stimulation could accelerate rigor and the aging process. In this study, 20 out of 40 young steers received no beta agonist (C), the remaining twenty steers received a beta agonist (zilpaterol hydrochloride) (Z) for the 30 days prior to slaughter followed by 4 days withdrawal. After slaughter carcasses were split, the left side electrically stimulated (ES) and the right side not stimulated (NES). Samples were aged for 3 or 14 days post mortem. Parameters included Warner Bratzler shear force (WBSF), myofibril filament length (MFL), sarcomere length and calpastatin and calpain enzyme activity. Zilpaterol resulted in increased (P<0.001) WBSF mainly due to an increased (P<0.001) calpastatin activity. ES improved tenderness (P<0.001) in general by early onset of rigor triggering the activity of calpains. ES also reduced the calpastatin activity (P<0.001), which partially countered the effect of high calpastatin activity on the aging potential of Z loins. ES can therefore be implemented to improve meat tenderness in zilpaterol supplemented steers, although steers without zilpaterol will still have an advantage in final tenderness.

Topics: Adrenergic beta-Agonists; Animal Feed; Animals; Calcium-Binding Proteins; Calpain; Cattle; Electric Stimulation; Food Technology; Male; Meat; Muscle Proteins; Muscle, Skeletal; Myofibrils; Rigor Mortis; Sarcomeres; Stress, Mechanical; Trimethylsilyl Compounds

We examined whether single-nucleotide polymorphisms (SNPs) in the calpain (CAPN) and calpastatin (CAST) genes, described from Bos primigenius taurus, are polymorphic in Nellore cattle. We also looked for a possible association of linkage disequilibrium of this polymorphism with tenderness of the longissimus dorsi muscle after 7, 14 and 21 days of postmortem aging in 638 purebred Nellore bulls. Meat tenderness was measured as Warner-Bratzler shear force. Additive and dominance effects were tested for SNPs of the three genotypic classes; the substitution effect was tested for SNPs with missing genotypic classes. Genotypic and gene frequencies were also calculated for the different SNPs. An increase in tenderness was observed from 7 to 21 days; the average values for shear force at 7, 14 and 21 days of aging were 5.92 +/- 0.06, 4.92 +/- 0.05, and 4.38 +/- 0.04 kg, respectively. All markers showed polymorphism, but there was no CC genotype for CAPN316, and few animals showed the AA genotype for CAPN530. The alleles CAPN4751, UOGCAST1, and WSUCAST were found to have additive and dominance effects for shear force at 7, 14 and 21 days, while CAPN316 showed a substitution effect for shear force at 7 and 21 days. An additive-by-additive epistatic interaction was observed between CAPN4751 and markers on the CAST gene. In conclusion, these markers should be considered for use in breeding programs.

Topics: Amino Acid Substitution; Animals; Calcium-Binding Proteins; Calpain; Cattle; Epistasis, Genetic; Gene Frequency; Genetic Markers; Genotype; Meat; Models, Genetic; Polymorphism, Single Nucleotide; Postmortem Changes

Calpains (CAPN) are intracellular, non-lysosomal cytoplasmic cysteine endopeptidases and they are expressed ubiquitously. Their endogenous specific inhibitor is calpastatin. When calcium is present, calpastatin and calpain attach to each other, inhibiting the protease. The calpain system plays an important role in many processes including apoptosis, necrosis, ischaemia and exocytosis. The role of calpains in pathogenesis or further tumour progression has been proved in related studies. This study focused on the expression of the enzymes calpain 1, calpain 2 and the inhibitor calpastatin in normal and malignant endometrial tissue.. Immunohistochemical stainings were performed on paraffin slices and staining intensity, percentage of positive cells and international ratio score were evaluated.. The endometrial carcinoma showed a higher expression of calpastatin than benign endometrial tissue.

Topics: Calcium-Binding Proteins; Calpain; Endometrial Neoplasms; Female; Humans; Immunohistochemistry; Middle Aged; Neoplasm Staging

The objective of this study was to investigate the effects of bovine GH1, CAPN1 and CAST gene polymorphisms on carcass and meat traits in Nellore and Nellore x Bos taurus beef cattle. Three hundred animals were genotyped for GH1/MspI (TC/G in intron 3), CAPN316 (AF_252504.2:g.5709C>G) and CAST/RsaI (AY_008267.1:g282C>G) and phenotyped for rib eye area, backfat thickness, intramuscular fat, shear force (SF), and myofibrillar fragmentation index (MFI). No significant associations were observed between the GH1/MspI and CAST/RsaI polymorphisms and phenotypes, although the relation between the CAST/RsaI genotypes and meat tenderness evaluated by MFI approached significant. The fact that the CAPN316 polymorphism did not show adequate segregation in Nellore cattle confirms the difficulty of using this marker in breeding programs of different Bos indicus breeds. However, the positive results of the association analysis obtained for Nellore x B. taurus crosses contributed to the validation of previous findings.

Topics: Adipose Tissue; Animals; Breeding; Calcium-Binding Proteins; Calpain; Cattle; Genotype; Growth Hormone; Meat; Muscle, Skeletal; Myofibrils; Phenotype; Polymorphism, Genetic; Stress, Mechanical

Research on fertilization in mammalian species has revealed that Ca(2+) is an important player in biochemical and physiological events enabling the sperm to penetrate the oocyte. Ca(2+) is a signal transducer that particularly mediates capacitation and acrosome reaction (AR). Before becoming fertilization competent, sperm must experience several molecular, biochemical, and physiological changes where Ca(2+) plays a pivotal role. Calpain-1 and calpain-2 are Ca(2+)-dependent proteases widely studied in mammalian sperm; they have been involved in capacitation and AR but little is known about their mechanism. In this work, we establish the association of calpastatin with calpain-1 and the changes undergone by this complex during capacitation in guinea pig sperm. We found that calpain-1 is relocated and translocated from cytoplasm to plasma membrane (PM) during capacitation, where it could cleave spectrin, one of the proteins of the PM-associated cytoskeleton, and facilitates AR. The aforementioned results were dependent on the calpastatin phosphorylation and the presence of extracellular Ca(2+). Our findings underline the contribution of the sperm cytoskeleton in the regulation of both capacitation and AR. In addition, our findings also reveal one of the mechanisms by which calpain and calcium exert its function in sperm.

Topics: Acrosome Reaction; Animals; Blotting, Western; Calcium-Binding Proteins; Calpain; Cytoskeleton; Dipeptides; Guinea Pigs; Leupeptins; Male; Microscopy, Electron; Spectrin; Sperm Capacitation; Spermatozoa

Amyotrophic lateral sclerosis (ALS) is an adult-onset, rapidly progressing, fatal disease occurring in both familial and sporadic forms. Mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) cause ALS through a gain of toxic function. Calpain activity is increased in mutant SOD1 (SOD1(G93A)) transgenic mice and in models of ischemia because of increased cytosolic calcium, which has been documented in motor neurons in rodent models of familial ALS and in sporadic ALS patients. We report that inhibition of calpain activity using calpastatin prevented the toxicity of SOD1(G93A) in motor neurons of dissociated spinal cord cultures, prolonging viability of and reducing the proportion containing SOD1(G93A) inclusions. The data support the central role of calcium dysregulation in ALS and identify a potential therapeutic pathway.

Topics: Amyotrophic Lateral Sclerosis; Animals; Calcium-Binding Proteins; Calpain; Cell Aggregation; Cell Survival; Cells, Cultured; Cysteine Proteinase Inhibitors; Gene Transfer Techniques; Mice; Motor Neurons; Superoxide Dismutase

Photoreceptor degeneration is the hallmark of a group of inherited blinding diseases collectively termed retinitis pigmentosa (RP); a major cause of blindness in humans. RP is at present untreatable and the underlying neurodegenerative mechanisms are largely unknown, even though the genetic causes are often established. The activation of calpain-type proteases may play an important role in cell death in various neuronal tissues, including the retina. We therefore tested the efficacy of two different calpain inhibitors in preventing cell death in the retinal degeneration (rd1) human homologous mouse model for RP. Pharmacological inhibition of calpain activity in rd1 organotypic retinal explants had ambiguous effects on photoreceptor viability. Calpain inhibitor XI had protective effects when applied for short periods of time (16 h) but demonstrated substantial levels of toxicity in both wild-type and rd1 retina when used over several days. In contrast, the highly specific calpain inhibitor calpastatin peptide reduced photoreceptor cell death in vitro after both short and prolonged exposure, an effect that was also evident after in vivo application via intravitreal injection. These findings highlight the importance of calpain activation for photoreceptor cell death but also for photoreceptor survival and propose the use of highly specific calpain inhibitors to prevent or delay RP.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Death; Glycoproteins; Humans; Mice; Mice, Inbred C3H; Mice, Transgenic; Organ Culture Techniques; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Retinitis Pigmentosa

Calpain activation has been implicated in the disease pathology of Duchenne muscular dystrophy. Inhibition of calpain has been proposed as a promising therapeutic target, which could lessen the protein degradation and prevent progressive fibrosis. At the same time, there are conflicting reports as to whether elevation of calpastatin, an endogenous calpain inhibitor, alters pathology. We compared the effects of pharmacological calpain inhibition in the mdx mouse using leupeptin and a proprietary compound (C101) that linked the inhibitory portion of leupeptin to carnitine (to increase uptake into muscle). Administration of C101 for 4 wk did not improve muscle histology, function, or serum creatine kinase levels in mdx mice. Mdx mice injected daily with leupeptin (36 mg/kg) for 6 mo also failed to show improved muscle function, histology, or creatine kinase levels. Biochemical analysis revealed that leupeptin administration caused an increase in m-calpain autolysis and proteasome activity, yet calpastatin levels were similar between treated and untreated mdx mice. These data demonstrate that pharmacological inhibition of calpain is not a promising intervention for the treatment of Duchenne muscular dystrophy due to the ability of skeletal muscle to counter calpain inhibitors by increasing multiple degradative pathways.

Topics: Animals; Biomarkers; Calcium-Binding Proteins; Calpain; Creatine Kinase; Cysteine Proteinase Inhibitors; Diaphragm; Disease Models, Animal; Dose-Response Relationship, Drug; Genotype; Leupeptins; Mice; Mice, Inbred mdx; Muscle Contraction; Muscle Strength; Muscular Dystrophy, Duchenne; Necrosis; Phenotype; Proteasome Endopeptidase Complex; Time Factors

In the course of chronic kidney disease (CKD) the intracellular erythrocyte calcium (Ca (i) (2+) ) level increases along with the progression of the disease. The decreased activity of Ca(2+)-Mg(2+)-dependent ATP-ase (PMCA) and its endogenous modulators calmodulin (CALM), calpain (CANP), and calpastatin (CAST) are all responsible for disturbed calcium metabolism. The aim of the study was to analyze the activity of PMCA, CALM, and the CANP-CAST system in the red blood cells (RBCs) of hemodialyzed (HD) children and to estimate the impact of a single HD session on the aforementioned disturbances. Eighteen patients on maintenance HD and 30 healthy subjects were included in the study. CALM, Ca (i) (2+) levels and basal PMCA (bPMCA), PMCA, CANP, and CAST activities were determined in RBCs before HD, after HD, and before the next HD session. Prior to the HD session, the level of Ca (i) (2+) and the CAST activity were significantly higher, whereas bPMCA, PMCA, and CANP activities and the CALM level were significantly lower than in controls. After the HD session, the Ca (i) (2+) concentration and the CAST activity significantly decreased compared with the basal values, whereas the other parameters significantly increased, although they did not reach the levels of healthy children. The values observed prior to both HD sessions were similar. Ca (i) (2+) homeostasis is severely disturbed in HD children, which may be caused by the reduction in the PMCA activity, CALM deficiency, and CANP-CAST system disturbances. A single HD session improved these disturbances but the effect is transient.

Topics: Adolescent; Ca(2+) Mg(2+)-ATPase; Calcium; Calcium-Binding Proteins; Calmodulin; Calpain; Case-Control Studies; Child; Erythrocytes; Female; Homeostasis; Humans; Kidney Failure, Chronic; Male; Poland; Renal Dialysis; Time Factors; Treatment Outcome

The aim of the present study was to investigate the expressions of calpain and calpastatin in the myocardium of simulated weightlessness rats, and to elucidate the underlying mechanism of cardiac troponin I (cTnI) degradations. Tail-suspended (SUS) rats were used as a simulated weightlessness model on the ground. The myocardium of rats was homogenized, and the expressions of calpain-1, calpain-2, calpastatin and cTnI were analyzed by Western blotting technique. Calpastatin expression was significantly decreased in 2- and 4-week SUS groups compared with that in the synchronous controls (P<0.05). Calpain-2 expression was slightly decreased, whereas calpain-1 expression was unaltered in SUS groups. However, calpain-1/calpastatin and calpain-2/calpastatin ratios were increased after tail-suspension, being significantly higher in 2- and 4-week SUS groups than those in the synchronous controls (P<0.05, P<0.01). Cardiac TnI degradation was significantly increased after tail-suspension (P<0.01), but cTnI degradation in both SUS and control groups was significantly inhibited by a non-specific inhibitor of calpain, PD150606 (P<0.01). These results suggest that an increase in calpain activity may enhance cTnI degradation in the myocardium of tail-suspended rats.

Topics: Animals; Calcium-Binding Proteins; Calpain; Hindlimb Suspension; Myocardium; Proteolysis; Rats; Troponin I; Weightlessness Simulation

Changes in connexins and calpains of the myocardial sleeve of the pulmonary vein and the left atrium were investigated in chronic atrial fibrillation (AF) animal models.. There are no reports of changes in the calpain system and connexins in the pulmonary vein where AF is initiated.. An AF animal model was prepared by rapid pacing of the right atrium for 8 weeks. Histological changes of pulmonary veins were analyzed by Masson trichrome staining, and mRNA as well as protein expression of connexins and calpains were measured by real-time fluorescence quantitative PCR and Western blotting.. In AF dogs, the fibrous collagen reticulum surrounding individual myocardial cells was reduced or disrupted. In the myocardial sleeve of the AF dogs, Cx40 protein expression was significantly downregulated compared to the control group (60.78 +/- 10.91 vs. 88.31 +/- 14.73, p < 0.05), but calpain 1 was significantly upregulated (94.00 +/- 7.24 vs. 81.77 +/- 5.82, p < 0.05), and they were negatively correlated (r = -0.66, p < 0.05). Cx40 protein expression was significantly lower in the myocardial sleeve tissue than in the left atrium in the AF dogs (60.78 +/- 10.91 vs. 91.38 +/- 17.16, p < 0.05).. Varied gap junctional remodeling around the pulmonary vein may be one of the underlying mechanisms for pulmonary vein-left atrial reentry. During AF, the calpain system of the myocardial sleeve tissue is activated and may hydrolyze Cx40 protein, which is a possible important molecular mechanism for gap junctional remodeling that merits further investigation.

Topics: Animals; Atrial Fibrillation; Calcium-Binding Proteins; Calpain; Connexin 43; Connexins; Disease Models, Animal; Dogs; Female; Gap Junction alpha-5 Protein; Gap Junctions; Heart Atria; Male; Pacemaker, Artificial; Pulmonary Veins; RNA, Messenger

Taurine has been shown to prevent cardiomyocyte apoptosis. This study investigated the effects of taurine on NADPH oxidase and calpain activation in mediating apoptosis in cardiomyocytes. Apoptosis was induced by norepinephrine (NE) in cultured adult rat ventricular cardiomyocytes. NE (5 microM) increased NADPH oxidase activation and reactive oxygen species (ROS) production and induced apoptosis. These effects of NE on cardiomyocytes were diminished by taurine (0.5 mg/kg) but not beta-alanine. Inhibition of gp91(phox)-NADPH oxidase or ROS production protected cardiomyocytes from apoptosis. NE also induced calpain-1 activation in cardiomyocytes. This effect of NE on calpain was abrogated by gp91(phox)-NADPH oxidase inhibition or ROS scavengers and was mimicked by H(2)O(2) (25 microM) in cardiomyocytes. Pharmacological inhibitors of calpain or overexpression of calpastatin, a specific calpain inhibitor, blocked calpain activation and prevented cardiomyocyte apoptosis during NE stimulation. Furthermore, taurine treatment inhibited NE- or H(2)O(2)-induced calpain activation in cardiomyocytes. In conclusion, NADPH oxidase induces calpain activation, leading to apoptosis in NE-induced cardiomyocytes. Taurine inhibits NADPH oxidase and calpain activation. Thus, inhibition of NADPH oxidase-mediated calpain activation may be an important mechanism for taurine's antiapoptotic action in cardiomyocytes.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Death; Cells, Cultured; Cytoprotection; Enzyme Activation; Gene Expression Regulation, Enzymologic; Heart Ventricles; Hydrogen Peroxide; Male; Microscopy, Confocal; Myocytes, Cardiac; NADPH Oxidases; Norepinephrine; Rats; Rats, Sprague-Dawley; Taurine; Transgenes

The neurotoxin 6-hydroxydopamine has been widely used to model aspects of Parkinson's disease in rodents, but the mechanisms underlying toxin-induced dopaminergic degeneration and functional impairment have not been fully elucidated. The main aim of the present study was to assess a possible role for calpains in neurochemical and behavioral deficits following unilateral infusion of intrastriatal 6-hydroxydopamine in adult rats. Toxin administration produced a profound dopaminergic denervation, as indicated by a 90-95% reduction in dopamine transporter radiolabeling measured in the caudate-putamen at 2 weeks post-lesion. Treatment with 6-hydroxydopamine also resulted in calpain activation in both caudate-putamen and substantia nigra, as measured by the appearance of calpain-specific spectrin breakdown products. Calpain activation peaked at 24 h after 6-hydroxydopamine infusion and remained elevated at later time points. In contrast, caspase-3-mediated spectrin cleavage subsided within 48 h in both brain areas. In a subsequent experiment, calpain inhibition was achieved by intrastriatal infusion of an adenovirus expressing the endogenous calpain inhibitor, calpastatin. Calpastatin delivery abolished the lesion-induced calpain-mediated spectrin cleavage and alleviated forelimb asymmetries resulting from unilateral intrastriatal 6-hydroxydopamine. Unexpectedly, dopamine transporter and tyrosine hydroxylase labeling revealed significant neuroprotection, not in the nigrostriatal pathway but rather in the ventral tegmental area. These findings support a role for calpain activation in 6-hydroxydopamine-induced degeneration of dopaminergic neurons. However, after near-total dopaminergic depletion, the primary benefit of calpain inhibition may not occur within the nigrostriatal dopaminergic pathway itself.

Topics: Adrenergic Agents; Animals; Autoradiography; Calcium-Binding Proteins; Calpain; Caspase 3; Cocaine; Corpus Striatum; Dopamine; Dopamine Plasma Membrane Transport Proteins; Functional Laterality; Green Fluorescent Proteins; Male; Motor Activity; Oxidopamine; Psychomotor Performance; Rats; Rats, Sprague-Dawley; Spectrin; Time Factors; Tyrosine 3-Monooxygenase

An experiment was conducted using 200 beef carcasses to evaluate the effects of feeding zilpaterol hydrochloride with or without monensin and tylosin on carcass cutability and meat sensory variables. The experiment was conducted using a randomized complete block design with treatments arranged as a 2 (no zilpaterol vs. zilpaterol) x 2 (monensin and tylosin withdrawn vs. monensin and tylosin fed) factorial. Cattle (n=3,757) were fed zilpaterol hydrochloride, a beta(2)-adrenergic agonist, for 30 d at the end of the finishing period and withdrawn from zilpaterol hydrochloride for the last 5 d on feed. Five carcasses (weighing between 305 and 421 kg and free of slaughter defects) were selected from each of 40 feedlot treatment pens. Strip loins from the left sides were collected for sensory analysis and Warner-Bratzler shear force (WBSF) testing, and the rib was collected for 9th, 10th, 11th-rib dissections. A subsample of 3 carcass right sides per pen was fabricated into boneless subprimals according to Institutional Meat Purchase Specifications. Carcasses from zilpaterol-fed steers had greater (P or= 0.26). For the main effect of monensin and tylosin, withdrawal of monensin and tylosin decreased (P=0.01) consumer juiciness scores, although other yield and compositional measurements were not affected (P >or= 0.07). Zilpaterol is a strong repartitioning agent that increases meat yield through increased protein and decreased fat deposition.

Topics: Animals; Body Composition; Calcium-Binding Proteins; Calpain; Cattle; Demography; Dietary Supplements; Humans; Hydrogen-Ion Concentration; Male; Meat; Monensin; Muscle, Skeletal; Random Allocation; Sensation; Shear Strength; Trimethylsilyl Compounds; Tylosin

Treatment of bovine pulmonary artery smooth muscle mitochondria with the calcium ionophore, A23187 (0.2 microM) stimulates mu-calpain activity and subsequently cleaves Na(+)/Ca(2+) exchanger (NCX). Pretreatment of the A23187 treated mitochondria with the calpain inhibitors, calpeptin or MDL28170 or with Ca(2+) chelator, EGTA does not cleave NCX. Treatment of the mitochondria with A23187 increases Ca(2+) level in the mitochondria, which subsequently dissociates mu-calpain-calpastatin association leading to the activation of mu-calpain. Immunoblot study of the A23187 treated mitochondria with the NCX polyclonal antibody indicates the degradation of mitochondrial inner membrane NCX (110kDa) resulting in the doublet of approximately 54-56kDa NCX fragments. Moreover, in vitro cleavage of mitochondrial purified NCX by mitochondrial purified mu-calpain supports our conclusion. This cleavage of NCX may be interpreted as the main cause of Ca(2+) overload and could lay a key role in the activation of apoptotic process in pulmonary smooth muscle.

Topics: Animals; Calcimycin; Calcium; Calcium-Binding Proteins; Calpain; Cattle; Dipeptides; Intracellular Membranes; Mitochondria, Muscle; Mitochondrial Membranes; Muscle, Smooth, Vascular; Phosphatidylcholines; Phospholipid Ethers; Pulmonary Artery; Sodium-Calcium Exchanger

To establish the physiological role of calpain, it is necessary to define how the protease can escape from the effect of its natural inhibitor calpastatin, since both proteins co-localize into the cell cytosol.. To answer this question, we have overexpressed four fluorescent calpastatin constructs, differing in the composition of their XL- and L-domains, and the intracellular trafficking of this protein inhibitor has been followed by single cell fluorescence imaging.. By the use of these calpastatin forms differing in the type of exon-derived sequences contained in the XL- and L-domains, we have demonstrated that the sequence coded by exon 6, containing multiple phosphorylation sites, is directly involved in determining the cell localization of calpastatin. In fact, exposure to cAMP promotes the recruitment into aggregates of those calpastatin forms containing the exon 6 sequence. These protein movements are directly related to the level of cytosolic inhibitory capacity and thereby to the extent of intracellular calpain activation.. The recruitment of calpastatin into aggregates allows the translocation and activation of the protease to the membranes; on the contrary, the presence of large amounts of calpastatin in the cytosol prevents both processes, protecting the cell from undesired proteolysis.

Topics: Base Sequence; Calcium-Binding Proteins; Calpain; Cell Line; Cyclic AMP; DNA Primers; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Exons; Humans; Microscopy, Fluorescence; Subcellular Fractions

Contrary to globular proteins, intrinsically disordered proteins (IDPs) lack a folded structure and they do not lose solubility at elevated temperatures. Although this should also be true at low temperatures, cold stability of IDPs has not been addressed in any scientific work so far. As direct characterization of cold-denaturation is difficult, we approached the problem through a freezing-induced loss-of-function model of globular-disordered functional protein pairs (m-calpain-calpastatin, tubulin-Map2c, Hsp90-ERD14). Our results affirm that in contrast with globular proteins IDPs are resistant to cold treatment. The theoretical and functional aspects of this observation are discussed.

Topics: Animals; Arabidopsis Proteins; Calcium-Binding Proteins; Calpain; Cattle; Cold Temperature; HSP90 Heat-Shock Proteins; Humans; Microtubule-Associated Proteins; Protein Denaturation; Protein Folding; Tubulin

It is well known that rapid gain of muscle mass in neonatal pigs is highly related to protein synthesis. However, the role of protein degradation in muscle gain of the neonatal period has not been well established. Calpains and their endogenous inhibitors, calpastatins, play a significant role in early-stage myofibrillar protein degradation. To investigate the role of calpain-calpastatin system in muscle protein accumulation, we studied the expressions of their mRNA in muscle tissue sampled at days 1, 4, 6, 12, 20 and 28 from a total of 36 neonatal pigs. The steady-state mRNA levels of calpains 1A, 2 and 3A, calpastatin types 1, 2 and 3, obtained by quantitative real-time PCR analysis, decreased by 2-4 folds at the age of 4 to 6 days compared to 1-day-old piglets. Then, the relatively low expression level was maintained through 28 days of age. Expressions of calpains 1A, 3A and calpastatin type 1 were significantly correlated with the measurements of muscle protein accumulations such as muscle protein content and RNA/protein ratio. Expressions of calpain 1A, calpastatin types 1 and 3 were negatively correlated with birth weight and fractional rate of growth. The levels of calpains 1A and 2 mRNA were correspondent to their protease activities. In conclusion, decreased levels of calpain and calpastatin expressions over development in neonatal pigs are associated with high protein accumulations, suggesting that dramatic muscle growth during the neonatal period may be partially controlled by down-regulated calpain-calpastatin system.

Topics: Animals; Animals, Newborn; Calcium-Binding Proteins; Calpain; Gene Expression Profiling; Male; Muscle, Skeletal; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sus scrofa

Intermittent hypoxia (IH) occurs in many pathological conditions including recurrent apneas. Hypoxia-inducible factors (HIFs) 1 and 2 mediate transcriptional responses to low O(2). A previous study showed that HIF-1 mediates some of the IH-evoked physiological responses. Because HIF-2alpha is an orthologue of HIF-1alpha, we examined the effects of IH on HIF-2alpha, the O(2)-regulated subunit expression, in pheochromocytoma 12 cell cultures. In contrast to the up-regulation of HIF-1alpha, HIF-2alpha was down-regulated by IH. Similar down-regulation of HIF-2alpha was also seen in carotid bodies and adrenal medullae from IH-exposed rats. Inhibitors of calpain proteases (ALLM, ALLN) prevented IH-evoked degradation of HIF-2alpha whereas inhibitors of prolyl hydroxylases or proteosome were ineffective. IH activated calpain proteases and down-regulated the endogenous calpain inhibitor calpastatin. IH-evoked HIF-2alpha degradation led to inhibition of SOD2 transcription, resulting in oxidative stress. Over-expression of transcriptionally active HIF-2alpha prevented IH-evoked oxidative stress and restored SOD2 activity. Systemic treatment of IH-exposed rats with ALLM rescued HIF-2alpha degradation and restored SOD2 activity, thereby preventing oxidative stress and hypertension. These observations demonstrate that, unlike continuous hypoxia, IH leads to down-regulation of HIF-2alpha via a calpain-dependent signaling pathway and results in oxidative stress as well as autonomic morbidities.

Topics: Animals; Apnea; Autonomic Nervous System; Basic Helix-Loop-Helix Transcription Factors; Calcium Signaling; Calcium-Binding Proteins; Calpain; Cell Hypoxia; Down-Regulation; Enzyme Activation; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Oligopeptides; Oxidative Stress; PC12 Cells; Protein Binding; Protein Processing, Post-Translational; Rats; Rats, Sprague-Dawley; Superoxide Dismutase

To address the pathophysiological relevance of ovarian hormones in chronic beta-adrenergic stimulation-induced myocardial injury, we assessed impairments of Ca(2+)-mediated cell signaling in the left ventricle of ovariectomized female rats.. Female Wistar rats were subjected to bilateral ovariectomy and sham operation. Six weeks after ovariectomy (OVX), both OVX and sham rats were treated with isoproterenol (5mg/kg, intraperitoneally), a nonselective beta-adrenergic agonist, once a day for 28 days.. We found that chronic beta-adrenergic stimulation caused enhanced breakdown of sarcolemmal proteins such as dystrophin and utrophin in OVX rats compared to sham-operated rats. Generation of calpain-mediated 150 kDa-breakdown product of spectrin confirmed calpain activation following isoproterenol treatment. Marked breakdown of endogenous calpain inhibitor, calpastatin, in OVX rats was consistent with the calpain activation following chronic beta-adrenergic stimulation. In addition to calpain activation, we also found marked reduction of endothelial nitric oxide synthase (eNOS) activity with concomitant deregulation by heat shock proteins 90 kDa and caveolin 3, both of which are eNOS-associated proteins. Finally, we documented decreased Akt phosphorylation with concomitant increased glycogen synthase kinase 3beta phosphorylation underlying cell injury following chronic beta-adrenergic stimulation.. Taken together chronic beta-adrenergic stimulation caused severe cardiac injury in OVX rats through calpain activation and impairments of Akt and eNOS signaling pathways.

Topics: Adrenergic beta-Agonists; Animals; Calcium; Calcium-Binding Proteins; Calpain; Female; Heart Ventricles; Isoproterenol; Nitric Oxide Synthase Type III; Ovariectomy; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Receptors, Adrenergic, beta; Signal Transduction

Taurine has potent protective function against glutamate-induced neuronal injury presumably through its function in regulation of intracellular free calcium level, [Ca2+]i. In this communication, we report that taurine exerts its protective function through one or more of the following mechanisms: 1. Inhibition of glutamate-induced calcium influx through L-, N- and P/Q-type voltage-gated calcium channels and NMDA receptor calcium channel; 2. Attenuation of glutamate-induced membrane depolarization; 3. Prevention of glutamate-induced apoptosis via preventing glutamate-mediated down-regulation of Bcl-2; 4. Prevention of cleavage of Bcl-2 by calpain. This action of taurine is due to its inhibition on glutamate induced calpain activation. Based on these observations, we propose that taurine protects neurons against glutamate-induced neurotoxicity in part, by preventing glutamate-induced membrane depolarization, elevation of [Ca2+]i, activation of calpain, reduction of Bcl-2 and apoptosis.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Calcium Channels; Calcium-Binding Proteins; Calpain; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Glutamic Acid; Ion Channel Gating; Neurons; Neuroprotective Agents; Proto-Oncogene Proteins c-bcl-2; Rats; Receptors, N-Methyl-D-Aspartate; Taurine

Lipopolysaccharide (LPS) induces cardiomyocyte caspase-3 activation and proinflammatory factors, in particular tumour necrosis factor-alpha (TNF-alpha) production, both of which contribute to myocardial dysfunction during sepsis. The present study was to investigate the roles of calpain/calpastatin system in cardiomyocyte caspase-3 activation, TNF-alpha expression, and myocardial dysfunction during LPS stimulation.. In cultured adult rat cardiomyocytes, LPS (1 microg/mL) induced calpain and caspase-3 activity, and up-regulated TNF-alpha expression. These effects of LPS were abrogated by over-expression of calpastatin, an endogenous calpain inhibitor, transfection of calpain-1 siRNA, or various pharmacological calpain inhibitors. Furthermore, blocking gp91(phox)-NADPH oxidase prevented calpain and caspase-3 activation and decreased TNF-alpha expression in LPS-stimulated cardiomyocytes. To investigate the role of calpastatin in endotoxaemia, transgenic mice with calpastatin over-expression (CAST-Tg) and wild-type mice were treated with LPS (4 mg/kg, i.p.) or saline in the presence of calpain inhibitor-III (10 mg/kg, i.p.) for 4 h, and their heart function was measured with a Langendorff system. Over-expression of calpastatin significantly attenuated myocardial dysfunction (P < 0.05). Consistently, calpain activity, caspase-3 activity, and TNF-alpha expression were also reduced in CAST-Tg and calpain inhibitor-III compared with wild-type and vehicle-treated hearts, respectively.. gp91(phox)-NADPH oxidase-mediated calpain-1 activation induces caspase-3 activation and TNF-alpha expression in cardiomyocytes during LPS stimulation. Over-expression of calpastatin inhibits calpain activation and improves myocardial function in endotoxaemia. The present study suggests that targeting calpain/calpastatin system may be a potential therapeutic intervention for septic hearts.

Topics: Acrylates; Animals; Calcium-Binding Proteins; Calpain; Caspase 3; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; Disease Models, Animal; Endotoxemia; Heart; Lipopolysaccharides; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myocardium; Myocytes, Cardiac; NADPH Oxidase 2; NADPH Oxidases; Rats; Rats, Sprague-Dawley; RNA, Small Interfering; Tumor Necrosis Factor-alpha

Calpastatin, the endogenous inhibitor of calpain, a cysteine protease in eukaryotic cells, is an intrinsically unstructured protein, which upon binding to the enzyme goes through a conformational change. Peptides calpA (SGKSGMDAALDDLIDTLGG) and calpC (SKPIGPDDAIDALSSDFTS), corresponding to the two conserved subdomains of calpastatin, are known to activate calpain and increase the Ca(2+) sensitivity of the enzyme. Using solution NMR spectroscopy, here we show that calpA and calpC are disordered in water but assume an alpha-helical conformation in 50% CD(3)OH. The position and length of the helices are in agreement with those described in the literature for the bound state of the corresponding segments of calpastatin suggesting that the latter might be structurally primed for the interaction with its target. According to our data, the presence of Ca(2+) induces a backbone rearrangement in the peptides, an effect that may contribute to setting the fine conformational balance required for the interaction of the peptides with calpain.

Topics: Calcium; Calcium-Binding Proteins; Calpain; Enzyme Activation; Magnetic Resonance Spectroscopy; Peptides; Protein Binding; Protein Conformation; Protein Folding; Protein Structure, Secondary

Caspase-8 is a proapoptotic protease that suppresses neuroblastoma metastasis by inducing programmed cell death. Paradoxically, caspase-8 can also promote cell migration among nonapoptotic cells; here, we show that caspase-8 can promote metastasis when apoptosis is compromised. Migration is enhanced by caspase-8 recruitment to the cellular migration machinery following integrin ligation. Caspase-8 catalytic activity is not required for caspase-8-enhanced cell migration; rather, caspase-8 interacts with a multiprotein complex that can include focal adhesion kinase and calpain 2 (CPN2), enhancing cleavage of focal adhesion substrates and cell migration. Caspase-8 association with CPN2/calpastatin disrupts calpastatin-mediated inhibition of CPN2. In vivo, knockdown of either caspase-8 or CPN2 disrupts metastasis among apoptosis-resistant tumors. This unexpected molecular collaboration provides an explanation for the continued or elevated expression of caspase-8 observed in many tumors.

Topics: Alstrom Syndrome; Animals; Calcium-Binding Proteins; Calpain; Caspase 8; Cell Line, Tumor; Cell Movement; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesions; Humans; Lung Neoplasms; Mice; Mice, Transgenic; Neoplasm Metastasis; Neuroblastoma; Talin

The objectives of this work were to study the segregation of single nucleotide polymorphisms of the calpain 1, large subunit (CAPN1) and calpastatin (CAST) genes in Nellore (Bos indicus) and Nellore xBos taurus beef cattle, as well as to evaluate their effects on meat traits. For this, 300 animals, including 114 Nellore, 67 Angus x Nellore, 44 Rubia Gallega x Nellore, 41 Canchim, 19 Brangus three-way crosses and 15 Braunvieh three-way crosses, were genotyped for the CAPN4751 [AF_248054.2:g.6545C>T (GenBank accession AF248054.2)] and CAST/DdeI [AF_159246.1:g.2959A>G (GenBank accession AF159246.1)] polymorphisms and phenotyped for Ribeye Area, Backfat Thickness, Intramuscular Fat, Shear Force (SF) and Myofibrillar Fragmentation Index (MFI). In relation to the CAPN4751 polymorphism, a frequency of 10.5% was observed for the C allele in the Nellore breed. In the total sample of studied animals, a significant association was found between genotypes and meat tenderness, assessed by SF (P = 0.005) and MFI (P = 0.008), with genotype CT being more favourable than TT. For the CAST/DdeI polymorphism, a frequency of 55.7% was found for the A allele in the Nellore breed. In the total sample, a significant association was observed between genotypes and meat tenderness - SF (P = 0.004) and MFI (P = 0.006), with genotype AA being more favourable than AG. The relationship between genotypes and aged meat tenderness in confluence with the distribution of favourable alleles shows great potential for application of the CAPN4751 and CAST/DdeI polymorphisms in the genetic improvement of the Nellore breed, whilst contributing to the validation, in this breed and in its crosses with B. taurus, of the association results previously described in the literature.

Topics: Alleles; Animals; Body Composition; Calcium-Binding Proteins; Calpain; Cattle; Crosses, Genetic; Gene Frequency; Genotype; Least-Squares Analysis; Meat; Polymorphism, Single Nucleotide

Caspase-independent cell death, an important death pathway in many cells including neurons, is executed via apoptosis-inducing factor (AIF), an oxidoreductase, localized to the mitochondrial intermembrane space. AIF is processed and released from mitochondria following mitochondrial permeability transition pore (mPTP) formation, and translocates to the nucleus to induce DNA fragmentation and cell death. The release of AIF requires cleavage of its N-terminus anchored in the inner mitochondrial membrane. The protease responsible for this AIF truncation has not been established, although there is considerable evidence suggesting a role for micro-calpain. We previously found that a pool of micro-calpain is localized to the mitochondrial intermembrane space, the submitochondrial compartment in which AIF truncation occurs. The close submitochondrial proximity of mitochondrial micro-calpain and AIF gives support to the hypothesis that mitochondrial micro-calpain may be the protease responsible for processing AIF prior to its release. In the present study, AIF was released from rat liver mitochondria following mPTP induction by atractyloside. This release was inhibited by the cysteine protease inhibitor MDL28170, but not by more specific calpain inhibitors PD150606 and calpastatin. Atractyloside caused swelling in rat brain mitochondria, but did not induce AIF release. In a mitochondrial fraction from SH-SY5Y neuroblastoma cells, incubation with 5 mM Ca(2+) resulted in the activation of micro-calpain but not in AIF truncation. In summary, the localization of micro-calpain to the mitochondrial intermembrane space is suggestive of its possible involvement in AIF processing, but direct experimental evidence supporting such a role has been elusive.

Topics: Acrylates; Animals; Apoptosis Inducing Factor; Atractyloside; Blotting, Western; Calcium; Calcium-Binding Proteins; Calpain; Cell Line, Tumor; Cerebral Cortex; Cysteine Proteinase Inhibitors; Dipeptides; Enzyme Inhibitors; Liver; Male; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Swelling; Rats; Rats, Sprague-Dawley

Calpastatin, a naturally occurring protein, is the only inhibitor that is specific for calpain. A novel blood-brain barrier (BBB)-permeant calpastatin-based calpain inhibitor, named B27-HYD, was developed and used to assess calpain's contribution to neurological dysfunction after stroke in rats. Postischemic administration of B27-HYD reduced infarct volume and neurological deficits by 35% and 44%, respectively, compared to untreated animals. We also show that the pharmacologic intervention has engaged the intended biologic target. Our data further demonstrates the potential utility of SBDP145, a signature biomarker of acute brain injury, in evaluating possible mechanisms of calpain in the pathogenesis of stroke and as an adjunct in guiding therapeutic decision making.

Topics: Animals; Blood-Brain Barrier; Brain; Calcium-Binding Proteins; Calpain; Cerebral Infarction; Cysteine Proteinase Inhibitors; Disease Models, Animal; Male; Peptide Fragments; Rats; Rats, Wistar; Spectrin

Cardiomyocyte apoptosis contributes to cardiac complications of diabetes. The aim of this study was to investigate the role of calpain in cardiomyocyte apoptosis induced by hyperglycaemia.. In cultured adult rat ventricular cardiomyocytes, high glucose (33 mM) increased calpain activity and induced apoptosis, concomitant with the impairment of Na+/K+ ATPase activity. These effects of high glucose on cardiomyocytes were abolished by various pharmacological calpain inhibitors, knockdown of calpain-1 but not calpain-2 using siRNA, or over-expression of calpastatin, a specific endogenous calpain inhibitor. The effect of calpain inhibition on cardiomyocyte apoptosis was abrogated by ouabain, a selective inhibitor of Na+/K+ ATPase. Furthermore, blocking gp91(phox)-NADPH oxidase activation, L-type calcium channels, or ryanodine receptors prevented calpain activation and apoptosis in high glucose-stimulated cardiomyocytes. In a mouse model of streptozotocin-induced diabetes, administration of different calpain inhibitors blocked calpain activation, increased the Na+/K+ ATPase activity, and decreased apoptosis in the heart.. Calpain-1 activation induces apoptosis through down-regulation of the Na+/K+ ATPase activity in high glucose-stimulated cardiomyocytes and in vivo hyperglycaemic hearts. High glucose-induced calpain-1 activation is mediated through the NADPH oxidase-dependent pathway and associated with activation of L-type calcium channels and ryanodine receptors. Our data suggest that calpain activation may be important in the development of diabetic cardiomyopathy and thus may represent a potential therapeutic target for diabetic heart diseases.

Topics: Animals; Apoptosis; Calcium; Calcium Channels; Calcium-Binding Proteins; Calpain; Caspase 3; Enzyme Activation; Hyperglycemia; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Myocytes, Cardiac; NADPH Oxidase 2; NADPH Oxidases; Sodium-Potassium-Exchanging ATPase

Calpains and calpastatin can degrade muscle proteins, but no research has investigated the expression pattern of calpains and calpastatin after exhaustive exercise.. To investigate the alterations in expression of micro-, m-, and n-calpain and calpastatin after exhaustive exercise and its association with muscle injury.. 64 rats divided into 2 groups, a nonexercise control group and an acute-exhaustive-exercise (AEE) group. Biopsies in the AEE group were taken at different times after exercise.. Calpastatin protein expression and m-calpain activity increased early after exercise, but both n-calpain protein expression and micro-calpain activity generally decreased with time. n-Calpain mRNA expression was down-regulated from late after exercise.. The increased m-calpain activity might promote muscle-protein degradation and muscle injury. On the contrary, calpastatin might execute a protective function against muscle injury. The change in micro-calpain activity was found earlier than muscle injury and therefore might serve as a useful predictor of muscle injury.

Topics: Animals; Biopsy; Calcium-Binding Proteins; Calpain; Male; Muscle, Skeletal; Physical Conditioning, Animal; Rats; Rats, Sprague-Dawley; RNA, Messenger

Multiple sclerosis (MS) is a T-cell mediated autoimmune disease of the CNS, possessing both immune and neurodegenerative events that lead to disability. Adoptive transfer (AT) of myelin basic protein (MBP)-specific T cells into naïve female SJL/J mice results in a relapsing-remitting (RR) form of experimental autoimmune encephalomyelitis (EAE). Blocking the mechanisms by which MBP-specific T cells are activated before AT may help characterize the immune arm of MS and offer novel targets for therapy. One such target is calpain, which is involved in activation of T cells, migration of immune cells into the CNS, degradation of axonal and myelin proteins, and neuronal apoptosis. Thus, the hypothesis that inhibiting calpain in MBP-specific T cells would diminish their encephalitogenicity in RR-EAE mice was tested. Incubating MBP-specific T cells with the calpain inhibitor SJA6017 before AT markedly suppressed the ability of these T cells to induce clinical symptoms of RR-EAE. These reductions correlated with decreases in demyelination, inflammation, axonal damage, and loss of oligodendrocytes and neurons. Also, calpain : calpastatin ratio, production of truncated Bid, and Bax : Bcl-2 ratio, and activities of calpain and caspases, and internucleosomal DNA fragmentation were attenuated. Thus, these data suggest calpain as a promising target for treating EAE and MS.

Topics: Animals; Axons; Boron Compounds; Calcium-Binding Proteins; Calpain; Cell Survival; Demyelinating Diseases; Dipeptides; Disease Models, Animal; DNA Fragmentation; Dose-Response Relationship, Drug; Encephalomyelitis, Autoimmune, Experimental; Female; In Situ Nick-End Labeling; L-Lactate Dehydrogenase; Mice; Myelin Basic Protein; Statistics, Nonparametric; T-Lymphocytes; Time Factors

The neurotoxic amyloid-beta-peptide (Abeta) is important in the pathogenesis of Alzheimer's disease (AD). Calpain (Ca(2+)-dependent protease) and caspase-8 (the initiating caspase for the extrinsic, receptor-mediated apoptosis pathway) have been implicated in AD/Abeta toxicity. We previously found that Abeta promoted degradation of calpastatin (the specific endogenous calpain inhibitor); calpastatin degradation was prevented by inhibitors of either calpain or caspase-8. The results implied a cross-talk between the two proteases and suggested that one protease was responsible for the activity of the other one. We now report on the previously unrecognized caspase-8 activation by calpain. In neuron-like differentiated PC12 cells, calpain promotes active caspase-8 formation from procaspase-8 via the Abeta and CD95 pathways, along with degradation of the procaspase-8 processing inhibitor caspase-8 (FLICE)-like inhibitory protein, short isoform (FLIP(S)). Inhibition of calpain (by pharmacological inhibitors and by overexpression of calpastatin) prevents the cleavage of procaspase-8 to mature, active caspase-8, and inhibits FLIP(S) degradation in the Abeta-treated and CD95-triggered cells. Increased cellular Ca(2+) per se results in calpain activation but does not lead to caspase-8 activation or FLIP(S) degradation. The results suggest that procaspase-8 and FLIP(S) association with cell membrane receptor complexes is required for calpain-induced caspase-8 activation. The results presented here add to the understanding of the roles of calpain, caspase-8, and CD95 pathway in AD/Abeta toxicity. Calpain-promoted activation of caspase-8 may have implications for other types of CD95-induced cell damage, and for nonapoptotic functions of caspase-8. Inhibition of calpain may be useful for modulating certain caspase-8-dependent processes.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Apoptosis; Binding, Competitive; Calcium-Binding Proteins; Calpain; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 8; Cell Differentiation; Disease Models, Animal; fas Receptor; Fas-Associated Death Domain Protein; Neurons; PC12 Cells; Peptide Fragments; Rats; Signal Transduction

The cytosolic cysteine protease calpain is implicated in a multitude of cellular functions but also plays a role in cell damage. Our previous results suggest that an activation of calpain accompanied by a decrease in its endogenous inhibitor calpastatin may contribute to pancreatic damage during cerulein-induced acute pancreatitis. The present study aimed at the time course of secretagogue-induced calpain activation and cellular substrates of the protease. Isolated rat pancreatic acini were incubated with a supramaximal concentration of cholecystokinin (0.1 microM CCK) for 30 min in the presence or absence of the calpain inhibitor Z-Val-Phe methyl ester (100 microM ZVP). The activation of calpain and the expression of calpastatin and the actin cytoskeleton-associated proteins alphaII-spectrin, E-cadherin and vinculin were studied by immunoblotting. The cell damage was assessed by lactate dehydrogenase release and ultrastructural analysis including fluorescence-labelled actin filaments. Immediately after administration, CCK led to activation of both calpain isoforms, mu- and m-calpain. The protease activation was accompanied by a decrease in the E-cadherin level and formation of calpain-specific breakdown products of alphaII-spectrin. A calpain-specific cleavage product of vinculin appeared concomitantly with changes in the actin filament organization. No effect of CCK on calpastatin was found. Inhibition of calpain by ZVP reduced CCK-induced damage of the actin-associated proteins and the cellular ultrastructure including the actin cytoskeleton. The results suggest that CCK-induced acinar cell damage requires activation of calpain and that the actin cytoskeleton belongs to the cellular targets of the protease.

Topics: Actins; Acute Disease; Animals; Blotting, Western; Cadherins; Calcium-Binding Proteins; Calpain; Ceruletide; Cholecystokinin; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Cytoskeleton; Dipeptides; Enzyme Activation; Female; Gene Expression; Microscopy, Confocal; Microscopy, Electron; Models, Animal; Organ Culture Techniques; Pancreas; Pancreatitis; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spectrin; Stimulation, Chemical; Time Factors; Vinculin

Calpain is a calcium ion-dependent cysteine protease, consisting of two primary isoforms (calpain1/calpain2) which mediate crucial cellular functions. The activity of the calpains is tightly regulated by the endogenous inhibitor calpastatin. Calpains have been detected in several studies during the embryonic and foetal stages. The aim of this study is to investigate the temporal transition of typical calpains and their inhibitor calpastatin during odontogenesis.. We used the first molar of foetal ICR mice from embryonic day (E) 14 to postnatal day (PN) 7. Using laser microdissection and semi-quantitative real-time PCR, we investigated calpain1, calpain2 and calpastatin expressions in each enamel epithelium, inner enamel epithelium, stellate reticulum and outer enamel epithelium.. We found calpain1 and calpain2 mRNA increased in the all enamel epithelia between E18 and PN1. In addition calpastatin mRNA expression increased in the ameloblasts from PN1 to PN7. The immunohistochemistry results demonstrated that calpain1/calpain2 was present in the distal side of ameloblasts from PN1 to PN7, and calpastatin was present in the extracellular enamel matrix from E16 to PN1. Furthermore calpain1/calpain2 was present in the dentin, and calpastatin was detected in dentin producing odontoblasts and predentin at PN7.. In this study the temporal transition of calpain1, calpain2 and calpastatin mRNA and the immunolocalization are identified during tooth development. Our results indicate that the calcium-dependent proteases may play an important role in mouse molar development and extracellular calpain and calpastatin may be involved in molar mineralization.

Topics: Ameloblasts; Amelogenesis; Animals; Calcium-Binding Proteins; Calpain; Dental Enamel; Enamel Organ; Epithelial Cells; Gene Expression; Immunohistochemistry; Mice; Mice, Inbred ICR; Molar

Marfan syndrome (MFS) is a heritable disorder of connective tissue, affecting principally skeletal, ocular, and cardiovascular systems. The most life-threatening manifestations are aortic aneurysm and dissection. We investigated changes in the proteome of aortic media in patients with and without MFS to gain insight into molecular mechanisms leading to aortic dilatation.. Aortic samples were collected from 46 patients. Twenty-two patients suffered from MFS, 9 patients had bicuspid aortic valve, and 15 patients without connective tissue disorder served as controls. Aortic media was isolated and its proteome was analyzed in 12 patients with the use of 2-dimensional difference gel electrophoresis and mass spectrometry. We found higher amounts of filamin A C-terminal fragment, calponin 1, vinculin, microfibril-associated glycoprotein 4, and myosin-10 heavy chain in aortic media of MFS aneurysm samples than in controls. Regulation of filamin A C-terminal fragmentation was validated in all patient samples by immunoblotting. Cleavage of filamin A and the calpain substrate spectrin was increased in the MFS and bicuspid aortic valve groups. Extent of cleavage correlated positively with calpain 2 expression and negatively with the expression of its endogenous inhibitor calpastatin.. Our observation demonstrates for the first time upregulation of the C-terminal fragment of filamin A in dilated aortic media of MFS and bicuspid aortic valve patients. In addition, our results present evidence that the cleavage of filamin A is highly likely the result of the protease calpain. Increased calpain activity might explain, at least in part, histological alterations in dilated aorta.

Topics: Adult; Aorta; Aortic Aneurysm; Calcium-Binding Proteins; Calpain; Contractile Proteins; Enzyme Activation; Female; Filamins; Humans; Male; Marfan Syndrome; Microfilament Proteins; Middle Aged; Protein Structure, Tertiary; Proteomics; Spectrin; Tunica Media

Retinitis pigmentosa is a heterogeneous group of inherited retinal dystrophies in which the loss of photoreceptor cells via apoptosis leads to blindness. In this study we have experimentally mimicked this condition by treating 661W cells and wild-type mouse retinal explants with a Ca(2+) ionophore. Ca(2+) overload induced apoptosis, which was correlated with calpain-2 activation, loss of calpastatin, its endogenous inhibitor, as well as the loss of its transcriptional activator, phospho-cAMP response element binding (CREB). All are similar changes to those observed in the rd1 mouse model of retinitis pigmentosa. Insulin like-growth factor-I (IGF-I) attenuated this Ca(2+)-induced apoptosis, as well as decreased the activation of calpain-2 and maintained calpastatin levels through the activation of the Akt-CREB pathway. Similarly, IGF-I decreased photoreceptor apoptosis in rd1 mouse retinal explants in parallel with reduced activation of calpain-2 and increased levels of calpastatin and activation of phospho-CREB. In conclusion, IGF-I seems to protect neural cells following a physiopathological or an experimental increase in intracellular Ca(2+), an observation that may have therapeutic consequences in neurodegenerative diseases such as retinitis pigmentosa.

Topics: Analysis of Variance; Animals; Apoptosis; Blotting, Western; Calcium; Calcium-Binding Proteins; Calpain; Cell Line; Cells, Cultured; Cyclic AMP Response Element-Binding Protein; Cytoprotection; Fluorescent Antibody Technique; Fluorescent Dyes; In Situ Nick-End Labeling; Insulin-Like Growth Factor I; Mice; Phosphorylation; Photoreceptor Cells; Proto-Oncogene Proteins c-akt; Retina; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Calpain had been shown to be highly activated at one day after exposure to the damaging light (Perche et al. (2007)Caspase-dependent apoptosis in light-induced retinal degeneration. Invest Ophthalmol Vis Sci 48:2753-2759.), suggesting that they might play a critical role in photoreceptor apoptosis induced by light. Therefore in the present study we investigate the role of calpain in light-induced photoreceptor cell death. In a first set of experiments, untreated albino Wistar rats were sacrificed at 0, 2, 4, 6, 12, 24 h of light exposure and at one day after the light was turned off (D1) to measure retinal calpain activity and to study calpain expression. In a second set of experiments, after control electroretinograms (ERGs), rats were uninjected or injected intravitreally with DMSO or the calpain inhibitor Mu-Phe-hPhe-FMK, before being exposed to the damaging light for 24 h. ERGs were then recorded at one day (D1) and fifteen days (D15) after the end of light exposure. Rats were sacrificed at D1 for apoptotic cell detection or D15 for histological analysis (ONL thickness). Calpain activity and expression significantly increased in Untreated retinas, from 0 h to D1. DMSO has no effect on calpain activity. Mu-Phe-hPhe-FMK significantly inhibited retinal calpain activity by 85% at 2 h of light exposure and still 48% at D1. However, Mu-Phe-hPhe-FMK has no effect on light-induced retinal degeneration as evidence by equivalent loss of function, equivalent loss of photoreceptor cells and an equivalent number of apoptotic cells in Mu-Phe-hPhe-FMK and DMSO retinas. Therefore, calpains are up-regulated by light stress but they do not have a pivotal role in photoreceptor apoptosis.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Electroretinography; Light; Morpholines; Neuroprotective Agents; Photic Stimulation; Radiation Injuries, Experimental; Rats; Rats, Wistar; Retina; Retinal Degeneration

Calpains and caspases are cysteine endopeptidases which share many similar substrates. Caspases are essential for caspase-dependent apoptotic death where calpains may play an augmentive role, while calpains are strongly implicated in necrotic cell death morphologies. Previous studies have demonstrated a down-regulation in the expression of many components of the caspase-dependent cell death pathway during CNS development. We therefore sought to determine if there is a corresponding upregulation of calpains. The major CNS calpains are the mu-and m-isoforms, composed of the unique 80 kDa calpain 1 and 2 subunits, respectively, and the shared 28 kDa small subunit. In rat brain, relative protein and mRNA levels of calpain 1, calpain 2, caspase 3, and the endogenous calpain inhibitor-calpastatin, were evaluated using western blot and real-time RT-PCR. The developmental time points examined ranged from embryonic day 18 until postnatal day 90. Calpain 1 and calpastatin protein and mRNA levels were low at early developmental time points and increased dramatically by P30. Conversely, caspase-3 expression was greatest at E18, and was rapidly downregulated by P30. Calpain 2 protein and mRNA levels were relatively constant throughout the E18-P90 age range examined. The inverse relationship of calpain 1 and caspase 3 levels during CNS development is consistent with the shift from caspase-dependent to caspase-independent cell death mechanisms following CNS injury in neonatal vs. adult rat brain.

Topics: Aging; Animals; Animals, Newborn; Blotting, Western; Brain; Brain Chemistry; Calcium-Binding Proteins; Calpain; Caspase 3; Mitochondria; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Many factors contribute to nervous system dysfunction and failure to regenerate after injury or disease. Here, we describe a previously unrecognized mechanism for nervous system injury. We show that neuronal injury causes rapid, irreversible, and preferential proteolysis of the axon initial segment (AIS) cytoskeleton independently of cell death or axon degeneration, leading to loss of both ion channel clusters and neuronal polarity. Furthermore, we show this is caused by proteolysis of the AIS cytoskeletal proteins ankyrinG and betaIV spectrin by the calcium-dependent cysteine protease calpain. Importantly, calpain inhibition is sufficient to preserve the molecular organization of the AIS both in vitro and in vivo. We conclude that loss of AIS ion channel clusters and neuronal polarity are important contributors to neuronal dysfunction after injury, and that strategies to facilitate recovery must preserve or repair the AIS cytoskeleton.

Topics: Analysis of Variance; Animals; Axons; Calcium-Binding Proteins; Calpain; Cell Adhesion Molecules; Cell Death; Cells, Cultured; Cerebral Cortex; Cysteine Proteinase Inhibitors; Cytoskeleton; Disease Models, Animal; Embryo, Mammalian; Glucose; Green Fluorescent Proteins; Hypoxia; Infarction, Middle Cerebral Artery; Mice; Mice, Inbred C57BL; Nerve Growth Factors; Nerve Tissue Proteins; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Transfection

The activity of calpain is controlled by the free intracellular calcium level and by the protein's intrinsically disordered endogenous inhibitor, calpastatin, mediated by short conserved segments: subdomains A-C. The exact binding mode of calpastatin to the enzyme has until now been unclear. Our NMR data of the 141 amino acid long inhibitor, with and without calcium and calpain, have revealed structural changes and a tripartite binding mode, in which the disordered inhibitor wraps around, and contacts, the enzyme at three points, facilitated by flexible linkers. This unprecedented binding mode permits a unique combination of specificity, speed and binding strength in regulation.

Topics: Calcium; Calcium-Binding Proteins; Calpain; Humans; Nuclear Magnetic Resonance, Biomolecular; Protein Conformation

Calpain, the calcium-activated intracellular cysteine protease, is under the tight control of its intrinsically unstructured inhibitor, calpastatin. Understanding how potent inhibition by calpastatin can be reconciled with its unstructured nature provides deeper insight into calpain function and a more general understanding of how proteins devoid of a well-defined structure carry out their function. To this end, we performed a full NMR assignment of hCSD1 to characterize it in its solution state. Secondary chemical shift values and NMR relaxation data, R 1, R 2, and hetero-NOE, as well as spectral density function analysis have shown that conserved regions of calpastatin, subdomains A and C, which are responsible for calcium-dependent anchoring of the inhibitor to the enzyme, preferentially sample partially helical backbone conformations of a reduced flexibility. Moreover, the linker regions between subdomains are more flexible with no structural preference. The primary determinant of calpain inhibition, subdomain B, also has a non-fully random conformational preference, resembling a beta-turn structure also ascertained by prior studies of a 27-residue peptide encompassing the inhibitory region. This local structural preference is also confirmed by a deviation in chemical shift values between full-length calpastatin domain 1 and a truncated construct cut in the middle of subdomain B. At the C-terminal end of the molecule, a nascent helical region was found, which in contrast to the overall structural properties of the molecule may indicate a previously unknown functional region. Overall, these observations provide further evidence that supports previous suggestions that intrinsically unstructured proteins use preformed structural elements in efficient partner recognition.

Topics: Amino Acid Sequence; Calcium-Binding Proteins; Calpain; Crystallography, X-Ray; Enzyme Inhibitors; Humans; Hydrogen-Ion Concentration; Models, Molecular; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Protein Structure, Secondary; Protein Structure, Tertiary; Sequence Alignment; Sequence Homology, Amino Acid; Temperature

The c-Myc transcription factor is commonly dysregulated in cancer. c-Myc also sensitizes cells to apoptosis induced by a variety of toxic events. c-Myc turnover is rapid and mediated by the proteasome and intracellular calpains. Therefore, c-Myc accumulation could contribute to cell death associated with protease inhibitors. We investigated the response of c-Myc-positive and c-Myc-negative rat fibroblast cells to proteasome and calpain inhibitors. Apoptosis induced by the proteasome inhibitor, epoxomycin, was c-Myc-independent, whereas apoptosis induced by the calpain inhibitor, PD150606, or by knockdown of calpain small subunit 1 (CPNS1) was strongly dependent on c-Myc. HL60 cells knocked down for c-Myc expression exhibited reduced calpain activity and decreased sensitivity to PD150606 but not epoxomycin. Calpain inhibitor- or CPNS1 knockdown-induced apoptosis in c-Myc-positive fibroblasts was associated with cell detachment and could be prevented by plating cells on fibronectin, suggesting an anoikis phenomenon. c-Myc stimulated calpain activity by suppressing calpastatin expression, the endogenous calpain inhibitor. Knockdown of calpastatin in c-Myc-negative cells led to a restoration of calpain activity, enhanced cell growth, cell cycle redistribution, anchorage independence, and tumorigenicity in immunodeficient mice. Taken together, these results indicate that c-Myc regulates calpain activity through calpastatin; apoptosis induced by calpain inhibition is dependent on c-Myc, and calpastatin knockdown promotes transformation in c-Myc-negative cells.

Topics: Acrylates; Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Cell Adhesion; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fibroblasts; Fibronectins; HL-60 Cells; Humans; Proto-Oncogene Proteins c-myc; Rats; RNA, Small Interfering

Calpastatin is an endogenous inhibitor of calpain, which has been implicated in various physiological and pathological processes. In the present study we determined the molecular and inhibitory properties of HMWCaMBP, calpastatin I, and calpastatin II. Western blot analysis with antibodies raised against either full length HMWCaMBP or internal peptides that are common to all isoforms showed that all three homologs have common antigenic epitopes. However, additional Western blot analysis with N-terminal specific antibodies showed that all three proteins are different at the N-terminal end. HMWCaMBP is clearly different from two other homologues, calpastatin I and II, at the N-terminal end. In addition, HMWCaMBP also showed the same affinities for m-calpain as calpastatin I and calpastatin II. Our findings suggest that HMWCaMBP is the homolog of calpastatin and may be a CaM-binding form of calpastatin.

Topics: Amino Acid Sequence; Animals; Binding Sites; Calcium-Binding Proteins; Calmodulin-Binding Proteins; Calpain; Cattle; Heart; Molecular Sequence Data; Molecular Weight; Peptide Mapping; Sequence Alignment; Sequence Analysis, Protein

Dystrophin deficiency is the underlying molecular cause of progressive muscle weakness observed in Duchenne muscular dystrophy (DMD). Loss of functional dystrophin leads to elevated levels of intracellular Ca(2+), a key step in the cellular pathology of DMD. The cysteine protease calpain is activated in dystrophin-deficient muscle, and its inhibition is regarded as a potential therapeutic approach. In addition, previous work has shown that the ubiquitin-proteasome system also contributes to muscle protein breakdown in dystrophic muscle and, therefore, also qualifies as a potential target for therapeutic intervention in DMD. The relative contribution of calpain- and proteasome-mediated proteolysis induced by increased Ca(2+) levels was characterized in cultured muscle cells and revealed initial Ca(2+) influx-dependent calpain activity and subsequent Ca(2+)-independent activity of the ubiquitin-proteasome system. We then set out to optimize novel small-molecule inhibitors that inhibit both calpain as well as the 20S proteasome in a cellular system with impaired Ca(2+) homeostasis. On administration of such inhibitors to mdx mice, quantitative histological parameters improved significantly, in particular with compounds strongly inhibiting the 20S proteasome. To investigate the role of calpain inhibition without interfering with the ubiquitin-proteasome system, we crossed mdx mice with transgenic mice, overexpressing the endogenous calpain inhibitor calpastatin. Although our data show that proteolysis by calpain is strongly inhibited in the transgenic mdx mouse, this calpain inhibition did not ameliorate muscle histology. Our results indicate that inhibition of the proteasome rather than calpain is required for histological improvement of dystrophin-deficient muscle. In conclusion, we have identified novel proteasome inhibitors that qualify as potential candidates for pharmacological intervention in muscular dystrophy.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Cells, Cultured; Humans; Mice; Mice, Inbred mdx; Mice, Transgenic; Muscles; Muscular Dystrophy, Duchenne; Myoblasts; Oligopeptides; Protease Inhibitors; Proteasome Inhibitors

Amyloid beta peptide (Abeta) plays a major role in the pathogenesis of Alzheimer's disease (AD). Abeta is toxic to neurons, possibly through causing initial synaptic dysfunction and neuronal membrane dystrophy, promoted by increased cellular Ca(2+). Calpain (Ca(2+)-dependent protease) and caspase have been implicated in AD. Previously, we used calpain and caspase pharmacological inhibitors to study effects of Abeta25-35 (sAbeta) on neuronal-like differentiated PC12 cells. We reported that sAbeta-treated cells exhibited calpain activation and protein degradation (due to both calpain and caspase-8). We have now found that overexpression of the calpain specific inhibitor calpastatin in differentiated PC12 cells significantly inhibited the sAbeta-induced calpain activation and decreased the protease activity. Calpastatin overexpression inhibited the sAbeta-promoted degradation of fodrin, protein kinase Cepsilon, beta-catenin (membrane structural proteins and proteins involved in signal transduction pathways), and prevented the sAbeta-induced alteration of neurite structure (manifested by varicosities). Overexpression of calpastatin also inhibited Ca(2+)-promoted calpain activation and protein degradation; this is consistent with the notion that the Abeta-induced increase in calpain activity results from a rise in cellular Ca(2+), provided the calpastatin level is not so high as to strongly inhibit calpain. Carrying out transfection without selection allowed the comparison in the same culture of calpastatin-overexpressing with non-overexpressing cells. In cultures transfected with green fluorescent protein (GFP)-calpastatin plasmid, calpastatin overexpression (indicated by GFP-labeling) led to inhibition in sAbeta-induced membrane propidium iodide (PI) permeability, whereas non-transfected, GFP-unlabeled cells exhibited PI permeability. Overall, the results demonstrate that the effects of Abeta-toxicity studied here were attenuated to a large extent by calpastatin overexpression, indicating that the protease calpain is involved in Abeta-toxicity (obviating a primary, direct role for caspases). Increased expression of calpastatin and/or decrease in calpain may serve as one of the means for ameliorating some of the early symptoms of AD.

Topics: Amyloid beta-Peptides; Animals; beta Catenin; Calcium; Calcium-Binding Proteins; Calpain; Carrier Proteins; Cell Differentiation; Gene Expression; Microfilament Proteins; Neurons; PC12 Cells; Peptide Fragments; Propidium; Protein Kinase C-epsilon; Rats; Transfection

The specific, calcium-dependent, high affinity interaction between calpain and its endogenous inhibitor calpastatin was exploited to selectively detect the calcium-bound, catalytically competent, conformation of calpain in vitro. Modification of calpastatin domain-1 (Val(114)-Ser(270)) or its N-terminal fragment (Val(114)-Pro(202)), at selected unique cysteine residues with maleimide-AlexaFluor546 did not compromise calpastatin function (inhibition of calpain) or its binding with calpain. Ca(2+)-dependent binding between catalytically dead calpain-2 (Cys(105)Ala) fused with eGFP and these fluorigenic calpastatin peptides generates fluorescent resonance energy transfer (FRET). The FRET signal documents proximity of calpain-2, C-terminally linked fluorophore to specific sites within calpastatin when the proteins form a complex. These results provide important insights into the calcium-dependent interaction between calpain and calpastatin and for holo-calpain-2 in solution experimentally validate some key features of their predicted interactions. These data also provide proof of concept that the calpastatin-based reagents may be useful to selectively detect the active conformation of calpain.

Topics: Amino Acid Sequence; Calcium; Calcium-Binding Proteins; Calpain; Catalytic Domain; Cysteine Proteinase Inhibitors; Enzyme Activation; Humans; Models, Biological; Models, Molecular; Molecular Sequence Data; Protein Conformation; Protein Structure, Tertiary; Substrate Specificity

In dividing cells calpastatin diffuses from aggregates into cytosol, indicating the requirement for a tight regulation of calpain. Accordingly, the involvement of the calpain-calpastatin system in cell proliferation and in the density-dependent growth arrest was studied in JA3 cells stably transfected with a calpastatin form permanently localized in cytosol. In calpastatin overexpressing cells, cell cycle rate is 50% reduced, and cells enter the ungrowing, still fully reversible, stage at a 3-fold higher cell density. Furthermore, in cell density growth arrest phase, down regulation of alpha- and theta-PKC isoforms, as well as FAK and talin occurs. In calpastatin overexpressing cells, degradation of these calpain substrate proteins is prevented and delayed. Thus, calpain activity plays a crucial role in inducing the cell entry into a functional quiescent phase.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Proliferation; Cytosol; Focal Adhesion Kinase 1; Gene Expression; Humans; Isoenzymes; Jurkat Cells; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-theta; Rats

The objective of this study was to investigate the effects of supplementing swine finishing diets with two levels of magnesium aspartate (MgAsp) and short-term transportation stress on blood parameters, pork quality and the mRNA abundance of p-calpain and calpastatin in muscles of finishing pigs. Thirty-six crossbred finishing pigs (mean BW 90 kg) were assigned randomly to 0, 1000, or 2000 mg supplemental Mg from MgAsp per kg of diet for five days before slaughter. Then six pigs from each dietary treatment were subjected either to no transportation stress (NTS) or 2 h of transportation stress (TS). Transportation stress resulted in higher concentrations (p < 0.01) of serum calcium, glucose and cortisol, lower pH (p < 0.01), higher Warner-Bratzler shear force (WBSF) (p < 0.05) and higher calpastatin mRNA abundance (p = 0.05) of longissimus muscle (LM) compared with NTS treatments. Supplementation of MgAsp in TS treatments increased serum Mg concentration (p < 0.05) at 2000 mg of Mg/kg, reduced drip loss (p < 0.05) and improved pork quality colour (p < 0.05) at 2000 mg of Mg/kg, and decreased 1-day and 3-day WBSF (p < 0.05) at 1000 mg of Mg/kg compared with TS treatments fed the control diet. It is concluded that supplementation of MgAsp improves water-holding capacity and pork colour, and alleviates the negative effects of transportation stress on meat tenderness.

Topics: Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Aspartic Acid; Calcium-Binding Proteins; Calpain; Dietary Supplements; Dose-Response Relationship, Drug; Male; Meat; Muscle, Skeletal; Random Allocation; RNA, Messenger; Stress, Psychological; Swine; Time Factors; Transportation

Increased activity of calpains is implicated in synaptic dysfunction and neurodegeneration in Alzheimer's disease (AD). The molecular mechanisms responsible for increased calpain activity in AD are not known. Here, we demonstrate that disease progression is propelled by a marked depletion of the endogenous calpain inhibitor, calpastatin (CAST), from AD neurons, which is mediated by caspase-1, caspase-3, and calpains. Initial CAST depletion focally along dendrites coincides topographically with calpain II and ERK 1/2 activation, tau cleavage by caspase-3, and tau and neurofilament hyperphosphorylation. These same changes, together with cytoskeletal proteolysis and neuronal cell death, accompany CAST depletion after intrahippocampal kainic acid administration to mice, and are substantially reduced in mice overexpressing human CAST. Moreover, CAST reduction by shRNA in neuronal cells causes calpain-mediated death at levels of calcium-induced injury that are sublethal to cells normally expressing CAST. Our results strongly support a novel hypothesis that CAST depletion by multiple abnormally activated proteases accelerates calpain dysregulation in AD leading to cytoskeleton disruption and neurodegeneration. CAST mimetics may, therefore, be neuroprotective in AD.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Animals; Brain; Calcium-Binding Proteins; Calpain; Case-Control Studies; Caspases; Cell Death; Cell Line, Transformed; Cytoskeleton; Excitatory Amino Acid Agonists; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation; Hippocampus; Humans; Kainic Acid; Male; Mice; Mice, Transgenic; Middle Aged; Nerve Degeneration; Postmortem Changes; RNA, Small Interfering; Transfection

Topics: Animals; Biocatalysis; Calcium; Calcium-Binding Proteins; Calpain; Catalytic Domain; Crystallography, X-Ray; Models, Molecular; Peptide Fragments; Protein Binding; Protein Multimerization; Rats

Calpains are non-lysosomal calcium-dependent cysteine proteinases that selectively cleave proteins in response to calcium signals and thereby control cellular functions such as cytoskeletal remodelling, cell cycle progression, gene expression and apoptotic cell death. In mammals, the two best-characterized members of the calpain family, calpain 1 and calpain 2 (micro-calpain and m-calpain, respectively), are ubiquitously expressed. The activity of calpains is tightly controlled by the endogenous inhibitor calpastatin, which is an intrinsically unstructured protein capable of reversibly binding and inhibiting four molecules of calpain, but only in the presence of calcium. To date, the mechanism of inhibition by calpastatin and the basis for its absolute specificity have remained speculative. It was not clear how this unstructured protein inhibits calpains without being cleaved itself, nor was it known how calcium induced changes that facilitated the binding of calpastatin to calpain. Here we report the 2.4-A-resolution crystal structure of the calcium-bound calpain 2 heterodimer bound by one of the four inhibitory domains of calpastatin. Calpastatin is seen to inhibit calpain by occupying both sides of the active site cleft. Although the inhibitor passes through the active site cleft it escapes cleavage in a novel manner by looping out and around the active site cysteine. The inhibitory domain of calpastatin recognizes multiple lower affinity sites present only in the calcium-bound form of the enzyme, resulting in an interaction that is tight, specific and calcium dependent. This crystal structure, and that of a related complex, also reveal the conformational changes that calpain undergoes on binding calcium, which include opening of the active site cleft and movement of the domains relative to each other to produce a more compact enzyme.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Catalytic Domain; Crystallography, X-Ray; Models, Molecular; Protein Binding; Protein Multimerization; Rats; Structure-Activity Relationship

The Ca(2+)-dependent cysteine proteases, calpains, regulate cell migration, cell death, insulin secretion, synaptic function and muscle homeostasis. Their endogenous inhibitor, calpastatin, consists of four inhibitory repeats, each of which neutralizes an activated calpain with exquisite specificity and potency. Despite the physiological importance of this interaction, the structural basis of calpain inhibition by calpastatin is unknown. Here we report the 3.0 A structure of Ca(2+)-bound m-calpain in complex with the first calpastatin repeat, both from rat, revealing the mechanism of exclusive specificity. The structure highlights the complexity of calpain activation by Ca(2+), illustrating key residues in a peripheral domain that serve to stabilize the protease core on Ca(2+) binding. Fully activated calpain binds ten Ca(2+) atoms, resulting in several conformational changes allowing recognition by calpastatin. Calpain inhibition is mediated by the intimate contact with three critical regions of calpastatin. Two regions target the penta-EF-hand domains of calpain and the third occupies the substrate-binding cleft, projecting a loop around the active site thiol to evade proteolysis.

Topics: Animals; Biocatalysis; Calcium; Calcium-Binding Proteins; Calpain; Catalytic Domain; Crystallography, X-Ray; EF Hand Motifs; Enzyme Activation; Protein Binding; Protein Multimerization; Protein Processing, Post-Translational; Rats; Structure-Activity Relationship; Substrate Specificity

Taurine as an endogenous substance possesses a number of cytoprotective properties. In the study, we have evaluated the neuroprotective effect of taurine and investigated whether taurine exerted neuroprotection through affecting calpain/calpastatin or caspase-3 actions during focal cerebral ischemia, since calpain and caspase-3 play central roles in ischemic neuronal death.. Male Sprague-Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAo), and 22 h of reperfusion. Taurine was administrated intravenously 1 h after MCAo. The dose-responses of taurine to MCAo were determined. Next, the effects of taurine on the activities of calpain, calpastatin and caspase-3, the levels of calpastatin, microtubule-associated protein-2 (MAP-2) and alphaII-spectrin, and the apoptotic cell death in penumbra were evaluated.. Taurine reduced neurological deficits and decreased the infarct volume 24 h after MCAo in a dose-dependent manner. Treatment with 50 mg/kg of taurine significantly increased the calpastatin protein levels and activities, and markedly reduced the m-calpain and caspase-3 activities in penumbra 24 h after MCAo, however, it had no significant effect on mu-calpain activity. Moreover, taurine significantly increased the MAP-2 and alphaII-spectrin protein levels, and markedly reduced the ischemia-induced TUNEL staining positive score within penumbra 24 h after MCAo.. Our data demonstrate the dose-dependent neuroprotection of taurine against transient focal cerebral ischemia, and suggest that one of protective mechanisms of taurine against ischemia may be blocking the m-calpain and caspase-3-mediated apoptotic cell death pathways.

Topics: Animals; Apoptosis; Brain Ischemia; Calcium-Binding Proteins; Calpain; Caspase 3; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Activation; Male; Microfilament Proteins; Microtubule-Associated Proteins; Models, Biological; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Taurine; Up-Regulation; Vesicular Transport Proteins

Right ventricular contractile failure from acute RV pressure overload is an important cause of morbidity and mortality, but the mechanism of RV failure in this setting is incompletely defined. We hypothesized that RV dysfunction from acute RV pressure overload is, in part, due to activation of calpain, and that calpain inhibition would therefore attenuate RV dysfunction. Anesthetized, open chest pigs were treated with the calpain inhibitor MDL-28170 or with inactive vehicle, and then subjected to acute RV pressure overload for 90 min. RV contractile function was assessed by the regional Frank-Starling relation. RV myocardial tissue was analyzed for evidence of calpain activation and calpain-mediated proteolysis. RV pressure overload caused severe contractile dysfunction, along with significant alterations in the endogenous calpain inhibitor calpastatin typical of calpain activation. MDL-28170 attenuated RV free wall dysfunction by more than 50%. However, there were no differences in degradation of spectrin, desmin, troponin-I or SERCA2 between SHAM operated pigs and pigs subjected to acute RV pressure overload, or between vehicle and MDL-28170 treated pigs. Acute RV pressure overload causes calpain activation, and RV contractile dysfunction from acute RV pressure overload is attenuated by the calpain inhibitor MDL-28170; however, the effect is not explained by inhibition of calpain-mediated degradation of spectrin, desmin, troponin-I or SERCA2. Because this is the first report of any agent that can directly attenuate RV contractile dysfunction in acute RV pressure overload, further investigation of the mechanism of action of MDL-28170 in this setting is warranted.

Topics: Animals; Calcium-Binding Proteins; Calpain; Densitometry; Desmin; Dipeptides; Female; Hemodynamics; Male; Myocardial Contraction; Protein Processing, Post-Translational; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Spectrin; Swine; Troponin; Ventricular Dysfunction, Right; Ventricular Pressure

Recently, we have reported the presence of calpain-calpastatin system in mitochondria of bovine pulmonary smooth muscle [P. Kar, T. Chakraborti, S. Roy, R. Choudhury, S. Chakraborti, Arch. Biochem. Biophys. 466 (2007) 290-299]. Herein, we report its localization in the mitochondria. Immunoblot, immunoelectron microscopy and casein zymographic studies suggest that mu-calpain and calpastatin are present in the inner mitochondrial membrane; but not in the outer mitochondrial membrane or in the inter membrane space or in the matrix of the mitochondria. Co-immunoprecipitation studies suggest that mu-calpain-calpastatin is associated in the inner mitochondrial membrane. Additionally, the proteinase K and sodium carbonate treatments of the mitoplasts revealed that mu-calpain is integrally and calpastatin is peripherally embedded to the outer surface of inner mitochondrial membrane. These studies indicate that an association between mu-calpain and calpastatin occurs in the inner membrane towards the inter membrane space of the mitochondria, which provides better insight about the protease regulation towards initiation of apoptotic processes mediated by mitochondria.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Cells, Cultured; Humans; Mitochondria, Muscle; Muscle, Smooth, Vascular

The 27-mer peptide CP1B-[1-27] derived from exon 1B of calpastatin stands out among the known inhibitors for mu- and m-calpain due to its high potency and selectivity. By systematical truncation, a 20-mer peptide, CP1B-[4-23], was identified as the core sequence required to maintain the affinity/selectivity profile of CP1B-[1-27]. Starting with this peptide, the turn-like region Glu(10)(i)-Leu(11)(i+1)-Gly(12)(i+2)-Lys(13)(i+3) was investigated. Sequence alignment of subdomains 1B, 2B, 3B and 4B from different mammalians revealed that the amino acid residues in position i+1 and i+2 are almost invariably flanked by oppositely charged residues, pointing towards a turn-like conformation stabilized by salt bridge/H-bond interaction. Accordingly, using different combinations of acidic and basic residues in position i and i+3, a series of conformationally constrained variants of CP1B-[4-23] were synthesized by macrolactamization utilizing the side chain functionalities of these residues. With the combination of Glu(i)/Dab(i+3), the maximum of conformational rigidity without substantial loss in affinity/selectivity was reached. These results clearly demonstrate that the linear peptide chain corresponding to subdomain 1B reverses its direction in the region Glu(10)-Lys(13) upon binding to mu-calpain, and thereby adopts a loop-like rather than a tight turn conformation at this site.

Topics: Amino Acid Sequence; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Drug Design; Humans; Kinetics; Lactams; Models, Molecular; Molecular Sequence Data; Peptides; Protein Conformation; Structure-Activity Relationship; Substrate Specificity

Some data have shown the functional connection between calpain and caspase-3. Here, we investigated the cross-talk between calpain and caspase-3 in penumbra and core during focal cerebral ischemia-reperfusion.. The activities of calpain and the levels of calpastatin, microtubule-associated protein-2 (MAP-2), and spectrin in penumbra and core at 3 or 23 h of reperfusion (R 3 h or R 23 h) after 1-h focal cerebral ischemia in rats were determined in sham- or caspase-3 inhibitor z-DEVD-CHO-treated rats.On the other hand, the determination of the activities of caspase-3 and the levels of MAP-2 and spectrin was done in sham- or calpain-inhibitor I-treated rats.. z-DEVD-CHO (600 ng/rat, i.c.v.) markedly reduced the mu- and m-calpain activities in penumbra and the m-calpain activities in core at R 3 h and R 23 h, and enhanced the calpastatin levels in penumbra at R 3 h and in core at R 3 h and R 23 h significantly; however, it had no significant effects on the mu-calpain activities in core and the calpastatin levels in penumbra at R 23 h. Calpain inhibitor I (0.8 mg/rat, i.c.v.) markedly reduced the caspase-3 activities in core at R 3 h and R 23 h, but not in penumbra. Both calpain and caspase-3 inhibitors increased the levels of MAP-2 and spectrin in penumbra and core significantly after focal cerebral ischemia-reperfusion.. Our data provide direct evidence to demonstrate the cross-talk between calpain and caspase-3 in penumbra and core during focal cerebral ischemia-reperfusion.

Topics: Animals; Brain Ischemia; Calcium-Binding Proteins; Calpain; Caspase 3; Caspase Inhibitors; Glycoproteins; Male; Microtubule-Associated Proteins; Models, Biological; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Reperfusion; Spectrin

The invariant characteristic features associated with Alzheimer's disease (AD) brain include the presence of extracellular neuritic plaques composed of amyloid beta (Abeta) peptide, intracellular neurofibrillary tangles containing hyper-phosphorylated tau protein and the loss of basal forebrain cholinergic neurons. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that in vivo accumulation of Abeta(1-42) may initiate the process of neurodegeneration observed in AD brains. However, the cause of degeneration of the basal forebrain cholinergic neurons and their association to Abeta peptides or phosphorylated tau protein have not been clearly established. In the present study, using rat primary septal cultures, we have shown that Abeta(1-42), in a time (1-48 h) and concentration (0.01-20 microM)-dependent manner, induce toxicity in cultured neurons. Subsequently, we have demonstrated that Abeta toxicity is mediated via activation of cysteine proteases, i.e., calpain and caspase, and proteolytic breakdown of their downstream substrates tau, microtubule-associated protein-2 and alpha II-spectrin. Additionally, Abeta-treatment was found to induce phosphorylation of tau protein along with decreased levels of phospho-Akt and phospho-Ser(9)glycogen synthase kinase-3beta. Exposure to specific inhibitors of caspase or calpain can partially protect cultured neurons against Abeta-induced toxicity but their effects are not found to be additive. These results, taken together, suggest that Abeta peptide can induce toxicity in rat septal cultured neurons by activating multiple intracellular signaling molecules. Additionally, evidence that inhibitors of caspase and calpains can partially protect the cultured basal forebrain neurons raised the possibility that their inhibitors could be of therapeutic relevance in the treatment of AD pathology.

Topics: Amyloid beta-Peptides; Animals; Calcium-Binding Proteins; Calpain; Caspases; Cell Death; Cells, Cultured; Dose-Response Relationship, Drug; Embryo, Mammalian; Enzyme Inhibitors; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Microscopy, Electron, Scanning; Microtubule-Associated Proteins; Neurofibrillary Tangles; Neurons; Oligopeptides; Peptide Fragments; Pregnancy; Rats; Septum of Brain; Tetrazolium Salts; Thiazoles; Time Factors

In hypertension, angiotensin (Ang) II is a critical mediator of cardiovascular remodeling, whose prominent features include myocardial and vascular media hypertrophy, perivascular inflammation, and fibrosis. The signaling pathways responsible for these alterations are not completely understood. Here, we investigated the importance of calpains, calcium-dependent cysteine proteases. We generated transgenic mice constitutively expressing high levels of calpastatin, a calpain-specific inhibitor. Chronic infusion of Ang II led to similar increases in systolic blood pressure in wild-type and transgenic mice. In contrast, compared with wild-type mice, transgenic mice displayed a marked blunting of Ang II-induced hypertrophy of left ventricle. Ang II-dependent vascular remodeling, ie, media hypertrophy and perivascular inflammation and fibrosis, was also limited in both large arteries (aorta) and small kidney arteries from transgenic mice as compared with wild type. In vitro experiments using vascular smooth muscle cells showed that calpastatin transgene expression blunted calpain activation by Ang II through epidermal growth factor receptor transactivation. In vivo and in vitro models of inflammation showed that impaired recruitment of mononuclear cells in transgenic mice was attributable to a decrease in both the release of and the chemotactic response to monocyte chemoattractant protein-1. Finally, results from collagen synthesis assay and zymography suggested that limited fibrogenesis was attributable to a decrease in collagen deposition rather than an increase in collagen degradation. These results indicate a critical role for calpains as downstream mediators in Ang II-induced cardiovascular remodeling and, thus, highlight an attractive therapeutic target.

Topics: Angiotensin II; Animals; Aorta; Blood Pressure; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Disease Models, Animal; Fibrosis; Genetic Therapy; Hypertension; Hypertrophy; Hypertrophy, Left Ventricular; Inflammation; Infusion Pumps, Implantable; Mice; Mice, Transgenic; Muscle, Smooth, Vascular; Myocardium; NF-kappa B; NFATC Transcription Factors; Renal Artery; Time Factors; Ventricular Remodeling

The objective of this study was to compare carcass characteristics of a newly introduced breed, the Waguli (Wagyu x Tuli), with the carcass characteristics of the Brahman breed. Brahman cattle are used extensively in the Southwest of the United States because of their tolerance to adverse environmental conditions. However, Brahman carcasses are discounted according to the height of their humps because of meat tenderness issues. The Waguli was developed in an attempt to obtain a breed that retained the heat tolerance of the Brahman but had meat quality attributes similar to the Wagyu. Twenty-four animals were used. Six steers from each breed were fed a 94% concentrate diet and 6 steers from each breed were fed an 86% concentrate diet. Eight steers, 2 from each group, were harvested after 128 d, after 142 d, and after 156 d on feed. Waguli steers had larger LM, greater backfat thickness, greater marbling scores, and greater quality grades than the Brahman steers (P < 0.05). The Japanese Wagyu breed is well known for its highly marbled and tender meat, and these traits are also present in the Waguli. The Waguli had significantly lower Warner-Bratzler shear force values than the Brahman steers after 7 and 10 d of postmortem aging (P < 0.05); this difference decreased after 14 d postmortem (P = 0.2), when tenderness of the slower aging Brahman had increased to acceptable levels. Toughness of the Brahman has been associated with high levels of calpastatin in Brahman muscle, and the Waguli LM had significantly less calpastatin activity (P = 0.02) at 0 h postmortem than the Brahman LM. At 0-h postmortem, the total LM calpain activity did not differ between the Brahman and Waguli (P = 0.57). Neither diet nor days on feed had any significant effect on the 0-h postmortem calpain or at 0-h postmortem calpastatin activity, nor an effect on Warner-Bratzler shear-force values. In conclusion, LM muscle from the Waguli steers had a high degree of marbling, lower shear force values, and low calpastatin activity, all of which are related to more tender meat.

Topics: Adipose Tissue; Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Body Composition; Breeding; Calcium-Binding Proteins; Calpain; Cattle; Food Technology; Male; Meat; Muscle, Skeletal; Phenotype; Random Allocation; Time Factors

The purpose of this study was to compare the responsiveness of changes in Ca(2+)-content and calpain-calpastatin gene expression to concentric and eccentric single-bout and repeated exercise. An exercise group (n = 14) performed two bouts of bench-stepping exercise with 8 weeks between exercise bouts, and was compared to a control-group (n = 6). Muscle strength and soreness and plasma creatine kinase and myoglobin were measured before and during 7 days following exercise bouts. Muscle biopsies were collected from m. vastus lateralis of both legs prior to and at 3, 24 h and 7 days after exercise and quantified for muscle Ca(2+)-content and mRNA levels for calpain isoforms and calpastatin. Exercise reduced muscle strength and increased muscle soreness predominantly in the eccentric leg (P < 0.05). These responses as well as plasma levels of creatine kinase and myoglobin were all attenuated after the repeated eccentric exercise bout (P < 0.05). Total muscle Ca(2+)-content did not differ between interventions. mRNA levels for calpain 2 and calpastatin were upregulated exclusively by eccentric exercise 24 h post-exercise (P < 0.05), with no alteration in expression between bouts. Calpain 1 and calpain 3 mRNA did not change at any specific time point post-exercise for either intervention. Our mRNA results suggest a regulation on the calpain-calpastatin expression response to muscle damaging eccentric exercise, but not concentric exercise. Although a repeated bout effect was demonstrated in terms of muscle function, no immediate support was provided to suggest that regulation of expression of specific system components is involved in the repeated bout adaptation.

Topics: Adaptation, Physiological; Adult; Calcium; Calcium-Binding Proteins; Calpain; Creatine Kinase; Exercise; Humans; Male; Muscle Contraction; Muscle Proteins; Muscle Strength; Myoglobin; Pain; Pain Measurement; Quadriceps Muscle; RNA, Messenger; Time Factors; Up-Regulation

Amyloid beta peptide (Abeta) is implicated in the pathogenesis of Alzheimer's disease (AD). The peptide is toxic to neurons, possibly by causing initial synaptic dysfunction and neuronal membrane dystrophy, promoted by increased cellular Ca(2+). Calpain (Ca(2+)-dependent protease) and caspase have also been implicated in AD. There is little information on communication between the two proteases or on the involvement of calpastatin (the specific calpain inhibitor) in Abeta toxicity. We studied the effects of Abeta25-35 (sAbeta) on calpain, calpastatin, and caspase in neuronal-like differentiated PC12 cells. sAbeta-treated cells exhibited primarily cell membrane damage (varicosities along neurites, enhanced membrane permeability to propidium iodide, without apparent nuclear changes of apoptosis, and little poly (ADP-ribose) polymerase [PARP] degradation). The sAbeta-induced membrane damage is in contrast with staurosporine-induced damage (nuclear apoptotic changes, PARP degradation, without membrane propidium iodide permeability). sAbeta led to activation of caspase-8 and calpain, promotion of calpastatin degradation (by caspase-8 and by calpain), and enhanced degradation of fodrin (mainly by calpain). The results support the idea that Abeta causes primarily neuronal membrane dysfunction, and point to cross-talk between calpain and caspase (protease activation and degradation of calpastatin) in Abeta toxicity. Increased expression of calpastatin and/or decrease in calpain and caspase-8 may serve as means for ameliorating early symptoms of AD.

Topics: Amyloid beta-Peptides; Animals; Blotting, Western; Calcium-Binding Proteins; Calpain; Carrier Proteins; Caspases; Cell Membrane; Cell Membrane Permeability; Electrophoresis, Polyacrylamide Gel; Microfilament Proteins; Neurons; PC12 Cells; Rats; Receptor Cross-Talk

Zinc and copper are trace elements essential for proper folding, stabilization and catalytic activity of many metalloenzymes in living organisms. However, disturbed zinc and copper homeostasis is reported in many types of cancer. We have previously demonstrated that copper complexes induced proteasome inhibition and apoptosis in cultured human cancer cells. In the current study we hypothesized that zinc complexes could also inhibit the proteasomal chymotrypsin-like activity responsible for subsequent apoptosis induction. We first showed that zinc(II) chloride was able to inhibit the chymotrypsin-like activity of a purified 20S proteasome with an IC(50) value of 13.8 microM, which was less potent than copper(II) chloride (IC(50) 5.3 microM). We then compared the potencies of a pyrrolidine dithiocarbamate (PyDT)-zinc(II) complex and a PyDT-copper(II) complex to inhibit cellular proteasomal activity, suppress proliferation and induce apoptosis in various human breast and prostate cancer cell lines. Consistently, zinc complex was less potent than copper complex in inhibiting the proteasome and inducing apoptosis. Additionally, zinc and copper complexes appear to use somewhat different mechanisms to kill tumor cells. Zinc complexes were able to activate calpain-, but not caspase-3-dependent pathway, while copper complexes were able to induce activation of both proteases. Furthermore, the potencies of these PyDT-metal complexes depend on the nature of metals and also on the ratio of PyDT to the metal ion within the complex, which probably affects their stability and availability for interacting with and inhibiting the proteasome in tumor cells.

Topics: Animals; Apoptosis; Blotting, Western; Calcium-Binding Proteins; Calpain; Caspase 3; Cell Line, Tumor; Chymotrypsin; Copper; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Female; Humans; In Situ Nick-End Labeling; Kinetics; Proteasome Inhibitors; Pyrrolidines; Rabbits; Tetrazolium Salts; Thiazoles; Thiocarbamates; Time Factors; Zinc

The genotypes for Beta-Lactoglobulin (BLG) and calpastatin (CAST) were determined by Polymerase Chain Reaction (PCR) and restriction enzyme digestion and genotyped for calpain (CAPN) by PCR-SSCP method in an Iranian breed sheep, Kurdi. Blood samples were collected from 100 pure bred Kurdi sheep from Kurdi breeding station located in Shirvan, Mashhad. The extraction of genomic DNA was based on Guanidin Thiocyanate-Silica gel method. After PCR reaction, amplicons were digested with restriction enzymes MspI and RsaI for beta-lactoglobulin and calpastatin genes, respectively. The beta-lactoglobulin locus had 3 genotypes with frequencies of 0.24, 0.54 and 0.22 for AA, AB and BB, respectively; calpastatin genotypes had 2 genotypes with frequencies of 0.76 and 0.24 for MM and MN genotypes, respectively. Calpain genotypes were analyzed with SSCP method, which had 2 genotypes with frequencies of 0.92 and 0.08 for AA and AB, respectively. Heterozygosity value for beta-lactoglobulin locus was 49% and for calpastatin and calpain loci was very low (24 and 8%, respectively). chi2 test confirmed the Hardy-Weinberg equilibrium for three loci in this population. These data provide evidence that Iranian's Kurdi sheep breed have a variability, which opens interesting prospects for future selection programs, especially marker-assistant selection between different genotypes of different locus and milk and cheese characteristics, gain and meat traits and also for preservation strategies.

Topics: Animals; Calcium-Binding Proteins; Calpain; Female; Genetic Variation; Genetics, Population; Genotype; Iran; Lactoglobulins; Meat; Polymorphism, Single-Stranded Conformational; Pregnancy; Sheep

This study is aimed at demonstrating the role played by a calpastatin isoform (Xcalp3) in Xenopus embryos. A specific monoclonal antibody (mAb) was raised against a glutathione S-transferase (GST)-Xcalp3 fusion protein and characterized by immunoblotting and confocal fluorescence microscopy on stage 20-36 embryos. Under these conditions, calpastatin reactivity is associated with a major 110kDa protein fraction and preferentially expressed by notochord and somitic cells. In notochord cells, anti-calpastatin reactive sites were initially restricted to the luminal space of the vacuoles and later became diffused throughout the cytoplasm. In contrast, anti-calpastatin reactive sites in somitic cells were initially diffused throughout the cytoplasm and became restricted to a few intracellular granules in the later developmental stages. At the ultrastructural level, notochord cells appeared as flattened discs containing several vacuoles and numerous electron-dense granules. During transition from stages 26 to 32, electron-dense granules were gradually reduced in number as vacuoles enlarged in size and losed their calpastatin reactivity. Electron-dense granules were also present in myoblast cells and their number gradually reduced during development. To determine whether these observations bear any causal relationship to the calpain/calpastatin system, a number of Xenopus embryos were examined both ultrastructurally and histochemically following exposure to a specific calpain inhibitor (CI3). Under these conditions, Xenopus embryos exhibited an altered right-left symmetry and an abnormal axial shortening. In CI3-treated stage 32 embryos, notochord cells had a reduced vacuolar extension and exhibited at the same time an increase in granular content. The overall morphology of the somites was also distorted and myoblasts were altered both in shape and granular content. Based on these findings, it is concluded that the calpain/calpastatin may play an important role in the control of notochord elongation and somite differentiation during Xenopus embryogenesis.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cytoplasm; Dipeptides; Embryo, Nonmammalian; Enzyme Inhibitors; Immunoblotting; Immunohistochemistry; Microscopy, Confocal; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Myoblasts; Notochord; Protein Isoforms; Somites; Vacuoles; Xenopus laevis

The ubiquitous calpains, mu- and m-calpain, are implicated in a variety of vital (patho)physiological processes and therefore cell-permeable specific inhibitors represent important tools for defining the role of calpains in cells and animal models. A synthetic N-acetylated 27-mer peptide derived from exon B of the human calpastatin inhibitory domain 1 is known to be the most potent and selective reversible inhibitor of calpains. To improve the membrane permeability of this peptidic inhibitor, it was N-terminally extended with or disulfide-linked to the C-terminal 7-mer fragment of penetratin, a well-established vector for cell membrane translocation of bioactive compounds. Despite the shorter penetratin sequence, both constructs showed increased cell permeability and retained their full calpain inhibitory potency.

Topics: Amino Acid Sequence; Calcium-Binding Proteins; Calpain; Carrier Proteins; Cell Line, Tumor; Cell Membrane Permeability; Cell-Penetrating Peptides; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Glycoproteins; Humans; Molecular Sequence Data

The mechanism for the decrease in cardiomyocyte size with mechanical unloading is unknown. The calpain system regulates cardiomyocyte atrophy. We obtained samples from failing human hearts at the time of implantation and explantation of a left ventricular assist device. For mechanical unloading, we also heterotopically transplanted rat or mouse hearts for 1 week. The effect of calpain inhibition on cardiac atrophy was assessed in transplanted hearts overexpressing calpastatin. We measured transcript levels of calpain 1 and 2 in the human and the rodent model, as well as calpain activity, a calpain-specific degradation product and cardiomyocyte size in the two rodent models. Mechanical unloading of the failing human heart significantly increased calpain 2 gene expression. Transcript levels of calpain 1 and 2, calpain activity and a calpain-specific degradation product all significantly increased in the unloaded rat heart. Unexpectedly, in hearts of animals overexpressing calpastatin, cardiomyocyte size also decreased. Mechanical unloading of the mammalian heart activates the calpain system, although other proteolytic systems may compensate for decreased calpain activity when calpastatin is overexpressed.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Size; Disease Models, Animal; Gene Expression Regulation; Heart Failure; Heart Transplantation; Heart-Assist Devices; Humans; Male; Mice; Middle Aged; Myocardium; Myocytes, Cardiac; Rats; Time Factors; Transplantation, Heterotopic

Previous studies from this laboratory have shown that catecholamines exert an inhibitory effect on muscle protein degradation through a pathway involving the cAMP cascade. The present work investigated the systemic effect of pentoxifylline (PTX; cAMP-phosphodiesterase inhibitor) treatment on the rate of overall proteolysis, the activity of proteolytic systems, and the process of protein synthesis in extensor digitorum longus muscles from normal and acutely diabetic rats. The direct in vitro effect of this drug on the rates of muscle protein degradation was also investigated. Muscles from diabetic rats treated with PTX showed an increase (22%) in the cAMP content and reduction in total rates of protein breakdown and in activity of Ca2+-dependent (47%) and ATP proteasome-dependent (23%) proteolytic pathways. The high content of m-calpain observed in muscles from diabetic rats was abolished by PTX treatment. The addition of PTX (10(-3) M) to the incubation medium increased the cAMP content in muscles from normal (22%) and diabetic (51%) rats and induced a reduction in the rates of overall proteolysis that was accompanied by decreased activity of the Ca2+-dependent and ATP proteasome-dependent proteolytic systems, in both groups. The in vitro addition of H-89, an inhibitor of protein kinase A (PKA), completely blocked the effect of PTX on the reduction of proteolysis in muscles from normal and diabetic rats. The present data suggest that PTX exerts a direct inhibitory effect on protein degradative systems in muscles from acutely diabetic rats, probably involving the participation of cAMP intracellular pathways and activation of PKA, independently of tumor necrosis factor-alpha inhibition.

Topics: Adenosine Triphosphate; Animals; Calcium; Calcium-Binding Proteins; Calpain; Cyclic AMP; Diabetes Mellitus, Experimental; Male; Muscle Proteins; Muscle, Skeletal; Pentoxifylline; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Rats; Rats, Wistar; Tumor Necrosis Factor-alpha

Although the calpain system has been studied extensively in mammalian animals, much less is known about the properties of mu-calpain, m-calpain, and calpastatin in lower vertebrates such as fish. These three proteins were isolated and partly characterized from rainbow trout, Oncorhynchus mykiss, muscle. Trout m-calpain contains an 80-kDa large subunit, but the approximately 26-kDa small subunit from trout m-calpain is smaller than the 28-kDa small subunit from mammalian calpains. Trout mu-calpain and calpastatin were only partly purified; identity of trout mu-calpain was confirmed by labeling with antibodies to bovine skeletal muscle mu-calpain, and identity of trout calpastatin was confirmed by specific inhibition of bovine skeletal muscle mu- and m-calpain. Trout mu-calpain requires 4.4+/-2.8 microM and trout m-calpain requires 585+/-51 microM Ca(2+) for half-maximal activity, similar to the Ca(2+) requirements of mu- and m-calpain from mammalian tissues. Sequencing tryptic peptides indicated that the amino acid sequence of trout calpastatin shares little homology with the amino acid sequences of mammalian calpastatins. Screening a rainbow trout cDNA library identified three cDNAs encoding for the large subunit of a putative m-calpain. The amino acid sequence predicted by trout m-calpain cDNA was 65% identical to the human 80-kDa m-calpain sequence. Gene duplication and polyploidy occur in fish, and the amino acid sequence of the trout m-calpain 80-kDa subunit identified in this study was 83% identical to the sequence of a trout m-calpain 80-kDa subunit described earlier. This is the first report of two isoforms of m-calpain in a single species.

Topics: Amino Acid Sequence; Animals; Base Sequence; Calcium; Calcium-Binding Proteins; Calpain; Cattle; Cloning, Molecular; DNA, Complementary; Molecular Sequence Data; Muscle, Skeletal; Oncorhynchus mykiss; Sequence Homology, Amino Acid

Regulating the balance between synthesis and proteasomal degradation of cellular proteins is essential for tissue growth and maintenance, but the critical pathways regulating protein ubiquitination and degradation are incompletely defined. Although participation of calpain calcium-activated proteases in post-necrotic myocardial autolysis is well characterized, their importance in homeostatic turnover of normal cardiac tissue is controversial. Hence, we evaluated the consequences of physiologic calpain (calcium-activated protease) activity in cultured cardiomyocytes and unstressed mouse hearts. Comparison of in vitro proteolytic activities of cardiac-expressed calpains 1 and 2 revealed calpain 1, but not calpain 2, activity at physiological calcium concentrations. Physiological calpain 1 activation was evident in adenoviral transfected cultured cardiomyocytes as proteolysis of specific substrates, generally increased protein ubiquitination, and accelerated protein turnover, that were each inhibited by coexpression of the inhibitor protein calpastatin. Conditional forced expression of calpain 1, but not calpain 2, in mouse hearts demonstrated substrate-specific proteolytic activity under basal conditions, with hyperubiquitination of cardiac proteins and increased 26S proteasome activity. Loss of myocardial calpain activity by forced expression of calpastatin diminished ubiquitination of 1 or more specific myocardial proteins, without affecting overall ubiquitination or proteasome activity, and resulted in a progressive dilated cardiomyopathy characterized by accumulation of intracellular protein aggregates, formation of autophagosomes, and degeneration of sarcomeres. Thus, calpain 1 is upstream of, and necessary for, ubiquitination and proteasomal degradation of a subset of myocardial proteins whose abnormal accumulation produces autophagosomes and degeneration of cardiomyocytes with functional decompensation.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Cardiomyopathy, Dilated; Cells, Cultured; Heart Failure; Homeostasis; Mice; Mice, Transgenic; Microscopy, Electron; Myocardium; Myocytes, Cardiac; Osmolar Concentration; Proteasome Endopeptidase Complex; Protein Isoforms; Proteins; Substrate Specificity; Transfection; Ubiquitin

Disruption of axonal transport may represent a final common pathway leading to neurological dysfunction in cerebral malaria (CM). Calpains are calcium (Ca2+)-activated cysteine proteases which have been implicated in axonal injury in neurological diseases of various aetiologies. In this study we examined the association between mu- and m-calpain, the specific inhibitor calpastatin, and axonal injury in post mortem brain tissue from patients who died from severe malaria. Calpains were associated with axons labelled for the beta-amyloid precursor protein that detects impaired axonal transport. Elevated levels of calpastatin were rarely observed in injured axons. There were increased numbers of neurones with mu-calpain in the nuclear compartment in severe malaria cases compared with non-neurological controls, and increased numbers of glia with nuclear mu-calpain in CM patients compared with non-CM malaria cases and non-neurological controls. There was marked redistribution of calpastatin in the sequestered Plasmodium falciparum-infected erythrocytes. Responses specific to malaria infection were ascertained following analysis of brain samples from fatal cases with acute axonal injury, HIV encephalitis, and progressive multifocal leucoencephalopathy. Our findings implicate a role for calpains in the modulation of disease progression in CM.

Topics: Adult; Aged; AIDS Dementia Complex; Axonal Transport; Axons; Calcium-Binding Proteins; Calpain; Endothelium, Vascular; Erythrocytes; Female; Humans; Immunohistochemistry; Leukoencephalopathy, Progressive Multifocal; Malaria, Falciparum; Male; Middle Aged; Neuroglia; Neurons

Unloading of skeletal muscle by hindlimb unweighting (HU) is characterized by atrophy, protein loss, and an elevation in intracellular Ca(2+) levels that may be sufficient to activate Ca(2+)-dependent proteases (calpains). In this study, we investigated the time course of calpain activation and the depletion pattern of a specific structural protein (desmin) with unloading and subsequent reweighting. Rats underwent 12 h, 24 h, 72 h or 9 days of HU, followed by reweighting for either 0, 12 or 24 h. Total calpain-like activity was elevated with HU in skeletal muscle (P < 0.05) and was further enhanced with reweighting (P < 0.05). The increases in calpain-like activity were associated with a proportional increase in activity of the particulate fraction (P < 0.05). Activity of the mu-calpain isoform was elevated with 12 and 24 h of HU (P < 0.05) and returned to control levels thereafter. With reweighting, activities of mu-calpain were elevated above control levels for all HU groups except 9 days (P < 0.05). In contrast, minimal changes in m-calpain and calpastatin activity were observed with HU and reweighting. Although desmin depletion levels did not reach statistical significance, a significant inverse relationship was found between the mu-calpain/calpastatin ratio and the amount of desmin in isolated myofibrils (R = -0.83, P < 0.001). The results suggest that calpain activation is an early event during unloading in skeletal muscle, and that the majority of the increase in calpain activity can be attributed to the micro-isoform.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Desmin; Hindlimb Suspension; Isoenzymes; Male; Models, Animal; Muscle, Skeletal; Protein Isoforms; Rats; Rats, Wistar; Time Factors

In this study, we investigated whether there is a signalling interaction between calpain and caspase-3 during apoptosis in Jurkat T cells by Entamoeba histolytica. When Jurkat cells were co-incubated with E. histolytica, phosphatidylserine externalisation and DNA fragmentation markedly increased compared with results for cells incubated with medium alone. In addition, E. histolytica strongly induced cleavage of caspases-3, -6, -7 and poly(ADP-ribose) polymerase. A rise in intracellular calcium levels and activation of calpain were seen in Jurkat cells after exposure to E. histolytica. Pretreatment of Jurkat cells with calpain inhibitor calpeptin effectively blocked E. histolytica-triggered cleavage of caspase-3 as well as calpain. In contrast, pan-caspase inhibitor did not affect E. histolytica-induced calpain activation. In addition, incubation with E. histolytica resulted in multiple fragmented bands of calpastatin, which is an endogenous inhibitor of calpain, in Jurkat T cells. Moreover, Entamoeba-induced calpastatin degradation was dramatically suppressed by pretreatment with calpeptin, but not by z-VAD-fmk. Entamoeba-induced DNA fragmentation was strongly retarded by z-VAD-fmk, but not calpeptin. Our results suggest that calpain-mediated calpastatin degradation plays a crucial role in regulation of caspase-3 activation during apoptosis of Jurkat T cells by E. histolytica.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Calcium; Calcium-Binding Proteins; Calpain; Caspase 3; Caspase Inhibitors; Dipeptides; DNA Cleavage; Entamoeba histolytica; Entamoebiasis; Enzyme Activation; Host-Parasite Interactions; Humans; Jurkat Cells; Parasitology; T-Lymphocytes

Calpastatin is the endogenous, specific protein inhibitor of the calcium-dependent protease, calpain. Using an active site knock-out m-calpain mutant we have studied the enzyme's calcium-dependent binding to calpastatin by surface plasmon resonance without the complication of proteolysis. Calpastatin was capable of simultaneously binding four molecules of calpain. Its four inhibitory domains (CAST1, 2, 3, and 4) were individually expressed in Escherichia coli and the kinetics of their interaction with calpain was separately compared. Their K(d) values ranged from picomolar to nanomolar in the order CAST1>4>3>2. They have similar k(on) values but the k(off) values ranged over three orders of magnitude and can account for the differences in affinity.

Topics: Animals; Calcium-Binding Proteins; Calpain; In Vitro Techniques; Kinetics; Models, Biological; Protein Binding; Protein Structure, Tertiary; Rats; Recombinant Proteins

The calpain gene family and its inhibitors have diverse effects, many related to protein turnover, which appear to affect a range of phenotypes such as diabetes, exercise-induced muscle injury, and pathological events associated with degenerative neural diseases in humans, fertility, longevity, and postmortem effects on meat tenderness in livestock species. The calpains are inhibited by calpastatin, which binds directly to calpain. Here we report the direct measurement of epistatic interactions of causative mutations for quantitative trait loci (QTL) at calpain 1 (CAPN1), located on chromosome 29, with causative mutations for QTL variation at calpastatin (CAST), located on chromosome 7, in cattle. First we identified potential causative mutations at CAST and then genotyped these along with putative causative mutations at CAPN1 in >1500 cattle of seven breeds. The maximum allele substitution effect on the phenotype of the CAPN1:c.947G>C single nucleotide polymorphism (SNP) was 0.14 sigma(p) (P = 0.0003) and of the CAST:c.155C>T SNP was also 0.14 sigma(p) (P = 0.0011) when measured across breeds. We found significant epistasis between SNPs at CAPN1 and CAST in both taurine and zebu derived breeds. There were more additive x dominance components of epistasis than additive x additive and dominance x dominance components combined. A minority of breed comparisons did not show epistasis, suggesting that genetic variation at other genes may influence the degree of epistasis found in this system.

Topics: Animals; Base Sequence; Breeding; Calcium-Binding Proteins; Calpain; Cattle; DNA Primers; Epistasis, Genetic; Humans; Linkage Disequilibrium; Mutation; Phenotype; Polymorphism, Single Nucleotide; Quantitative Trait Loci; Species Specificity

Agrin induces, whereas acetylcholine (ACh) disperses, ACh receptor (AChR) clusters during neuromuscular synaptogenesis. Such counteractive interaction leads to eventual dispersal of nonsynaptic AChR-rich sites and formation of receptor clusters at the postjunctional membrane. However, the underlying mechanisms are not well understood. Here we show that calpain, a calcium-dependent protease, is activated by the cholinergic stimulation and is required for induced dispersion of AChR clusters. Interestingly, the AChR-associated protein rapsyn interacted with calpain in an agrin-dependent manner, and this interaction inhibited the protease activity of calpain. Disrupting the endogenous rapsyn/calpain interaction enhanced CCh-induced dispersion of AChR clusters. Moreover, the loss of AChR clusters in agrin mutant mice was partially rescued by the inhibition of calpain via overexpressing calpastatin, an endogenous calpain inhibitor, or injecting calpeptin, a cell-permeable calpain inhibitor. These results demonstrate that calpain participates in ACh-induced dispersion of AChR clusters, and rapsyn stabilizes AChR clusters by suppressing calpain activity.

Topics: Agrin; Animals; Calcium-Binding Proteins; Calpain; Carbachol; Cell Line; Cholinergic Agonists; Humans; Mice; Mice, Transgenic; Muscle Fibers, Skeletal; Muscle Proteins; Neuromuscular Junction; Receptor Aggregation; Receptors, Cholinergic; Signal Transduction; Synaptic Membranes

MYOC, a gene involved in different types of glaucoma, encodes myocilin, a secreted glycoprotein of unknown function, consisting of an N-terminal leucine-zipper-like domain, a central linker region, and a C-terminal olfactomedin-like domain. Recently, we have shown that myocilin undergoes an intracellular endoproteolytic processing. We show herein that the proteolytic cleavage in the linker region splits the two terminal domains. The C-terminal domain is secreted to the culture medium, whereas the N-terminal domain mainly remains intracellularly retained. In transiently transfected 293T cells, the cleavage was prevented by calpain inhibitors, such as calpeptin, calpain inhibitor IV, and calpastatin. Since calpains are calcium-activated proteases, we analyzed how changes in either intra- or extracellular calcium affected the cleavage of myocilin. Intracellular ionomycin-induced calcium uptake enhanced myocilin cleavage, whereas chelation of extracellular calcium by EGTA inhibited the proteolytic processing. Calpains I and II cleaved myocilin in vitro. However, in cells in culture, only RNA interference knockdown of calpain II reduced myocilin processing. Subcellular fractionation and digestion of the obtained fractions with proteinase K showed that full-length myocilin resides in the lumen of the endoplasmic reticulum together with a subpopulation of calpain II. These data revealed that calpain II is responsible for the intracellular processing of myocilin in the lumen of the endoplasmic reticulum. We propose that this cleavage might regulate extracellular interactions of myocilin, contributing to the control of intraocular pressure.

Topics: Calcium-Binding Proteins; Calpain; Cell Line; Cytoskeletal Proteins; Dipeptides; Egtazic Acid; Endopeptidase K; Eye Proteins; Glaucoma; Glycoproteins; Humans; Ionomycin; Mutation, Missense; Protein Conformation; Protein Structure, Tertiary; Transfection

Calpain and calpastatin have been demonstrated to play many physiological roles in a variety of systems. It, therefore, appears important to study their localization and association in different suborganelles. Using immunoblot studies, we have identified 80 kDa m-calpain in both lumen and membrane of ER isolated from bovine pulmonary artery smooth muscle. Treatment of the ER with Na(2)CO(3) and proteinase K demonstrated that 80 kDa catalytic subunit and 28 kDa regulatory subunit (Rs) of m-calpain, and the 110-kDa and 70-kDa calpastatin (Cs) forms are localized in the cytosolic side of the ER membrane. Coimmunoprecipitation studies revealed that m-calpain is associated with calpastatin in the cytosolic face of the ER membrane. We have also identified m-calpain activity both in the ER membrane and lumen by casein-zymography. The casein-zymogram has also been utilized to demonstrate differential pattern of the effects of reversible and irreversible cysteine protease inhibitors on m-calpain activity. Thus, a potential site of Cs regulation of m-calpain activity is created by positioning Cs, 80 kDa and 28 kDa m-calpain in the cytosolic face of ER membrane. However, such is not the case for the 80-kDa m-calpain found within the lumen of the ER because of the conspicuous absence of 28 kDa Rs of m-calpain and Cs in this locale.

Topics: Animals; Calcium-Binding Proteins; Calpain; Carbonates; Caseins; Cattle; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum; Muscle, Smooth, Vascular; Pulmonary Artery

The slow-channel myasthenic syndrome (SCS) is a hereditary disorder of the acetylcholine receptor (AChR) of the neuromuscular junction (NMJ) that leads to prolonged AChR channel opening, Ca(2+) overload, and degeneration of the NMJ. We used an SCS transgenic mouse model to investigate the role of the calcium-activated protease calpain in the pathogenesis of synaptic dysfunction in SCS. Cleavage of a fluorogenic calpain substrate was increased at the NMJ of dissociated muscle fibers. Inhibition of calpain using a calpastatin (CS) transgene improved strength and neuromuscular transmission. CS caused a 2-fold increase in the frequency of miniature endplate currents (MEPCs) and an increase in NMJ size, but MEPC amplitudes remained reduced. Persistent degeneration of the NMJ was associated with localized activation of the non-calpain protease caspase-3. This study suggests that calpain may act presynaptically to impair NMJ function in SCS but further reveals a role for other cysteine proteases whose inhibition may be of additional therapeutic benefit in SCS and other excitotoxic disorders.

Topics: Animals; Calcium-Binding Proteins; Calpain; Disease Models, Animal; Humans; Mice; Mice, Transgenic; Muscle, Skeletal; Myasthenic Syndromes, Congenital; Neuromuscular Junction; Synaptic Transmission

Using calpastatin antibody we have identified a 145 kDa major band along with two relatively minor bands at 120 kDa and 110 kDa calpastatin molecules in bovine pulmonary artery smooth muscle mitochondria. To the best of our knowledge this is first report regarding the identification of calpastatin in mitochondria. We also demonstrated the presence of micro-calpain in the mitochondria by immunoblot and casein zymogram studies. Immunoblot studies identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of mu-calpain. Additionally 76 kDa, 40 kDa and 18 kDa immunoreactive bands have also been detected. Purification and N-terminal amino acid sequence analysis of the identified proteins confirmed their identity as mu-calpain and calpastatins. Immunoprecipitation study revealed molecular association between mu-calpain and calpastatin in the mitochondria indicating that calpastatin could play an important role in preventing uncontrolled activity of mu-calpain which otherwise may facilitate pulmonary hypertension, smooth muscle proliferation and apoptosis.

Topics: Amino Acid Sequence; Animals; Calcium-Binding Proteins; Calpain; Cattle; In Vitro Techniques; Mitochondria, Muscle; Molecular Sequence Data; Muscle, Smooth; Pulmonary Artery

Evidence has indicated that mu-calpain, m-calpain, and calpastatin have important roles in the proteolytic degradation that results in postmortem tenderization. Simple assays of these 3 proteins at different times postmortem, however, has shown that calpastatin and mu-calpain both rapidly lose their activity during postmortem storage, so that proteolytic activity of mu-calpain is nearly zero after 3 d postmortem, even when assayed at pH 7.5 and 25 degrees C, and ability of calpastatin to inhibit the calpains is 30% or less of its ability when assayed at death. m-Calpain, however, retains much of its proteolytic activity during postmortem storage, but the Ca(2+) requirement of m-calpain is much higher than that reported to exist in postmortem muscle. Consequently, it is unclear how the calpain system functions in postmortem muscle. To clarify this issue, we have initiated attempts to purify the 2 calpains and calpastatin from bovine semitendinosus muscle after 11-13 d postmortem. The known properties of the calpains and calpastatin in postmortem muscle have important effects on approaches that can be used to purify them. A hexyl-TSK hydrophobic interaction column is a critical first step in separating calpastatin from the 2 calpains in postmortem muscle. Dot-blot assays were used to detect proteolytically inactive mu-calpain. After 2 column chromatographic steps, 5 fractions can be identified: 1) calpastatin I that does not bind to an anion-exchange matrix, that does not completely inhibit the calpains, and that consists of small polypeptides <60 kDa; 2) calpastatin II that binds weakly to an anion-exchange matrix and that contains polypeptides <60 kDa; all these polypeptides are smaller than the native 115- to 125-kDa skeletal muscle calpastatin; 3) proteolytically active mu-calpain even though very little mu-calpain activity can be detected in zymogram assays of muscle extracts from 11- to 13-d postmortem muscle; this mu-calpain has an autolyzed 76-kDa large subunit but the small subunit consists of 24-, 26- and a small amount of unautolyzed 28-kDa polypeptides; 4) proteolytically active m-calpain that is not autolyzed; and 5) proteolytically inactive mu-calpain whose large subunit is autolyzed to a 76-kDa polypeptide and whose small subunit contains polypeptides similar to the proteolytically active mu-calpain. Hence, loss of calpastatin activity in postmortem muscle is due to its degradation, but the cause of the loss of mu-calpain activity remains unk

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Food Handling; Food Preservation; Meat; Molecular Weight; Muscle, Skeletal; Postmortem Changes; Time Factors

Fast, efficiently growing animals have increased protein synthesis and/or reduced protein degradation relative to slow, inefficiently growing animals. Consequently, minimizing the energetic cost of protein turnover is a strategic goal for enhancing animal growth. Characterization of gene expression profiles associated with protein turnover would allow us to identify genes that could potentially be used as molecular biomarkers to select for germplasm with improved protein accretion.. We evaluated changes in hepatic global gene expression in response to 3-week starvation in rainbow trout (Oncorhynchus mykiss). Microarray analysis revealed a coordinated, down-regulated expression of protein biosynthesis genes in starved fish. In addition, the expression of genes involved in lipid metabolism/transport, aerobic respiration, blood functions and immune response were decreased in response to starvation. However, the microarray approach did not show a significant increase of gene expression in protein catabolic pathways. Further studies, using real-time PCR and enzyme activity assays, were performed to investigate the expression of genes involved in the major proteolytic pathways including calpains, the multi-catalytic proteasome and cathepsins. Starvation reduced mRNA expression of the calpain inhibitor, calpastatin long isoform (CAST-L), with a subsequent increase in the calpain catalytic activity. In addition, starvation caused a slight but significant increase in 20S proteasome activity without affecting mRNA levels of the proteasome genes. Neither the mRNA levels nor the activities of cathepsin D and L were affected by starvation.. These results suggest a significant role of calpain and 20S proteasome pathways in protein mobilization as a source of energy during fasting and a potential association of the CAST-L gene with fish protein accretion.

Topics: Animals; Calcium-Binding Proteins; Calpain; Computer Systems; Energy Metabolism; Fish Diseases; Gene Expression Profiling; Gene Expression Regulation; Oligonucleotide Array Sequence Analysis; Oncorhynchus mykiss; Peptide Hydrolases; Polymerase Chain Reaction; Proteasome Endopeptidase Complex; Protein Biosynthesis; Proteins; Starvation

Aging is associated with a progressive and involuntary loss of muscle mass also known as sarcopenia. This condition represents a major public health concern with high socio-economics implications. Although sarcopenia is well documented, the aetiology of this condition still remains poorly understood. Calpains are ubiquitous proteases regulated in part by a specific inhibitor, calpastatin. They are well known to have major implications in muscle growth and differentiation. The aim of the present study was to determine if this proteolytic system could be involved in the phenotype associated with sarcopenia. Calpains and calpastatin levels, subcellular distributions and activities were compared between muscles from 3 and 24 months old rats. Altogether, the results we obtained showed an overall increase in calpain activities associated with muscle aging. These findings suggest that the calcium-dependent proteolytic system is indeed involved in sarcopenia.

Topics: Aging; Animals; Biomarkers; Calcium; Calcium-Binding Proteins; Calpain; Male; Muscle, Skeletal; Protein Processing, Post-Translational; Rats; Rats, Sprague-Dawley

Ca2+ loading of Jurkat and bovine aorta endothelium cells induces the degradation of the neuronal and endothelial nitric oxide synthases that are selectively expressed in these cell lines. For neuronal nitric oxide synthase, this process involves a conservative limited proteolysis without appreciable loss of catalytic activity. By contrast, endothelial nitic oxide synthase digestion proceeds through a parallel loss of protein and catalytic activity. The chaperone heat shock protein 90 (HSP90) is present in a large amount in Jurkat cells and at significantly lower levels in bovine aorta endothelium cells. The differing ratios of HSP90/nitric oxide synthase (NOS) occurring in the two cell types are responsible for the conservative or nonconservative digestion of NOS isozymes. Consistently, we demonstrate that, in the absence of Ca2+, HSP90 forms binary complexes with NOS isozymes or with calpain. When Ca2+ is present, a ternary complex containing the three proteins is produced. In this associated state, HSP90 and NOS forms are almost completely resistant to calpain digestion, probably due to a structural hindrance and a reduction in the catalytic efficiency of the protease. Thus, the recruitment of calpain in the HSP90-NOS complexes reduces the extent of the proteolysis of these two proteins. We have also observed that calpastatin competes with HSP90 for the binding of calpain in reconstructed systems. Digestion of the proteins present in the complexes can occur only when free active calpain is present in the system. This process can be visualized as a novel mechanism involving the association of NOS with HSP90 and the concomitant recruitment of active calpain in ternary complexes in which the proteolysis of both NOS isozymes and HSP90 is significantly reduced.

Topics: Animals; Aorta; Calcium-Binding Proteins; Calpain; Cattle; Cells, Cultured; Cysteine Proteinase Inhibitors; Endothelial Cells; Endothelium, Vascular; Erythrocytes; HSP90 Heat-Shock Proteins; Humans; Hydrolysis; Isoenzymes; Jurkat Cells; Models, Biological; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; Precipitin Tests

Calpains (calcium-activated neutral proteases) of sea bass (Dicentrarchus labrax L.) muscle may participate in the degradation of muscle tissue during postmortem storage. These enzymes are regulated by calpastatin, their endogenous specific inhibitor. The objective of this study was to evaluate the changes encountered by the calpain system during the postmortem storage of fish muscle after high-pressure treatment. From 100 MPa, high-pressure treatment of purified calpains results in a loss of their activity as well as in the dissociation of the heterodimeric form. In muscle, the high-pressure processing decreases the initial activity of calpain. This loss in activity may be due to an inactivation by a change of structure. Initial calpastatin activity is not modified by the high-pressure treatment, but it decreases during the storage from the beginning for a treatment at 300 MPa after which calpastatin is stable during 2 d. Therefore, this study also suggests that high-pressure treatment could be a useful way to improve fish flesh quality.

Topics: Animals; Bass; Calcium-Binding Proteins; Calpain; Food Handling; Food Preservation; Muscle, Skeletal; Postmortem Changes; Pressure; Seafood

Limited proteolysis of multiple intracellular proteins by endogenous Ca-dependent cysteine proteases--calpains--is an important regulatory mechanism for cell proliferation, apoptosis etc. Its importance for cellular functions is stressed by existence of endogenous calpain inhibitors--calpastatins. The calpain-calpastatin system within living cells is in a fragile balance, which depends on both partners. The interdependence of calpain--a protease--and calpastatin--an endogenous inhibitor and at the same time a substrate for this enzyme makes any assessment of actual activity of this enzyme in the cells very difficult. In this work we made an attempt to estimate and compare the activity of calpain in human peripheral blood lymphocytes by assessing the levels of limited proteolysis of calpastatin in these cells by western blot, while at the same time the levels of calpain protein inside these cells was measured by flow cytometry. Our results indicate that it is possible to compare (semi-quantitatively) the activities of calpain in peripheral blood CD4+ and CD19+ lymphocytes from various donors that way. Preliminary results showed that calpain activity is increased in the CD4+ T cells isolated from peripheral blood of rheumatoid arthritis patients as compared to control lymphocytes. Extremely high intrinsic activity of calpain was detected in chronic lymphocytic leukemia (CD19+) cells. All this confirms the detection of immunoreactive products of calpastatin as a good maker of endogenous calpain activity.

Topics: Antigens, CD19; Blotting, Western; Calcium-Binding Proteins; Calpain; CD4 Antigens; Enzyme Activation; Flow Cytometry; Humans; Lymphocytes; Peptide Fragments; Protein Processing, Post-Translational

Lens-specific Lp82 and ubiquitous m-calpain are neutral, calcium-activated, cysteine proteases. Both calpains are activated during rodent lens maturation and cataract formation. Lp85 calpain (Lens protein with MW=85 kDa) is a slightly larger splice variant of Lp82. Lp85 contains a 28 amino acid insert peptide (IS3) in calcium binding domain IV. Theoretically, the insert could alter the properties of Lp85 and influence proteolytic activity. The purpose of the present experiment was to compare the biochemical properties of Lp85 to Lp82 and m-calpain. Recombinant Lp85 and Lp82 were separately expressed using the baculovirus system and partially purified using Co2+ affinity and DEAE chromatographies. Calcium activation, pH dependency, and susceptibility to calpain inhibitors were assessed in a protease assay using BODIPY fluorescence-labeled casein substrate. Hydrolysis of lens proteins was assessed by SDS-PAGE and immunoblotting. Cleavage site analysis was performed by mass spectroscopy and Edman sequencing. Computer-based homology modeling was used to predict the influence of the IS3 region on the 3-dimensional structure of Lp85. Compared to m-calpain, Lp85 showed a lower calcium-activation requirement (K(50%act)=20 microM), marked insensitivity to, and cleavage of, the endogenous tissue inhibitor of calpains-calpastatin, and different preferred cleavage sites on alphaA-crystallin (five amino acid C-terminal truncation) and on aquaporin 0 (G239 and N246). Although the IS3 insert was predicted to form a loop protruding from the calcium binding region of Lp85, the biochemical properties of Lp85 studied were nearly identical to those of Lp82. Lp85 and Lp82 did not catalyze hydrolysis of each other, but both hydrolyzed m-calpain. Lp85 seems to be the enzymatic equivalent of Lp82. Both calpains could become active at lower cellular calcium levels than m-calpain. Lp85/Lp82 may have different functions than m-calpain since they cleave substrates at different sites. Lp85/Lp82 may regulate m-calpain activity by catalyzing the hydrolysis of calpastatin. The function of the IS3 insert on Lp85 remains unknown but is speculated to control subcellular distribution.

Topics: Animals; Baculoviridae; Calcium; Calcium-Binding Proteins; Calpain; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Genetic Vectors; Humans; Immunoblotting; Insecta; Lens, Crystalline; Mass Spectrometry; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Sequence Analysis, DNA; Sequence Homology

The goal of this study was to assess if neurons exposed to amyloid-beta peptide (Abeta) exclusively in distal axons, undergo apoptosis. This is relevant to the loss of cholinergic neurons in Alzheimer's disease. Using a three-compartmented culture system for rat sympathetic neurons, we demonstrate that exposure of axons to Abeta1-42 activates an independent destruction program in axons, which leads to nuclear apoptosis. Abeta-induced axonal degeneration does not involve local caspase activation, but causes caspase activation in cell bodies. Accordingly, inhibition of caspase activation blocks Abeta-induced apoptosis but not axonal degeneration. In agreement with previous suggestions that disruption of nerve growth factor (NGF)-mediated signaling might contribute to the loss of cholinergic neurons, we found that provision of NGF to cell bodies protects sympathetic neurons from Abeta-induced apoptosis. However, our data indicate that Abeta-induced axonal degeneration follows a mechanism different than that activated by NGF withdrawal. Only Abeta-induced axonal degeneration is prevented by the calpain inhibitor calpastatin and is insensitive to the inhibitor of the ubiquitin-proteasome system MG132. Importantly, inhibition of Abeta-induced axonal degeneration by calpastatin prevents nuclear apoptosis.

Topics: Amyloid beta-Peptides; Animals; Animals, Newborn; Antidotes; Apoptosis; Axons; Blotting, Western; Calcium-Binding Proteins; Calpain; Cells, Cultured; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Drug Interactions; Egtazic Acid; Nerve Degeneration; Nerve Growth Factor; Neurons; Peptide Fragments; Prosencephalon; Rats; Rats, Sprague-Dawley; Superior Cervical Ganglion; Tetrazolium Salts; Thiazoles

The Ca2+-activated protease calpain has been shown to play a deleterious role in the heart during ischemia-reperfusion (I/R). We tested the hypothesis that exercise training would minimize I/R-induced calpain activation and provide cardioprotection against I/R-induced injury. Hearts from adult male rats were isolated in a working heart preparation, and myocardial injury was induced with 25 min of global ischemia followed by 45 min of reperfusion. In sedentary control rats, I/R significantly increased calpain activity and impaired cardiac performance (cardiac work during reperfusion = 24% of baseline). Compared with sedentary animals, exercise training prevented the I/R-induced rise in calpain activity and improved cardiac work (recovery = 80% of baseline). Similar to exercise, pharmacological inhibition of calpain activity resulted in comparable cardioprotection against I/R injury (recovery = 86% of baseline). The exercise-induced protection against I/R-induced calpain activation was not due to altered myocardial protein levels of calpain or calpastatin. However, exercise training was associated with increased myocardial antioxidant enzyme activity (Mn-SOD, catalase) and a reduction in oxidative stress. Importantly, exercise training also prevented the I/R-induced degradation of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a. These findings suggest that increases in endogenous antioxidants may diminish the free radical-mediated damage and/or degradation of Ca2+ handling proteins (such as SERCA2a) typically observed after I/R. In conclusion, these results support the concept that calpain activation is an important component of I/R-induced injury and that exercise training provides cardioprotection against I/R injury, at least in part, by attenuating I/R-induced calpain activation.

Topics: Animals; Calcium-Binding Proteins; Calcium-Transporting ATPases; Calpain; Enzyme Activation; Heart Function Tests; L-Lactate Dehydrogenase; Male; Myocardial Reperfusion Injury; Myocardium; Physical Conditioning, Animal; Rats; Sarcoplasmic Reticulum Calcium-Transporting ATPases

Neuronal calpains appear to be activated uncontrollably by sustained elevation of cytosolic calcium levels under pathological conditions as well as neurodegenerative diseases. In the present study, we have characterized calpain activation in cytosolic extract of mice cerebral cortex and cerebellum using an experimental model of fatal murine cerebral malaria (FMCM). Pathology of FMCM resulted in the increase in activity of calpains in both cerebral cortex and cerebellum. Western blot analysis revealed an increase in the levels of mu-calpain (calpain-1) in the cytosolic fraction of infected cerebral cortex and cerebellum although a decrease in the level of m-calpain was observed in the cytosolic fraction of infected cerebellum and cerebral cortex. Calpain activation was further confirmed by monitoring the formation of calpain-specific spectrin breakdown products (SBDP). Protease-specific SBDP revealed the formation of calpain-generated 150kDa product in the infected cerebral cortex and cerebellum. The specific signature fragment of calpain activation and spectrin breakdown after Plasmodium berghei ANKA infection provide a strong evidence of the role of calpains during the cell death in cerebral cortex and cerebellum. Given the role of calpains in neurodegeneration and cell death, our results strongly suggest that calpains are important mediators of cell injury and neurological sequelae associated with FMCM.

Topics: Animals; Blotting, Western; Brain; Calcium-Binding Proteins; Calpain; Enzyme Activation; Female; Hydrolysis; Immunohistochemistry; Malaria, Cerebral; Male; Mice; Spectrin

The effects of low intensity, low frequency magnetic fields (MFs) on catalytic activity of the calcium dependent protease calpain was determined following the enzyme activation both in "in vitro" and "in vivo" conditions. We have observed that a 0.3 mT MF induces a significant increase in the requirement of the protease for this metal ion. This change is detectable at low [Ca(2+)] and disappears when the level of Ca(2+) is raised to saturating amounts. The observed effects are not due to transient MF(-) induced conformational changes occurring in calpain, but to direct effects of the MF on Ca(2+) ions, which become less available for the binding sites present in calpain. Altogether, these results indicate that exposure to low intensity, low frequency MFs alters the intracellular Ca(2+) "availability," thereby modifying the related cell response.

Topics: Animals; Anion Exchange Protein 1, Erythrocyte; Calcium; Calcium-Binding Proteins; Calpain; Electromagnetic Fields; Enzyme Activation; Erythrocytes; Hemolysis; Humans; Rats

Calpastatin is an intrinsic intracellular inhibitor of calpain, a Ca(2+)-dependent thiol protease. The calpain-calpastatin system constitutes one functional proteolytic unit whose presence and function has already been investigated in various cell types, but not in the egg. We have previously shown that calpain is expressed in rat eggs and is activated upon egg activation. The present study was designed to investigate the calpain-calpastatin interplay throughout the process. Western blot analysis revealed two main calpastatin isoforms, the erythrocyte type (77 kDa) and the muscle tissue type (110 kDa). By immunohistochemistry and confocal laser scanning microscopy, we demonstrated that the 110 kDa calpastatin was localized at the membrane area and highly abundant at the meiotic spindle in eggs at the first and second meiotic divisions. The 77 kDa calpastatin isoform appeared to be localized as a cortical sphere of clusters. The 110 kDa calpastatin and beta-tubulin have both been localized to the spindle of metaphase II eggs, both being scattered all through the cytoplasm following spindle disruption by nocodazole treatment, implying a dynamic interaction between calpastatin and microtubule elements. Upon egg activation, membranous calpastatin translocated to the cortex whereas cortical millimolar (m)-calpain shifted towards the membrane. Spindle calpastatin and calpain remained static. We suggest that calpastatin serves as a regulator of m-calpain. The counter translocation of m-calpain and calpastatin could serve as a means of calpain escape from calpastatin inhibition and may reflect a step in the process of calpain activation, throughout egg activation, that is required for calpain to exert its proteolytic activity.

Topics: Animals; Biological Transport; Blotting, Western; Calcium-Binding Proteins; Calpain; Cell Membrane; Female; Fertilization; Ionomycin; Ionophores; Microscopy, Confocal; Molecular Weight; Ovum; Parthenogenesis; Protein Isoforms; Rats; Rats, Wistar

The retinal degeneration (rd)1 mouse displays an inherited retinal degeneration and therefore allows studies of the molecular mechanisms behind the blinding disease retinitis pigmentosa. Activation of the calcium-dependent protease calpain has been suggested to play an important role in cell death in various tissues, but little is known about the expression and activity of calpain during inherited retinal degeneration. Using microarray techniques, transcript levels of cyclic AMP response element-binding protein (CREB)-1, calpastatin and of various calpain genes were analysed in the rd1 mouse compared with its wild-type control. Expression of distinct calpain isoforms and calpastatin was investigated using immunofluorescence and immunoblotting. Gene transcription and protein expression levels were compared with calpain activity using an enzymatic assay that allowed monitoring of calpain activity at the cellular level. We found that CREB-1 and calpastatin expression was reduced in rd1 retinas, whereas calpain activity was substantially increased in rd1 photoreceptors. Calpain activity peaked at postnatal day 13, together with rd1 photoreceptor cell death. Calpain-specific inhibitors decreased calpain activity in situ. These results indicate that activation of calpains correlates with rd1 photoreceptor cell death, which raises the possibility of using calpain inhibitors to prevent or delay photoreceptor degeneration.

Topics: Age Factors; Animals; Animals, Newborn; Blotting, Western; Calcium-Binding Proteins; Calpain; Cyclic AMP Response Element-Binding Protein; Enzyme Activation; Fluorescent Antibody Technique; Gene Expression Regulation, Developmental; Glycoproteins; In Situ Nick-End Labeling; Mice; Mice, Inbred C3H; Microarray Analysis; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Transcription, Genetic

The mechanisms underlying dopamine neuron loss in Parkinson's disease (PD) are not clearly defined. Here, we delineate a pathway by which dopaminergic loss induced by 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine (MPTP) is controlled in vivo. We reported previously that calpains play a central required role in dopamine loss after MPTP treatment. Here, we provide evidence that the downstream effector pathway of calpains is through cyclin-dependent kinase 5 (cdk5)-mediated modulation of the transcription factor myocyte enhancer factor 2 (MEF2). We show that MPTP-induced conversion of the cdk5 activator p35 to a pathogenic p25 form is dependent on calpain activity in vivo. In addition, p35 deficiency attenuates MPTP-induced dopamine neuron loss and behavioral outcome. Moreover, MEF2 is phosphorylated on Ser444, an inactivating site, after MPTP treatment. This phosphorylation is dependent on both calpain and p35 activity, consistent with the model that calpain-mediated activation of cdk5 results in phosphorylation of MEF2 in vivo. Finally, we provide evidence that MEF2 is critical for dopaminergic loss because "cdk5 phosphorylation site mutant" of MEF2D provides neuroprotection in an MPTP mouse model of PD. Together, these data indicate that calpain-p35-p25/cdk5-mediated inactivation of MEF2 plays a critical role in dopaminergic loss in vivo.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Corpus Striatum; Cyclin-Dependent Kinase 5; Dopamine; Enzyme Activation; Male; MEF2 Transcription Factors; Mice; Mice, Inbred C57BL; Myogenic Regulatory Factors; Nerve Tissue Proteins; Neurons; Parkinsonian Disorders; Phosphorylation; Point Mutation; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Signal Transduction; Substantia Nigra

Muscle wasting in sepsis is a significant clinical problem because it results in muscle weakness and fatigue that may delay ambulation and increase the risk for thromboembolic and pulmonary complications. Treatments aimed at preventing or reducing muscle wasting in sepsis, therefore, may have important clinical implications. Recent studies suggest that sepsis-induced muscle proteolysis may be initiated by calpain-dependent release of myofilaments from the sarcomere, followed by ubiquitination and degradation of the myofilaments by the 26S proteasome. In the present experiments, treatment of rats with one of the calpain inhibitors calpeptin or BN82270 inhibited protein breakdown in muscles from rats made septic by cecal ligation and puncture. The inhibition of protein breakdown was not accompanied by reduced expression of the ubiquitin ligases atrogin-1/MAFbx and MuRF1, suggesting that the ubiquitin-proteasome system is regulated independent of the calpain system in septic muscle. When incubated muscles were treated in vitro with calpain inhibitor, protein breakdown rates and calpain activity were reduced, consistent with a direct effect in skeletal muscle. Additional experiments suggested that the effects of BN82270 on muscle protein breakdown may, in part, reflect inhibited cathepsin L activity, in addition to inhibited calpain activity. When cultured myoblasts were transfected with a plasmid expressing the endogenous calpain inhibitor calpastatin, the increased protein breakdown rates in dexamethasone-treated myoblasts were reduced, supporting a role of calpain activity in atrophying muscle. The present results suggest that treatment with calpain inhibitors may prevent sepsis-induced muscle wasting.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Line; Cysteine Proteinase Inhibitors; Dexamethasone; Dipeptides; Gene Expression; Glycoproteins; Hydrogen Peroxide; Male; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Myoblasts, Skeletal; Pepstatins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Rats, Sprague-Dawley; Sepsis; SKP Cullin F-Box Protein Ligases; Transfection; Tripartite Motif Proteins; Ubiquitin-Protein Ligases

Microsphere embolism (ME)-induced cerebral ischemia can elicit various pathological events leading to neuronal death. Western blotting and immunohistochemical studies revealed that expression of calpastatin, an endogenous calpain inhibitor, decreased after ME induction. Calpain activation after ME was apparently due to, in part, a decrease in calpastatin in a late phase of neuronal injury. The time course of that decrease also paralleled caspase-3 activation. In vitro studies demonstrated that calpastatin was degraded by caspase-3 in a Ca(2+)/calmodulin (CaM)-dependent manner. Because CaM binds directly to calpastatin, we asked whether a novel CaM antagonist, 3-[2-[4-(3-chloro-2-methylphenylmethyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydro-chloride 3.5 hydrate (DY-9760e), inhibits caspase-3-induced calpastatin degradation during ME-induced neuronal damage. We also tested the effect of DY-9760e on degradation of fodrin, a calpain substrate. Consistent with our hypothesis, DY-9760e (25 or 50 mg/kg i.p.) treatment inhibited degradation of calpastatin and fodrin in a dose-dependent manner. Because DY-9760e showed powerful neuroprotective activity with concomitant inhibition of calpastatin degradation, cross-talk between calpain and caspase-3 through calpastatin possibly accounts for ME-induced neuronal injury. Taken together, both inhibition of caspase-3-induced calpastatin degradation and calpain-induced fodrin breakdown by DY-9760e in part mediate its neuroprotective action.

Topics: Animals; Brain Ischemia; Calcium-Binding Proteins; Calmodulin; Calpain; Caspase 3; Caspases; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Indazoles; Intracranial Embolism; Male; Microspheres; Neurons; Neuroprotective Agents; Protein Binding; Rats; Rats, Wistar

The objective of this study was to assess the association of single nucleotide polymorphisms (SNP) developed at the calpastatin (CAST) and mu-calpain (CAPN1) genes with meat tenderness and palatability traits in populations with diverse genetic backgrounds. Three populations were used in the study. One population consisted of Bos taurus that included crossbred animals derived from Hereford, Angus, Red Angus, Limousin, Charolais, Gelbvieh, and Simmental (GPE7; n = 539). Another population consisted of Bos taurus with Bos indicus influence, including crossbred animals from Hereford, Angus, Brangus, Beefmaster, Bonsmara, and Romosinuano (GPE8; n = 580). The third population was Bos indicus and consisted of purebred Brahman (STARS; n = 444). Traits evaluated were meat tenderness measured as Warner-Bratzler shear force (WBSF; kg) at 14 d postmortem, and traits evaluated by trained sensory panels that included tenderness score, juiciness, and flavor intensity. A SNP at the CAST gene had a significant (P < 0.003) effect on WBSF and tenderness score in the GPE7 and GPE8 populations. Animals inheriting the TT genotype at CAST had meat that was more tender than those inheriting the CC genotype. The marker at the CAPN1 gene was significant (P < 0.03) for tenderness score in GPE7 and GPE8. Animals inheriting the CC genotype at CAPN1 had meat that was more tender than those inheriting the TT genotype. Markers at the CAST and CAPN1 genes were associated with flavor intensity in the GPE8 population. Animals inheriting the CC genotype at CAST and the TT genotype at CAPN1 produced steaks with an intense flavor when compared with the other genotypes. An interaction between CAST and CAPN1 was detected (P < 0.05) for WBSF on GPE8. The statistical significance of the interaction is questionable because of the limited number of observations in some cells. Markers developed at the CAST and CAPN1 genes are suitable for use in identifying animals with the genetic potential to produce meat that is more tender.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Female; Genetic Markers; Genetic Variation; Genotype; Male; Meat; Phenotype; Polymorphism, Single Nucleotide; Shear Strength

The calcium-dependent proteolytic system is a large family of well-conserved ubiquitous and tissue-specific proteases, known as calpains, and an endogenous inhibitor, calpastatin. Ubiquitous calpains are involved in many physiological phenomena, such as the cell cycle, muscle cell differentiation, and cell migration. This study investigates the regulation of crucial steps of cell motility, myoblast adhesion and spreading, by calpains. Inhibition of each ubiquitous calpain isoform by antisense strategy pinpointed the involvement of each of these proteases in myoblast adhesion and spreading. Moreover, the actin cytoskeleton and microtubules were observed in transfected cells, demonstrating that each ubiquitous calpain could be involved in the actin fiber organization. C2C12 cells with reduced mu- or m-calpain levels have a rounded morphology and disorganized stress fibers, but no modification in the microtubule cytoskeleton. Antisense strategy directed against MARCKS, a calpain substrate during C2C12 migration, showed that this protein could play a role in stress fiber polymerization. A complementary proteomic analysis using C2C12 cells over-expressing calpastatin indicated that two proteins were under-expressed, while six, which are involved in the studied phenomena, were overexpressed after calpain inhibition. The possible role of these proteins in adhesion, spreading, and migration was discussed.

Topics: Actins; Animals; Calcium-Binding Proteins; Calpain; Cell Adhesion; Cell Fusion; Cell Line; Cell Movement; Cytoskeleton; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Microscopy, Confocal; Microtubules; Myoblasts; Myristoylated Alanine-Rich C Kinase Substrate; Oligonucleotides, Antisense; Proteomics; Stress Fibers; Time Factors; Transfection

The objective of this study was to evaluate the effect of oxidation on mu- and m-calpain activity at varying pH and ionic strength conditions in the presence of calpastatin. In 2 separate experiments, purified porcine skeletal muscle mu- or m-calpain (0.45 units of caseinolytic activity) was incubated in the presence of calpastatin (0, 0.15, or 0.30 units) at pH 7.5, 6.5, or 6.0 with either 165 or 295 mM NaCl. The reactions were initiated with the addition of CaCl2 (100 microM for mu-calpain; 1 mM for m-calpain). In Experiment 1, mu- or m-calpain was incubated with the calpain substrate Suc-Leu-Leu-Val-Tyr-AMC (170 microM). Either 0 or 16 mu microM H2O2 was added to each assay. Activity was measured at 60 min. In Experiment 2, calpain was incubated with highly purified porcine myofibrils (4 mg/mL) under conditions described. Either 0 or 100 microM H2O2 was added immediately prior to the addition of calpain. Degradation of desmin was determined on samples collected at 2, 15, 60, and 120 min. Results from Experiment 1 indicated that oxidation decreased (P < 0.01) activity of mu-calpain. Mu-calpain had the greatest (P < 0.01) activity at pH 6.5, and m-calpain had the greatest (P < 0.01) activity at pH 7.5 at 60 min. m-Calpain activity was not detected at pH 6.0. Mu- and m-calpain activity were lower (P < 0.01) at 295 mM NaCl than at 165 mM NaCl at all pH conditions. Oxidation lowered (P < 0.01) calpastatin inhibition of mu-and m-calpain at all pH and ionic strength combinations. In Experiment 2, oxidation decreased proteolytic activity of mu-calpain against desmin at pH 6.0 (P < 0.05 at 15, 60, and 120 min) and decreased m-calpain at all pH conditions. However, desmin degradation by mu-calpain was not as efficiently inhibited by calpastatin at pH 7.5 and as at pH 6.5 (P = 0.03 at 60 min) when oxidizing conditions were created. This is consistent with the results from Experiment 1, which indicated that oxidation decreased the ability of calpastatin to inhibit mu-calpain. These studies provide evidence that oxidation influences calpain activity and inhibition of calpains by calpastatin differently under varying environmental conditions. The results suggest that, at the higher pH conditions used, calpastatin may limit the possibility of oxidation-induced inactivation of mu-calpain.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cathepsin A; Desmin; Hydrogen-Ion Concentration; Muscle, Skeletal; Myofibrils; Osmolar Concentration; Oxidation-Reduction; Swine

Previously we have shown that 90% of streptozotocin (STZ)-induced type-1 diabetic (DB) mice survive from acute renal failure (ARF) and death induced by a normally LD(90) dose (75 mg/kg, i.p.) of the nephrotoxicant S-1,2-dichlorovinyl-l-cysteine (DCVC). This remarkable protection is due to a combination of slower progression of DCVC-initiated renal injury and increased compensatory nephrogenic tissue repair in the DB kidneys. BRDU immunohistochemistry revealed that the DB condition led to 4-fold higher number of proximal tubular cells (PTC) entering S-phase of cell cycle. In the present study, we tested the hypothesis that DB-induced augmentation of PTC into S-phase is accompanied by overexpression of the calpain-inhibitor calpastatin, which endogenously prevents the progression of DCVC-initiated renal injury mediated by the calpain escaping out of damaged PTCs. Immunohistochemical detection of renal calpain and its activity in the urine, over a time course after treatment with the LD(90) dose of DCVC, indicated progressive increase in leakage of calpain into the extracellular spaces of the injured PTCs of the non-diabetic (NDB) kidneys as compared to the DB kidneys. Calpastatin expression was minimally detected in the NDB kidneys, using immunohistochemistry, over the time course. On the other hand, consistently higher number of tubules in the DB kidney showed calpastatin expression over the time course. The lower leakage of calpain in the DB kidneys was commensurate with constitutively higher expression of calpastatin in the S-phase-laden PTCs of these mice. To test the protective role of newly divided/dividing PTCs, DB mice were given the anti-mitotic agent colchicine (CLC) (2 mg/kg and 1.5 mg/kg, i.p., on days 8 and 10 after STZ injection) prior to challenge with a LD(90) dose of DCVC, which led to 100% mortality by 48 h. Mortality was due to rapid progression of DCVC-initiated renal injury, suggesting that newly divided/dividing cells are instrumental in mitigating the progression of DCVC-initiated renal injury in DB. The anti-mitotic effect of CLC in DB kidney is associated with lower expression of calpastatin and higher leakage of calpain in the injured tubules. These findings suggest that constitutively higher cell division in the DB kidney is associated with overexpression of calpastatin, which reduces the progression of DCVC-initiated renal injury mediated by calpain on the one hand and accelerates nephrogenic tissue repair on the other, thereby r

Topics: Acetylcysteine; Acute Kidney Injury; Animals; Antimitotic Agents; Calcium-Binding Proteins; Calpain; Colchicine; Cysteine Proteinase Inhibitors; Diabetes Mellitus, Experimental; Drug Therapy, Combination; Kidney Function Tests; Kidney Tubules, Proximal; Longevity; Male; Mice; Regeneration

Amyotrophic lateral sclerosis (ALS) is characterized by the selective degeneration of motor neurons. The cause for nerve cell demise is not clear but involves activation of the caspase family of cysteine proteases. We have shown that ER stress and caspase-12 activation occur in ALS transgenic mice carrying the mutant copper/zinc superoxide dismutase (SOD1) gene. In these mice, we found that the antiapoptotic proteins, X-linked Inhibitor of Apoptosis Protein (XIAP) and the related protein, MIAP2 were decreased. To study the role of this, we generated double transgenic mice expressing XIAP in ALS spinal cord neurons using the Thy1 promoter. Overexpression of XIAP inhibited caspase-12 cleavage and reduced calpain activity in the ALS mice. XIAP also reduced the breakdown of calpastatin that is an inhibitor of calpain. In the double transgenic mice, life span was increased by about 12%. These data support the view that XIAP has beneficial effects in ALS and extends survival. The neuroprotective effect of XIAP involves inhibition of caspases and the stabilization of the calpastatin/calpain system that is altered in the ALS mice.

Topics: Amyotrophic Lateral Sclerosis; Animals; Baculoviral IAP Repeat-Containing 3 Protein; Calcium-Binding Proteins; Calpain; Caspase 12; Caspases; Cell Survival; Cysteine Proteinase Inhibitors; Humans; Inhibitor of Apoptosis Proteins; Mice; Mice, Transgenic; Motor Neurons; Spinal Cord; Superoxide Dismutase; Superoxide Dismutase-1; Survival Rate; Ubiquitin-Protein Ligases; X-Linked Inhibitor of Apoptosis Protein

Conformational changes in the calpain molecule following interaction with natural ligands can be monitored by the binding of a specific monoclonal antibody directed against the catalytic domain of the protease. None of these conformational states showed catalytic activity and probably represent intermediate forms preceding the active enzyme state. In its native inactive conformation, calpain shows very low affinity for this monoclonal antibody, whereas, on binding to the ligands Ca(2+), substrate or calpastatin, the affinity increases up to 10-fold, with calpastatin being the most effective. This methodology was also used to show that calpain undergoes similar conformational changes in intact cells exposed to stimuli that induce either a rise in intracellular [Ca(2+)] or extensive diffusion of calpastatin into the cytosol without affecting Ca(2+) homeostasis. The fact that the changes in the calpain state are also observed under the latter conditions indicates that calpastatin availability in the cytosol is the triggering event for calpain-calpastatin interaction, which is presumably involved in the control of the extent of calpain activation through translocation to specific sites of action.

Topics: Animals; Antibodies, Monoclonal; Binding Sites; Brain; Calcium; Calcium-Binding Proteins; Calpain; Catalysis; Cysteine Proteinase Inhibitors; Cytosol; Erythrocytes; Humans; Jurkat Cells; Ligands; Neutrophils; Protein Conformation; Rats

Changes in proteolytic activity of the myocardium during the development of heart failure after left coronary artery ligation (CAL) of rats were examined. Hemodynamics of the rats at the eighth week (8w-CAL rat), but not at the second week (2w-CAL rat), after CAL showed the symptoms of chronic heart failure. Contents of mu-calpin and m-calpain, but not an intrinsic calpain inhibitor calpastatin, in the viable left ventricular muscle (viable LV) and the right ventricular muscle (RV) of the 2w-CAL and 8w-CAL rats were increased, which was associated with an elevation of intrinsic activities of leupeptin-sensitive, Ca(2+)-activated proteolysis in the cytosolic fractions of the viable LV and RV. Oral administration of 3 mg/kg/d trandolapril or 1 mg/kg/d candesartan from the second to eighth week after CAL improved the hemodynamics of 8w-CAL rats. The drug treatment attenuated the increases in mu-calpain and m-calpain contents and the elevation of the proteolytic activity of the viable LV and RV in the 8w-CAL rat. The drug treatment increased calpastatin content of the RV in the 8w-CAL rat. These results suggest that sustained activation of calpain is involved in the development of chronic heart failure and that trandolapril and candesartan prevent the activation of calpains after CAL.

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Pressure; Calcium-Binding Proteins; Calpain; Cytosol; Enzyme Activation; Heart Failure; Male; Myocardial Infarction; Myocardium; Organ Size; Rats; Rats, Wistar

The human genome contains 14 genes for 80 kDa catalytic subunit of the calcium-activated protease calpain (EC 34.22.17), yet no calpain-like cleavage sites have been detected on human lens crystallins in vivo. The purpose of the present study was to provide a comprehensive study of calpain activation in human and macaque lenses developing experimental cataract due to lens culture in ionophore A23187. Zymography was used to measure calpain activity; SDS-PAGE and immunoblotting were used to detect hydrolysis of potential lens protein substrates. Quantitative PCR was used to measure transcripts for calpains and the endogenous inhibitor calpastatin. We found that the lack of appreciable calpain-induced proteolysis in primate lenses is most likely due to relatively low levels of endogenous calpain activity compared to the high levels of endogenous calpain inhibitor, calpastatin.

Topics: Aged; Aged, 80 and over; Animals; Apoptosis; Calcimycin; Calcium; Calcium-Binding Proteins; Calpain; Caseins; Cataract; Eye Proteins; Humans; Immunoblotting; In Situ Nick-End Labeling; Ionophores; Lens, Crystalline; Macaca mulatta; Models, Animal; Reverse Transcriptase Polymerase Chain Reaction; Staining and Labeling

It was previously found that transgenic mice that overexpress the calpain inhibitor calpastatin (CsTg) have an approximately 3-fold increase in GLUT4 protein in their skeletal muscles. Despite the increase in GLUT4, which appears to be due to inhibition of its proteolysis by calpain, insulin-stimulated glucose transport is not increased in CsTg muscles. PKB (Akt) protein level is reduced approximately 60% in CsTg muscles, suggesting a possible mechanism for the relative insulin resistance. Muscle contractions stimulate glucose transport by a mechanism that is independent of insulin signaling. The purpose of this study was to test the hypothesis that the threefold increase in GLUT4 in CsTg would result in a large increase in contraction-stimulated glucose transport. CAMKII and AMPK mediate steps in the contraction-stimulated pathway. The protein levels of AMPK and CAMKII were increased three- to fourfold in CsTg muscles, suggesting that these proteins are also calpain substrates. Despite the large increases in GLUT4, AMPK, and CAMKII, contraction-stimulated GLUT4 translocation and glucose transport were not increased above wild-type values. These findings suggest that inhibition of calpain results in impairment of a step in the GLUT4 translocation process downstream of the insulin- and contraction-signaling pathways. They also provide evidence that CAMKII and AMPK are calpain substrates.

Topics: AMP-Activated Protein Kinases; Animals; Calcium-Binding Proteins; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calpain; Deoxyglucose; Gene Expression; Glucose Transporter Type 4; Glycogen; Mice; Mice, Transgenic; Multienzyme Complexes; Muscle Contraction; Muscle, Skeletal; Phosphorylation; Protein Serine-Threonine Kinases; Protein Transport

In this study, we adopted a model of tenderness classification in order to determine the factors affecting the tenderness and tenderization characteristics of beef longissimus, using cluster analysis on the basis of Warner-Bratzler shear force and myofibril fragmentation index, at 1, 7, and 14 d. The rate of tenderization was effectively differentiated by pH, R-values, mu-calpain activity, and calpastatin activity. Differences among tenderness classes were generally detected at 3 and 9 h postmortem for metabolic rate, and at 9 and 24 h for the activities of mu-calpain and calpastatin. Early postmortem metabolic rate and calpain system activities were verified as important factors with regard to longissimus tenderization.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Cluster Analysis; Meat Products; Myofibrils; Postmortem Changes

Podosomes, highly dynamic adhesion structures implicated in cell motility and extracellular matrix degradation, are characteristic of certain cells of the myeloid lineage and a limited range of other cell types. The nature and the mechanisms that regulate their high turnover are unknown at present. The cysteine protease calpain is involved in the regulation of cell migration in part by promoting either formation or disassembly of adhesion sites. Despite the fact that many known substrates of calpain are also structural components of the podosome complex, no studies have yet demonstrated that calpain participates in the regulation of podosome dynamics. In the present work, we show that inhibition of calpain in primary mouse dendritic cells leads to enhanced accumulation of actin filaments, the Wiskott Aldrich Syndrome protein (WASP), beta(2) integrins, talin, paxillin and vinculin in podosomes. This accumulation of components is associated with stabilisation of podosome turnover, overall reduction in velocity of cell locomotion and impaired transmigration across an endothelial monolayer. We also demonstrate that calpain cleaves the podosome components talin, Pyk2 and WASP in dendritic cells. In summary, our results provide evidence that calpain regulates podosome composition and turnover and that this process is required for efficient migration of dendritic cells.

Topics: Actin Cytoskeleton; Animals; Calcium-Binding Proteins; Calpain; Cell Adhesion; Cell Line; Cell Membrane Structures; Cell Movement; Cells, Cultured; Dendritic Cells; Enzyme Activation; Focal Adhesion Kinase 2; Glycoproteins; Mice; Mice, Inbred Strains; Oligopeptides; Peptide Fragments; Structure-Activity Relationship; Talin; Wiskott-Aldrich Syndrome Protein

The aim of the present study was to detect the expression of calpain-I, calpastatin, caspase-3 and apoptosis in the left atria of patients with rheumatic heart disease (RHD), and to find the association of these factors. Also, it was intended to investigate the effect of the above factors on the mechanism of atrial fibrillation (AF).. 43 patients with RHD undergoing valve-replacement were included, 15 patients with regular sinus rhythm (Group RSR), 8 patients with paroxysmal AF (Group AF1) and 20 patients with permanent AF (Group AF2). Western blot was used to examine the content of calpain-I, caspase-3 and calpastatin. The apoptosis index (AI) was measured by TUNEL.. (1) Expression of calpain-I in group AF2 was increased to (344.0 +/- 101.9)%, and caspase-3 was increased to (394.0 +/- 99.4)% compared to group RSR (P < 0.01, respectively). Amount of calpastatin was reduced to (27.0 +/- 12.8)% (P < 0.01). The expressions of these proteins were unchanged in group AF1. (2) AI in group AF2 was higher than that in groups RSR and AF1 (P < 0.01). (3) In group AF2, the levels of calpain-I, caspase-3 and AI were positively relative to left atrial dimension and AF duration, P < 0.05 - 0.01, respectively, whereas calpastatin was negatively correlated with left atrial dimension and AF duration (P = 0.007 and P = 0.001, respectively). (4) The protein content of calpain-I was positively related with that of caspase-3 and AI (P < 0.01, respectively), and the content of calpastatin was negatively related with that of calpain-I and caspase-3 (P < 0.01, respectively).. Apoptosis of atrial cell increased in left atria and the protein contents of calpain-I, caspase-3 and calpastatin significantly altered during AF in humans with RHD. The observed interactions suggest that these factors compose a system to cause the structural remodeling and dysfunction of atria. The course may play a key role in promoting the onset and maintenance of AF.

Topics: Adult; Apoptosis; Atrial Fibrillation; Atrial Function; Calcium-Binding Proteins; Calpain; Caspase 3; Female; Heart Atria; Humans; Male; Middle Aged; Myocytes, Cardiac; Rheumatic Heart Disease

It is generally accepted that the Ca(2+)-dependent interaction of calpain with calpastatin is the most relevant mechanism involved in the regulation of Ca(2+)-induced proteolysis. We now report that a calpain-calpastatin association can occur also in the absence of Ca(2+) or at very low Ca(2+) concentrations, reflecting the physiological conditions under which calpain retains its inactive conformational state. The calpastatin binding region is localized in the non-inhibitory L-domain containing the amino acid sequences encoded by exons 4-7. This calpastatin region recognizes a calpain sequence located near the end of the DII-domain. Interaction of calpain with calpastatins lacking these sequences becomes strictly Ca(2+)-dependent because, under these conditions, the transition to an active state of the protease is an obligatory requirement. The occurrence of the molecular association between Ca(2+)-free calpain and various recombinant calpastatin forms has been demonstrated by the following experimental results. Addition of calpastatin protected calpain from trypsin digestion. Calpain was coprecipitated when calpastatin was immunoprecipitated. The calpastatin molecular size increased following exposure to calpain. The two proteins comigrated in zymogram analysis. Furthermore, calpain-calpastatin interaction was perturbed by protein kinase C phosphorylation occurring at sites located at the exons involved in the association. At a functional level, calpain-calpastatin interaction at a physiological concentration of Ca(2+) represents a novel mechanism for the control of the amount of the active form of the protease potentially generated in response to an intracellular Ca(2+) influx.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Cattle; Enzyme Activation; Exons; Humans; Protein Structure, Tertiary; Rats; Recombinant Proteins; Substrate Specificity

Angiogenesis is a complex process involving endothelial cell migration, proliferation, and differentiation as well as tube formation. These processes are stimulated by a variety of growth factors such as vascular endothelial growth factor (VEGF). VEGF-induced cytoskeletal reorganization plays a crucial role in the angiogenic processes. In the present study, we evaluated the role of calpain in VEGF-induced angiogenesis in vitro and in vivo. Human pulmonary microvascular endothelial cells (PMEC) were incubated with VEGF (10-60 ng/ml) for 2-24 h, after which we measured calpain activities, protein contents of the calpain subunits and of calpastatin, endothelial monolayer wound repair, tube formation, and actin cytoskeleton changes. Incubation of PMEC with VEGF resulted in dose- and time-dependent increases in calpain activity and protein content of calpain-2. VEGF did not change the protein contents of calpain-1 and the small subunit or of calpastatin. Incubation of PMEC with a VEGF receptor blocker prevented the VEGF-induced increase in calpain activity. Inhibition of calpain activity by siRNA directed against calpain-2 and by overexpression of calpastatin prevented VEGF-induced increases in actin stress fibers in endothelial cells and angiogenesis. Overexpression of calpastatin also inhibits vessel formation in subcutaneous (s.c.) matrigel plugs in mice. These results indicate that calpain mediates VEGF-induced angiogenic effects by modulating actin cytoskeletal organization.

Topics: Adenoviridae; Calcium-Binding Proteins; Calpain; Cells, Cultured; Endothelium, Vascular; Genetic Vectors; Humans; Microcirculation; Neovascularization, Physiologic; Pulmonary Circulation; Vascular Endothelial Growth Factor A

Calpains are intracellular Ca2+-dependent cysteine proteases that are released in the extracellular milieu by tubular epithelial cells following renal ischemia. Here we show that externalized calpains increase epithelial cell mobility and thus are critical for tubule repair. In vitro, exposure of human tubular epithelial cells (HK-2 cells) to mu-calpain limited their adhesion to extracellular matrix and increased their mobility. Calpains acted primarily by promoting the cleavage of fibronectin, thus preventing fibronectin binding to the integrin alphavbeta3. Analyzing downstream integrin effects, we found that the cyclic AMP-dependent protein kinase A pathway was activated in response to alphavbeta3 disengagement and was essential for calpain-mediated increase in HK-2 cell mobility. In a murine model of ischemic acute renal failure, injection of a fragment of calpastatin, which specifically blocked calpain activity in extracellular milieu, markedly delayed tubule repair, increasing functional and histological lesions after 24 and 48 h of reperfusion. These findings suggest that externalized calpains are critical for tubule repair process in acute renal failure.

Topics: Animals; Calcium-Binding Proteins; Calnexin; Calpain; Cell Movement; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cysteine Proteinase Inhibitors; Epithelial Cells; Fibronectins; Humans; Integrin alphaVbeta3; Kidney Tubules, Proximal; Mice; Mice, Inbred C57BL; Protein Isoforms; Reperfusion Injury

Our recent study suggested involvement of calpain-induced proteolysis in retinal degeneration and dysfunction in acute ocular hypertensive rats. The purpose of the present study was to determine if an orally available form of calpain inhibitor, ((1S)-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid 5-methoxy-3-oxapentyl ester (SNJ-1945), ameliorated retinal degeneration induced by acute hypertension in rats. To help extrapolate the effect of SNJ-1945 from the rat model to the human glaucomatous patient, in vitro inhibition of calpain-induced proteolysis by SNJ-1945 in monkey and human retinal proteins was compared with proteolysis in rat proteins.. Intraocular pressure (IOP) in rats was elevated to 110 mm Hg for 50 min. SNJ-1945 was administrated i.p. or orally before ocular hypertension. Retinal degeneration was evaluated by hematoxylin and eosin (H&E) staining and cell counting. Transcripts for calpains and calpastatin in rat, monkey, and human retinas were measured by quantitative RT-PCR. Calpain activities were determined by casein zymography. Soluble retinal proteins from rat, monkey, and humans were incubated with calcium to activate calpains, with or without SNJ-1945. Proteolysis of calpain substrate alpha-spectrin was analyzed by immunoblotting.. Elevated IOP caused retinal degeneration and proteolysis of alpha-spectrin. Both i.p. and oral administration of SNJ-1945 inhibited proteolysis of alpha-spectrin and ameliorated retinal degeneration. Transcript levels for calpain 1 and calpastatin were similar in rat, monkey, and human retinas. Calpain 2 transcript levels were higher in rats compared with monkey and human. Appreciable caseinolytic activities due to calpains were observed in monkey and human retinas. Incubation of retinal soluble proteins with calcium led to proteolysis of alpha-spectrin due to calpains in rat, monkey, and human samples. SNJ-1945 similarly inhibited proteolysis in all species.. Our results suggested that orally available calpain inhibitor SNJ-1945 might be a possible candidate drug for testing in preventing progression of glaucomatous retinal degeneration.

Topics: Animals; Calcium-Binding Proteins; Calpain; Carbamates; Disease Models, Animal; Drug Administration Routes; Glycoproteins; Haplorhini; Humans; Intraocular Pressure; Ocular Hypertension; Rats; Rats, Sprague-Dawley; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Staining and Labeling; Time Factors

The activity of calpain, a calcium-activated protease, is required during the mitotic clonal expansion phase of 3T3-L1 embryonic preadipocyte differentiation. Here we examined the role of calpain in the adipogenesis of ST-13 preadipocytes established from adult primitive mesenchymal cells, which do not require mitotic clonal expansion. After exposure to the calpain inhibitor, N-benzyloxycarbonyl-L-leucyl-L-leucinal or overexpression of calpastatin, a specific endogenous inhibitor of calpain, ST-13 preadipocytes acquired the adipocyte phenotype. Overexpression of calpastatin in ST-13 adipocytes stimulated the expression of adipocyte-specific CCAAT/enhancer-binding protein-alpha (C/EBPalpha), peroxisome proliferator-activated receptor (PPAR)-gamma, sterol regulatory element-binding protein 1, and the insulin signaling molecules, insulin receptor alpha, insulin-receptor substrates, and GLUT4. However, insulin-stimulated glucose uptake was reduced by approximately 52%. The addition of calpain to the nuclear fraction of ST-13 adipocytes resulted in the Ca(2+)-dependent degradation of PPARgamma and C/EBPalpha but not sterol regulatory element-binding protein 1. Exposing ST-13 adipocytes to A23187 also led to losses of endogenous PPARgamma and C/EBPalpha. Under both conditions, calpain inhibitors almost completely prevented C/EBPalpha cleavage but partially blocked the decrease of PPARgamma. Two ubiquitous forms of calpain, mu- and m-calpain, localized to the cytosol and the nucleus, whereas the activated form of mu- but not m-calpain was found in the nucleus. Finally, stable dominant-negative mu-calpain transfectants showed accelerated adipogenesis and increase in the levels of PPARgamma and C/EBPalpha during adipocyte program. These results support evidence that the calpain system is involved in regulating the differentiation of adult primitive mesenchymal ST-13 preadipocytes.

Topics: Adipocytes; Adipose Tissue; Antimetabolites; Blotting, Northern; Calcimycin; Calcium-Binding Proteins; Calpain; CCAAT-Enhancer-Binding Protein-alpha; Cell Differentiation; Deoxyglucose; Dipeptides; DNA, Complementary; Humans; Stem Cells; Sterol Regulatory Element Binding Protein 1; Subcellular Fractions

Acute liver failure induced by hepatotoxic drugs results from rapid progression of injury. Substantial research has shown that timely liver regeneration can prevent progression of injury leading to a favorable prognosis. However, the mechanism by which compensatory regeneration prevents progression of injury is not known. We have recently reported that calpain released from necrotic hepatocytes mediates progression of liver injury even after the hepatotoxic drug is cleared from the body. By examining expression of calpastatin (CAST), an endogenous inhibitor of calpain in three liver cell division models known to be resistant to hepatotoxicity, we tested the hypothesis that increased CAST in the dividing hepatocytes affords resistance against progression of injury. Liver regeneration that follows CCl(4)-induced liver injury, 70% partial hepatectomy, and postnatal liver development were used. In all three models, CAST was upregulated in the dividing/newly divided hepatocytes and declined to normal levels with the cessation of cell proliferation. To test whether CAST overexpression confers resistance against hepatotoxicity, CAST was overexpressed in the livers of normal SW mice using adenovirus before challenging them with acetaminophen (APAP) overdose. These mice exhibited markedly attenuated progression of liver injury and 57% survival. Whereas APAP-bioactivating enzymes and covalent binding of the APAP-derived reactive metabolites remained unaffected, degradation of calpain specific target substrates such as fodrin was significantly reduced in these mice. In conclusion, CAST overexpression could be used as a therapeutic strategy to prevent progression of liver injury where liver regeneration is severely hampered.

Topics: Acetaminophen; Animals; Animals, Newborn; Calcium-Binding Proteins; Calpain; Carbon Tetrachloride; Cell Division; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP2E1; Disease Models, Animal; Disease Progression; Hepatocytes; Immunohistochemistry; Liver; Liver Failure, Acute; Liver Regeneration; Male; Mice; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; RNA, Messenger; Up-Regulation

Hyperhomocysteinemia (HHcy) is associated with atherosclerosis, stroke, and dementia. Hcy causes extracellular matrix remodeling by the activation of matrix metalloproteinase-9 (MMP-9), in part, by inducing redox signaling and modulating the intracellular calcium dynamics. Calpains are the calcium-dependent cysteine proteases that are implicated in mitochondrial damage via oxidative burst. Mitochondrial abnormalities have been identified in HHcy. The mechanism of Hcy-induced extracellular matrix remodeling by MMP-9 activation via mitochondrial pathway is largely unknown. We report a novel role of calpains in mitochondrial-mediated MMP-9 activation by Hcy in cultured rat heart microvascular endothelial cells. Our observations suggested that calpain regulates Hcy-induced MMP-9 expression and activity. We showed that Hcy activates calpain-1, but not calpain-2, in a calcium-dependent manner. Interestingly, the enhanced calpain activity was not mirrored by the decreased levels of its endogenous inhibitor calpastatin. We presented evidence that Hcy induces the translocation of active calpain from cytosol to mitochondria, leading to MMP-9 activation, in part, by causing intramitochondrial oxidative burst. Furthermore, studies with pharmacological inhibitors of calpain (calpeptin and calpain-1 inhibitor), ERK (PD-98059) and the mitochondrial uncoupler FCCP suggested that calpain and ERK-1/2 are the major events within the Hcy/MMP-9 signal axis and that intramitochondrial oxidative stress regulates MMP-9 via ERK-1/2 signal cascade. Taken together, these findings determine the novel role of mitochondrial translocation of calpain-1 in MMP-9 activation during HHcy, in part, by increasing mitochondrial oxidative stress.

Topics: Animals; Calcium-Binding Proteins; Calpain; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cells, Cultured; Coronary Vessels; Cytosol; Dipeptides; Endothelium, Vascular; Flavonoids; Gene Expression Regulation, Enzymologic; Homocysteine; Hyperhomocysteinemia; Matrix Metalloproteinase 9; Mitochondria; Mitogen-Activated Protein Kinase 3; Oxidative Stress; Rats; Rats, Wistar; Uncoupling Agents

Single nucleotide polymorphisms (SNPs) in the calpain 1 (CAPN1) and calpastatin (CAST) genes were studied to determine their effects on meat tenderness in Bos taurus cattle. Strip loins (M. longissimus dorsi) were removed from cattle in four resource populations after slaughter (n = 1042), aged under controlled conditions until fixed times after rigor mortis, cooked and measured using a tenderometer. Animals were genotyped for the CAPN1 SNP c.947C>G (p.Ala316Gly; AF252504) and for the CAST SNP c.2959A>G (AF159246). Frequencies of CAPN1 C alleles ranged from 23% to 68%, and CAST A alleles from 84% to 99.5%. From all data combined, the CAPN1 CC genotype (compared with the GG genotype) was associated with a 20.1 +/- 1.7% reduced average shear force at intermediate stages of ageing (P < 0.001) and with a 9.5 +/- 1.3% reduction near ultimate tenderness (P < 0.001). The heterozygote was intermediate. For CAST, corresponding values for AA compared with AG genotypes were reductions of 8.6 +/- 2.0% and 5.1 +/- 1.6% respectively (both P < 0.001), but there were too few GG genotypes for comparison. There were small interactions between the CAPN1 and CAST genotypes. For the CAPN1 and CAST genotypes combined, the maximal genotype effect in average shear force was 25.7 +/- 5.5% (P < 0.001) at intermediate stages and 15.2 +/- 4.8% near ultimate tenderness (P < 0.01).

Topics: Alleles; Animals; Calcium-Binding Proteins; Calpain; Cattle; Crosses, Genetic; Genotype; Meat; Polymorphism, Single Nucleotide

Previously, we reported that l-glutamic acid decarboxylase isoform 65 (GAD65) could be cleaved in vitro to release a stable truncated form which lacks amino acid 1-69 from the N-terminus, GAD65(Delta1-69). However, whether such a truncated form is also present under certain physiological conditions remains elusive. In the present study, we showed that, upon sustained neuronal stimulation, GAD65 could be cleaved into a truncated form in a rat synaptosomal preparation. This truncated form had similar electrophoretic mobility to purified recombinant human GAD65(Delta1-69). Furthermore, we demonstrated that this conversion was calcium dependent. Calcium-chelating reagents such as EDTA and 1,2-bis-(o-aminphenoxy)-ethane-N,N,N',N'-tetra-acetic acid tetra-acetoxy-methyl ester prevented the cleavage of GAD65. In addition, our data suggested that calpain, a calcium-dependent cysteine protease, is activated upon neuronal stimulation and could be responsible for the conversion of full-length GAD65 to truncated GAD65 in the brain. Moreover, calpain inhibitors such as calpain inhibitor I or calpastatin could block the cleavage. Results of our in vitro cleavage assay using purified calpain and immunopurified rat GAD65 also supported the idea that GAD65 could be directly cleaved by calpain.

Topics: Animals; Blotting, Western; Brain; Calcium; Calcium-Binding Proteins; Calpain; Cells, Cultured; Chelating Agents; Dose-Response Relationship, Drug; Drug Interactions; Edetic Acid; Embryo, Mammalian; Enzyme Activation; Female; Glutamate Decarboxylase; Glutamic Acid; Immunoprecipitation; Ionomycin; Ionophores; Isoenzymes; Neurons; Pregnancy; Rats; Rats, Sprague-Dawley; Synaptosomes; Time Factors

Disturbances of erythrocyte calcium homeostasis in the course of chronic renal failure (CRF) may result from impaired function of a membrane calcium pump (PMCA). Plasma membrane Ca+2-transport ATP-ase is responsible for maintaining cytoplasmic level of intracellular Ca+2. PMCA activity is regulated by high Ca levels and calmodulin, in case of Ca+2 deficiency--by intracellular cysteine protease--calpain, and its natural inhibitor--calpastatin. The aim of the study was to assess the relationship between the activity of PMCA and calpain-calpastatin system in erythrocytes of children with CRF treated conservatively.. The study was performed on 21 CRF children treated conservatively (gr. I) aged 6-18 years. The control group consisted of 18 healthy age matched children. The parameters evaluated in erythrocytes were as follows: calpain-calpastatin system (Cp-Cs) activity, PMCA activity, level of intracellular Ca (Cai+2) and calmodulin (Ca) activity.. Our investigation revealed: calmodulin concentrations decrement in CRF children (2.08 +/- 0.51 mg/L) vs. controls (4.54 +/- 1.09 nmol/min/mg) (p<0.01), calpain activity in CRF children (60.56 +/- 12.43 U/mg) higher than in controls (45.34 +/- 5.65 U/mg) (p<0.01) and calpastatin activity increment in gr. I (40.03 +/- 10.24 U/mg) vs. control group (20.123.05 U/mg) (p<0.01). Decreased activity of a system containing an enzyme and its inhibitor (calpain-calpastatin) was also observed: 1 : 0.66 ratio in gr. I vs 1:0.4 ratio in controls.. There is a decrease inPMCA activity and an increase of intracellular calcium in erythrocytes of children with mild CRF. The concomitant increment of erythrocyte calpastatin is the cause of disturbances in calpain-calpastatin system, thus unabling normalization of PMCA pump activity.

Topics: Adolescent; Calcium; Calcium-Binding Proteins; Calpain; Child; Erythrocytes; Female; Humans; Kidney Failure, Chronic; Male; Plasma Membrane Calcium-Transporting ATPases

Previous research in our laboratory has already shown the importance of the role played by ubiquitous calpains during myoblast migration. The aim of this study was to investigate calpain expression during myoblast migration and, to enhance this phenomenon via calpain stimulation. Ubiquitous calpains are members of a large family of calcium-dependent cysteine proteases. They play an important role in numerous biological and pathological phenomena, such as signal transduction, apoptosis, cell-cycle regulation, cell spreading, adhesion, invasion, myogenesis, and motility. Myoblast migration is a crucial step in myogenesis, as it is necessary for myoblast alignment and fusion to form myotubes. This study started by examining changes in calpain expression during migration, then investigated the possibility of activating myoblast migration via the stimulation of calpain expression and/or activity. The migration rate of myoblasts overexpressing mu- or milli-calpain was quantified. The results showed that calpain overexpression dramatically inhibited myoblast migration. Growth-factor treatments were then used to enhance myoblast migration. The results showed that treatment with IGF-1, TGF-beta1, or insulin induced a major increase in migration and caused a significant increase in m-calpain expression and activity. The increase in migration was totally inhibited by adding calpeptin, a calpain-specific inhibitor. These findings suggest that milli-calpain is involved in growth factor-mediated migration.

Topics: Animals; Calcium-Binding Proteins; Calpain; Caseins; Cell Movement; Dipeptides; Gene Expression; Growth Substances; Insulin; Mice; Myoblasts; Stress Fibers

Glomerular injury and albuminuria in acute glomerulonephritis are related to the severity of inflammatory process. Calpain, a calcium-activated cysteine protease, has been shown to participate in the development of the inflammatory process. Therefore, for determination of the role of calpain in the pathophysiology of acute glomerulonephritis, transgenic mice that constitutively express high levels of calpastatin, a calpain-specific inhibitor protein, were generated. Wild-type mice that were subjected to anti-glomerular basement membrane nephritis exhibited elevated levels of calpain activity in kidney cortex at the heterologous phase of the disease. This was associated with the appearance in urine of calpain activity, which originated potentially from inflammatory cells, abnormal transglomerular passage of plasma proteins, and tubular secretion. In comparison with nephritic wild-type mice, nephritic calpastatin-transgenic mice exhibited limited activation of calpain in kidney cortex and limited secretion of calpain activity in urine. This was associated with less severe glomerular injury (including capillary thrombi and neutrophil activity) and proteinuria. There was a reduction in NF-kappaB activation, suggesting that calpain may participate in inflammatory lesions through NF-kappaB activation. There also was a reduction in nephrin disappearance from the surface of podocytes, indicating that calpain activity would enhance proteinuria by affecting nephrin expression. Exposure of cultured podocytes to calpain decreased nephrin expression, and, conversely, exposure of these cells to calpastatin prevented TNF-alpha from decreasing nephrin expression, demonstrating a role for the secreted form of calpain. Thus, both activation and secretion of calpains participate in the development of immune glomerular injury.

Topics: Albuminuria; Animals; Anti-Glomerular Basement Membrane Disease; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Disease Models, Animal; Female; Glomerulonephritis; Inflammation; Kidney; Mice; Mice, Inbred C57BL; Mice, Transgenic; NF-kappa B

As previously suggested by PCR analysis [R. DeTullio, R. Stifanese, F. Salamino, S. Pontremoli, E. Melloni, Characterization of a new p94-like calpain form in human lymphocytes, Biochem. J. 375 (2003) 689-696], a p94-like calpain was now established to be present in six different human cells resembling the various peripheral blood cell types. This protease resulted to be the predominant calpain isoforms whereas the conventional mu- and m-calpains are also expressed although at lower or almost undetectable amounts. The p94-like calpain has been identified by a specific mAb and displays unique features such as: Ca2+ requirement for half maximum activity around 30 microM; no autolytic conversion to a low Ca2+ requiring form and lower sensitivity to calpastatin inhibition. Following cell stimulation, the p94-like calpain undergoes inactivation, a process indicating that the protease is activated and participates in the cell responses to stimuli. The involvement of this protease isoform in immunocompetent cell activation is further supported by its partial recruitment on plasma membranes, the site of action of the conventional calpain forms. The amount of calpain translocated to the membranes correlates to the level of calpastatin which has been shown to control this process through the formation of a complex with calpain, which maintains the protease in the cytosol. These results provide new information on the calpain/calpastatin system expressed in immunocompetent cells and on the functional relationship between the p94-like calpain and the biological function of these cells.

Topics: Calcium-Binding Proteins; Calpain; Cell Line; Hematopoietic Stem Cells; Humans; Subcellular Fractions

The activation of the [Ca(2+)]-dependent cysteine protease calpain plays an important role in ischemic injury. Here, the levels of two calpain-specific substrates, p35 protein and eukaryotic initiation factor 4G (eIF4G), as well as its physiological regulator calpastatin, were investigated in a rat model of transient global cerebral ischemia with or without ischemic tolerance (IT). Extracts of the cerebral cortex, whole hippocampus and hippocampal subregions after 30 min of ischemia and different reperfusion times (30 min and 4 h) were used. In rats without IT, the p35 levels slightly decreased after ischemia or reperfusion, whereas the levels of p25 (the truncated form of p35) were much higher than those in sham control rats after ischemia and remained elevated during reperfusion. The eIF4G levels deeply diminished after reperfusion and the decrease was significantly greater in CA1 and the rest of the hippocampus than in the cortex. By contrast, the calpastatin levels did not significantly decrease during ischemia or early reperfusion, but were upregulated after 4 h of reperfusion in the cortex. Although IT did not promote significant changes in p35 and p25 levels, it induced a slight increase in calpastatin and eIF4G levels in the hippocampal subregions after 4 h of reperfusion.

Topics: Animals; Brain; Brain Chemistry; Calcium-Binding Proteins; Calpain; Cerebral Cortex; Eukaryotic Initiation Factor-4G; Hippocampus; HSP70 Heat-Shock Proteins; Ischemic Attack, Transient; Ischemic Preconditioning; Mice; Phosphotransferases; Rats; Rats, Wistar; Reperfusion Injury; Tissue Extracts

We examined the influence of sepsis on the expression and activity of the calpain and caspase systems in skeletal muscle. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham operated. Calpain activity was determined by measuring the calcium-dependent hydrolysis of casein and by casein zymography. The activity of the endogenous calpain inhibitor calpastatin was measured by determining the inhibitory effect on calpain activity in muscle extracts. Protein levels of mu- and m-calpain and calpastatin were determined by Western blotting, and calpastatin mRNA was measured by real-time PCR. Caspase-3 activity was determined by measuring the hydrolysis of the fluorogenic caspase-3 substrate Ac-DEVD-AMC and by determining protein and mRNA expression for caspase-3 by Western blotting and real-time PCR, respectively. In addition, the role of calpains and caspase-3 in sepsis-induced muscle protein breakdown was determined by measuring protein breakdown rates in the presence of specific inhibitors. Sepsis resulted in increased muscle calpain activity caused by reduced calpastatin activity. In contrast, caspase-3 activity, mRNA levels, and activated caspase-3 29-kDa fragment were not altered in muscle from septic rats. Sepsis-induced muscle proteolysis was blocked by the calpain inhibitor calpeptin but was not influenced by the caspase-3 inhibitor Ac-DEVD-CHO. The results suggest that sepsis-induced muscle wasting is associated with increased calpain activity, secondary to reduced calpastatin activity, and that caspase-3 activity is not involved in the catabolic response to sepsis.

Topics: Animals; Bacterial Infections; Calcium-Binding Proteins; Calpain; Caspase 3; Caspases; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; Enzyme Activation; Male; Muscle Fibers, Skeletal; Muscle Proteins; Muscle, Skeletal; Oligopeptides; Rats; Rats, Sprague-Dawley; RNA, Messenger

Psoriasis is believed to be a T cell-mediated autoimmune disease, but also exhibits autoantibody production. Calpastatin is an endogenous inhibitor of calpain, a ubiquitous protease that regulates inflammatory processes. Anti-calpastatin autoantibody was first identified as an autoantibody specific to rheumatoid arthritis, but has been also detected in other autoimmune diseases. In this study, we examined the presence and levels of anti-calpastatin antibody in 77 psoriasis patients by enzyme-linked immunosorbent assay. Compared with normal controls, psoriasis patients exhibited significantly elevated IgG anti-calpastatin antibody levels that were similar to those found in rheumatoid arthritis patients. Remarkably, IgG anti-calpastatin autoantibody in sera from psoriasis patients inhibited calpastatin activity. Calpain II expression was up-regulated in psoriasis skin lesions compared with normal skin while calpastatin expression was normal. The results of this study reveal the presence of anti-calpastatin autoantibody in psoriasis.

Topics: Adult; Antibodies, Monoclonal; Autoantibodies; Calcium-Binding Proteins; Calpain; Case-Control Studies; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunoglobulin G; Male; Middle Aged; Psoriasis; Skin

Calpains are a family of 14 intracellular calcium-dependent proteases, which have been implicated in cardiovascular diseases. We aimed to analyze specifically the expressional regulation of the different calpain isoforms in hypertensive target organ damage. Using real-time PCR, we found calpain 6 and 9 down-regulated by more than 50% and the endogenous calpain inhibitor calpastatin up-regulated by 225%, respectively, in the hearts of Dahl salt-sensitive rats on a high salt (4% NaCl) compared to normal salt diet. On the protein level, calpain 9 but not calpastatin was regulated in the hypertensive target organs heart and kidney. Moreover, the myocardial expression of calpain 9 protein was inversely linked to left ventricular mass (r= -0.93, p<0.01), and renal expression of calpain 9 protein correlated inversely with albuminuria (r= -0.82, p<0.05). In the aorta, there was no regulation of calpain 9 on the protein level. We conclude that differential regulation of calpain 9 may play a role in hypertensive target organ damage.

Topics: Animals; Calcium-Binding Proteins; Calpain; Down-Regulation; Gene Expression Regulation, Enzymologic; Heart Diseases; Heart Ventricles; Hypertension; Isoenzymes; Kidney Diseases; Male; Rats; Rats, Inbred Dahl; RNA, Messenger

In brain ischemia, gating of postsynaptic glutamate receptors and other membrane channels triggers intracellular Ca2+ overload and cell death. In excitotoxic settings, the initial Ca2+ influx through glutamate receptors is followed by a second uncontrolled Ca2+ increase that leads to neuronal demise. Here we report that the major plasma membrane Ca2+ extruding system, the Na+/Ca2+ exchanger (NCX), is cleaved during brain ischemia and in neurons undergoing excitotoxicity. Inhibition of Ca2+-activated proteases (calpains) by overexpressing their endogenous inhibitor protein, calpastatin or the expression of an NCX isoform not cleaved by calpains, prevented Ca2+ overload and rescued neurons from excitotoxic death. Conversely, down-regulation of NCX by siRNA compromised neuronal Ca2+ handling, transforming the Ca2+ transient elicited by non-excitotoxic glutamate concentrations into a lethal Ca2+overload. Thus, proteolytic inactivation of NCX-driven neuronal Ca2+ extrusion is responsible for the delayed excitotoxic Ca2+ deregulation and neuronal death.

Topics: Amino Acid Sequence; Animals; Brain Ischemia; Calcium; Calcium-Binding Proteins; Calpain; Cell Death; Cell Membrane; Cells, Cultured; Cysteine Proteinase Inhibitors; Ion Channel Gating; Membrane Transport Proteins; Molecular Sequence Data; Neurons; Rats; Rats, Wistar; Receptors, Glutamate; Sodium-Calcium Exchanger

Calpain has been implicated in excitotoxic neurode-generation, but its mechanism of action particularly in adult brains remains unclear. We generated mutant mice lacking or overexpressing calpastatin, the only solely calpain-specific inhibitor ever identified or synthesized. Modulation of calpastatin expression caused no defect in the mice under normal conditions, indicating that calpastatin functions as a negative regulator of calpain only under pathological conditions. Kainate-evoked excitotoxicity in hippocampus resulted in proteolytic activation of a proapoptotic Bcl-2 subfamily member (Bid), nuclear translocation of mitochondria-derived DNA fragmentation factors (apoptosis-inducing factor and endonuclease G), DNA fragmentation, and nuclear condensation in pyramidal neurons. These apoptotic responses were significantly augmented by calpastatin deficiency. Consistently calpastatin overexpression suppressed them. No evidence of caspase-3 activation was detected. Our results demonstrated that calpain mediates excitotoxic signals through mobilization of proapoptotic factors in a caspase-independent manner. These mutant mice will serve as useful tools for investigating calpain involvement in various diseases.

Topics: Animals; Brain; Calcium-Binding Proteins; Calpain; Cattle; DNA Fragmentation; Mice; Mice, Knockout; Mice, Transgenic; Mitochondria

We show calcium-dependent, direct binding between the N-terminal portion of the PTH/PTHrP receptor (PTH1R) C-terminal intracellular tail and the calpain small subunit. Binding requires, but may not be limited to, amino acids W474, S475, and W477. The wild-type, full-length rat (r) PTH1R, but not rPTH1R with W474A/W477A substitutions, copurifies with the endogenous calpain small subunit in HEK293 cells. Calpain hydrolyzes delta Nt-rPTH1R, a receptor with a 156-amino acid N-terminal deletion, in a calcium-dependent manner in vitro and in intact cells. Most importantly, PTH stimulation increases the cleavage of delta Nt-rPTH1R and rPTH1R-yellow fluorescent protein in HEK293 cells, and of talin in HEK293 cells expressing rPTH1R-yellow fluorescent protein and in ROS17/2.8 osteoblast-like cells that express rPTH1R endogenously. The absence of calpain in Capn4-null embryonic fibroblasts and the lowered calpain activity in MC3T3-E1 osteoblastic cells due to stable expression of the calpain inhibitor, calpastatin, reduce PTH-stimulated cAMP accumulation. The calpain small subunit is the second protein, in addition to the sodium-hydrogen exchanger regulatory factor, and the first enzyme that binds the PTH1R; PTH1R bound to both of these proteins results in altered PTH signaling.

Topics: Bacterial Proteins; Calcium; Calcium-Binding Proteins; Calpain; Cell Line; Cyclic AMP; Embryo, Mammalian; Fibroblasts; Glutathione Transferase; Humans; Hydrolysis; Luminescent Proteins; Osteoblasts; Parathyroid Hormone; Peptide Fragments; Receptor, Parathyroid Hormone, Type 1; Recombinant Fusion Proteins; Signal Transduction; Transfection

We have previously reported that calpastatin, an endogenous inhibitory protein of calpain, is cleaved by a caspase-3-like protease during apoptosis in human Jurkat T cells [Kato, M. et al. (2000) J. Biochem. 127, 297-305]. In this study, we found that nonmuscle myosin heavy chain-A (NMHC-A) is cleaved during apoptosis in Jurkat cells by using a cleavage-site-directed antibody for calpastatin. The cleavage-site-directed antibody was raised against the amino-terminal fragment of calpastatin, and this antibody detected the in vitro cleaved calpastatin fragment. Although cleaved calpastatin was not detected, a 95-kDa polypeptide (p95) was detected in apoptotic cells by this antibody. This p95 was identified as the carboxyl-terminal fragment of NMHC-A based on the results of peptide mass spectrometry fingerprinting and amino-terminal sequencing. Furthermore, two cleavage sites on NMHC-A, Asp-1153 and Asp-1948, were determined, and three cleaved fragments of NMHC-A, one cleaved at Asp-1153 and the other two cleaved at Asp-1948, were detected by cleavage-site-directed antibodies against each cleavage site. The results of confocal immunofluorescence microscopic analysis show that the cleavage at Asp-1948 occurs faster than that at Asp-1153 during apoptosis. In addition, the Asp-1153 cleaved fragment was distributed diffusely in the cytoplasm of apoptotic cells, whereas the Asp-1948 cleaved fragments were detected as condensed dots. In conclusion, our findings can be summarized as follows: (i) NMHC-A is cleaved at two sites during apoptosis, (ii) the timing of cleavage is different between these two cleavage sites, and (iii) the distribution of cleaved fragments is different in apoptotic cells.

Topics: Amino Acid Sequence; Antibodies; Apoptosis; Calcium-Binding Proteins; Calpain; Humans; Jurkat Cells; Microscopy, Confocal; Molecular Motor Proteins; Molecular Sequence Data; Myosin Heavy Chains; Peptides; Proteasome Inhibitors

Beta-catenin is a multifunctional protein serving both as a structural element in cell adhesion and as a signaling component in the Wnt pathway, regulating embryogenesis and tumorigenesis. The signaling fraction of beta-catenin is tightly controlled by the adenomatous polyposis coli-axin-glycogen synthase kinase 3beta complex, which targets it for proteasomal degradation. It has been recently shown that Ca(2+) release from internal stores results in nuclear export and calpain-mediated degradation of beta-catenin in the cytoplasm. Here we have highlighted the critical relevance of constitutive calpain pathway in the control of beta-catenin levels and functions, showing that small interference RNA knock down of endogenous calpain per se (i.e. in the absence of external stimuli) induces an increase in the free transcriptional competent pool of endogenous beta-catenin. We further characterized the role of the known calpain inhibitors, Gas2 and Calpastatin, demonstrating that they can also control levels, function, and localization of beta-catenin through endogenous calpain regulation. Finally we present Gas2 dominant negative (Gas2DN) as a new tool for regulating calpain activity, providing evidence that it counteracts the described effects of both Gas2 and Calpastatin on beta-catenin and that it works via calpain independently of the classical glycogen synthase kinase 3beta and proteasome pathway. Moreover, we provide in vitro biochemical evidence showing that Gas2DN can increase the activity of calpain and that in vivo it can induce degradation of stabilized/mutated beta-catenin. In fact, in a context where the classical proteasome pathway is impaired, as in colon cancer cells, Gas2DN biological effects accounted for a significant reduction in proliferation and anchorage-independent growth of colon cancer.

Topics: Adenomatous Polyposis Coli Protein; Animals; Axin Protein; beta Catenin; Blotting, Western; Calcium-Binding Proteins; Calpain; Cell Adhesion; Cell Line; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cytoskeletal Proteins; Genes, Reporter; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Green Fluorescent Proteins; Humans; Intercellular Signaling Peptides and Proteins; Mice; Mice, Inbred BALB C; Microfilament Proteins; Microscopy, Fluorescence; Models, Biological; Mutation; Plasmids; Proteasome Endopeptidase Complex; Repressor Proteins; RNA Interference; RNA, Small Interfering; Signal Transduction; Subcellular Fractions; Time Factors; Trans-Activators; Transfection; Wnt Proteins

Previously, we have found that caspase-1 activity is increased during myoblast differentiation to myotubes. Here we show that caspase-1 activity is required for PC12 differentiation to neuronal-like cells. Caspase-1 is shown to be activated (by immunoblotting and by assessing activity in cell extracts) in the PC12 cells following the initial stage of differentiation. The inhibition of caspase-1 arrests PC12 cells at an intermediate stage of differentiation and prevents neurite outgrowth in these cells; the inhibition is reversed upon the removal of the inhibitor. Calpastatin (calpain endogenous specific inhibitor, and a known caspase substrate) is diminished at the later stages of PC12 cell differentiation, and diminution is prevented by caspase-1 inhibition. The degradation of fodrin (a known caspase and calpain substrate) is found in the advanced stage of differentiation. Caspase-1 has been implicated in the activation of proinflammatory cytokines, and in cell apoptosis. The involvement of caspase-1 in two distinct differentiation processes (myoblast fusion and neuronal differentiation of PC12 cells) indicates a function for this caspase in differentiation processes, and suggests some common mechanisms underlying caspase roles in such processes.

Topics: Animals; Calcium-Binding Proteins; Calpain; Carrier Proteins; Caspase 1; Caspase Inhibitors; Cell Differentiation; Immunoblotting; Microfilament Proteins; PC12 Cells; Protease Inhibitors; Rats

Epidemiological studies have indicated that malnutrition during early life may programme chronic degenerative disease in adulthood. In an animal model of fetal malnutrition, rats received an isoenergetic, low-protein (LP) diet during gestation. This reduced fetal beta-cell proliferation and insulin secretion. Supplementation during gestation with taurine prevented these alterations. Since proteases are involved in secretion and proliferation, we investigated which proteases were associated with these alterations and their restoration in fetal LP islets. Insulin secretion and proliferation of fetal control and LP islets exposed to different protease modulators were measured. Lactacystin and calpain inhibitor I, but not isovaleryl-L-carnitine, raised insulin secretion in control islets, indicating that proteasome and cysteinyl cathepsin(s), but not mu-calpain, are involved in fetal insulin secretion. Insulin secretion from LP islets responded normally to lactacystin but was insensitive to calpain inhibitor I, indicating a loss of cysteinyl cathepsin activity. Taurine supplementation prevented this by restoring the response to calpain inhibitor I. Control islet cell proliferation was reduced by calpain inhibitor I and raised by isovaleryl-L-carnitine, indicating an involvement of calpain. Calpain activity appeared to be lost in LP islets and not restored by taurine. Most modifications in the mRNA expression of cysteinyl cathepsins, calpains and calpastatin due to maternal protein restriction were consistent with reduced protease activity and were restored by taurine. Thus, maternal protein restriction affected cysteinyl cathepsins and the calpain-calpastatin system. Taurine normalised fetal LP insulin secretion by protecting cysteinyl cathepsin(s), but the restoration of LP islet cell proliferation by taurine did not implicate calpains.

Topics: Acetylcysteine; Animals; Calcium-Binding Proteins; Calpain; Cell Proliferation; Cells, Cultured; Diet, Protein-Restricted; Disease Models, Animal; Female; Fetal Nutrition Disorders; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Glycoproteins; Insulin; Insulin Secretion; Islets of Langerhans; Maternal-Fetal Exchange; Peptide Hydrolases; Pregnancy; Proteasome Endopeptidase Complex; Protein Array Analysis; Rats; Rats, Wistar; Taurine

The objectives of this study were to determine the extent to which pH and ionic strength influence mu- and m-calpain activity and the inhibition of calpains by calpastatin. Calpastatin, mu-calpain, and m-calpain were purified from at-death porcine semimembranosus. Mu-calpain or m-calpain (0.45 U) were incubated with the calpain substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl coumarin in the presence of calpastatin (0, 0.15, or 0.30 U of calpain inhibitory activity) under the following pH and ionic strength conditions: pH 7.5 and 165 mM NaCl or 295 mM NaCl; pH 6.5 and 165 mM NaCl or 295 mM NaCl; and pH 6.0 and 165 mM NaCl or 295 mM NaCl. The reactions were initiated with addition of 100 microM (mu-calpain) or 1 mM CaCl2 (m-calpain), and calpain activity was recorded at 30 and 60 min. Mu-calpain had the greatest (P < 0.01) activity at pH 6.5 at each ionic strength. Higher ionic strength decreased mu-calpain activity (P < 0.01) at all pH conditions. Inhibition percent of mu-calpain by calpastatin was not affected by pH; however, it was influenced by ionic strength. Inhibition of mu-calpain by calpastatin was higher (P < 0.01) at 295 mM NaCl than at 165 mM NaCl when 0.3 units of calpastatin were included in the assay. Activity of m-calpain was greater (P < 0.01) at pH 7.5 than at pH 6.5. m-Calpain activity was not detected at pH 6.0. Inhibition of m-calpain was greater (P < 0.01) when 0.15 and 0.3 U calpastatin were added at pH 6.5 than 7.5 at 165 mM NaCl, whereas percentage inhibition of m-calpain was greater (P < 0.01) at 295 mM than 165 mM NaCl at pH 7.5 and 6.5. These observations provide new evidence that defines further the influence of pH decline and increased ionic strength on mu-calpain, m-calpain, and calpastatin activity, thereby helping to more accurately define a role for these enzymes in the process of postmortem tenderization.

Topics: Animals; Blotting, Western; Calcium Chloride; Calcium-Binding Proteins; Calpain; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Least-Squares Analysis; Meat; Muscle, Skeletal; Osmolar Concentration; Swine; Time Factors

The formation of skeletal muscle fibers involves cessation of myoblast division, myoblast alignment, and fusion to multinucleated myofibers. Calpain is one of the factors shown to be involved in myoblast fusion. Using L8 rat myoblasts, we found that calpain levels did not change significantly during myoblast differentiation, whereas calpastatin diminished prior to myoblast fusion and reappeared after fusion. The transient diminution in calpastatin allows the Ca2+-promoted activation of calpain and calpain-induced membrane proteolysis, which is required for myoblast fusion. Here we show that calpastatin overexpression in L8 myoblasts does not inhibit cell proliferation and alignment, but prevents myoblast fusion and fusion-associated protein degradation. In addition, calpastatin appears to modulate myogenic gene expression, as indicated by the lack of myogenin (a transcription factor expressed in differentiating myoblasts) in myoblasts overexpressing calpastatin. These results suggest that, in addition to the role in membrane disorganization in the fusing myoblasts, the calpain-calpastatin system may also modulate the levels of factors required for myoblast differentiation.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cell Fusion; Cell Line; Cell Proliferation; Gene Expression; Gene Expression Regulation, Developmental; Myoblasts, Skeletal; Rats; Transfection

The pathogenesis of various acute and chronic neurodegenerative disorders has been linked to excitotoxic processes and excess generation of nitric oxide. We investigated the deleterious effects of calpain activation in nitric oxide-elicited neuronal apoptosis. In this model, nitric oxide triggers apoptosis of murine cerebellar granule cells by an excitotoxic mechanism requiring glutamate exocytosis and receptor-mediated intracellular calcium overload. Here, we found that calcium-dependent cysteine proteases, calpains, were activated early in apoptosis of cerebellar granule cells exposed to nitric oxide. Release of the proapoptogenic factors cytochrome c and apoptosis-inducing factor from mitochondria preceded neuronal death. However, caspases-3 was not activated. We observed that procaspase-9 was cleaved by calpains to proteolytically inactive fragments. Inhibition of calpains by different synthetic calpain inhibitors or by adenovirally mediated expression of the calpastatin inhibitory domain prevented mitochondrial release of cytochrome c and apoptosis-inducing factor, calpain-specific proteolysis and neuronal apoptosis. We conclude that (i) signal transduction pathways exist that prevent the entry of neurons into a caspase-dependent death after mitochondrial release of cytochrome c and (ii) that calpain activation links nitric oxide-triggered excitotoxic events with the execution of caspase-independent apoptosis in neurons.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Calcium-Binding Proteins; Calpain; Caspase 9; Caspases; Cells, Cultured; Cerebellum; Cytochromes c; Enzyme Activation; Flavoproteins; Membrane Proteins; Mice; Mice, Inbred BALB C; Mitochondria; Neurons; Neurotoxins; Nitric Oxide; Nitric Oxide Donors; Peptide Hydrolases; Protein Structure, Tertiary; S-Nitrosoglutathione

To investigate the expression of the calpain-calpastatin system in the human oocyte.. The expression of the calpain-calpastatin system was determined by immunohistochemistry and immunoblot analysis.. Academic research laboratory.. Twenty Israeli women who underwent IVF for fertility problems.. Oocytes that had no pronuclei 24 hours after insemination by either conventional IVF or intracytoplasmic sperm injection were retrieved for the study.. Analysis of calpain isoforms (m, mu) and calpastatin distribution within the human oocyte.. Western blot analysis confirmed the expression of calpain and calpastatin. Immunohistochemistry of fixed, permeabilized oocytes exhibited localization of both calpains to the cortical region of the oocyte, as well as the cytosol. Calpastatin seemed to be distributed throughout the cytosol, with a marked accumulation in the cell membrane. We have demonstrated a negative correlation between the occurrence of cortical granule exocytosis and the stability of the metaphase plate.. A complete calpain-calpastatin system is expressed in the human oocyte and might play a role in the various calcium-mediated processes occurring during activation of human oocytes.

Topics: Blotting, Western; Calcium-Binding Proteins; Calpain; Cell Membrane Permeability; Cysteine Proteinase Inhibitors; Female; Gene Expression Regulation, Enzymologic; Humans; Immunohistochemistry; Isoenzymes; Oocytes

Studies on the correlation between expression and/or autolysis of calpain and postmortem proteolysis in muscle have provided conflicting evidence regarding the possible role of calpain 3 in postmortem tenderization of meat. Thus, the objective of this research was to test the effect of postmortem storage on proteolysis and structural changes in muscle from normal and calpain 3 knockout mice. Knockout mice (n = 6) were sacrificed along with control mice (n = 6). Hind limbs were removed and stored at 4 degrees C; muscles were dissected at 0, 1, and 3 d postmortem and subsequently analyzed individually for degradation of desmin. Pooled samples for each storage time and mouse type were analyzed for degradation of nebulin, dystrophin, vinculin, and troponin-T. In a separate experiment, hind-limb muscles from knockout (n = 4) and control mice (n = 4) were analyzed for structural changes at 0 and 7 d postmortem using light microscopy. As an index of structural changes, fiber detachment, cracked or broken fibers, and the appearance of space between sarcomeres were quantified. Cumulatively, the results of the first experiment indicated that postmortem proteolysis of muscle occurred similarly in control and in calpain 3 knockout mice. Desmin degradation did not differ (P > 0.99), and there were no indications that degradation of nebulin, dystrophin, vinculin, and troponin-T were affected by the absence of calpain 3 in postmortem muscle. Structural changes were affected by time postmortem (P < 0.05), but not by the absence of calpain 3 from the muscles. In conclusion, these results indicate that calpain 3 plays a minor role, if any, in postmortem proteolysis in muscle.

Topics: Animals; Blotting, Western; Calcium-Binding Proteins; Calpain; Lower Extremity; Meat Products; Mice; Mice, Knockout; Muscle Proteins; Muscle, Skeletal; Myofibrils; Postmortem Changes; Time Factors

Modern processing techniques for turkey involve rapid chilling to slow microbial growth and early deboning of the economically important breast meat. This paper shows that these 2 processes lead to significantly tougher meat with higher cooking losses. The toughening appears to be due to less extensive proteolysis and shortening of the sarcomeres. Calpains I and II and their inhibitor, calpastatin, were quantified in turkey breast. Calpain II was the more common isoform but showed no evidence of activation during aging. In contrast, calpain I and calpastatin activities declined rapidly and were no longer detected 24 h postslaughter. There was no evidence of an association between calpain activity and processing conditions.

Topics: Animals; Bone and Bones; Calcium-Binding Proteins; Calpain; Food Handling; Freezing; Meat; Muscle, Skeletal; Peptide Hydrolases; Postmortem Changes; Quality Control; Time Factors; Turkeys

Our objective was to evaluate whether small (biopsy-sized) samples could be used to measure calpain and calpastatin activities in skeletal muscle. The accuracy of different separation and assay methods for the quantification of calpains and calpastatin from small (1.0 and 0.2 g) skeletal muscle samples was tested. In Exp. 1, the LM was removed from six lambs, and a 50-g subsample was processed using the reference method (DEAE-Sephacel chromatography and casein assay). Subsamples (1.0 and 0.2 g) also were processed using the two-step separation (1 mL DEAE-Sephacel and bulk elution using 200 and 400 mM NaCl) and heated calpastatin methods; in both cases, fractions were assayed with Bodipy-labeled and [14C]-labeled casein microassays. Finally, casein zymography was used to separate and quantify the calpain proteases from 1.0-and 0.2-g samples. The values obtained after processing the 50-g sample using the reference method were judged most accurate, and the alternative approaches were compared with these. For each extraction and assay approach, we considered: 1) the effect of the sample size on the mean activity; 2) increased or decreased variation of data; and 3) the correlation relative to the reference method. Where possible, we compared the ratio of calpain to calpastatin activities determined using the alternative approaches with the ratios found using the reference method. These methodologies were further investigated in Exp. 2, where single homogenates from different tissues (heart, spleen, lung, and muscle) were assayed using the alternative approaches. Experiment 1 established that most of the approaches suffered from poor correlations and/or unacceptable variation. By using a large, homogenous sample in Exp. 2, however, we determined that this error was not due to the methodologies themselves. Therefore, the unacceptable variation found in Exp. 1 resulted from the small sample size, and we recommend that large tissue samples (e.g., 50 g) should be used for calpain and calpastatin activity measurements in skeletal muscle instead of small tissue biopsies (e.g., 0.2 and 1.0 g).

Topics: Animals; Biopsy, Needle; Boron Compounds; Calcium-Binding Proteins; Calpain; Carbon Isotopes; Chemistry Techniques, Analytical; Chromatography, DEAE-Cellulose; Lung; Muscle, Skeletal; Myocardium; Reference Values; Sheep; Spleen

We reported that cigarette smoke extract (CSE) causes decreases in the activity and expression of endothelial nitric oxide synthase (eNOS) and calpain activity in pulmonary artery endothelial cells (PAECs). Calpains are a family of calcium-dependent endopeptidases, and their specific endogenous inhibitor is calpastatin. In this study, we evaluated the role of calpain-calpastatin in CSE-induced decrease in eNOS gene expression. PAEC were incubated with 5-10% CSE for 2-24 h. eNOS gene transcription rate, eNOS messenger ribonucleic acid (mRNA) half-life, and the activity and protein contents of calpain and calpastatin were measured. Incubation of PAEC with CSE caused significant decreases in eNOS gene transcription and calpain activity and an increase in calpastatin protein content. eNOS mRNA half-life was not significantly altered by CSE. To investigate whether CSE-induced inhibition of eNOS gene expression is caused by decreased calpain activity due to an increase in calpastatin protein content, we cloned calpastatin gene from PAEC and constructed adenovirus vectors containing calpastatin. Overexpression of calpastatin mimics the inhibitory effects of CSE on calpain activity and on the activity, protein, and mRNA of eNOS. The cell-permeable calpain inhibitor, calpastatin peptide, inhibits acetylcholine-induced endothelium-dependent relaxation of the pulmonary artery. Incubation of PAEC with an antisense oligodeoxyribonucleotide of calpastatin prevented CSE-induced increases in calpastatin protein and CSE-induced decreases in calpain activity, eNOS gene transcription, activity and protein content of eNOS, and NO release. These results indicate that CSE-induced inhibition of eNOS expression in PAEC is caused by calpain inhibition due to an increase in calpastatin protein content.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cells, Cultured; Endothelium; Half-Life; Lung; Molecular Sequence Data; Nitric Oxide Synthase Type III; Oligodeoxyribonucleotides, Antisense; Peptides; Pulmonary Artery; RNA, Messenger; Smoking; Swine; Transcription, Genetic

We examined the effects of trandolapril and candesartan on changes in the levels of sarcoglycans and dystrophin in the right ventricle of rats with the left coronary artery ligation. Hemodynamic and morphological alterations suggested the development of hypertrophy of the right ventricle and chronic heart failure by the 8th week. By the end of the 8th week, alpha- and beta-sarcoglycans and dystrophin were decreased. Increases in mu- and m-calpains in the hypertrophied right ventricle were associated with an elevation of casein-proteolytic activity in the cytosolic fraction. Oral administration of 3 mg/kg/day trandolapril or 1 mg/kg/day candesartan from the 2nd to 8th week after the left coronary artery ligation attenuated decreases in alpha-sarcoglycan and dystrophin and reduced the increased proteolytic activity. The results suggest that attenuation of decreases in sarcoglycans and dystrophin is a possible mechanism underlying trandolapril- and candesartan-mediated improvement of structural and functional alterations of the right ventricle in the coronary artery-ligated rat.

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Benzimidazoles; Biphenyl Compounds; Blood Pressure; Blotting, Western; Body Weight; Calcium-Binding Proteins; Calpain; Coronary Vessels; Cytosol; Dystrophin; Gene Expression; Heart Rate; Heart Ventricles; Indoles; Ligation; Male; Myocardial Infarction; Protein Isoforms; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sarcoglycans; Tetrazoles; Time Factors; Transcription, Genetic

In the current study, the involvement of calpain, a cysteine proteinase in the regulation of melanogenesis was examined using mouse B16 melanoma cells. In response to alpha-melanocyte-stimulating hormone (a-MSH), B16 melanoma cells underwent differentiation characterized by increased melanin biosynthesis. The total calapain activity was decreased within 2 h following alpha-MSH-treatment, and restored to the initial level in 6-12 h. To further investigate the involvement of calpain in the regulation of melanogenesis, the effect of calpain inhibitors on alpha-MSH-induced melanogenesis was examined. Inhibition of calpain by either N-acetyl-Leu-Leu-norleucinal (ALLN) or calpastatin (CS) peptide blocked alpha-MSH-induced melanogenesis. The magnitude of inhibition of melanin biosynthesis was well correlated with a decrease in the activity of tyrosinase, a key regulatory enzyme in melanogenesis. Treatment of B16 cells with ALLN caused marked decrease in both tyrosinase protein and mRNA levels. These results indicate that calpain would be involved in the melanogenic signaling by modulating the expression of tyrosinase in mouse B16 melanoma cells.

Topics: alpha-MSH; Animals; Blotting, Western; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Hormones; Indolequinones; Leupeptins; Melanins; Melanocytes; Melanoma, Experimental; Mice; Monophenol Monooxygenase; RNA, Messenger; Tumor Cells, Cultured

p94 (also called calpain 3) is the skeletal muscle-specific calpain and is considered to be a "modulator protease" in various cellular processes. Analysis of p94 at the protein level is an urgent issue because the loss of p94 protease activity causes limb-girdle muscular dystrophy type 2A. In this study, we enzymatically characterized one alternatively spliced variant of p94, p94:exons 6(-)15(-)16(-) (p94delta), which lacks two of the p94-specific insertion sequences. In contrast to p94, which has hardly been studied enzymatically due to its rapid, thorough, and apparently Ca(2+)-independent autolytic activity, p94delta was stably expressed in COS and insect cells. p94delta showed Ca(2+)-dependent caseinolytic and autolytic activities and an inhibitor spectrum similar to those of the conventional calpains. However, calpastatin did not inhibit p94delta and is a substrate for p94delta, which is consistent with the properties of p94, presenting p94 as a possible regulator of the conventional calpain system. We also established a semi-quantitative fluorescence resonance energy transfer assay using the calpastatin sequence specifically to measure p94 activity. This method detects the activity of COS-expressed p94 and p94delta, suggesting that it has potential to evaluate p94 activity in vivo and in the diagnosis of limb-girdle muscular dystrophy type 2A.

Topics: Alternative Splicing; Amino Acid Sequence; Animals; Biological Assay; Calcium-Binding Proteins; Calpain; Chlorocebus aethiops; COS Cells; Enzyme Activation; Fluorescence; Mice; Molecular Sequence Data; Muscle, Skeletal; Sequence Deletion

In the absence and in the resolution of inflammatory responses, neutrophils rapidly undergo spontaneous apoptosis. Here we report about a new apoptosis pathway in these cells that requires calpain-1 activation and is essential for the enzymatic activation of the critical effector caspase-3. Decreased levels of calpastatin, a highly specific intrinsic inhibitor of calpain, resulted in activation of calpain-1, but not calpain-2, in neutrophils undergoing apoptosis, a process that was blocked by a specific calpain-1 inhibitor or by intracellular delivery of a calpastatin peptide. Further support for the importance of the calpastatin-calpain system was obtained by analyzing neutrophils from patients with cystic fibrosis that exhibited delayed apoptosis, associated with markedly increased calpastatin and decreased calpain-1 protein levels compared with neutrophils from control individuals. Additional studies were designed to place calpain-1 into the hierarchy of biochemical events leading to neutrophil apoptosis. Pharmacological calpain inhibition during spontaneous and Fas receptor-induced neutrophil apoptosis prevented cleavage of Bax into an 18-kDa fragment unable to interact with Bcl-xL. Moreover, calpain blocking prevented the mitochondrial release of cytochrome c and Smac, which was indispensable for caspase-3 processing and enzymatic activation, both in the presence and absence of agonistic anti-Fas receptor antibodies. Taken together, calpastatin and calpain-1 represent critical proximal elements in a cascade of pro-apoptotic events leading to Bax, mitochondria, and caspase-3 activation, and their altered expression appears to influence the life span of neutrophils under pathologic conditions.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-X Protein; Calcium-Binding Proteins; Calpain; Carrier Proteins; Caspase 3; Caspases; Cell Death; Cells, Cultured; Cystic Fibrosis; Cytochromes c; Densitometry; Dose-Response Relationship, Drug; Enzyme Activation; fas Receptor; Humans; Immunoblotting; Intracellular Signaling Peptides and Proteins; Microscopy, Confocal; Mitochondrial Proteins; Models, Biological; Models, Molecular; Neutrophils; Precipitin Tests; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Subcellular Fractions; Time Factors

Cell migration is a fundamental cellular function particularly during skeletal muscle development. Ubiquitous calpains are well known to play a pivotal role during muscle differentiation, especially at the onset of fusion. In this study, the possible positive regulation of myoblast migration by calpains, a crucial step required to align myoblasts to permit them to fuse, was investigated. Inhibition of calpain activity by different pharmacological inhibitors argues for the involvement of these proteinases during the migration of myoblasts. Moreover, a clonal cell line that fourfold overexpresses calpastatin, the endogenous inhibitor of calpains, and that exhibits deficient calpain activities was obtained. The results showed that the migratory capacity of C2C12 and fusion into multinucleated myotubes were completely prevented in these clonal cells. Calpastatin-overexpressing myoblasts unable to migrate were characterized by rounded morphology, the loss of membrane extensions, the disorganization of stress fibers and exhibited a major defect in new adhesion formation. Surprisingly, the proteolytic patterns of desmin, talin, vinculin, focal adhesion kinase (FAK) and ezrin, radixin, moesin (ERM) proteins are the same in calpastatin-overexpressing myoblasts as compared to control cells. However, an important accumulation of myristoylated alanine-rich C kinase substrate (MARCKS) was observed in cells showing a reduced calpain activity, suggesting that the proteolysis of this actin-binding protein is calpain-dependent and could be involved in both myoblast adhesion and migration.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Adhesion; Cell Fusion; Cell Line; Cell Movement; Clone Cells; Cysteine Proteinase Inhibitors; Cytoskeleton; Dipeptides; Dose-Response Relationship, Drug; Glucosidases; Intracellular Signaling Peptides and Proteins; Leupeptins; Membrane Proteins; Mice; Muscle Fibers, Skeletal; Myoblasts; Myristoylated Alanine-Rich C Kinase Substrate; Oligopeptides; Phosphoproteins; Stress Fibers

Uncontrolled activation of calpain has been linked to tissue damage after neuronal and cardiac ischemias, traumatic spine and brain injuries, and multiple sclerosis and Alzheimer's disease. In vivo, the activity of calpain is regulated by its endogenous inhibitor calpastatin. The pathological role of calpain has been attributed to an imbalance between the activities of the protease and its inhibitor. Thus, it is possible that by reimposing functional control on the protease, the progression of calpain-mediated diseases could be slowed or eliminated. B27-WT is a 27-residue peptide (DPMSSTYIEELGKREVTIPPKYRELLA) derived from calpastatin that was previously shown to be a potent inhibitor of mu- and m-calpain. Recently, we identified two hot spots (Leu(11)-Gly(12) and Thr(17)-Ile(18)-Pro(19)) within which the amino acid residues that are key to B27-WT's bioactivity are clustered. In the work described here, the most critical residues of B27-WT, Leu(11) and Ile(18), were further probed to determine the nature of their interaction with calpain. Our results demonstrate that the side chains of both residues interact with hydrophobic pockets in calpain and that each of these interactions is indispensable for effective inhibition of calpain. Direct interactions involving the beta- and gamma-CH(2)- of the Leu(11) and Ile(18) side chains, respectively, rather than the degree of side chain branching or hydrophobicity, seemed to play a significant role in the peptide's ability to inhibit calpain. Furthermore, the minimum peptide sequence that still retained the calpain-inhibitory potency of B27-WT was found to be MSSTYIEELGKREVTIPPKYRELL.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Autolysis; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Humans; Hydrophobic and Hydrophilic Interactions; Isoleucine; Leucine; Molecular Sequence Data; Peptide Fragments; Peptide Library; Protein Structure, Secondary; Protein Structure, Tertiary; Structure-Activity Relationship; Swine

The forkhead box transcription factor Foxj1 is required for cilia formation and left-right axis determination. To define the role of Foxj1 in ciliogenesis, microarray analysis was performed to identify differentially expressed genes in the pulmonary epithelium of foxj1(+/+) and foxj1(-/-) mice. In the absence of Foxj1, the expression of calpastatin, an inhibitor of the protease calpain, decreased. RNase protection confirmed the decrease in calpastatin expression and decreased calpastatin was detected in the proximal pulmonary epithelium of foxj1(-/-) mice by immunohistochemistry. No change was detected in the expression of calpain 2 in the pulmonary epithelium by western blot or immunohistochemistry. By western blot and immunofluorescence, ezrin, a substrate for calpain, was also found to decrease in the pulmonary epithelium of foxj1(-/-) mice. No change in ezrin gene expression was found by RT-PCR. A decrease in ezrin binding phosphoprotein-50 (EBP-50) was also detected by immunofluorescence in the foxj1(-/-) mouse pulmonary epithelium. Immunoelectron microscopy demonstrated ezrin associated with the basal bodies of cilia in the pulmonary epithelium. Treatment of tracheal explants from foxj1(-/-) mice with a calpain inhibitor resulted in a partial reappearance of cilia observed in these mice. Additionally, following treatment of foxj1(-/-) tracheal explants with calpain inhibitor, basal bodies were observed in an apical location along with relocalization of ezrin and EBP-50. Regulation of calpain activity by calpastatin thus provides a mechanism for regulating the anchoring of basal bodies to the apical cytoskeleton in ciliated cells. In the absence of Foxj1, decreased calpastatin expression with decreased ezrin and EBP-50 results in an inability of basal bodies to anchor to the apical cytoskeleton and subsequent failure of axonemal formation.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cilia; Cytoskeletal Proteins; Cytoskeleton; DNA-Binding Proteins; Fluorescent Antibody Technique, Direct; Forkhead Transcription Factors; Gene Expression Regulation; Immunohistochemistry; Lung; Mice; Mice, Knockout; Organ Culture Techniques; Phosphoproteins; Respiratory Mucosa; Trachea; Transcription Factors

The experiments in this study were undertaken to determine whether inhibition of calpain activity in skeletal muscle is associated with alterations in muscle metabolism. Transgenic mice that overexpress human calpastatin, an endogenous calpain inhibitor, in skeletal muscle were produced. Compared with wild type controls, muscle calpastatin mice demonstrated normal glucose tolerance. Levels of the glucose transporter GLUT4 were increased more than 3-fold in the transgenic mice by Western blotting while mRNA levels for GLUT4 and myocyte enhancer factors, MEF 2A and MEF 2D, protein levels were decreased. We found that GLUT4 can be degraded by calpain-2, suggesting that diminished degradation is responsible for the increase in muscle GLUT4 in the calpastatin transgenic mice. Despite the increase in GLUT4, glucose transport into isolated muscles from transgenic mice was not increased in response to insulin. The expression of protein kinase B was decreased by approximately 60% in calpastatin transgenic muscle. This decrease could play a role in accounting for the insulin resistance relative to GLUT4 content of calpastatin transgenic muscle. The muscle weights of transgenic animals were substantially increased compared with controls. These results are consistent with the conclusion that calpain-mediated pathways play an important role in the regulation of GLUT4 degradation in muscle and in the regulation of muscle mass. Inhibition of calpain activity in muscle by overexpression of calpastatin is associated with an increase in GLUT4 protein without a proportional increase in insulin-stimulated glucose transport. These findings provide evidence for a physiological role for calpains in the regulation of muscle glucose metabolism and muscle mass.

Topics: Animals; Calcium-Binding Proteins; Calpain; Female; Glucose Transporter Type 4; Humans; Male; Mice; Mice, Transgenic; Monosaccharide Transport Proteins; Muscle Proteins; Muscle, Skeletal; Organ Size; RNA, Messenger; Sex Characteristics

Previous studies using post-mortem human brain extracts demonstrated that PrP in Creutzfeldt-Jakob disease (CJD) brains is cleaved by a cellular protease to generate a C-terminal fragment, referred to as C2, which has the same molecular weight as PrP-(27-30), the protease-resistant core of PrP(Sc) (1). The role of this endoproteolytic cleavage of PrP in prion pathogenesis and the identity of the cellular protease responsible for production of the C2 cleavage product has not been explored. To address these issues we have taken a combination of pharmacological and genetic approaches using persistently infected scrapie mouse brain (SMB) cells. We confirm that production of C2 is the predominant cleavage event of PrP(Sc) in the brains of scrapie-infected mice and that SMB cells faithfully recapitulate the diverse intracellular proteolytic processing events of PrP(Sc) and PrP(C) observed in vivo. While increases in intracellular calcium (Ca(2+)) levels in prion-infected cell cultures stimulate the production of the PrP(Sc) cleavage product, pharmacological inhibitors of calpains and overexpression of the endogenous calpain inhibitor, calpastatin, prevent the production of C2. In contrast, inhibitors of lysosomal proteases, caspases, and the proteasome have no effect on C2 production in SMB cells. Calpain inhibition also prevents the accumulation of PrP(Sc) in SMB and persistently infected ScN2A cells, whereas bioassay of inhibitor-treated cell cultures demonstrates that calpain inhibition results in reduced prion titers compared with control-treated cultures assessed in parallel. Our observations suggest that calpain-mediated endoproteolytic cleavage of PrP(Sc) may be an important event in prion propagation.

Topics: Animals; Biological Assay; Brain; Calcium; Calcium-Binding Proteins; Calpain; Cell Division; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Ionophores; Kinetics; Mice; Prions; Protein Structure, Secondary; Protein Structure, Tertiary; PrPC Proteins; PrPSc Proteins; Scrapie; Time Factors

Using both in vitro and in vivo approaches, numerous studies have provided evidence that mu-calpain is responsible for postmortem proteolysis. This paper reports the effect of overexpression of calpastatin on postmortem proteolysis in transgenic mice. Transgenic mice (n = 8) with a human calpastatin gene, whose expression was driven by the human skeletal muscle actin promoter, were killed along with control nontransgenic littermates (n = 5). Hind limbs were removed and stored at 4 degrees C, and muscle samples were dissected at 0, 1, 3, and 7 d postmortem and analyzed individually. At time 0, active human calpastatin was expressed in transgenic murine skeletal muscle at a level 370-fold greater (P < 0.001) than calpastatin in control mice. Although the native isoform of this protein was degraded with storage, at 7 d postmortem, approximately 78% of at-death activity remained, indicating that degraded calpastatin retains activity. Calpain (mu- and m-) expression was unaffected (P > 0.05) by the transgene as assessed by immunoreactivity at d 0. Over 7 d, 33% of at-death 80-kDa isoform immunoreactivity of mu-calpain was lost in transgenics compared to an 87% loss in controls, indicating that autolysis of mu-calpain was slowed in transgenic mice. Desmin degradation was also inhibited (P < 0.05) in transgenics when compared to controls. Control mice lost 6, 78, and 91% of at-death native desmin at 1, 3, and 7 d postmortem, respectively; conversely, transgenic mice lost only 1, 3, and 17% at the same times. A similar trend was observed when examining the degradation of troponin-T. Interestingly, m-calpain seemed to undergo autolysis in control mice, which in postmortem tissue is indicative of proteolysis. Further investigation revealed that both mu- and m-calpain are active postmortem in normal murine skeletal muscle. In conclusion, a high level of expression of active calpastatin was achieved, which, by virtue of its inhibitory specificity, was determined to be directly responsible for a decrease in postmortem proteolysis.

Topics: Animals; Blotting, Western; Calcium-Binding Proteins; Calpain; Desmin; DNA; Electrophoresis, Polyacrylamide Gel; Female; Male; Meat; Mice; Mice, Transgenic; Molecular Weight; Muscles; Myofibrils; Polymerase Chain Reaction; Postmortem Changes; Troponin T; Vinculin

The objective of this study was to determine whether differences in pork tenderness and water-holding capacity could be explained by factors influencing calpain activity and proteolysis. Halothane-negative (HAL-1843 normal) Duroc pigs (n = 16) were slaughtered, and temperature and pH of the longissimus dorsi (LD), semimembranosus (SM), and psoas major (PM) were measured at 30 and 45 min and 1, 6, 12, and 24 h postmortem. Calpastatin activity; mu-calpain activity; and autolysis and proteolysis of titin, nebulin, desmin, and troponin-T were determined on muscle samples from the LD, SM, and PM at early times postmortem. Myofibrils from each muscle were purified to assess myofibril-bound (mu-calpain. Percentage drip loss was determined, and Warner-Bratzler shear (WBS) force was analyzed. Myosin heavy-chain (MHC) isoforms were examined using SDS-PAGE. The pH of PM was lower (P < 0.01) than the pH of LD and SM at 30 and 45 min and 1 h postmortem. The PM had a higher (P < 0.01) percentage of the MHC type IIa/IIx isoforms than the LD. The-LD had the greatest proportion of (P < 0.01) MHC IIb isoforms of any of the muscles. The PM had the lowest (P < 0.01) percentage of MHC IIb isoforms and a greater (P < 0.05) percentage of type I MHC isoforms than the LD and SM. The PM had less (P < 0.01) drip loss after 96 h of storage than the SM and LD. The PM had more desmin degradation (P < 0.01) than the LD and SM at 45 min and 6 h postmortem. Degradation of titin occurred earlier in the PM than the LD and SM. At 45 min postmortem, the PM consistently had some autolysis of mu-calpain, whereas the LD and SM did not. At 6 h postmortem, some autolysis of mu-calpain (80-kDa subunit) was observed in all three muscles. The rapid pH decline and increased rate of autolysis in the PM paralleled an earlier appearance of myofibril-bound mu-calpain. The SM had higher calpastatin activity (P < 0.05) at 45 min, 6 h, and 24 h and had higher WBS values at 48 h (P < 0.01) and 120 h (P < 0.05) postmortem than the LD. At 48 and 120 h postmortem, more degradation of desmin, titin, and nebulin were observed in the LD than in the SM. These results show that mu-calpain activity, mu-calpain autolysis, and protein degradation are associated with differences in pork tenderness and water-holding capacity observed in different muscles.

Topics: Animals; Calcium-Binding Proteins; Calpain; Desmin; Hydrogen-Ion Concentration; Isoenzymes; Male; Meat; Muscle Proteins; Muscle, Skeletal; Postmortem Changes; Swine; Temperature; Time Factors

Calpain, a calcium-dependent cytosolic cysteine protease, is implicated in a multitude of cellular functions but also plays a role in cell death. Recently, we have shown that two ubiquitous isoforms, termed micro-calpain and m-calpain, are expressed in rat pancreatic acinar cells and that calcium ionophore-induced calpain activation leads to acinar cell injury. On the basis of these observations, we have now investigated the role of both calpain forms and the endogenous calpain inhibitor calpastatin in acute pancreatitis. After treatment of rats either without or with calpain inhibitor Z-Val-Phe methyl ester (ZVP; 60 mg/kg i.p.), pancreatitis was induced by cerulein injections (10 microg/kg i.p.; 5 times at hourly intervals). Calpain activation and calpastatin expression in the pancreatic tissue were studied by Western blot analysis. Pancreatic injury was assessed by plasma amylase activity, pancreatic wet/dry weight ratio (edema), histological and electron-microscopic analyses, as well as fluorescence labeling of actin filaments. Cerulein caused an activation of both micro-calpain and m-calpain, accompanied by degradation of calpastatin. Prophylactic administration of ZVP reduced the cerulein-induced calpain activation but had no effect on calpastatin alterations. In correlation to the diminished calpain activity, the severity of pancreatitis decreased as indicated by a decline in amylase activity (P < 0.01), pancreatic edema formation (P < 0.05), histological score for eight parameters (P < 0.01), and actin filament alterations. Our findings support the hypothesis that dysregulation of the calpain-calpastatin system may play a role in the onset of acute pancreatitis.

Topics: Acute Disease; Animals; Calcium-Binding Proteins; Calpain; Ceruletide; Dipeptides; Enzyme Activation; Female; Pancreas; Pancreatitis; Rats; Rats, Inbred Lew; Severity of Illness Index

Postmortem changes in osmotic pressure; ionic strength; pH; temperature; mu- and m-calpain; calpastatin; desmin degradation; and myofibril fragmentation index (MFI) were determined in ovine longissimus muscle. Our objectives were to characterize changes in these variables and to identify postmortem time points at which significant proteolysis and tenderization (as measured by change in MFI) could be detected. Seven crossbred (Dorset x Romanov) lambs were slaughtered, and samples of the longissimus muscle were removed at 0, 3, 6, 9, 12, 24, 72, and 360 h postmortem. Osmotic pressure increased (P < 0.05) from 379 to 528 mOsm during the postmortem storage period, with two-thirds of the increase occurring within the first 24 h. By measuring conductivity, we showed that ionic strength increased (P < 0.05) from 8.13 to 9.78 mS/cm during the storage period, which is equivalent to 79 and 97 mM NaCl solutions, respectively. In accordance with pH and temperature, conductivity reached ultimate levels at 24 h postmortem. Within 9 h postmortem, mu-calpain activity had decreased (P < 0.05) from at-death values and continued to decrease until 72 h, at which time it was undetectable. It was still possible to detect the 76-kDa isoforms (a product of the autolysis of the 80-kDa subunit of mu-calpain) immunologically, which implies that the loss of activity was not caused by extensive autolysis. In contrast, m-calpain activity remained constant throughout the aging period, whereas calpastatin activity was stable until 24 h postmortem, after which it gradually decreased. Autolysis products of mu-calpain were detected at 3 h postmortem, indicating that mu-calpain was activated some time between 0 and 3 h postmortem. Moreover, the effect of mu-calpain activity on myofibrillar substrates was first observed at 9 h postmortem, when a 23% loss of native desmin was detected. This degradation translated into an increase in MFI at 12 h. Collectively, these results imply that mu-calpain is active in postmortem muscle in the presence of calpastatin, and that effects of mu-calpain activity as determined by increased MFI are detectable during the first 12 h postmortem.

Topics: Animals; Body Temperature; Calcium-Binding Proteins; Calpain; Desmin; Hydrogen-Ion Concentration; Immunoblotting; Meat; Muscle, Skeletal; Myofibrils; Osmolar Concentration; Osmotic Pressure; Postmortem Changes; Sheep; Time Factors

Angiogenesis is an integral part of both the pulmonary inflammatory response to chronic exposure to cigarette smoke and the lung tissue remodeling associated with cigarette smoke-induced chronic obstructive pulmonary disease (COPD). To investigate the role of angiogenesis in the pathogenesis of COPD, we evaluated the effect of cigarette smoke extract (CSE) on angiogenesis of pulmonary artery endothelial cells (PAEC). Incubation of PAEC with 2.5-10% CSE resulted in a dose-dependent inhibition of endothelial monolayer wound repair. CSE also caused inhibition of tube formation on Matrigel, migration in a Boyden chamber, and proliferation of PAEC. Because calpain, a family of calcium-dependent intracellular proteases, mediates cytoskeletal signaling in endothelial motility, we explored the role of calpain in the CSE-induced inhibition of endothelial angiogenesis. Incubation of CSE resulted in a dose-dependent decrease in calpain activity. Calpain inhibitor-1, a specific inhibitor of calpain, potentiates inhibitory effect of CSE on the endothelial monolayer wound repair, tube formation, cell migration, and cell proliferation. Transfection of PAEC with antisense oligodeoxyribonucleotides of calpastatin, the major endogenous calpain inhibitor, prevented CSE-induced increase in calpastatin protein content and CSE-induced decreases in calpain activity. It also prevented CSE-induced decreases in monolayer wound repair, tube formation, and migration. These results suggest that CSE attenuates angiogenesis of PAEC and the mechanism involves inhibition of calpain. Impaired angiogenesis may impede the repair process in the lungs of cigarette smokers and contribute to the altered structural remodeling observed in the lungs of patients with cigarette smoke-related COPD.

Topics: Animals; Base Sequence; Calcium-Binding Proteins; Calpain; Cell Division; Endothelium, Vascular; Enzyme Inhibitors; Gene Expression Regulation; Models, Animal; Neovascularization, Pathologic; Oligodeoxyribonucleotides, Antisense; Pulmonary Artery; RNA, Messenger; Smoking; Swine; Transfection; Wound Healing

Excessive activation of calpains (calcium-activated neutral proteases) is observed following spinal cord contusion injury, traumatic brain injury, stroke, and in neurodegenerative disorders including Alzheimer's disease. Calpain inhibition represents an attractive therapeutic target, but current calpain inhibitors possess relatively weak potency, poor specificity, and in many cases, limited cellular and blood-brain barrier permeability. We developed novel calpain inhibitors consisting of the endogenous inhibitor, calpastatin or its inhibitory domain I, fused to the protein transduction domain of the HIV trans-activator (Tat) protein (Tat(47-57)). The Tat-calpastatin fusion proteins were potent calpain inhibitors in a cell-free activity assay, but did not inhibit cellular calpain activity in primary rat cortical neurons when applied exogenously at concentrations up to 5 microM. The fusion proteins were able to transduce neurons, but were localized within endosome-like structures. A similar endosomal uptake was observed for Tat-GFP. Together, the results suggest that endosomal uptake of the Tat-calpastatin prevents its interaction with calpain in other cellular compartments. Endosomal uptake of proteins fused to the Tat protein transduction domain severely limits the applications of this methodology.

Topics: Animals; Brain Diseases; Calcium-Binding Proteins; Calpain; Cell Compartmentation; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; Endosomes; Fetus; Gene Products, tat; Humans; Nerve Degeneration; Neurons; Protein Structure, Tertiary; Rats; Recombinant Fusion Proteins; Transduction, Genetic

Sixteen multiparous Jersey cows were assigned at calving to one of 4 dietary treatments. An 18% crude protein (CP) diet was fed as a total mixed ration through 30 d in milk (DIM), and beginning at 31 DIM a 9, 12, 15, or 18% CP diet was fed through 58 DIM (depletion). All cows were then fed the 18% CP diet until 84 DIM (repletion). Muscle biopsies were taken under local anesthesia at 49 and 84 DIM from the semitendinosus muscle. Milk production, DMI, and milk component contents were measured. Calpain and calpastatin contents of muscle taken at biopsy were evaluated using Western blotting techniques. Milk production and milk protein content were reduced during the depletion period by decreasing dietary protein. Diet had no effect on milk fat content or DMI. During repletion, DMI was affected by dietary treatment. Western blots of muscle extracts indicated no differences in calpain content at any stage of the experiment or in calpastatin content of muscle at 49 DIM. However, at 84 DIM, calpastatin (135 kDa) was lower or undetectable in cows fed either the 9 or 12% CP diets from 31 to 59 DIM. Bands for a 110-kDa degradation product of calpastatin were present in some cows fed the 9, 12, and 15% CP diets during the depletion period. Results indicate a change in skeletal muscle calpain/calpastatin proteolytic system during protein repletion following depletion with diets of less than 15% CP during early to peak lactation in dairy cows.

Topics: Animals; Biopsy; Blotting, Western; Calcium-Binding Proteins; Calpain; Cattle; Dietary Proteins; Female; Lactation; Muscle, Skeletal

Calpain, a calcium-activated cysteine protease, is involved in modulating a variety of cell activities such as shape change, mobility, and apoptosis. The two ubiquitous isoforms of this protease, calpain I and II, are considered to be cytosolic proteins that can translocate to various sites in the cell. The activity of calpain is modulated by two regulatory proteins, calpastatin, the specific endogenous inhibitor of calpain, and the 28-kDa regulatory subunit. Using velocity gradient centrifugation, the results of this study confirm and greatly expand upon our previous finding that the calpain/calpastatin network is associated with the endoplasmic reticulum and Golgi apparatus in cells. Moreover, confocal microscopy demonstrates that calpain II colocalizes with specific proteins found in these organelles. Additional experiments reveal that hydrophobic rather than electrostatic interactions are responsible for the association of the calpain/calpastatin network with these organelles. Treatment of the organelles with Na2CO3 or deoxycholate reveal that calpain I, 78-kDa calpain II, and the regulatory subunit are "embedded" within the organelle membranes similar to integral membrane proteins. Proteinase K treatment of the organelles shows that calpain I and II, calpastatin, and the regulatory subunit localize to the cytosolic surface of the organelle membranes, and a subset of calpain II and the regulatory subunit are also found within the lumen of these organelles. These results provide a new and novel explanation for how the calpain/calpastatin network is organized in the cell.

Topics: Calcium-Binding Proteins; Calpain; Cell Line, Tumor; Centrifugation, Density Gradient; Chelating Agents; Cytoplasm; Cytosol; Deoxycholic Acid; Detergents; Edetic Acid; Egtazic Acid; Endopeptidase K; Endoplasmic Reticulum; Golgi Apparatus; Humans; Microscopy, Confocal; Microscopy, Fluorescence; Microsomes; Models, Biological; Potassium Chloride; Protein Isoforms; Sodium Chloride; Static Electricity; Time Factors; Ubiquitin

Calpains are crucial for the degradation of myofibrillar proteins in muscle. Calpastatin is a specific inhibitor of calpains. The objective of this study was to elucidate the effect of nutrient restriction on the activity of calpains and calpastatin in the skeletal muscle of both cows and fetuses. Beginning 30 d after conception, 20 cows were fed either a control diet consisting of native grass hay fortified with vitamins and minerals at recommendations for a mature cow to gain 0.72 kg/d or half the vitamins and minerals and millet straw at 68.1% of NEm requirements. Cows were slaughtered on d 125 of gestation, and the LM was sampled at the 12th rib for calpain and calpastatin measurement. When comparing the muscle samples from nutrient-restricted and control cows, no difference in the activity of calpain I and II was observed; however, there was a significant difference (P < 0.05) in calpastatin activity. Muscle samples from control cows had greater calpastatin content than those of nutrient-restricted cows (P < 0.05); in contrast, the calpastatin content of fetal muscle was greater in fetuses gestated by nutrient-restricted cows than those of control cows (P < 0.05). Further, there were three calpastatin isoforms of 125, 110, and 70 kD detected in fetal muscle, whereas only the110-kD isoform was detected for cow muscle. These results indicate that the activity of the calpain system in skeletal muscle is mainly controlled through the expression of calpastatin. Alternating the calpastatin content in muscle and thereby modulating calpain activity may provide a mechanism for the maintenance of fetal muscle growth during nutrient restriction, whereas skeletal muscle loss in cows is upregulated.

Topics: Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Calcium-Binding Proteins; Calpain; Cattle; Female; Fetal Development; Fetus; Food Deprivation; Isoenzymes; Molecular Weight; Muscle, Skeletal; Nutritional Requirements; Pregnancy; Random Allocation

While the average age for patients admitted with spinal cord injury is 32 years, patients under the age of 16 account for 5% of spinal cord injured persons. For these younger patients, an increased mortality up to 24 h post-injury has been reported, however, survivors may regain more function than their adult counterparts, suggesting that age may play a role in injury tolerance. While the use of growth factors as a therapy for spinal cord injury is well researched, the response of the developing cord to secondary injury has not been thoroughly investigated. Following spinal cord injury, Ca(2+) influx can activate enzymes such as calpain, a Ca(2+)-dependent protease, which plays a role in the pathogenesis of spinal cord injury in rats. The present investigation revealed that following spinal cord injury, calpain upregulation was significantly less (15.3%) in the 21-day-old rats than in either 45-day-old (70%) or 90-day-old (99.6%) rats, as shown by Western blot and in situ immunofluorescent studies. Expression of the endogenous calpain inhibitor, calpastatin, was significantly higher in juvenile rats than adult rats. Juvenile rats with spinal cord injury also showed a reduced Bax:Bcl-2 ratio (4:1 vs. 6:1), reduced caspase-3 staining, reduced myelin loss (3% vs. 18%), and less neuronal DNA damage, as compared to older rats. These results suggest that increased calpastatin levels found in juvenile rats muted calpain activity and neuronal apoptosis, following spinal cord injury.

Topics: Age Factors; Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Female; Hydrolysis; Neurons; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries

Calpains represent a well-conserved family of Ca2+ -dependent proteolytic enzymes. Recently, the importance of calpain in the metastatic process has received great attention. To investigate whether m-calpain contributes to the pathogenesis of colorectal cancer, we investigated the expression of m-calpain and its inhibitors, calpastatin and high-molecular-weight calmodulin-binding protein (HMWCaMBP), in human colorectal surgical specimens.. Fifty cases of colon carcinoma were evaluated for this study. Of 50 cases evaluated, we presented in this report six cases for m-calpain, calpastatin and HMWCaMBP protein expression by Western blot analyses was done in both normal and invasive tumor components of human samples. In addition, immunohistochemistry analysis was also carried out in all patients.. The activity and protein expression of m-calpain was significantly higher in colorectal adenocarcinoma than in normal colonic mucosa. This finding was corroborated by immunohistochemical studies that showed strong cytoplasmic staining in the colon tumors with m-calpain antibody. The decreased expression of these calpain inhibitors (calpastatin and HMWCaMBP) paralleled increased activity and expression of calpain in colorectal adenocarcinoma and the well-documented involvement of this Ca2+ -dependent protease in colon tumor.. Increased activity and moderate staining of m-calpain in polyps show the usage of this enzyme as a marker for the early detection of colorectal adenocarcinoma using immunologic approaches. These findings represent the first description of calpain overexpression in colorectal cancer. This has implications with regard to the design of chemotherapeutic drugs as well as in monitoring colorectal cancer in early stages of the metastatic process.

Topics: Adenocarcinoma; Biomarkers, Tumor; Calcium-Binding Proteins; Calmodulin-Binding Proteins; Calpain; Colorectal Neoplasms; Cysteine Proteinase Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry

We previously proposed that the calpain-mediated proteolytic pathway is activated in cultured cardiomyocytes following exposure to hypoxia (Mol Cell Biochem 214: 47, 2000). The potential role of calpastatin, the endogenous specific inhibitor of calpain, and its expression in the hypoxic state were investigated here. Hypoxia induced the expression of two calpastatin and multiple VEGF splice variants. Although cardiomyocytes and fibroblasts responded to hypoxia differentially, both cell types exhibited hypoxia-induced calpastatin transcription. The two functional calpastatin splice variants encoding the 593- and 654-amino acid calpastatin isoforms differed only in their N-terminal leader domain sequences. In spite of the increased mRNA expression, levels of the calpastatin protein doublet were not increased, but rather slightly decreased under the hypoxic condition. Cardiac hypoxia was accompanied by preferential proteolytic cleavage of troponin I (TnI), one of the major myofibrillar proteins. Forced expression of calpastatin through an adenoviral vector effectively prevented the hypoxia- and calpain-mediated TnI proteolysis. Our results highlight the discordant expression pattern of cardiac calpastatin mRNA and protein in the hypoxic state. We suggest that although induction of calpastatin gene transcription may constitute a compensatory mechanism coping with the hypoxic stress, a sustained high calpastatin protein level appears to be essential in the intervention of the activated calpain proteolytic cascade.

Topics: Adenoviridae; Alternative Splicing; Amino Acid Sequence; Animals; Animals, Newborn; Calcium-Binding Proteins; Calpain; Cell Hypoxia; Cells, Cultured; DNA, Complementary; Fibroblasts; Humans; Models, Biological; Molecular Sequence Data; Myocytes, Cardiac; Protein Structure, Tertiary; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sequence Homology, Amino Acid; Transcription, Genetic; Troponin I

Results of our recent studies in rats suggested that calpains play an important role in retinal cell death induced by ischemia-reperfusion in vivo and by hypoxia in vitro. Study of spontaneous animal models could help determine the involvement of calpains in human retinopathy. The WBN/Kob rat is such a model for spontaneous retinal degeneration. The purpose of the study reported here was to determine the involvement of calpain isoforms during retinal degeneration in WBN/Kob rats. Histologic and functional retinal degeneration in WBN/Kob rats was observed by use of light microscopy and electroretinography, respectively. Proteolysis of alpha-spectrin in the retina was detected by use of immunoblot analysis in aging WBN/Kob rats. This proteolysis was associated with the increases of retinal calcium content and caseinolytic activity for calpains 1 and 2. Expression of calpain 1, calpain 2, and calpastatin mRNAs in the retina, as measured by use of reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, were only slightly up-regulated at 24 weeks of age. In contrast, expression of retina-specific calpains, such as Rt88, Rt88', and Rt90 mRNA, was markedly down-regulated at 12 weeks of age. Expression of calpain 10 mRNA in the retina was only slightly down-regulated at 12 weeks of age. In contrast to mRNA expression, various expression patterns of calpain 10 proteins were observed. Increased retinal calcium content, leading to activation of calpains 1 and 2, may be an important event in the sequential changes leading to degeneration of the retina in WBN/Kob rats. Activated calpain causing proteolysis of alpha-spectrin and changes in Rt88, Rt88', Rt90 and calpain 10 may also contribute to retinal degeneration.

Topics: Animals; Calcium-Binding Proteins; Calpain; Disease Models, Animal; Down-Regulation; Electroretinography; Isoenzymes; Rats; Rats, Inbred Strains; Rats, Wistar; Retina; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spectrin; Up-Regulation

Calpastatin is the natural specific inhibitor of calpain. Recent research has linked uncontrolled calpain activation to tissue damage after neuronal and cardiac ischemias, traumatic spine and brain injuries, as well as Alzheimer's disease and cataract formation. An imbalance between the activities of calpain and calpastatin is believed to be responsible for the pathological role of calpain. An important key to understanding calpain regulation by calpastatin is to determine, at the molecular level, how calpastatin interacts with calpain to inhibit its enzymatic activity. A 27-residue peptide (DPMSSTYIEELGKREVTIPPKYRELLA) derived from subdomain 1B of the repetitive domains of calpain, named peptide B27-WT, was previously shown to be a potent inhibitor of mu- and m-calpain. In this report, a combination of beta-alanine scanning mutagenesis and kinetic measurements was used to probe, in a quantitative, systematic, and simultaneous fashion, the relative contribution of the amino acid side chain and backbone functionalities to the overall calpain-inhibitory activity of B27-WT. The study identified two "hot spots," Leu(11)-Gly(12) and Thr(17)-Ile(18)-Pro(19), in B27-WT within which the residues critical for inhibitory function are clustered. Mutation of any one of the key residues in either of the two hot spots resulted in a dramatic loss of inhibitory activity. Furthermore, it was shown that a restricted conformation of the Leu(11)-Gly(12) and Thr(17)-Ile(18)-Pro(19) backbones is required for the peptide inhibitory function. These results suggest a plausible model in which the two hot spots are situated at or near the interface(s) of the calpain-calpastatin complex and act in a concerted fashion to inhibit calpain. The information on the specific contribution of the amide bond and side chain of each key residue to the bioactivity of B27-WT will contribute to a better understanding of the mechanism of calpain inhibition and lead to novel and effective therapies based on the specific inhibition of dysregulated or overactivated calpain.

Topics: Amino Acid Sequence; Animals; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Humans; Kinetics; Molecular Sequence Data; Peptide Fragments; Protein Conformation; Sequence Homology, Amino Acid; Swine

Cross-talk between calpain and caspase proteolytic systems has complicated efforts to determine their distinct roles in apoptotic cell death. This study examined the effect of overexpressing calpastatin, the specific endogenous calpain inhibitor, on the activity of the two proteolytic systems following an apoptotic stimulus. Human SH-SY5Y neuroblastoma cells were stably transfected with full-length human calpastatin cDNA resulting in 20-fold overexpression based on Western blot and 5-fold greater calpain inhibitory activity in cell extracts. Wild type and calpastatin overexpressing (CST1) cells were neuronally differentiated and apoptosis-induced with staurosporine (0.1-1.0 microm). Calpastatin overexpression decreased calpain activation, increased caspase-3-like activity, and accelerated the appearance of apoptotic nuclear morphology. Following 0.1-0.2 microm staurosporine, plasma membrane integrity based on calcein-acetoxymethyl fluorescence was significantly greater at 24 h in differentiated CST1 compared with differentiated wild type cells. However, this protective effect was lost at higher staurosporine doses (0.5-1.0 microm), which resulted in pronounced caspase-mediated degradation of the overexpressed calpastatin. These results suggest a dual role for calpains during neuronal apoptosis. In the early execution phase, calpain down-regulates caspase-3-like activity and slows progression of apoptotic nuclear morphology. Subsequent calpain activity, facilitated by caspase-mediated degradation of calpastatin, contributes to plasma membrane disruption and secondary necrosis.

Topics: Apoptosis; Blotting, Western; Calcium-Binding Proteins; Calpain; Caspase 3; Caspases; Cell Line; Cell Membrane; Cell Nucleus; DNA, Complementary; Dose-Response Relationship, Drug; Humans; Microscopy, Fluorescence; Necrosis; Neurons; Staurosporine; Time Factors; Transfection; Tumor Cells, Cultured; Up-Regulation

Ubiquitously expressed calpains are Ca(2+)-dependent, intracellular cysteine proteases comprising a large catalytic subunit (domains DI-DIV) and a noncovalently bound small regulatory subunit (domains DV and DVI). It is unclear whether Ca(2+)-induced calpain activation is followed by subunit dissociation or not. Here, we have applied advanced fluorescence microscopy techniques to study calpain subunit interactions in living cells using recombinant calpain subunits or domains fused to enhanced cyan and enhanced yellow fluorescent reporter proteins. All of the overexpressed variants of the catalytic subunit (DI-IV, DI-III, and DI-IIb) were active and Ca(2+)-dependent. The intact large subunit, but not its truncated variants, associates with the small subunit under resting and ionomycin-activated conditions. All of the variants were localized in cytoplasm and nuclei, except DI-IIb, which accumulates in the nucleus and in nucleoli as shown by microscopy and cell fractionation. Localization studies with mutated and chimeric variants indicate that nuclear targeting of the DI-IIb variant is conferred by the two N-terminal helices of DI. Only those variants that contain DIII migrated to membranes upon the addition of ionomycin, suggesting that DIII is essential for membrane targeting. We propose that intracellular localization and in particular membrane targeting of activated calpain, but not dissociation of its intact subunits, contribute to regulate its proteolytic activity in vivo.

Topics: Amino Acid Motifs; Animals; Blotting, Western; Calcium; Calcium-Binding Proteins; Calpain; Cell Membrane; Cell Nucleus; COS Cells; Enzyme Activation; Fluorescent Antibody Technique; Humans; Protein Subunits; Recombinant Fusion Proteins

The cysteine proteases calpain and caspase-3 are known mediators of cell death. The aim of this study was to assess their contribution to the tissue damage found in experimental uremia.. Calpain and caspase-3 activities were measured in the hearts of rats that were sham-operated (control), sham-operated and spontaneously hypertensive (SHR), and those rendered uremic by 5/6 nephrectomy (uremic). In an in vitro study, heart myoblasts (Girardi) were incubated with human serum from healthy subjects (control serum conditioned media, CSCM) or uremic patients (uremic serum conditioned media, USCM), in the presence and absence of calpain and caspase-3 inhibitors. After 48 hours the activity of calpain and caspase-3 was measured, and cell injury determined by DNA fragmentation (ELISA) and lactate dehydrogenase (LDH) release. An in situ assay was designed to study how USCM affects calpain activity over time.. In the in vivo study, mean calpain activities were almost identical in the control and SHR groups, but calpain and caspase-3 activities were much elevated in the uremic group (P < 0.01 and 0.001 respectively vs. control). The SHR group had significantly higher mean arterial blood pressure (P < 0.001 vs. control, 0.01 vs. uremic). In the in vitro study calpain activity and DNA fragmentation were markedly higher in USCM treated cells compared to CSCM (both P<0.05). Both were reduced in USCM cells containing calpain inhibitors (E64d, calpastatin, or PD 150606). LDH release was raised also in USCM treated cultures (P < 0.05), which only the E64d treatment could significantly reduce (P < 0.02). Caspase-3 activities were similar in USCM and CSCM groups. The in situ assay showed significant increases in calpain activity in USCM treated cells compared to CSCM after just 3.5 hours (P<0.01).. In vivo results suggest that the increases in calpain and caspase-3 activity in uremic rat hearts were primarily due to uremia and not to hypertension. In vitro data demonstrate that uremia-induced cell injury can be attenuated by calpain inhibition. Therefore, it is likely that calpain is a mediator of uremia-induced myocardial injury.

Topics: Acrylates; Animals; Calcium-Binding Proteins; Calpain; Caspase 3; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Disease Models, Animal; Humans; Hypertrophy, Left Ventricular; Leucine; Male; Nephrectomy; Oligopeptides; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Uremia

Vitamin D-elicited hypercalcemia/hypercalciuria is associated with polyuria in humans and in animal models. In rats, dihydrotachysterol (DHT) induces AQP2 water channel downregulation despite unaltered AQP2 mRNA expression and thus we investigated the mechanism of AQP2 degradation. Incubation of AQP2-containing inner medullary collecting duct (IMCD) endosomes with Ca(2+) or calpain elicited AQP2 proteolysis, an effect abolished by leupeptin. This endogenous, Ca(2+)-sensitive protease activity exhibited a different proteolytic digest pattern from trypsin, which also degraded AQP2 in vitro. IMCDs contain abundant micro-calpain protein and functional calpain proteolytic activity as demonstrated by immunohistochemistry, immunoblotting, and gel zymography. Furthermore, by small particle flow cytometry we demonstrated that micro-calpain colocalizes with apical IMCD endosomes. DHT does not appear to elicit general proteolysis, however, in addition to AQP2 degradation, DHT treatment also diminished micro-calpain and calpastatin expression although whether these changes contributed to the AQP2 instability remains unclear. Together, these data show for the first time that AQP2 is a substrate for calpain-mediated proteolysis and that furthermore, micro-calpain, like AQP2, is both highly expressed in renal inner medulla and localized to apical IMCD endosomes.

Topics: Animals; Aquaporin 2; Aquaporin 6; Aquaporins; Calcium; Calcium-Binding Proteins; Calpain; Caseins; Cysteine Proteinase Inhibitors; Dextrans; Dihydrotachysterol; Dose-Response Relationship, Drug; Down-Regulation; Endosomes; Flow Cytometry; Immunoblotting; Immunohistochemistry; Kidney Tubules, Collecting; Leupeptins; Male; Protein Binding; Rats; Rats, Sprague-Dawley; Trypsin

Degradation of troponin I (TnI) by calpain occurs with myocardial stunning in ischemia-reperfusion injury. Glucocorticoids attenuate myocardial ischemia-reperfusion injury, but their effect on TnI degradation is unknown. A piglet model was used to test the hypotheses that cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA) are associated with TnI degradation and that TnI alterations could be prevented by glucocorticoid treatment. Piglets were cooled to 18 degrees C, subjected to 2 h of circulatory arrest, rewarmed to 37 degrees C, and allowed to recover for 2 h. Methylprednisolone was administered 6 h before surgery (3 0 mg/kg) and at initiation of CPB (30 mg/kg). The untreated group received saline. Left ventricular tissue was collected after recovery and analyzed by Western blot for TnI, calpain, and calpastatin (the natural inhibitor of calpain). CPB/DHCA animals had 27.4 +/- 0.2% of total detected TnI present in degraded form. Glucocorticoid treatment significantly decreased the percentage of degraded TnI (12.0 +/- 0.1%, p < 0.05). Calpain I and calpain II increased after CPB/DHCA compared with non-CPB/DHCA controls (p < 0.05), with or without glucocorticoid treatment. Calpastatin significantly decreased in untreated CPB/DHCA animals compared with non-CPB/DHCA controls (p < 0.05), but levels were preserved by glucocorticoids. Glucocorticoids were associated with preservation of maximum rate of increase of left ventricular pressure at 95 +/- 10% of baseline, whereas maximum rate of increase of left ventricular pressure decreased to 62 +/- 12% of baseline without steroids. TnI degradation occurs after CPB/DHCA in neonatal pigs. Reduction in reperfusion injury by glucocorticoids may depend partly on preservation of calpastatin activity and intact TnI.

Topics: Age Factors; Animals; Calcium-Binding Proteins; Calpain; Cardiac Output; Cardiopulmonary Bypass; Cells, Cultured; Glucocorticoids; Myocytes, Cardiac; Swine; Troponin I; Vascular Resistance

Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.

Topics: Animals; Anti-Bacterial Agents; Calcium-Binding Proteins; Calpain; Cattle; Cells, Cultured; Creatine Kinase; Creatine Kinase, MM Form; Cysteine Proteinase Inhibitors; DNA Primers; Doxycycline; Female; Gene Expression Regulation; Genes, Reporter; Immunoenzyme Techniques; Isoenzymes; Luciferases; Male; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Transgenic; Muscle, Skeletal; Plasmids; Polymerase Chain Reaction; Promoter Regions, Genetic

Treatment of OM10.1 cells latently infected with human immunodeficiency virus type 1 (HIV-1) with phorbol ester and calcium ionophore (A23187) induced virus replication which was blocked by N-Ac-Leu-Leu-norleucinal (ALLnL), a calpain inhibitor I, and not by lactacystin, a specific proteasome inhibitor. When the purified NF-kappa B/I kappa B complex was treated with mu-calpain, the specific DNA-binding activity was demonstrated by using electrophoretic mobility shift assay in vitro. This effect of mu-calpain was inhibited by ALLnL and calpastatin and not by lactacystin. In fact, we found that mu-calpain efficiently degraded I kappa B alpha. Furthermore, our Western blotting analysis has revealed that mu-calpain cleaves I kappa B alpha at its N-terminal and C-terminal regions that were previously reported to be involved in the interaction with NF-kappa B p65. These observations indicate that in monocyte/macrophage cells calcium signaling is involved in NF-kappa B activation through activation of calpain and thus calpain inhibitors may be effective in inhibiting the activation of latently infected HIV.

Topics: Acetylcysteine; Binding Sites; Calcimycin; Calcium Signaling; Calcium-Binding Proteins; Calpain; Cell Line; Glycoproteins; HIV-1; Humans; I-kappa B Proteins; In Vitro Techniques; Ionophores; Leucine; NF-kappa B; NF-KappaB Inhibitor alpha; Recombinant Fusion Proteins; Tetradecanoylphorbol Acetate; Virus Replication

The Ca(2+)-dependent cysteine protease calpain along with its endogenous inhibitor calpastatin is widely distributed. The interactions between calpain and calpastatin have been studied to better understand the nature of calpain inhibition by calpastatin, which can aid the design of small molecule inhibitors to calpain. Here we present the crystal structure of a complex between a calpastatin peptide and the calcium-binding domain VI of calpain. DIC19 is a 19 residue peptide, which corresponds to one of the three interacting domains of calpastatin, which is known to interact with domain VI of calpain. We present two crystal structures of DIC19 bound to domain VI of calpain, determined by molecular replacement methods to 2.5A and 2.2A resolution. In the process of crystallizing the inhibitor complex, a new native crystal form was identified which had the homodimer 2-fold axis along a crystallographic axis as opposed to the previously observed dimer in the asymmetric unit. The crystal structures of the native domain VI and its inhibitor PD150606 (3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid) complex were determined with the help of molecular replacement methods to 2.0A and 2.3A resolution, respectively. In addition, we built a homology model for the complex between domain IV and DIA19 peptide of calpastatin. Finally, we present a model for the calpastatin-inhibited calpain.

Topics: Acrylates; Amino Acid Sequence; Binding Sites; Calcium-Binding Proteins; Calpain; Crystallography, X-Ray; Cysteine Proteinase Inhibitors; Models, Molecular; Molecular Sequence Data; Protein Conformation; Protein Structure, Tertiary

The ubiquitous calpains, mu- and m-calpain, have been implicated in essential physiological processes and various pathologies. Cell-permeable specific inhibitors are important tools to elucidate the roles of calpains in cultivated cells and animal models. The synthetic N-acetylated 27-mer peptide derived from exon B of the inhibitory domain 1 of human calpastatin (CP1B) is unique as a potent and highly selective reversible calpain inhibitor, but is poorly cell-permeant. By addition of N-terminal cysteine residues we have generated a disulfide-conjugated CP1B with the cell-penetrating 16-mer peptide penetratin derived from the third helix of the Antennapedia homeodomain protein. The inhibitory potency and selectivity of CP1B for calpain versus cathepsin B and L, caspase 3 and the proteasome was not affected by the conjugation with penetratin. The conjugate was shown to efficiently penetrate into living LCLC 103H cells, since it prevents ionomycin-induced calpain activation at 200-fold lower concentration than the non-conjugated inhibitor and is able to reduce calpain-triggered apoptosis of these cells. Penetratin-conjugated CP1B seems to be a promising alternative to the widely used cell-permeable peptide aldehydes (e.g. calpain inhibitor 1) which inhibit the lysosomal cathepsins and partially the proteasome as well or even better than the calpains.

Topics: Apoptosis; Calcium-Binding Proteins; Calpain; Carrier Proteins; Cathepsins; Cell Line; Cell Membrane Permeability; Cell-Penetrating Peptides; Cysteine Proteinase Inhibitors; Erythrocytes; Exons; Humans; Ionomycin; Peptide Fragments

DNA damage is an initiator of neuronal death implicated in neuropathological conditions such as stroke. Previous evidence has shown that apoptotic death of embryonic cortical neurons treated with the DNA damaging agent camptothecin is dependent upon the tumor suppressor p53, an upstream death mediator, and more distal death effectors such as caspases. We show here that the calcium-regulated cysteine proteases, calpains, are activated during DNA damage induced by camptothecin treatment. Moreover, calpain deficiency, calpastatin expression, or pharmacological calpain inhibitors prevent the death of embryonic cortical neurons, indicating the important role of calpain in DNA damage-induced death. Calpain inhibition also significantly reduced and delayed the induction of p53. Consistent with the actions of calpains upstream of p53 and the proximal nature of p53 death signaling, calpain inhibition inhibited cytochrome c release and DEVD-AFC cleavage activity. Taken together, our results indicate that calpains are a key mediator of p53 induction and consequent caspase-dependent neuronal death due to DNA damage.

Topics: Adenoviridae; Animals; Blotting, Western; Calcium-Binding Proteins; Calpain; Camptothecin; Caspases; Cell Death; Cell Survival; Cells, Cultured; Cysteine Endopeptidases; Cytochrome c Group; DNA Damage; Green Fluorescent Proteins; Heterozygote; Immunohistochemistry; Luminescent Proteins; Mice; Microscopy, Fluorescence; Mitochondria; Neurons; Nuclear Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Time Factors; Tumor Suppressor Protein p53

The molecular mechanisms mediating degeneration of midbrain dopamine neurons in Parkinson's disease (PD) are poorly understood. Here, we provide evidence to support a role for the involvement of the calcium-dependent proteases, calpains, in the loss of dopamine neurons in a mouse model of PD. We show that administration of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) evokes an increase in calpain-mediated proteolysis in nigral dopamine neurons in vivo. Inhibition of calpain proteolysis using either a calpain inhibitor (MDL-28170) or adenovirus-mediated overexpression of the endogenous calpain inhibitor protein, calpastatin, significantly attenuated MPTP-induced loss of nigral dopamine neurons. Commensurate with this neuroprotection, MPTP-induced locomotor deficits were abolished, and markers of striatal postsynaptic activity were normalized in calpain inhibitor-treated mice. However, behavioral improvements in MPTP-treated, calpain inhibited mice did not correlate with restored levels of striatal dopamine. These results suggest that protection against nigral neuron degeneration in PD may be sufficient to facilitate normalized locomotor activity without necessitating striatal reinnervation. Immunohistochemical analyses of postmortem midbrain tissues from human PD cases also displayed evidence of increased calpain-related proteolytic activity that was not evident in age-matched control subjects. Taken together, our findings provide a potentially novel correlation between calpain proteolytic activity in an MPTP model of PD and the etiology of neuronal loss in PD in humans.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Adenoviridae; Aged; Aged, 80 and over; Animals; Behavior, Animal; Calcium; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Disease Models, Animal; Excitatory Postsynaptic Potentials; Genetic Vectors; Humans; Injections, Intraperitoneal; Male; Mice; Mice, Inbred C57BL; Middle Aged; Parkinson Disease; Proto-Oncogene Proteins c-fos; Radioimmunoassay; Striatonigral Degeneration; Substantia Nigra; Tyrosine 3-Monooxygenase

This study identifies calpain as being instrumental for brush border (BB) microvillus assembly during differentiation and effacement during bacterial pathogenesis. Calpain activity is decreased by 25-80% in Caco 2 lines stably overexpressing calpastatin, the physiological inhibitor of calpain, and the effect is proportional to the calpastatin/calpain ratio. These lines exhibit a 2.5-fold reduction in the rate of microvillus extension. Apical microvillus assembly is reduced by up to 50%, as measured by quantitative fluorometric microscopy (QFM) of ezrin, indicating that calpain recruits ezrin to BB microvilli. Calpain inhibitors ZLLYCHN2, MDL 28170, and PD 150606 block BB assembly and ezrin recruitment to the BB. The HIV protease inhibitor ritonavir, which inhibits calpain at clinically relevant concentrations, also blocks BB assembly, whereas cathepsin and proteasome inhibitors do not. Microvillus effacement is inhibited after exposure of calpastatin-overexpressing cells to enteropathogenic Escherichia coli. These results suggest that calpain regulates BB assembly as well as pathological effacement, and indicate that it is an important regulator involved in HIV protease inhibitor toxicity and host-microbial pathogen interactions.

Topics: Actins; Calcium-Binding Proteins; Calpain; Cell Membrane; Cytoplasm; Cytoskeletal Proteins; Down-Regulation; Enterocytes; Escherichia coli; Genetic Vectors; Humans; Microscopy, Confocal; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Microvilli; Phosphoproteins; Time Factors; Transfection; Tumor Cells, Cultured

Inappropriate imbalances between proteases and protease inhibitors are known to occur under cerebral ischemia and neurodegenerative processes, and could be contributors to various diseases that are characterized by excessive (ischemia, AIDS) or inadequate (cancer, autoimmunity) cell death. For instance, calpain is activated in various necrotic and apoptotic conditions, whereas caspase-3 is only activated in neuronal apoptosis. Caspases and calpains are cysteine proteases that require proteolytic cleavage for activation. The substrates cleaved by caspases include cytoskeletal and associated proteins, kinases, members of the Bcl-2 family of apoptosis-related proteins, presenilins, and DNA-modulating enzymes. Calpain substrates include cytoskeletal and associated proteins, kinases and phosphatases, membrane receptors and transporters, and steroid receptors. This article provides a review of the properties of caspases and calpains, their roles in cell death pathways following cerebral ischemia, and the substrates upon which they act. Because calpain inhibitors and caspase inhibitors appear to protect brain tissue by distinct mechanisms in cerebral ischemia, the possible therapeutic interactions between these drugs in a well-defined rodent model of global ischemia are briefly discussed and documented.

Topics: Animals; Apoptosis; Brain Ischemia; Calcium-Binding Proteins; Calpain; Caspases; Hippocampus; Necrosis; Neurons; Rats

Previously, we found that calpastatin diminished transiently prior to myoblast fusion (rat L8 myoblasts), allowing calpain-induced protein degradation, required for fusion. Here we show that the transient diminution in calpastatin is due to its degradation by caspase-1. Inhibition of caspase-1 prevents calpastatin diminution and prevents myoblast fusion. Caspase-1 activity is transiently increased during myoblast differentiation. Both calpain and caspase appear to be responsible for the fusion-associated membrane protein degradation. Caspase-1 has been implicated in the activation of proinflammatory cytokines, and in cell apoptosis. The involvement of caspase-1 in L8 myoblast fusion represents a novel function for this caspase in a non-apoptotic differentiation process, and points to cross-talk between the calpain and caspase systems in some differentiation processes.

Topics: Animals; Calcium-Binding Proteins; Calpain; Caspase 1; Cell Differentiation; Cell Fusion; Cell Line; Hydrolysis; Muscle, Skeletal; Rats

Inhibitors of the protease calpain are known to have selectively toxic effects on Plasmodium falciparum. The enzyme has a natural inhibitor calpastatin and in eukaryotes is responsible for turnover of proteins containing short sequences enriched in certain amino acids (PEST sequences). The genome of P. falciparum was searched for this protease, its natural inhibitor and putative substrates.. The publicly available P. falciparum genome was found to have too many errors to permit reliable analysis. An earlier annotation of chromosome 2 was instead examined. PEST scores were determined for all annotated proteins. The published genome was searched for calpain and calpastatin homologs.. Typical PEST sequences were found in 13% of the proteins on chromosome 2, including a surprising number of cell-surface proteins. The annotated calpain gene has a non-biological "intron" that appears to have been created to avoid an unrecognized frameshift. Only the catalytic domain has significant similarity with the vertebrate calpains. No calpastatin homologs were found in the published annotation.. A calpain gene is present in the genome and many putative substrates of this enzyme have been found. Calpastatin homologs may be found once the re-annotation is completed. Given the selective toxicity of calpain inhibitors, this enzyme may be worth exploring further as a potential drug target.

Topics: Amino Acid Sequence; Animals; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Databases, Genetic; Genome, Protozoan; Malaria, Falciparum; Plasmodium falciparum; Protozoan Proteins

To examine whether the calpain-calpastatin system is activated during the calcium paradox in the isolated perfused pigeon heart, we separated the protease from its inhibitor calpastatin and studied its kinetic properties. The protease exhibits kinetic properties similar to those of mammalian m-calpains. Ca(2+) requirements for half and maximum activities are 220 microM and 2 mM, respectively. In the absence of Ca(2+) the protease is strongly activated by Mn(2+) or Sr(2+). In the presence of Ca(2+), Mn(2+) and Sr(2+) exhibit a synergistic effect; Mg(2+) and Ba(2+) have no effect, whereas Co(2+), Ni(2+) and Cd(2+) completely inhibit its activation. Furthermore, we measured the activity of calpain and calpastatin under either conditions inducing a calcium paradox, or protecting the heart against this phenomenon. Although the calpain/calpastatin ratio is lowered during Ca(2+) depletion, during Ca(2+) repletion it is markedly inverted. Calpain activation during reperfusion is inhibited by the presence of 200 microM Mn(2+) or Ba(2+), in the Ca(2+)-free medium. Gel filtration of calpastatin, isolated from either untreated hearts or during Ca(2+) depletion, produces two main peaks of ñ150 and 40 kDa of molecular mass, respectively, whereas calpastatin isolated during the 2(nd) min of reperfusion appears to be shifted to the 150 kDa form. All the above data suggest that this system may be involved in the induction of the calcium paradox in pigeon heart.

Topics: Animals; Antipain; Barium; Calcium; Calcium-Binding Proteins; Calpain; Cobalt; Columbidae; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ethylmaleimide; Heart; In Vitro Techniques; Kinetics; Leucine; Leupeptins; Magnesium; Manganese; Molecular Weight; Myocardium; Nickel; Phenylmethylsulfonyl Fluoride; Strontium; Time Factors

The abundance of mRNAs transcribed from human genes of the calpain superfamily was studied in 72 human tissues and cell types by the Human Multiple Tissue Expression (MTE) Array technique. The analysis included the large subunits of mu- and m-calpains, the small subunits, calpastatin and calpain 3 (p94). Besides specific data on transcriptional activity, two major conclusions emerged: (i) 'ubiquitous' calpains are not expressed in all cell types, and (ii) a 'tissue-specific' calpain may be expressed in many cell types apart from the one in which it is particularly abundant. Therefore, the categoric classification of 'ubiquitous' vs. 'tissue-specific' calpains is a simplification.

Topics: Calcium-Binding Proteins; Calpain; Cell Line; Gene Expression Profiling; Humans; Organ Specificity; RNA, Messenger

Genetic defects in several sarcoglycans (SGs) and dystrophin (Dys) play a critical role in cardiomyopathy. The present study was designed to determine whether changes in SGs and Dys might occur in animals with chronic heart failure (CHF) induced by acute myocardial infarction (AMI), which have no genetic defects.. AMI was induced by the left coronary artery ligation (CAL) in rats. The hemodynamic parameters of the 2- and 8-week CAL (2w- and 8w-CAL) rats were measured and the myocardial SGs, Dys, calpain, and calpastatin levels were determined by the Western blot method. Myocardial calpain-like protease activity was evaluated as caseinolysis activity.. Increases in left ventricular end-diastolic pressure (LVEDP) and right ventricular systolic pressure, and a decrease in +/-dP/dt were observed at the 2nd week, whereas cardiac output index (COI) was preserved. In contrast, the 8w-CAL rats showed a further increment in LVEDP with low COI. alpha-SG of the viable left ventricle (LV), and septum (Sep) of the 8w-CAL rat decreased (60-70% of the control). The alpha- and beta-SGs of the right ventricle (RV) of the 2w- and 8w-CAL rats were reduced, while gamma- and delta-SGs in the three regions did not change significantly. Dys in the viable LV and RV of the 8w-CAL rat decreased (75% of the control). The amount of m-calpain in the three regions of the 2w- and 8w-CAL rats increased (140-200% of the control), whereas the endogenous calpain inhibitor, calpastatin, did not change significantly. The in vitro degradation studies using purified m-calpain or cytosolic fractions of the 8w-CAL rat heart suggested a reduction in SGs and Dys by calpain.. The results suggest that a decrease in SGs and Dys may play an important role in the pathophysiology of CHF following AMI.

Topics: Animals; Blood Pressure; Blotting, Western; Calcium-Binding Proteins; Calpain; Cardiac Output, Low; Culture Techniques; Cytoskeletal Proteins; Cytosol; Dystroglycans; Dystrophin; Heart Rate; Male; Membrane Glycoproteins; Models, Animal; Myocardial Infarction; Myocardium; Rats; Rats, Wistar; Sarcoglycans; Sarcolemma

Calpain, a Ca2+-dependent neutral protease, is highly related to the pathogenesis of a variety of disorders and its inhibitors offer potential for therapeutic intervention. General calpain inhibitors, however, have the disadvantage of a lack of specificity or poor cellular permeability or oxidization under physiological conditions. Here, we developed a membrane-permeable specific calpain inhibitor by fusing calpastatin peptide (CS) and 11 poly-arginine peptides (11R). The 11R-fused CS (11R-CS) effectively penetrated across the plasma membrane of living neurons and significantly inhibited calpain activity in the cells.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Death; Cell Membrane; Cell Membrane Permeability; Cells, Cultured; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Neurons; Peptides; Rats; Substrate Specificity

Eight Hampshire x Rambouillet crossbred wethers expressing the callipyge phenotype and eight Hampshire x Rambouillet half-sibling wethers with a normal phenotype were slaughtered when they reached 59 kg. The supraspinatus (SPM), longissimus (LM), and semitendinosus (STM) muscles were analyzed to determine callipyge effects on calpain and calpastatin activities, sarcomere length, percentage of muscle fiber types, and muscle fiber areas. After 14 d of aging, chops were frozen until analyses for trained sensory panel evaluations, Warner-Bratzler shear force values, and consumer perceptions of tenderness, flavor, juiciness, and overall satisfaction of chops were conducted. Calpastatin activity was 57% greater (P < 0.05) and m-calpain activity was 33% greater (P < 0.05) in muscles from carcasses of callipyge than normal sheep. Sarcomeres were shorter (P < 0.001) in the LM than the SPM or STM, regardless of phenotype. Muscle fiber area was 76% larger (P < 0.05) in the LM of callipyge than normal sheep, but muscle fiber area was not affected (P > 0.05) by phenotype in the SPM or STM. Phenotype had no effect (P = 0.12) on the percentage of slow-twitch, oxidative fiber types in any of the three muscles. In STM and LM from callipyge lambs, the percentage of fast-twitch, oxidative/glycolytic fibers was lower (P < 0.05) and that of fast-twitch-glycolytic fibers was higher (P < 0.05) than in their normal counterparts. Phenotype did not affect (P = 0.90) the fiber type percentage in the SPM. Callipyge LM were less tender and normal LM were more tender than other chops (P < 0.05). Callipyge loin chops had higher Warner-Bratzler shear force values than other chops (P < 0.001). Consumers rated fewer (P < 0.05) callipyge loin and shoulder chops acceptable in juiciness, tenderness, and overall acceptability than normal chops, but phenotype did not affect (P > 0.05) consumer acceptability of leg chops. These results indicate that LM from Hampshire x Rambouillet sheep displaying the callipyge phenotype had higher calpastatin activity and were less tender than the LM from normal sheep. In addition, consumer perceptions indicated that only one in 10 leg chops, one in five shoulder chops, and one in four loin chops from callipyge sheep were unacceptable.

Topics: Animals; Body Composition; Calcium-Binding Proteins; Calpain; Consumer Behavior; Cooking; Cysteine Proteinase Inhibitors; Male; Meat; Muscle Fibers, Skeletal; Muscle, Skeletal; Phenotype; Sheep; Taste

Calpastatin, the endogenous inhibitor of calpain, is an intrinsically unstructured protein proposed to undergo folding transitions upon binding to the enzyme. As this feature has never been experimentally tested, we have set out to characterize the conformation of two peptides corresponding to its conserved subdomains, A and C, known to interact with calpain in a Ca(2+)-dependent manner. The peptides are disordered in water but show a high propensity for alpha-helical conformation in the presence of trifluoroethanol. The conformational transition is sensitive to Ca(2+), and is clearly seen upon binding of the peptides to the enzyme. Secondary-structure prediction of all calpastatin sequences shows that the helix-forming potential within these regions is a conserved feature of the inhibitor. Furthermore, quantitative data on the binding strength of calpastatin fragments reveal that binding of the inhibitor is accompanied by a large decrease in its configurational entropy. Taken together, these observations point to significant binding-induced local folding transitions in calpastatin, in a way that ensures highly specific, yet reversible, action of the inhibitor.

Topics: Animals; Binding Sites; Calcium; Calcium-Binding Proteins; Calpain; Cattle; Circular Dichroism; Computational Biology; Databases, Protein; Entropy; Humans; Mice; Peptides; Protein Binding; Protein Folding; Protein Structure, Secondary; Rabbits; Rats; Recombinant Proteins; Sequence Alignment; Trifluoroethanol; Water

The objectives of this study were to assess the influence of sire on mu- and m-calpain activities, to evaluate the relationships of activities of these enzymes to other traits related to beef palatability, and to assess the influence of sire on the rate of tenderization (as measured by myofibril fragmentation index [MFI]) in Brahman longissimus muscle. Brahman calves (n = 87), sired by nine bulls, were born, weaned, fed, and slaughtered in central Florida. Traits evaluated were mu- and m-calpain activities and MFI after 1, 7, 14, and 21 d of aging. Other traits were analyzed to determine their associations with mu- and m-calpain activity and MFI, including calpastatin activity, percentage of raw and cooked lipids, Warner-Bratzler shear force (WBSF) values after 7, 14, and 21 d of aging, and sensory panel rating of tenderness, juiciness, and connective tissue amount after 14 d of aging. Data were analyzed using a model with sire, sex, year, and slaughter group (calves of the same sex slaughtered on the same date) as fixed effects, and adjusted to a constant adjusted 12th-rib fat thickness. Sire affected mu-calpain activity (P < 0.04), calpastatin activity (P < 0.01), d-14 MFI (P < 0.02), d-7 WBSF (P < 0.05), d-14 WBSF (P < 0.04), and sensory panel juiciness score (P < 0.01), but not (P < 0.75) m-calpain activity. Measures of tenderness and palatability were generally moderately to strongly correlated (both simple and residual correlations) with calpastatin and m-calpain activity. Myofibril fragmentation index residuals (adjusted for all model components except sire) after all aging periods were fitted using nonlinear regression to the exponential curve (MFI(i) = kappa0 + kappa1 exp[kappa2 t(i)] + epsilon(i), where t(i) represents aging in days, k0 is ultimate MFI after aging, kappa1 is the difference between initial and ultimate MFI, kappa2 is the rate of increase in MFI, and epsilon(i) is the error term associated with the ith observation, assumed to be independent and identically distributed normally). Sires had different estimates and combinations of estimates, which were used to plot MFI change with time. These curves visually differed for sires and suggested that postmortem tenderization extent and rate differ as well. Use of a combination of these estimated parameters in a selection/carcass sorting program represents an alternative consideration for tenderization improvement programs.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Consumer Behavior; Female; Food Handling; Male; Meat; Muscle, Skeletal; Myofibrils; Postmortem Changes; Taste; Time Factors

The calcium-activated cysteine protease calpain is intimately involved in modulating cell adhesion and migration. The two ubiquitous isoforms of this protease, calpain I and II, are considered to be cytosolic proteins that can translocate to both focal complexes/adhesions or the plasma membrane. Using confocal microscopy and isopycnic density centrifugation, the results demonstrate that calpain I and II, the 30kDa regulatory subunit, and calpastatin associate with the endoplasmic reticulum and Golgi apparatus. Confocal microscopy reveals that calpain II colocalizes with the subcellular proteins calnexin and Rab6 in cells bound to laminin. To further verify this association, cell lysates prepared from laminin stimulated and unstimulated cells were subjected to isopycnic density centrifugation. The results reveal an increased association of calpain I, II, calpastatin, and the 30kDa regulatory subunit with the endoplasmic reticulum and Golgi apparatus as evidenced by their position in the gradient relative to calnexin, Rab6, caveolin, and beta1 integrin after laminin stimulation. This correlates with the accumulation of inducible calpain activity at the endoplasmic reticulum-Golgi apparatus interface. Further experiments established that calpain II colocalizes with phosphatidylinositol 4,5-bisphosphate. Finally, calpain II associates with membrane lipid rafts. These results provide new insights into how the calpain/calpastatin network is spatially and temporally regulated in cells binding to the extracellular matrix.

Topics: Blotting, Western; Calcium-Binding Proteins; Calpain; Cell Line, Tumor; Cell Size; Cells, Cultured; Centrifugation; Humans; Membrane Lipids; Microscopy, Confocal; Organelles; rab GTP-Binding Proteins; Subcellular Fractions

In the CNS, where Ca(2+) overload has been established as a mechanism contributing to neuronal damage associated with excitotoxicity, stroke and ischemia, there is interest in understanding the role of calpain inhibition in rescuing neurons from death. In these settings, the activation of large stores of latent calpain may rapidly lead to the demise of the neuron within hours. The activity of calpain is strictly regulated by calcium concentrations and interactions with calpastatin (endogenous calpain inhibitor). The interaction between calpains and calpastatin is calcium dependent, and little is known about the regulation of the neuronal calpain-calpastatin system in vivo. It has been postulated that calpastatin can be modulated by nerve growth factors (NGFs). We have demonstrated in vitro as well as in vivo a neuroprotective effect of the beta(2)-adrenoceptor agonist clenbuterol (CLN) mediated through an increased NGF expression. In this study we attempt to find out whether CLN is capable (1) of modulating proteolysis regulated by the calpain-calpastatin system and (2) of attenuating DNA-fragmentation induced by cerebral ischemia. Rats received CLN daily for 1 week, were then subjected to ischemia and finally perfused at different times post-ischemia. The proteolytic activity of calpain was measured by the immunolocalisation of calpastatin and spectrin-breakdown products (SBP). The time course of apoptosis was assessed by terminal dUTP nick end-labeling (TUNEL)-staining. CLN reduced CA1-hippocampal cell damage by 23%, attenuated DNA-laddering and decreased proteolysis of spectrin by enhancing calpastatin activity. These results provide evidence that CLN is a potent neuroprotective substance, which through the enhancement of calpastatin synthesis attenuates the apoptotic machinery and modulates proteolysis.

Topics: Animals; Apoptosis; Blotting, Western; Brain Ischemia; Calcium-Binding Proteins; Calpain; Clenbuterol; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Hippocampus; Immunohistochemistry; In Situ Nick-End Labeling; Male; Neurons; Neuroprotective Agents; Rats; Rats, Wistar; Receptors, Adrenergic, beta-3

The amount of calpastatin directly available in cytosol is under the control of [Ca2+] and [cyclic AMP]. Prolonged calpain activation also promotes degradation of calpastatin. The fluctuation of calpastatin concentration in cell soluble fraction is accompanied by an initial decrease in calpastatin gene expression, followed by a fivefold increase in its expression when the inhibitor protein is degraded. This process can be conceptualized as a mechanism to regulate calpastatin availability in the cell. This conclusion is supported by the fact that calpain, the other component of this proteolytic system, undergoes changes in its levels of expression in a much more limited manner. Furthermore, this process can be observed both in cells exposed to different natural stimuli, or in other cell lines. Modification of calpastatin gene expression might represent a new tool for the in vivo control of the regulatory machinery required for the modulation of Ca(2+)-dependent proteolysis.

Topics: Calcium; Calcium-Binding Proteins; Calpain; Cyclic AMP; Humans; Peptide Hydrolases

Cell death in the core of human brain tumors is triggered by hypoxia and lack of nutrients, but the mode of cell death whether necrosis or apoptosis is not clearly defined. To identify the role of apoptosis in brain tumor cell death, we investigated macromolecular (RNA and protein) synthesis and activity in the central to peripheral region of benign [desmoplastic infantile ganglioglioma (DIG) and transitional meningioma (TMG)] and malignant [ependymoma (END), anaplastic astrocytoma (APA), and glioblastoma multiforme (GBM)] brain tumors derived from five patients who had not received previously radiotherapy or chemotherapy. Normal brain tissue (NBT) served as control. RT-PCR analysis of tumor tissues covering central to peripheral regions detected mRNA overexpression of pro-apoptotic gene bax in malignant tumors, indicating a commitment to apoptosis. The mRNA expression of calpain (a Ca(2+)-dependent cysteine protease) and calpastatin (endogenous calpain inhibitor) was altered resulting in an elevated calpain/calpastatin ratio. Calpain content and activity were increased, suggesting a role for calpain in cell death. In the mitochondria-dependent death pathway, caspase-9 and caspase-3 were also overexpressed in tumors. The increased caspase-3 activity cleaved poly(ADP-ribose) polymerase (PARP). Agarose gel electrophoresis detected a mixture of random and internucleosomal DNA fragmentation in malignant brain tumors. Overexpression of pro-apoptotic bax, upregulation of calpain and caspase-3, and occurrence of internucleosomal DNA fragmentation are now presented indicating that one mechanism of cell death in malignant brain tumors is apoptosis, and that enhancement of this process therapeutically may promote decreased tumor growth.

Topics: Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Brain Neoplasms; Calcium-Binding Proteins; Calpain; Caspase 3; Caspase 9; Caspases; DNA Fragmentation; Electrophoresis, Agar Gel; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Up-Regulation

Previous studies have led us to hypothesize that m-calpain plays a pivotal role in myoblast fusion through its involvement in cell membrane and cytoskeleton component reorganization. To support this hypothesis, a convenient and simple myoblast culture model using frozen embryonic myoblasts was developed, which resolved a number of problems inherent to cell primary culture. Biological assays on cultured myoblasts using different media to define the characteristics of the fusion process were first conducted. Proteinase was detectable before the initiation of the fusion process and was closely correlated to the phenomenon of fusion under each culture condition studied. In addition, the study of calpastatin showed that the initiation of fusion does not require a decrease in the level of this endogenous inhibitor of calpains and also confirmed that calpastatin may be implicated in the determination of the end of fusion. On the other hand, analysis of the evolution of myogenic factors revealed that myogenins, MyoD and Myf5, increase very significantly during the formation of multinucleated myotubes. Moreover, the antisense technique against myogenin is capable of preventing the process of fusion by 50%, confirming the pivotal role of this factor in the early stages of differentiation. The possible role of myogenic regulator factors on m-calpain gene expression is discussed.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Fusion; Cells, Cultured; Models, Biological; Muscle Development; Myoblasts; Myogenin; Rats; Rats, Wistar

Cell migration requires the regulated and dynamic turnover of adhesive complexes. We have previously demonstrated that the calcium-dependent protease, calpain, regulates the organization of adhesive complexes and cell detachment during cell migration. Evidence is now provided that inhibiting calpain through over-expression of the endogenous inhibitor of calpain, calpastatin, and pharmacological inhibitors results in an inhibition of adhesive complex disassembly with stabilization of GFP-vinculin and GFP/RFP-zyxin at the cell periphery. Calpain was also required for the microtubule-mediated turnover of adhesive complex sites after nocodazole wash-out, suggesting that calpain may mediate focal complex disassembly downstream of microtubules. Using dual imaging of RFP-zyxin and GFP-alpha-actinin, we observed a temporal and spatial relationship between alpha-actinin localization to focal contacts and the subsequent disassembly or translocation of RFP-zyxin containing focal complexes in areas of cell retraction. Calpain inhibition disrupted alpha-actinin localization to zyxin-containing focal contacts and focal complex disassembly or translocation to the cell center. In addition, disrupting alpha-actinin localization to focal complexes through expression of the alpha-actinin rod domain, but not the head domain, resulted in inhibition of focal adhesion disassembly similar to calpain inhibition. Our studies suggest a novel mechanism of action whereby calpain may modulate alpha-actinin localization into focal complexes and their subsequent disassembly or translocation.

Topics: Actinin; Animals; Calcium; Calcium-Binding Proteins; Calpain; Cell Adhesion; Cell Movement; Cells, Cultured; CHO Cells; Cricetinae; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Fibroblasts; Focal Adhesions; Glycoproteins; Goldfish; Green Fluorescent Proteins; Humans; Luminescent Proteins; Metalloproteins; Microscopy, Video; Microtubules; Nocodazole; Recombinant Fusion Proteins; Red Fluorescent Protein; Vinculin; Zyxin

In transformed cells, induction of apoptosis by adenovirus type 2 (Ad2) early region 4 ORF 4 (E4orf4) correlates with accumulation of E4orf4 in the cell membrane-cytoskeleton fraction. However, E4orf4 is largely expressed in nuclear regions before the onset of apoptosis. To determine the relative contribution of nuclear E4orf4 versus membrane-associated E4orf4 to cell death signaling, we engineered green fluorescent fusion proteins to target E4orf4 to specific cell compartments. The targeting of Ad2 E4orf4 to cell membranes through a CAAX-box or a myristylation consensus signal sufficed to mimic the fast Src-dependent apoptotic program induced by wild-type E4orf4. In marked contrast, the nuclear targeting of E4orf4 abolished the early induction of extranuclear apoptosis. However, nuclear E4orf4 still induced a delayed cell death response independent of Src-like activity and of E4orf4 tyrosine phosphorylation. The zVAD.fmk-inhibitable caspases were dispensable for execution of both cell death programs. Nevertheless, both pathways led to caspase activation in some cell types through the mitochondrial pathway. Finally, our data support a critical role for calpains upstream in the death effector pathway triggered by the Src-mediated cytoplasmic death signal. We conclude that Ad2 E4orf4 induces two distinct cell death responses, whose relative contributions to cell killing may be determined by the genetic background.

Topics: Adenoviridae; Adenoviridae Infections; Apoptosis; bcl-2-Associated X Protein; Calcium-Binding Proteins; Calpain; Caspase Inhibitors; Caspases; Cell Line, Transformed; Cell Membrane; Cytoplasm; DNA Fragmentation; Enzyme Inhibitors; Eukaryotic Cells; Green Fluorescent Proteins; Humans; Indicators and Reagents; Luminescent Proteins; Models, Biological; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; src-Family Kinases; Viral Proteins

The calpain system is involved in a number of human pathologies ranging from the muscular dystrophies to Alzheimer's disease. It is important, therefore, to be able to obtain and to characterize both mu-calpain and m-calpain from human tissue. Although human mu-calpain can be conveniently obtained from either erythrocytes or platelets, no readily available source of human m-calpain has been described. Human placenta extracts contain both mu-calpain and m-calpain in nearly equal proportions and in significant quantities (3-4 mg mu-calpain and 4-5 mg m-calpain/1000 g placenta tissue). Placenta also contains calpastatin that elutes off ion-exchange columns over a wide range of KCl concentrations completely masking the mu-calpain activity eluting off these columns and even partly overlapping m-calpain elution. Placenta mu-calpain requires 50-70 microM Ca2+ and placenta m-calpain requires 450-460 microM Ca2+ for half-maximal proteolytic activity. Western analysis of washed placenta tissue shows that placenta contains both mu- and m-calpain, although some of the mu-calpain in whole placenta extracts likely originates from the erythrocytes that are abundant in the highly vascularized placenta. Placenta calpastatin could not be purified with conventional methods. The most prominent form of calpastatin in Western analyses of placenta obtained as soon as possible after birth was approximately 48-51 kDa; partly purified preparations of placenta calpastatin also contained 48-51 and 70 kDa polypeptides. Human placenta extracts likely contain two different calpastatin isoforms, a 48-51 kDa "placenta calpastatin" and a 70 kDa erythrocyte calpastatin.

Topics: Adult; Blotting, Western; Calcium-Binding Proteins; Calpain; Centrifugation, Density Gradient; Chromatography, DEAE-Cellulose; Female; Humans; Isomerism; Placenta; Pregnancy; Subcellular Fractions

N-Methyl-D-aspartate (NMDA) receptors are calcium-permeable glutamate receptors that play putative roles in learning, memory, and excitotoxicity. NMDA receptor-mediated calcium entry can activate the calcium-dependent protease calpain, leading to substrate degradation. The major NMDA receptor 2 (NR2) subunits of the receptor are in vitro substrates for calpain at selected sites in the C-terminal region. In the present study, we assessed the ability of calpain-mediated proteolysis to modulate the NR1a/2A subtype in a heterologous expression system. Human embryonic kidney (HEK293t) cells, which endogenously express calpain, were cotransfected with NR1a/2A in addition to the calpain inhibitor calpastatin or empty vector as control. Receptor activation by glutamate and glycine as co-agonists led to calpain activation as measured by succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl-aminomethyl coumarin (Suc-LLVY-AMC). Calpain activation also resulted in the degradation of NR2A and decreased binding of (125)I-MK-801 ((125)I-dizocilpine) to NR1a/2A receptors. No stable N-terminal fragment of the NMDA receptor was formed after calpain activation, suggesting calpain regulation of NMDA receptor levels in ways distinct from that previously observed with in vitro cleavage. NR2 subunit constructs lacking the final 420 amino acids were not degraded by calpain. Agonist-stimulated NR1a/2A-transfected cells also had decreased calcium uptake and produced lower changes in agonist-stimulated intracellular calcium compared with cells cotransfected with calpastatin. Calpastatin had no effect on either calcium uptake or intracellular calcium levels when the NR2A subunit lacked the final 420 amino acids. These studies demonstrate that NR2A is a substrate for calpain in situ and that this proteolytic event can modulate NMDA receptor levels.

Topics: Blotting, Western; Calcium; Calcium-Binding Proteins; Calpain; Cell Line; Cell Survival; Cysteine Proteinase Inhibitors; Dizocilpine Maleate; DNA, Complementary; Excitatory Amino Acid Antagonists; Humans; Peptide Hydrolases; Receptors, N-Methyl-D-Aspartate; Transfection

Reduced sarcolemmal integrity in dystrophin-deficient muscles of mdx mice and Duchenne muscular dystrophy (DMD) patients has been reported to result in altered calcium homeostasis. Previous studies have shown a correlative relationship between calcium-dependent protease (calpain) activity in dystrophic muscle and muscle necrosis, but have not tested whether calpain activation precedes cell death or is a consequence of it. To test a causal relationship between calpain activation and muscle cell death in dystrophin deficiency, mdx mice were generated that overexpress a calpastatin transgene in muscle. Calpastatin (CS) is a specific, endogenous inhibitor of m- and micro -calpains that does not inhibit calpain 3 (p94). CS overexpression on a C57/BL 10 background produced no phenotype. Transgenic (Tg) mice crossed with mdx mice were tested for pathological indicators of necrosis, regeneration and membrane damage. Two lines of mice were examined, with different levels of CS overexpression. Both lines of Tg/mdx mice showed reductions in muscle necrosis at 4 weeks of age. These mice had fewer as well as smaller lesions. In addition, one line of mice had significantly less regeneration, indicating a reduction in previous necrosis. The extent of improvement correlated with the level of CS protein expression. Membrane damage, as assessed by procion orange and creatine kinase assays, was unchanged, supporting the idea that calpains act downstream of the primary muscle defect. These data suggest that calpains play an active role in necrotic processes in dystrophic muscle and that inhibition of calpains might provide a good therapeutic option for treatment of DMD.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Membrane; Down-Regulation; Mice; Mice, Inbred mdx; Mice, Transgenic; Muscle Cells; Muscle, Skeletal; Muscular Dystrophy, Animal

The cellular localization of calpain is important in understanding the roles that calpain may play in physiological function. We, therefore, examined calpain expression, activity, and immunofluorescent localization in primary cultures of rat oligodendrocytes. The mRNA expression of m-calpain was 64.8% (P = 0.0033) and 50.5% (P = 0.0254) higher than that of mu-calpain and calpastatin, respectively, in primary culture oligodendrocytes. The levels of mRNA expression of mu-calpain and calpastatin were not significantly different. As revealed by Western blotting, cultured oligodendrocytes contained a 70 kD major band identified by membrane m-calpain antibody, a 80 kD band recognized by cytosolic m-calpain antibody, and calpastatin bands ranging from 45 to 100 kD detected by a calpastatin antibody. Calpain activity in oligodendrocytes was determined by Ca(2+)-dependent 71.2% degradation of endogenous myelin basic protein compared with control; this activity was inhibited significantly (P = 0.0111) by EGTA and also substantially by calpeptin. Localization of calpain in cultured oligodendrocytes revealed strong membrane m-calpain immunofluorescence in the oligodendrocyte cell body and its processes. In contrast, the cytosolic antibody stained primarily the oligodendrocyte cell body, whereas the processes were stained very weakly or not at all. These results indicate that the major form of calpain in glial cells is myelin (membrane) m-calpain. The dissimilar localization of cytosolic and membrane m-calpain may indicate that each isoform has a unique role in oligodendrocyte function.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Compartmentation; Cell Membrane; Cell Surface Extensions; Cells, Cultured; Cytosol; Microscopy, Fluorescence; Myelin Basic Protein; Oligodendroglia; Rats; RNA, Messenger

Several behavioral and electrophysiological studies have suggested that a sustained activation of protein kinase C would be required to underlie persistent changes associated with memory formation. Limited proteolysis of PKCs by calpains, calcium-activated proteases, cleaves the catalytic and the regulatory domains, generating a free catalytic fragment termed PKM, constitutively active. In order to investigate the potential physiological importance of this limited proteolysis as a mechanism of PKC activation, we have studied the effect of the calpastatin peptide, a specific calpain inhibitor, on the learning of a spatial discrimination task in a radial maze. Thus, using osmotic micro-pumps, the calpastatin peptide was infused bilaterally into the dorsal hippocampus during the six sessions of training and the probe test. The treatment was shown to facilitate the performance of the mice on the two last training sessions and on the probe test. This behavioral effect was shown to correspond to the reduced calpain activity observed in the hippocampus at the very end of the 7-day infusion of the calpastatin peptide, suggesting a relation between both events. In addition, PKC activity measured immediately after the probe test was notably decreased in the membrane fraction of the hippocampus. Although protein levels of PKCs and calpains quantified by western blot were not affected by calpastatin infusion, we found a noticeable correlation between mu-calpain and PKCgamma levels confirming the particular relationship between both proteins. These results suggest that calpains influence on PKCs activity may affect cellular mechanisms during memory processes.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Membrane; Cysteine Proteinase Inhibitors; Cytosol; Discrimination Learning; Drug Implants; Habituation, Psychophysiologic; Hippocampus; Immunoblotting; Injections, Intraventricular; Male; Maze Learning; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Protein Kinase C; Space Perception

The effects of various growth rates in pigs induced by four different feeding strategies on the activity of the calpain system and on postmortem (PM) muscle proteolysis and tenderness development were studied. An increased growth rate may be caused by an increased protein turnover, which results in up-regulated levels of proteolytic enzymes in vivo that, in turn, possibly will affect PM tenderness development. It can be hypothesized that increased proteolytic activity pre-slaughter will increase the PM tenderization rate. From postnatal d 28 to d 90 (phase 1) the pigs were divided into two groups, given either ad libitum (A) or restricted (R, 60% of ad libitum) access to feed. The two groups were then divided into two subgroups, given either restricted or ad libitum access to feed from d 91 to slaughter at d 165 (phase 2). Measurements of the activity of mu-calpain, m-calpain, and calpastatin; concentrations of total collagen and the percent of soluble collagen; and RNA, DNA, and elongation factor-2 where made at slaugther. Myofibrillar fragmentation index (MFI) was determined at slaughter and 24 h PM. Warner-Braztler shear force was determined 1 d and 4 d PM. Pigs fed restricted diets in phase 1 and fed ad libitum in phase 2 (RA pigs) had increased growth rates in the last phase compared to pigs fed ad libitum during both phase 1 and phase 2 (AA pigs). The increased growth rate (compensatory growth) was followed by an increased proteolytic potential (mu-calpain:calpastatin ratio), increased MFI values, and higher tenderization rates. There was a positive correlation between the activities of m-calpain and growth rates (r = 0.35, P = 0.03), and between RNA levels and growth rates (r = 0.43, P = 0.006). The proposed hypothesis is largely supported by the results. The activities of both mu- and m-calpain at slaughter were highest in fast-growing pigs. The calpain activity was highest in RA pigs, which in turn also had the fastest growth rates prior tslaughter among the four groups. This implies that the synthesis of these enzymes was up-regulated during the second feeding period to a larger extent in RA pigs. The proteolytic potential and the MFI values indicate that the up-regulated in vivo calpain activity had an effect on PM protein degradation, which also is supported by the higher tenderization rate in RA pigs.

Topics: Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Calcium-Binding Proteins; Calpain; Diet, Reducing; Female; Meat; Muscle, Skeletal; Peptide Elongation Factor 2; Postmortem Changes; RNA; Swine; Taste

The purpose of this study was to test the hypothesis that myocardial ischemia-reperfusion (I/R) is accompanied by an early burst in calpain activity, resulting in decreased calpastatin activity and an increased calpain/calpastatin ratio, thereby promoting increased protein release. To determine the possibility of a 'calpain burst' impacting cardiac calpastatin inhibitory activity, rat hearts were subjected (Langendorff) to either 45 or 60 min of ischemia followed by 30 min of reperfusion with and without pre-administration (s.c.) of a cysteine protease inhibitor (E-64c). Myocardial function, calpain activities (casein release assay), calpastatin inhibitory activity and release of CK, LDH, cTnI and cTnT were determined (n = 8 for all groups). No detectable changes in calpain activities were observed following I/R with and without E-64c (p > 0.05). Both I/R conditions reduced calpastatin activity (p < 0.05) while E-64c pre-treatment was without effect, implicating a non-proteolytic event underlying the calpastatin changes. A similar result was noted for calpain-calpastatin ratios and the release of all marker proteins (p < 0.05). In regard to cardiac function, E-64c resulted in transient improvements (15 min) for left ventricular developed pressure (LVDP) and rate of pressure development (p < 0.05). E-64c had no effect on end diastolic pressure (LVEDP) or coronary pressure (CP) during I/R. These findings demonstrate that restricting the putative early burst in calpain activity, suggested for I/R, by pre-treatment of rats with E-64c does not prevent downregulation of calpastatin inhibitory activity and/or protein release despite a transient improvement in cardiac function. It is concluded that increases in calpain isoform activities are not a primary feature of l/R changes, although the role of calpastatin downregulation remains to be elucidated.

Topics: Animals; Calcium-Binding Proteins; Calpain; Male; Myocardial Ischemia; Rats; Rats, Wistar; Reperfusion Injury

Muscle wasting is a prominent feature of several systemic diseases, neurological damage and muscle disuse. The contribution of calpain proteases to muscle wasting in any instance of muscle injury or disease has remained unknown because of the inability to specifically perturb calpain activity in vivo. We have generated a transgenic mouse with muscle-specific overexpression of calpastatin, which is the endogenous inhibitor of calpains, and induced muscle atrophy by unloading hindlimb musculature for 10 days. Expression of the transgene resulted in increases in calpastatin concentration in muscle by 30- to 50-fold, and eliminated all calpain activity that was detectable on zymograms. Muscle fibres in ambulatory, transgenic mice were smaller in diameter, but more numerous, so that muscle mass did not differ between transgenic and non-transgenic mice. This is consistent with the role of the calpain-calpastatin system in muscle cell fusion that has been observed in vitro. Overexpression of calpastatin reduced muscle atrophy by 30 % during the 10 day unloading period. In addition, calpastatin overexpression completely prevented the shift in myofibrillar myosin content from slow to fast isoforms, which normally occurs in muscle unloading. These findings indicate that therapeutics directed toward regulating the calpain-calpastatin system may be beneficial in preventing muscle mass loss in muscle injury and disease.

Topics: Animals; Calcium-Binding Proteins; Calpain; Female; Gene Expression; Hindlimb Suspension; Humans; Male; Mice; Mice, Transgenic; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Atrophy; Myosins; Phenotype; Protein Isoforms; Transgenes

The anaemia associated with visceral leishmaniasis is accompanied by altered Ca(2+) homeostasis and degradation of the cytoskeletal and integral proteins of the erythrocytic membrane. In the present study, such changes were followed in hamsters that were anaemic as the result of their experimental infection with Leishmania donovani. At each stage of the infection, the blood concentration of haemoglobin was found to be negatively correlated with the concentration of Ca(2+) (R(2) = 0.91), the percentage of erythrocytes with Heinz bodies (R(2) = 0.98) and thiol depletion (R(2) = 0.96) in the erythrocytes. Calpain (Ca(2+)-activated protease; EC 3.4.22.17) and its natural inhibitor calpastatin are known to regulate the catabolism of membrane structural proteins. Densitometric scanning of SDS-PAGE gels showed that erythrocytic membranes from infected hamsters contained less calpain and calpastatin than those from control animals. The level of calpain autolysis was found to increase as the infection progressed. The addition of purified calpain (from control hamsters) to erythrocyte ghosts caused greater degradation of the membranes of erythrocytes from infected animals than of the corresponding membranes from control animals. Calpastatin from the control hamsters was more effective, at inhibiting calpain-induced membrane proteolysis, than calpastatin from the infected animals. The results indicate that the Ca(2+)-activated protease and its inhibitor are involved in the degradation of erythrocytic membranes observed during visceral leishmaniasis.

Topics: Anemia; Animals; Calcium; Calcium-Binding Proteins; Calpain; Cricetinae; Cysteine Proteinase Inhibitors; Disease Models, Animal; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Heinz Bodies; Leishmania donovani; Leishmaniasis, Visceral; Membrane Proteins; Mesocricetus; Protease Inhibitors; Sulfhydryl Compounds

v-Src-induced oncogenic transformation is characterized by alterations in cell morphology, adhesion, motility, survival, and proliferation. To further elucidate some of the signaling pathways downstream of v-Src that are responsible for the transformed cell phenotype, we have investigated the role that the calpain-calpastatin proteolytic system plays during oncogenic transformation induced by v-Src. We recently reported that v-Src-induced transformation of chicken embryo fibroblasts is accompanied by calpain-mediated proteolytic cleavage of the focal adhesion kinase (FAK) and disassembly of the focal adhesion complex. In this study we have characterized a positive feedback loop whereby activation of v-Src increases protein synthesis of calpain II, resulting in degradation of its endogenous inhibitor calpastatin. Reconstitution of calpastatin levels by overexpression of exogenous calpastatin suppresses proteolytic cleavage of FAK, morphological transformation, and anchorage-independent growth. Furthermore, calpastatin overexpression represses progression of v-Src-transformed cells through the G(1) stage of the cell cycle, which correlates with decreased pRb phosphorylation and decreased levels of cyclins A and D and cyclin-dependent kinase 2. Calpain 4 knockout fibroblasts also exhibit impaired v-Src-induced morphological transformation and anchorage-independent growth. Thus, modulation of the calpain-calpastatin proteolytic system plays an important role in focal adhesion disassembly, morphological transformation, and cell cycle progression during v-Src-induced cell transformation.

Topics: Actins; Animals; Calcium-Binding Proteins; Calpain; CDC2-CDC28 Kinases; Cell Division; Cell Size; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Cyclin A; Cyclin D; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Cyclins; Cytoskeleton; Feedback, Physiological; Fibroblasts; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesions; Genes, myc; Genes, ras; Immunohistochemistry; Mice; Mice, Knockout; Models, Biological; Oncogene Protein p65(gag-jun); Oncogene Protein pp60(v-src); Oncogene Proteins v-fos; Protein Isoforms; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Retinoblastoma Protein; Temperature

The inhibitory domains of calpastatin contain three highly conserved regions, A, B, and C, of which A and C bind calpain in a strictly Ca(2+)-dependent manner but have no inhibitory activity whereas region B inhibits calpain on its own. We synthesized the 19-mer oligopeptides corresponding to regions A and C of human calpastatin domain I and tested their effect on human erythrocyte mu-calpain and rat m-calpain. The two peptides significantly activate both calpains: the Ca(2+) concentration required for half-maximal activity is lowered from 4.3 to 2.4 microm for mu-calpain and from 250 to 140 microm for m-calpain. The EC(50) concentration of the peptides is 7.5 microm for mu-calpain and 25 microm for m-calpain. It is noteworthy that at low Ca(2+) concentrations (1-2 microm for mu-calpain and 70-110 microm for m-calpain) both enzymes are activated about 10-fold by the peptides. Based on these findings, it is suggested that calpastatin fragments may have a role in calpain activation in vivo. Furthermore, these activators open new avenues to cell biological studies of calpain function and eventually may alleviate pathological states caused by calpain malfunction.

Topics: Amino Acid Sequence; Animals; Calcium; Calcium-Binding Proteins; Calpain; Enzyme Activation; Humans; Molecular Sequence Data; Peptide Fragments; Rats

It has been reported that the equilibrium between the erythrocyte protease calpain I and its physiological inhibitor calpastatin is disrupted in patients with essential hypertension.. To investigate the activity of non-purified calpain I in hemolysates against the erythrocytic membrane proteins, rather than against other substrates.. Evaluation of calpain I red cell activity upon its own physiological substrates in hypertensive patients, in a near-physiological environment.. LIM-23 and LIM-40 of Hospital das Clinicas of the Faculty of Medicine of USP.. Patients with moderate primary hypertension over 21 years of age who were given amlodipine (n:10) and captopril (n:10) for 8 weeks, plus normal controls (n:10).. Red cell membrane proteins were incubated with and without protease inhibitors and with and without calcium chloride and underwent polyacrylamide gel electrophoresis.. Digestion of bands 2.1 and 4.1 was observed, indicating calpain I activity. No statistical differences regarding bands 2.1 and 4.1 were observed before treatment, between the controls and the hypertensive patients, either in ghosts prepared without calcium or with increasing concentrations of calcium. Nor were statistical differences observed after treatment, between the controls and the patients treated with amlodipine and captopril, or between the patients before and after treatment with both drugs.. The final activity of non-purified calpain I upon its own physiological substrate, which was the approach utilized in this study, may more adequately reflect what happens in red cells. Under such conditions no imbalance favoring calpain I activity increase was observed. The protective factor provided by calpastatin against calpain I activity may diminish under hypertension.

Topics: Adult; Amlodipine; Ankyrins; Calcium-Binding Proteins; Calpain; Captopril; Case-Control Studies; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Humans; Hypertension; Membrane Proteins

The calpains, a family of calcium-dependent cysteine proteinases, and calpastatin, their endogenous inhibitor protein, are involved in the proteolysis of amyloid precursor protein, which is thought to be abnormal in patients with Alzheimer's disease (AD). Specific inhibitors of calpains attenuate amyloid beta peptide-induced neuronal death. We hypothesized that some AD patients have functionally deficient mutation(s) of the CAST gene encoding calpastatin, and we screened 40 Japanese patients with AD for mutations in the coding region of CAST. Nine polymorphisms, -82A/G, IVS7-96A/G, 669A/G, 1223C/G (Ser408Cys), IVS20-10C/T, IVS21-65G/A, IVS22+31T/C, IVS24+38Ins/DelA, and IVS25-32A/G, were identified. The 669A allele causes skipping of exon 11, leading to the loss of 13 residues. Comparisons between 101 patients and 90 controls revealed no significant association between CAST polymorphisms and risk for AD, indicating that genomic variations of CAST are not likely to be substantially involved in the etiology of AD.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Calcium-Binding Proteins; Calpain; Cell Death; DNA Mutational Analysis; Female; Gene Frequency; Genotype; Humans; Japan; Male; Middle Aged; Mutation; Polymorphism, Genetic

Calpain, a calcium-activated cysteine protease, has been implicated in neuronal degeneration and death. In this study, we have characterized calpain activation in adult rat cerebral cortex and cerebellum, using an experimental paradigm of in vivo chronic ethanol exposure. Ethanol treatment increased the calpain activity in cortex and cerebellum, but to a higher extent in the cortex. Western blot analysis revealed a significant decrease in m-calpain levels while calpastatin levels were unaltered. Calpain activation was further monitored by the proteolysis of alpha-spectrin (fodrin) and protein kinase C-alpha (PKC-alpha). Protease specific spectrin breakdown products revealed calpain generated 150- and 145-kDa fragments. In addition, we also observed a 120-kDa fragment characteristic of caspase-3 activation in the cerebellum. PKC-alpha levels were decreased in the cortex and cerebellum by ethanol. Calpain activation, cleavage of alpha-spectrin into calpain specific signature fragments and decreased PKC-alpha protein levels after ethanol treatment provide the evidence of calpain involvement besides caspase-3-mediated cell death in the cortex and cerebellum. Given the role of calpains in cell death, increased calpain activity followed by alpha-spectrin cleavage in this study suggests that calpains are important effectors in ethanol-mediated cell injury and alcoholic neurodegeneration.

Topics: Alcohol-Induced Disorders, Nervous System; Animals; Brain; Calcium-Binding Proteins; Calpain; Caspase 3; Caspases; Cell Death; Cerebellum; Cerebral Cortex; Ethanol; Isoenzymes; Nerve Degeneration; Neurons; Peptide Fragments; Protein Kinase C; Protein Kinase C-alpha; Rats; Spectrin; Up-Regulation

The calpains, a family of calcium-requiring intracellular proteases, are proposed regulators of cell proliferation. However, ablation of the calpain small subunit gene necessary for function of the conventional calpains did not result in decreased rate of proliferative growth of mouse stem cells under routine culture conditions. To address the reasons for this discrepancy, Chinese hamster ovary cell lines were established that overexpress the calpain inhibitor protein, calpastatin, under control of the ecdysone congener, ponasterone A. Overexpression of calpastatin in these cell lines resulted in a decreased growth of isolated colonies adhering to tissue culture plates. However, when cells were plated at higher density, calpastatin overexpression had no influence on proliferative growth rate. Growth of colonies in soft agar was not inhibited by calpastatin overexpression. Cell adhesion, cell de-adhesion, and cell motility all appeared to be normal after calpastatin overexpression. Differential display analysis was initiated to detect possible alteration of gene expression upon calpastatin overexpression. Analysis of approximately 3000 differential display PCR signals resulted in identification of one band that was underexpressed. Northern blot analysis confirmed a decreased amount of approximately 1 kb mRNA in cells overexpressing calpastatin. Sequence analysis identified a putative protein, Csr, containing a region homologous to two ubiquitin transferases and a putative cation channel protein.

Topics: Amino Acid Sequence; Animals; Blotting, Northern; Blotting, Western; Calcium-Binding Proteins; Calpain; Cations; Cell Adhesion; Cell Division; Cell Movement; CHO Cells; Cricetinae; DNA, Complementary; Dose-Response Relationship, Drug; Down-Regulation; Ecdysterone; Fibroblasts; Gene Expression Profiling; Humans; Mice; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Messenger; Sequence Homology, Amino Acid; Time Factors; Transfection; Trypsin

Calcium-activated neutral proteinases (CANPs) and their endogenous specific inhibitor calpastatin are found in a wide variety of vertebrate and invertebrate tissues. The CANPs are cysteine proteinases that have an absolute requirement for Ca(2+) for activity. mu-Calpain and calpastatin were purified by successive chromatographic steps on Toyopearl-Super Q 650S and Pharmacia Mono Q HR 5/5 columns. The enzyme has a M(r) of 84KDa using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), a M(min) of 79KDa from amino acid analysis and an pI of 5.2. Calpastatin has a M(r) of 323KDa using denaturing gradient PAGE and a pI of 4.7. The amino acid composition of mu-calpain revealed 689 residues and the pH and temperature optima were found to be 7.5 and 37 degrees C, respectively. mu-Calpain underwent a Ca(2+)-dependent autoproteolysis producing a fragment of 82KDa. The N-terminal sequence of mu-calpain showed 24 and 18% sequence identity with human and bovine mu-calpain.

Topics: Amino Acid Sequence; Animals; Autolysis; Brain; Calcium-Binding Proteins; Calpain; Cattle; Chromatography; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Humans; Isoelectric Focusing; Isoelectric Point; Kinetics; Molecular Weight; Species Specificity; Struthioniformes; Swine; Temperature

Many studies have demonstrated that the calcium-dependent proteolytic system (calpains and calpastatin) is involved in myoblast differentiation. It is also known that myogenic differentiation can be studied in vitro. In the present experiments, using a mouse muscle cell line (C2C12) we have analyzed both the sequences of appearance and the expression profiles of calpains 1, 2, 3 and calpastatin during the course of myoblast differentiation. Our results mainly show that the expression of ubiquitous calpains (calpain 1 and 2) and muscle-specific calpain (calpain 3) at the mRNAs level as well as at the protein level do not change significantly all along this biological process. In the same time, the specific inhibitor of ubiquitous calpains, calpastatin, presents a stable expression at mRNAs level as well as protein level, all along myoblast to myotube transition. A comparison with other myogenic cells is presented.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cell Fusion; Cell Line; Gene Expression Regulation, Enzymologic; Mice; Muscle Development; Muscles; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors

Peripheral nerve injury results in a series of events culminating in degradation of the axonal cytoskeleton (Wallerian degeneration). In the time period between axotomy and cytoskeletal degradation (24-48 h in rodents), there is calcium entry and activation of calpains within the axon. The precise timing of these events during this period is unknown. In the present study, antibodies were generated to three distinct peptide epitopes of m-calpain, and a fusion protein antibody was generated to the intrinsic calpain inhibitor calpastatin. These antibodies were used to measure changes in these proteins in mouse sciatic nerves during Wallerian degeneration. In sciatic nerve homogenates and cultured dorsal root ganglion (DRG) neurites, m-calpain protein was significantly reduced in transected nerves very early after nerve injury, long before axonal degeneration occurred. Levels of m-calpain protein remained low as compared to control nerves for the remainder of the 72-h time course. No changes in calpastatin protein were evident. Systemic treatment of animals with the protease inhibitor leupeptin partially prevented the rapid loss of calpain protein. Removal of calcium in DRG cultures had the same effect. These data indicate that m-calpain protein is lost very early after axonal injury, and likely reflect activation and degradation of this protein long before the cytoskeleton is degraded. Calpain activation may be an early event in a proteolytic cascade that is initiated by axonal injury and culminates with axonal degeneration.

Topics: Animals; Axons; Calcium Signaling; Calcium-Binding Proteins; Calpain; Cell Membrane; Cytoskeleton; Down-Regulation; Leupeptins; Male; Mice; Mice, Inbred C57BL; Peptide Hydrolases; Peripheral Nerves; Peripheral Nervous System Diseases; Protease Inhibitors; Rabbits; Signal Transduction; Time Factors; Tubulin; Wallerian Degeneration

To clone a new gene related to human spermatogenesis.. cDNA probes of embryo and adult testis were used to hybridize the cDNA microarray of adult testis, and the clones of differential hybridization were sequenced and analyzed.. A novel isoform of calpastatin exclusively and highly expressed in human adult testis was found.. A novel isoform of calpastatin expresses in human testis and it is related to spermatogenesis.

Topics: Adult; Amino Acid Sequence; Base Sequence; Calcium-Binding Proteins; Calpain; Cloning, Molecular; Fetus; Humans; Male; Molecular Sequence Data; Protein Isoforms; Spermatogenesis; Testis

Calpains are neutral Ca2+-dependent cysteine proteases. In this study, we utilized casein zymography to detect such a proteolytic activity in Drosophila melanogaster extracts throughout the life of this organism. One calpain-like activity that was sensitive to the general cysteine protease inhibitors, E64 and calpain inhibitor I, but insensitive to the human calpain-specific inhibitor, calpastatin, is demonstrated. The relevance of this finding is discussed with respect to the absence of a corresponding Drosophila gene, homologous to the vertebrate calpastatin genes, as concluded from our unsuccessful attempts to clone such a gene and our Blast searches using the FlyBase. The mechanisms of Drosophila calpain regulation require further investigation. However, we suggest that single chain, non-heterodimeric calpains may be insensitive to calpastatin and that Drosophila cystatin-like molecules may play a role in negatively regulating Drosophila calpain.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cloning, Molecular; Cysteine Proteinase Inhibitors; Drosophila melanogaster; Gene Library

We examined effects of dietary soy protein isolate on muscle calpain activity and myosin heavy chain (MHC) degradation in rats performing an acute running exercise.. In rats fed a 20% casein diet, the treadmill running exercise, fixed at 80 kg/m, transiently increased calpain activity in gastrocnemius muscles in parallel with the release of creatine kinase into plasma. The fixed running also caused an accumulation of immunoreactive degradation fragments of MHC in the muscle. Feeding a 20% soy protein isolate diet as opposed to the control casein diet to rats significantly suppressed the running-induced activation of mu- and m-calpains, fragmentation of MHC, and release of creatine kinase into plasma (P < 0.05).. Rats fed the soy protein isolate diet had significantly higher calpastatin activity in gastrocnemius muscle than did rats fed the casein diet (P < 0.05), indicating that this increase inhibits the exercise-induced autoactivation of calpain. Activities of proteasome, cathepsin B + L, and antioxidant enzymes and the levels of glutathione and thiobarbituric acid-reactive substances in the muscle did not differ between the diet groups at the end of the exercise.. Our results suggest that diets containing soy protein prevent exercise-induced protein degradation in skeletal muscle, possibly through inhibiting the calpain-mediated proteolysis.

Topics: Animals; Calcium-Binding Proteins; Calpain; Creatine Kinase; Enzyme Activation; Lipid Peroxides; Male; Muscle Proteins; Muscle, Skeletal; Myosin Heavy Chains; Oxidation-Reduction; Physical Conditioning, Animal; Rats; Rats, Wistar; Soybean Proteins; Thiobarbituric Acid Reactive Substances; Time Factors

The class of Ca2+-permeable cation channels is composed of large families with six transmembrane segments including transient receptor potential, vanilloid receptor (VR), polycystin, epithelial calcium channels and melastatin (MLS). However, most of them are functionally silent and unexpressed in mammalian cells. An investigation of associated proteins made us believe that the blockade of calpain opens the silent channels. Using 1 microM of blockers in whole cellular patch pipette fill we measured currents of Chinese hamster ovary cells transfected by VR-like 1 and 2, polycystin-2, or a MLS-like new member (MLS3S). Significant conductance of every clone with a characteristic rectification by blockers was demonstrated. The permeability of Ca2+ to them is similar to that reported. Western blot suggested that blockers did not affect the assembly of the protein but enabled its cleavage. Therefore, investigation of these families with the blockers may boost our knowledge of electrophysiologic function.

Topics: Amino Acid Sequence; Animals; Calcium; Calcium Channels; Calcium-Binding Proteins; Calpain; Cell Membrane Permeability; CHO Cells; Cloning, Molecular; Cricetinae; Cysteine Proteinase Inhibitors; Electrophysiology; Ion Channel Gating; Leupeptins; Ligands; Membrane Proteins; Molecular Sequence Data; Patch-Clamp Techniques; Receptors, Drug; Transfection; TRPP Cation Channels

The conventional calpains, m- and micro-calpain, are suggested to be involved in apoptosis triggered by many different mechanisms. However, it has not been possible to definitively associate calpain function with apoptosis, largely because of the incomplete selectivity of the cell permeable calpain inhibitors used in previous studies. In the present study, Chinese hamster ovary (CHO) cell lines overexpressing micro-calpain or the highly specific calpain inhibitor protein, calpastatin, have been utilized to explore apoptosis signals that are influenced by calpain content. This approach allows unambiguous alteration of calpain activity in cells. Serum depletion, treatment with the endoplasmic reticulum (ER) calcium ATPase inhibitor thapsigargin, and treatment with calcium ionophore A23187 produced apoptosis in CHO cells, which was increased in calpain overexpressing cells and decreased by induced expression of calpastatin. Inhibition of calpain activity protected beta-spectrin, but not alpha-spectrin, from proteolysis. The calpains seemed not to be involved in apoptosis triggered by a number of other treatments. Calpain protected against TNF-alpha induced apoptosis. In contrast to previous studies, we found no evidence that calpains proteolyze I kappa B-alpha in TNF-alpha-stimulated cells. These studies indicate that the conventional calpains participate in some, but not all, apoptotic signaling mechanisms. In most cases, they contributed to apoptosis, but in at least one case, they were protective.

Topics: Animals; Apoptosis; Calcimycin; Calcium-Binding Proteins; Calcium-Transporting ATPases; Calpain; Cells, Cultured; CHO Cells; Cricetinae; Enzyme Inhibitors; Gene Expression; Humans; Ionophores; Mice; Rats; Recombinant Proteins; Signal Transduction; Thapsigargin; Tumor Necrosis Factor-alpha

Atrial fibrillation (AF) is accompanied by intracellular calcium overload. The purpose of this study was to assess the role of calcium-dependent calpains and cytokines during AF. Atrial tissue samples from 32 patients [16 with chronic AF and 16 in sinus rhythm (SR)] undergoing open heart surgery were studied. Atrial expression of calpain I and II, calpastatin, troponin T (TnT), troponin C (TnC), and cytokines [interleukin (IL)-1 beta, IL-2, IL-6, IL-8, IL-10, transforming growth factor (TGF)-beta 1, and tumor necrosis factor-alpha] were determined. Expression of calpain I was increased during AF (461 +/- 201% vs. 100 +/- 34%, P < 0.05). Amounts of calpain II and calpastatin were unchanged. Total calpain enzymatic activity was more than doubled during AF (35.2 +/- 17.7 vs. 12.4 +/- 9.2 units, P < 0.05). In contrast to TnC, TnT levels were reduced in fibrillating atria by 26% (P < 0.05), corresponding to the myofilament disintegration seen by electron microscopy. Small amounts of only IL-2 and TGF-beta 1 mRNA and protein were detected regardless of the underlying cardiac rhythm. In conclusion, atria of patients with permanent AF show evidence of calpain I activation that might contribute to structural remodeling and contractile dysfunction, whereas there is no evidence of activation of tissue cytokines.

Topics: Actin Cytoskeleton; Adult; Aged; Atrial Appendage; Atrial Fibrillation; Blotting, Western; Calcium-Binding Proteins; Calpain; Cytokines; Female; Humans; In Vitro Techniques; Interleukin-2; Male; Middle Aged; Myocardium; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

1. Recent studies demonstrated that the cardiac calpain system is activated during ischaemic events and is involved in cardiomyocyte injury. The aim of this study was to investigate the contribution of AT(1) and AT(2) receptors in the regulation of calpain-mediated myocardial damage following myocardial infarction (MI). 2. Infarcted animals were treated either with placebo, the ACE inhibitor ramipril (1 mg kg(-1) d(-1)), the AT(1) receptor antagonist valsartan (10 mg kg(-1) d(-1)) or the AT(2) receptor antagonist PD 123319 (30 mg kg(-1) d(-1)). Treatment was started 7 days prior to surgery. On day 1, 3, 7 and 14 after MI, gene expression and protein levels of calpain I, II and calpastatin were determined in left ventricular free wall (LVFW) and interventricular septum (IS). At day 3 and 14 post MI, morphological investigations were performed. 3. Calpain I mRNA expression and protein levels were increased in IS 14 days post MI, whereas mRNA expression and protein levels of calpain II were maximally increased in LVFW 3 days post MI. Ramipril and valsartan decreased mRNA and protein up-regulation of calpain I and II, and reduced infarct size and interstitial fibrosis. PD 123319 did not affect calpain I or II up-regulation in the infarcted myocardium, but decreased interstitial fibrosis. Calpastatin expression and translation were not affected by AT receptor antagonists or ACE inhibitor. 4. Our data demonstrate a distinct, temporary-spatial up-regulation of calpain I and II following MI confer with the hypothesis of calpain I being involved in cardiac remodelling in the late and calpain II contributing to cardiac tissue damage in the early phase of MI. The up-regulation of calpain I and II is partly mediated via the AT(1) receptor and can be reduced by ACE inhibitors and AT(1) receptor antagonists.

Topics: Angiotensin II; Angiotensin Receptor Antagonists; Angiotensin-Converting Enzyme Inhibitors; Animals; Antihypertensive Agents; Blood Pressure; Calcium-Binding Proteins; Calpain; Disease Models, Animal; Gene Expression Regulation; Heart; Heart Rate; Imidazoles; Male; Myocardial Infarction; Protein Biosynthesis; Pyridines; Rats; Rats, Wistar; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; RNA, Messenger; Tetrazoles; Transcription, Genetic; Valine; Valsartan

The biochemistry of intermuscular variation in tenderness is not fully understood. To investigate the role of the calpains in this process we performed two experiments using bovine and ovine species. In the bovine experiment, two distinct muscles, longissimus thoracis et lumborum (LT) and psoas major (PM), were used. In the ovine experiment, four muscles, LT, PM, semimembranosus (SM), and semitendinosus (ST), were used. Muscles were sampled at death for the determination of the steady-state mRNA level of calpains and calpastatin and the activities of calpain 1, 2, and calpastatin. Muscles were also sampled to determine the temporal changes in pH, tenderness, and the activity of the ubiquitous calpain system during postmortem aging. The results of the relative rate of tenderization in both species was found to be related to muscle type; LT had the highest value in both species. Within species, the mRNA steady-state levels of calpain 1 and calpastatin were similar in various bovine and ovine muscles. Bovine calpain 2 mRNA level was significantly lower in the LT than in the PM. Ovine calpain 2 mRNA level was lower, but not significantly different, in the LT compared to the other muscles. The mRNA level of bovine calpain 3 was significantly higher in the LT muscle than in the PM. In the ovine, the mRNA level of calpain 3 was highest in the LT, followed by SM, PM, and ST. Results on the activity of the ubiquitous calpain system in various muscles at death were dependent on muscle type and species. Temporal changes in the activity of calpains and calpastatin during the first 24 h of postmortem aging were similar in the muscles studied: calpain 1 and calpastatin declined significantly and calpain 2 remained relatively unchanged. The temporal changes in muscle pH in both experiments indicated that the extent and rate of pH decline during aging was related to muscle type. Correlation analysis between the relative rate of tenderization and mRNA expression of calpains revealed a strong relationship with calpain 3 in both species.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Cysteine Proteinase Inhibitors; Hydrogen-Ion Concentration; Male; Meat; Muscle, Skeletal; RNA, Messenger; Sheep; Single-Strand Specific DNA and RNA Endonucleases

The aim of the present study was to investigate whether muscle glycogen stores in slaughter pigs could be decreased through strategic finishing feeding before slaughter. Moreover, preliminary meat quality traits were measured to see whether such a regulation of muscle glycogen stores affected ultimate pH, color, and tenderness in the meat. The strategic finishing feeding was carried out the last 3 wk prior to slaughter. Seven experimental groups with eight animals per group were fed diets low in digestible carbohydrates. A control group with four animals was fed a traditional grower-finishing diet. The muscle glycogen stores were reduced in longissimus muscle (LM) 11 to 26% at the time of slaughter in pigs that were fed the experimental diets compared with the control group. Meat quality measured as ultimate pH and color on LM muscle in half the pigs 24 h postmortem showed that ultimate pH in LM was not affected by the reduction in glycogen stores in the muscles from pigs fed any of the experimental diets. However, the meat from pigs fed the experimental diets was darker than the meat from pigs that were fed the control diet with two of the experimental diets, resulting in significantly lower L* values. Activities of key enzymes in the glycolytic pathway, glycogen phoshorylase a and b, phosphofructokinase, and the fatty acid oxidative pathway, beta-hydrozyacyl-CoA-dehydrogenase, were not affected by the strategic feeding. In contrast, the activity of the proteolytic enzyme calpain as well as its inhibitor calpastatin was influenced by the strategic feeding. Lower activity of mu-calpain and greater activity of calpastatin in the muscle samples from the strategically fed pigs indicate a lesser muscle protein degradation in the muscles compared with muscles of control animals. The present study showed that the muscle glycogen stores in slaughter pigs can be reduced at the time of slaughter through strategic finishing feeding with diets low in digestible carbohydrate without compromising growth rate.

Topics: 3-Hydroxyacyl CoA Dehydrogenases; Animal Feed; Animals; Biopsy; Calcium-Binding Proteins; Calpain; Color; Cysteine Proteinase Inhibitors; Dietary Carbohydrates; Female; Glycogen; Hydrogen-Ion Concentration; Meat; Muscle, Skeletal; Myoglobin; Phosphofructokinase-1; Phosphorylases; Swine

Activities of mu- and m-calpain and of calpastatin were measured at four different times during postmortem storage (0, 1, 3, and 10 d) in three muscles from either callipyge or noncallipyge (normal) sheep. The weights of two muscles, the biceps femoris and the longissimus, are greater in the callipyge phenotype, whereas the weight of the infraspinatus is not affected. The activity of m-calpain was greater (P < 0.05) in the biceps femoris and longissimus from callipyge than in those from normal sheep, but it was the same in the infraspinatus in the two phenotypes. The extractable activity of m-calpain did not change (biceps femoris and infraspinatus) or decreased slightly (longissimus) during postmortem storage. Extractable activity of mu-calpain decreased to zero or nearly zero after 10 d postmortem in all muscles from both groups of sheep. The rate of decrease in mu-calpain activity was the same in muscles from the callipyge and normal sheep. At all time points during postmortem storage, calpastatin activity was greater (P < 0.05) in the biceps femoris and longissimus from the callipyge than from the normal sheep, but it was the same in the infraspinatus from callipyge and normal sheep. Calpastatin activity decreased (P < 0.05) in all three muscles from both phenotypes during postmortem storage; the rate of this decrease in the callipyge biceps femoris and longissimus and in the infraspinatus from both the callipyge and normal sheep was slow, especially after the first 24 h postmortem, whereas calpastatin activity in the biceps femoris and longissimus from the normal sheep decreased rapidly. During postmortem storage, the 125-kDa calpastatin polypeptide was degraded, but the 80-kDa subunit of mu-calpain was cleaved only to 76- and 78-kDa polypeptides even though extractable mu-calpain activity declined nearly to zero. Approximately 50 to 60% of total mu-calpain became associated with the nonextractable pellet after 1 d postmortem. The myofibril fragmentation index for the biceps femoris and longissimus from normal sheep increased significantly during postmortem storage. The fragmentation index for the infraspinatus from the callipyge and normal sheep increased to an intermediate extent, whereas the index for the biceps femoris and longissimus from the callipyge did not change during 10-d postmortem storage. The results suggest that postmortem tenderization is related to the rate of calpastatin degradation in postmortem muscle and that calpastatin inhibit

Topics: Animals; Blotting, Western; Calcium-Binding Proteins; Calpain; Chromatography, DEAE-Cellulose; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Female; Isoenzymes; Meat; Muscle, Skeletal; Myofibrils; Postmortem Changes; Sarcomeres; Sheep

1. Long-term (> or = 12 h) treatment of cultured bovine adrenal chromaffin cells with A23187 (a Ca(2+) ionophore) or thapsigargin (TG) [an inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)] caused a time- and concentration-dependent reduction of cell surface [(3)H]-saxitoxin (STX) binding capacity, but did not change the K:(D:) value. In A23187- or TG-treated cells, veratridine-induced (22)Na(+) influx was reduced (with no change in veratridine EC(50) value) while it was enhanced by alpha-scorpion venom, beta-scorpion venom, or Ptychodiscus brevis toxin-3, like in nontreated cells. 2. The A23187- or TG-induced decrease of [(3)H]-STX binding was diminished by BAPTA-AM. EGTA also inhibited the decreasing effect of A23187. A23187 caused a rapid, monophasic and persistent increase in intracellular concentration of Ca(2+) ([Ca(2+)](i)) to a greater extent than that observed with TG. 2,5-Di-(t-butyl)-1,4-benzohydroquinone (DBHQ) (an inhibitor of SERCA) produced only a rapid monophasic increase in [Ca(2+)](i), without any effect on [(3)H]-STX binding. 3. Reduction in [(3)H]-STX binding capacity induced by A23187 or TG was attenuated by Gö6976 (an inhibitor of conventional protein kinase C) or calpastatin peptide (an inhibitor of calpain). When the internalization rate of cell surface Na(+) channels was measured in the presence of brefeldin A (an inhibitor of vesicular exit from the trans-Golgi network), A23187 or TG accelerated the reduction of [(3)H]-STX binding capacity. 4. Six hours treatment with A23187 lowered Na(+) channel alpha- and beta(1)-subunit mRNA levels, whereas TG had no effect. 5. These results suggest that elevation of [Ca(2+)](i) caused by A23187, TG or DBHQ exerted differential effects on down-regulation of cell surface functional Na(+) channels and Na(+) channel subunit mRNA levels.

Topics: Animals; Binding, Competitive; Brefeldin A; Calcimycin; Calcium; Calcium-Binding Proteins; Calcium-Transporting ATPases; Calpain; Carbazoles; Cattle; Cells, Cultured; Chromaffin Cells; Dose-Response Relationship, Drug; Down-Regulation; Egtazic Acid; Enzyme Inhibitors; Hydroquinones; Indoles; Ionophores; Marine Toxins; Oxocins; Protein Subunits; RNA, Messenger; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Saxitoxin; Scorpion Venoms; Sodium; Sodium Channels; Thapsigargin; Time Factors; Tritium; Veratridine

Human cytomegalovirus (HCMV) stimulates arrested cells to enter the cell cycle by activating cyclin-dependent kinases (Cdks), notably Cdk2. Several mechanisms are involved in the activation of Cdk2. HCMV causes a substantial increase in the abundance of cyclin E and stimulates translocation of Cdk2 from the cytoplasm to the nucleus. Further, the abundance of the Cdk inhibitors (CKIs) p21cip1/waf1 (p21cip1) and p27kip1 is substantially reduced. The activity of cyclin E/Cdk2 increases as levels of CKIs, particularly p21cip1, fall. We have previously shown that these phenomena contribute to priming the cell for efficient replication of HCMV. In this study, the mechanisms responsible for the decrease in p21cip1 levels after HCMV infection were investigated by measuring p21cip1 RNA and protein levels in permissive human lung (LU) fibroblasts after HCMV infection. Northern blot analysis revealed that p21cip1 RNA levels increased briefly at 3 h after HCMV infection and then decreased to their nadir at 24 h; thereafter, RNA levels increased to about 60% of the preinfection level. Western blot analysis demonstrated that the relative abundance of p21cip1 protein roughly paralleled the observed changes in initial RNA levels; however, the final levels of protein were much lower than preinfection levels. After a transient increase at 3 h postinfection, p21cip1 abundance declined sharply over the next 24 h and remained at a very low level through 96 h postinfection. The disparity between p21cip1 RNA and protein levels suggested that the degradation of p21cip1 might be affected in HCMV-infected cells. Treatment of HCMV-infected cells with MG132, an inhibitor of proteasome-mediated proteolysis, provided substantial protection of p21cip1 in mock-infected cells, but MG132 was much less effective in protecting p21cip1 in HCMV-infected cells. The addition of E64d or Z-Leu-Leu-H, each an inhibitor of calpain activity, to HCMV-infected cells substantially increased the abundance of p21cip1 in a concentration-dependent manner. To verify that p21cip1 was a substrate for calpain, purified recombinant p21cip1 was incubated with either m-calpain or mu-calpain, which resulted in rapid proteolysis of p21cip1. E64d inhibited the proteolysis of p21cip1 catalyzed by either m-calpain or mu-calpain. Direct measurement of calpain activity in HCMV-infected LU cells indicated that HCMV infection induced a substantial and sustained increase in calpain activity, although there was no change in

Topics: Amino Acid Motifs; Calcium-Binding Proteins; Calpain; Cell Cycle; Cell Line; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Endopeptidases; Cytomegalovirus; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Leucine; Leupeptins; Multienzyme Complexes; Proteasome Endopeptidase Complex; RNA, Messenger; Time Factors; Ubiquitins

Cachexia is a syndrome characterized by profound tissue wasting that frequently complicates malignancies. In a cancer cachexia model we have shown that protein depletion in the skeletal muscle, which is a prominent feature of the syndrome, is mostly due to enhanced proteolysis. There is consensus on the views that the ubiquitin/proteasome pathway plays an important role in such metabolic response and that cytotoxic cytokines such as TNFalpha are involved in its triggering (Costelli and Baccino, 2000), yet the mechanisms by which the relevant extracellular signals are transduced into protein hypercatabolism are largely unknown. Moreover, little information is presently available as to the possible involvement in muscle protein waste of the Ca(2+)-dependent proteolysis, which may provide a rapidly activated system in response to the extracellular signals. In the present work we have evaluated the status of the Ca(2+)-dependent proteolytic system in the gastrocnemius muscle of AH-130 tumour-bearing rats by assaying the activity of calpain as well as the levels of calpastatin, the natural calpain inhibitor, and of the 130 kDa Ca(2+)-ATPase, both of which are known calpain substrates. After tumour transplantation, total calpastatin activity progressively declined, while total calpain activity remained unchanged, resulting in a progressively increasing unbalance in the calpain/calpastatin ratio. A decrease was also observed for the 130 kDa plasma membrane form of Ca(2+)-ATPase, while there was no change in the level of the 90 kDa sarcoplasmic Ca(2+)-ATPase, which is resistant to the action of calpain. Decreased levels of both calpastatin and 130 kDa Ca(2+)-ATPase have been also detected in the heart of the tumour-bearers. These observations strongly suggest that Ca(2+)-dependent proteolysis was activated in the skeletal muscle and heart of tumour-bearing animals and raise the possibility that such activation may play a role in sparking off the muscle protein hypercatabolic response that characterizes cancer cachexia.

Topics: Animals; Cachexia; Calcium-Binding Proteins; Calcium-Transporting ATPases; Calpain; Enzyme Activation; Liver Neoplasms, Experimental; Male; Muscle, Skeletal; Myocardium; Rats; Rats, Wistar

Long-term cultured pre-B cells are able to differentiate into immunoglobulin (Ig)M-positive B cells (IgM(+) cells) when transplanted into severe combined immunodeficient (SCID) mice. Based on previous studies, here we report the development of a reconstitution assay in nonobese diabetic/SCID (NOD/SCID) mice using pre-B cells, which allows us to study the role of calpains (calcium-activated endopeptidases) during B cell development as well as in B cell clonal deletion. Using this model, we show that calpastatin (the natural inhibitor of calpains) inhibits B cell receptor-induced apoptosis in IgM(+) cells derived from transplanted mice. We thus hypothesize an important function for calpain in sculpting the B cell repertoire.

Topics: Animals; Apoptosis; B-Lymphocytes; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cell Line; Clonal Deletion; Gene Expression; Hematopoiesis; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Immunoglobulin M; Interleukin-7; Mice; Mice, Inbred NOD; Mice, SCID; Models, Biological; Receptors, Antigen, B-Cell

Calpain is a heterodimeric Ca(2+)-dependent cysteine protease consisting of a large (80 kDa) catalytic subunit and a small (28 kDa) regulatory subunit. The effects of Ca(2+) on the enzyme include activation, aggregation, and autolysis. They may also include subunit dissociation, which has been the subject of some debate. Using the inactive C105S-80k/21k form of calpain to eliminate autolysis, we have studied its disassociation and aggregation in the presence of Ca(2+) and the inhibition of its aggregation by means of crystallization, light scattering, and sedimentation. Aggregation, as assessed by light scattering, depended on the ionic strength and pH of the buffer, on the Ca(2+) concentration, and on the presence or absence of calpastatin. At low ionic strength, calpain aggregated rapidly in the presence of Ca(2+), but this was fully reversible by EDTA. With Ca(2+) in 0.2 m NaCl, no aggregation was visible but ultracentrifugation showed that a mixture of soluble high molecular weight complexes was present. Calpastatin prevented aggregation, leading instead to the formation of a calpastatin-calpain complex. Crystallization in the presence of Ca(2+) gave rise to crystals mixed with an amorphous precipitate. The crystals contained only the small subunit, thereby demonstrating subunit dissociation, and the precipitate was highly enriched in the large subunit. Reversible dissociation in the presence of Ca(2+) was also unequivocally demonstrated by the exchange of slightly different small subunits between mu-calpain and m-calpain. We conclude that subunit dissociation is a dynamic process and is not complete in most buffer conditions unless driven by factors such as crystal formation or autolysis of active enzymes. Exposure of the hydrophobic dimerization surface following subunit dissociation may be the main factor responsible for Ca(2+)-induced aggregation of calpain. It is likely that dissociation serves as an early step in calpain activation by releasing the constraints upon protease domain I.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Catalysis; Chromatography, Liquid; Crystallography, X-Ray; Dimerization; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Ions; Light; Models, Molecular; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Rats; Recombinant Proteins; Scattering, Radiation; Time Factors; Ultracentrifugation; Water

The goals of this study were to determine 1) the expression of calpain isoforms in rabbit renal proximal tubules (RPT); 2) calpain autolysis and translocation, and calpastatin levels during RPT injury; and 3) the effect of a calpain inhibitor (PD-150606) on calpain levels, mitochondrial function, and ion transport during RPT injury. RT-PCR, immunoblot analysis, and FITC-casein zymography demonstrated the presence of only mu- and m-calpains in rabbit RPT. The mitochondrial inhibitor antimycin A decreased RPT mu- and m-calpain and calpastatin levels in conjunction with cell death and increased plasma membrane permeability. No increases in either mu- or m-calpain were observed in the membrane nor were increases observed in autolytic forms of either mu- or m-calpain in antimycin A-exposed RPT. PD-150606 blocked antimycin A-induced cell death, preserved calpain levels in antimycin A-exposed RPT, and promoted the recovery of mitochondrial function and active Na+ transport in RPT after hypoxia and reoxygenation. The present study suggests that calpains mediate RPT injury without undergoing autolysis or translocation, and ultimately they leak from cells subsequent to RPT injury/death. Furthermore, PD-150606 allows functional recovery after injury.

Topics: Acrylates; Animals; Anti-Bacterial Agents; Antimycin A; Autolysis; Biological Transport, Active; Calcium-Binding Proteins; Calpain; Caseins; Cell Death; Cell Membrane; Cysteine Proteinase Inhibitors; Cytosol; Female; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Gene Expression Regulation, Enzymologic; Immunoblotting; Isoenzymes; Kidney Tubules, Proximal; Mitochondria; Rabbits; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Sodium

The expression in porcine skeletal and cardiac muscle of calpastatin, the specific endogenous inhibitor of the calpain proteolytic system, was examined 16 h after a single dose of a specific beta(2)-agonist. Immunoblotting of extracts indicated that treatment increased skeletal calpastatin 135-kDa band intensity (P < 0.01), while in cardiac combined 145- and 135-kDa band intensity decreased (P < 0.05). Treatment increased skeletal (P < 0.01) but not cardiac calpastatin mRNA steady-state levels. Three types of cardiac calpastatin mRNA transcripts were identified by 5'-RACE. Types I and II encoded a putative XL region that originated either from exon 1x(A) or exon 1x(B), arranged in tandem. Type III predominated in skeletal muscle and originated from exon 1u, which was located 40-50 kb 3' to exons 1x(A) and 1x(B). The region 5' to exon 1u may act as an independent promoter regulated by a cAMP-dependent mechanisms, thereby explaining the differential response of calpastatin to adrenergic stimulation in cardiac and skeletal muscle.

Topics: 5' Flanking Region; Adrenergic beta-Agonists; Alternative Splicing; Animals; Base Sequence; Blotting, Northern; Calcium-Binding Proteins; Calpain; Clenbuterol; Exons; Immunoblotting; Molecular Sequence Data; Muscle, Skeletal; Myocardium; Organ Specificity; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Swine

Ubiquitous type m-calpain and lens specific Lp82 calpain were separated and partially purified from fetal bovine lens and the enzymatic characteristics were compared. Lens m-calpain required 200 microM calcium for 1/2 maximal activity, while Lp82 required 30 microM. Both types of calpains were inhibited by 0.1 mM E64, and 5 mM iodoacetamide, but not by 1 mM phenylmethylsulfonyl fluoride. Lp82 was insensitive to 1 microM calpastatin peptide while m-calpain was effectively inhibited. In the presence of calcium, m-calpain lost most of its activity within 2 hr, while Lp82 was continually active for 18 hr. Both calpains cleaved the natural substrates betaA3 and alphaB crystallins in a similar manner. However, incubation of alphaA crystallin with m-calpain removed ten amino acid residues from its C-terminus, while incubation with Lp82 removed only five residues. The latter truncation product of alphaA was also found in vivo. These data suggested that Lp82 may have a more important role than m-calpain in modification of crystallins during lens maturation.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Chromatography, Liquid; Crystallins; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Image Processing, Computer-Assisted; Immunoenzyme Techniques; Lens, Crystalline; Spectrometry, Mass, Electrospray Ionization; Time Factors

The use of 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BODIPY-FL) labeled casein in autoquenching assays of proteolytic activity has been recently described, and we have adapted this assay to measurement of calpain activity. BODIPY-FL coupled to casein at a ratio of 8 mol of BODIPY-FL/mol of casein or higher produces a BODIPY-FL-casein substrate that can be used in an autoquenching assay of calpain proteolytic activity. This assay has a number of advantages for measuring calpain activity. (1) The procedure does not require precipitation and removal of undegraded protein, so it is much faster than other procedures that require a precipitation step, and it can be used directly in kinetic assays of proteolytic activity. (2) The BODIPY-FL-casein assay is easily adapted to a microtiter plate format, so it can be used to screen large numbers of samples. (3) Casein is an inexpensive and readily available protein substrate that more closely mimics the natural substrates of endoproteinases, such as the calpains, than synthetic peptide substrates do. Casein has K(m) values for micro- and m-calpain that are similar to those of other substrates such as fodrin or MAP2 that may be "natural" substrates for the calpains, and there is no reason to believe that calpain hydrolysis of casein is inherently different from hydrolysis of fodrin or MAP2, which are much less accessible as substrates for protease assays. (4) The BODIPY-FL-casein assay is capable of detecting 10 ng ( approximately 5 nM) of calpain and is nearly as sensitive as the most sensitive calpain assay reported thus far. (5) The BODIPY-FL-casein assay is as reproducible as the FITC-casein assay, whose reproducibility is comparable to or better than the reproducibility of other methods used to assay calpain activity. The BODIPY-FL-casein assay is a general assay for proteolytic activity and can be used with any protease that cleaves casein.

Topics: Animals; Boron Compounds; Calcium-Binding Proteins; Calpain; Caseins; Cattle; Endopeptidases; Evaluation Studies as Topic; Fluorescent Dyes; Humans; Microchemistry; Reproducibility of Results

We have previously reported the activation of procalpain mu (precursor for low-calcium-requiring calpain) in apoptotic cells using a cleavage-site-directed antibody specific to active calpain [Kikuchi, H. and Imajoh-Ohmi, S. (1995) Cell Death Differ. 2, 195-199]. In this study, calpastatin, the endogenous inhibitor protein for calpain, was cleaved to a 90-kDa polypeptide during apoptosis in human Jurkat T cells. The limited proteolysis of calpastatin preceded the autolytic activation of procalpain. Inhibitors for caspases rescued the cells from apoptosis and simultaneously inhibited the cleavage of calpastatin. The full-length recombinant calpastatin was also cleaved by caspase-3 or caspase-7 at Asp-233 into the same size fragment. Cys-241 was also targeted by these caspases in vitro but not in apoptotic cells. Caspase-digested calpastatin lost its amino-terminal inhibitory unit, and inhibited three moles of calpain per mole. Our findings suggest that caspases trigger the decontrol of calpain activity suppression by degrading calpastatin.

Topics: Amino Acid Chloromethyl Ketones; Antibodies, Monoclonal; Apoptosis; Aspartic Acid; Calcium-Binding Proteins; Calpain; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; fas Receptor; Humans; Jurkat Cells; Leupeptins; Oligopeptides; Poly(ADP-ribose) Polymerases; Recombinant Proteins; Tumor Necrosis Factor-alpha

Calpastatin activity measured at 24 h postmortem in bovine longissimus muscle (PMLD24) is correlated with Warner-Bratzler shear force (WBS) measurements, an objective measure of tenderness. A live-animal measurement of calpastatin activity that correlates with 24-h postmortem activity would provide information for selection programs without the expense of progeny testing. The purpose of this study was to evaluate the effectiveness of calpastatin activity measurements obtained on tissue samples from live animals and to determine the relationship among various calpastatin activity measures and tenderness determined by WBS and sensory panel. Biopsies (approximately 10 g) were obtained surgically 2 d before slaughter from the supraspinatus muscle on the anterior surface of the scapula (LISH0) from contemporary purebred Angus bulls (n = 12) and steers (n = 17). Biopsies from a subset of these cattle (n = 12) were refrigerated at 4 degrees C to simulate the postmortem cooling process for 24 h (LISH24) prior to extraction. A rib section anterior to the 12 and 13th rib interface was collected from all animals at the commercial abattoir between 22 and 23 h postmortem for PMLD24, sensory panel, and WBS measurements. A postmortem shoulder muscle sample (PMSH24) was collected at the same time. Calpastatin was extracted from all muscle samples using a heated calpastatin activity protocol. Sensory panel tenderness, WBS, LISH0, LISH24, and PMSH24 were not different between bulls and steers. However, PMLD24 values were significantly different. Significant partial correlations were found between WBS and sensory panel tenderness (-.55), between WBS and PMLD24 (-.43), and between LISH24 and PMLD24 (.78). Therefore, similar calpastatin activity values are possible with ante- and postmortem tissue samples, suggesting the possibility of using measurements from live-tissue biopsies from other than the longissimus muscle to predict end product tenderness.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Cysteine Proteinase Inhibitors; Male; Muscles; Postmortem Changes

Topics: Animals; Calcium-Binding Proteins; Calpain; Chromatography, Agarose; Chromatography, DEAE-Cellulose; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Indicators and Reagents; Isoenzymes; Sepharose; Tissue Distribution

Topics: Animals; Anion Exchange Resins; Calcium-Binding Proteins; Calpain; Caseins; Chromatography, Agarose; Chromatography, Ion Exchange; Cysteine Proteinase Inhibitors; Dipeptides; Fluorescamine; Isoenzymes; Resins, Synthetic; Sepharose; Substrate Specificity

Topics: Calcium-Binding Proteins; Calpain; Chromatography, Affinity; Chromatography, Gel; Chromatography, Ion Exchange; Cysteine Proteinase Inhibitors; Escherichia coli; Humans; Protein Denaturation; Recombinant Proteins

Topics: 3T3 Cells; Animals; Blotting, Western; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Humans; Mice; Molecular Weight; Recombinant Proteins

Topics: Animals; Brain; Brain Chemistry; Calcium; Calcium-Binding Proteins; Calpain; Cattle; Chromatography, Gel; Enzyme Activation; Humans; In Vitro Techniques; Membrane Proteins; Rats

Topics: Animals; Base Sequence; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cell Division; Cell Fusion; Cells, Cultured; Chromatography, Ion Exchange; Cysteine Proteinase Inhibitors; Immunoblotting; Immunohistochemistry; Muscle, Skeletal; Oligonucleotides, Antisense; Rats; RNA, Messenger

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Fusion; Cell Line; Cysteine Proteinase Inhibitors; Electrophoresis; Erythrocytes; Humans; Immunoblotting; In Vitro Techniques; Membrane Proteins; Muscle, Skeletal; Rats

Topics: Animals; Calcium Signaling; Calcium-Binding Proteins; Calpain; Cell Hypoxia; Cells, Cultured; Coumarins; Cysteine Proteinase Inhibitors; Dipeptides; Fluorescent Dyes; Fura-2; Glutathione; Hypoxia; In Situ Hybridization, Fluorescence; Liver; Oligopeptides; Rats; Substrate Specificity

Topics: Animals; Ankyrins; Blotting, Western; Calcium-Binding Proteins; Calpain; Carrier Proteins; Enzyme Activation; Immunohistochemistry; Male; Membrane Proteins; Microfilament Proteins; Myocardial Reperfusion Injury; Rats; Spectrin

Topics: Animals; Base Sequence; Blotting, Western; Calcimycin; Calcium-Binding Proteins; Calpain; Carrier Proteins; Cell Line; Cysteine Proteinase Inhibitors; DNA Primers; DNA, Complementary; Gene Expression; Ionophores; Microfilament Proteins; Molecular Sequence Data; Muscle Proteins; Muscle, Skeletal; Mutation; Protein Structure, Tertiary; Rats; Reverse Transcriptase Polymerase Chain Reaction; Transfection

Calpain, a Ca(2+)-dependent cysteine protease, has been implicated in cytoskeletal protein degradation and neurodegeneration in the lesion and adjacent areas following spinal cord injury (SCI). To attenuate apoptosis or programmed cell death (PCD) in SCI, we treated injured rats with E-64-d, a cell permeable and selective inhibitor of calpain. SCI was induced on T12 by the weight-drop (40 g-cm force) method. Within 15 min, E-64-d (1 mg/kg) in 1.5% DMSO was administered i.v. to the SCI rats. Following 24 h treatment, a 5-cm long spinal cord section with the lesion in the center was collected. The spinal cord section was divided equally into five 1-cm segments (S1: distant rostral, S2: near rostral, S3: lesion or injury, S4: near caudal and S5: distant caudal) for analysis. Determination of mRNA levels by reverse transcriptase-polymerase chain reaction (RT-PCR) indicated that ratios of bax/bcl-2 and calpain/calpastatin were increased in spinal cord segments from injured rats compared to controls. Degradation of the 68-kD neurofilament protein and internucleosomal DNA fragmentation were also increased. All of these changes were maximally increased in the lesion and gradually decreased in the adjacent areas of SCI rats, while largely undetectable in E-64-d treated rats and absent in sham controls. The results indicate that apoptosis in rat SCI appears to be associated with calpain activity which can be attenuated by the calpain inhibitor E-64-d.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; DNA Fragmentation; DNA Primers; Female; Gene Expression; Leucine; Neurofilament Proteins; Nucleosomes; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; RNA, Messenger; Spinal Cord Injuries

In neuroblastoma LAN-5 cells during calpain activation, in addition to the two expressed 70 kDa and 30 kDa calpastatin forms, other inhibitory species are produced, having molecular masses of 50 kDa and 15 kDa. At longer times of incubation, both native and new calpastatin species disappear. The formation of these new calpastatins as well as the decrease in intracellular total calpastatin activity are mediated by calpain itself, as indicated by the effect of the synthetic calpain inhibitor I, which prevents both degradative processes. Analysis of the calcium concentrations required for the two processes indicates that the first conservative proteolytic event is mediated by micro-calpain, whereas the second one is preferentially carried out by m-calpain. The appearance of the 15 kDa form, containing only the calpastatin repetitive inhibitory domain and identified also in red cells of hypertensive rats as the major inhibitor form, can be considered a marker of intracellular calpain activation, and it can be used for the monitoring of the involvement of calpain in pathological situations.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Humans; Neuroblastoma; Rats; Tumor Cells, Cultured

Abnormal proteolytic processing of beta-amyloid precursor protein (APP) underlies the formation of amyloid plaques in aging and Alzheimer's disease. The proteases involved in the process have not been identified. Here we found that spontaneous proteolysis of intact APP in detergent-lysed human platelets generated a N-terminal fragment that was immunologically indistinguishable from secreted APP, reminiscent of the action of a putative alpha-secretase. This proteolysis of APP was inhibited by EDTA, suggesting that a metal-dependent protease was involved. Among the several metals tested, calcium was the only one that enhanced APP proteolysis and the reaction was blocked by EGTA as well as by several calpain inhibitors. The APP fragments generated by spontaneous proteolysis in platelet lysates were identical to those produced by exposure of partially purified APP to exogenous calpain. Finally, the secretion of APP from intact platelets was inhibited by cell-permeable calpain inhibitors. Taken together, these results suggest that normal processing of APP in human platelets is mediated by a calcium-dependent protease that exhibits calpain-like properties.

Topics: Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Blood Platelets; Calcimycin; Calcium; Calcium-Binding Proteins; Calpain; Cations, Divalent; Cell Extracts; Edetic Acid; Egtazic Acid; Endopeptidase K; Humans; Hydrogen-Ion Concentration; Inhibitory Concentration 50; Molecular Weight; Peptide Fragments; Protease Inhibitors; Protein Processing, Post-Translational; Trypsin

We isolated three Xenopus cDNA clones, Xcalp1, Xcalp2 and Xcalp3, which encode different forms of calpastatin mRNA. Compared to the canonical form of mammalian calpastatin, the predicted Xcalp3 protein contained a very long N-terminal domain L and an additional inhibitory domain. The other two deduced calpastatin proteins were truncated forms, both lacking domain L and containing four (Xcalp2) and two (Xcalp1) inhibitory domains, respectively. The presence of Xcalp1, Xcalp2 and Xcalp3 transcripts was detected by in situ hybridization in the notochord from the embryonic stage 20 to stage 36, afterwards the expression was only present in the growing tailbud. As shown by RT-PCR, the three calpastatin mRNAs were also expressed in the adult brain.

Topics: Amino Acid Sequence; Animals; Calcium-Binding Proteins; Calpain; Gene Expression Regulation, Developmental; Molecular Sequence Data; Notochord; RNA, Messenger; Sequence Alignment; Xenopus laevis

Fiber-type transitions in adult skeletal muscle induced by chronic low-frequency stimulation (CLFS) encompass coordinated exchanges of myofibrillar protein isoforms. CLFS-induced elevations in cytosolic Ca(2+) could activate proteases, especially calpains, the major Ca(2+)-regulated cytosolic proteases. Calpain activity determined by a fluorogenic substrate in the presence of unaltered endogenous calpastatin activities increased twofold in low-frequency-stimulated extensor digitorum longus (EDL) muscle, reaching a level intermediate between normal fast- and slow-twitch muscles. micro- and m-calpains were delineated by a calpain-specific zymographical assay that assessed total activities independent of calpastatin and distinguished between native and processed calpains. Contrary to normal EDL, structure-bound, namely myofibrillar and microsomal calpains, were abundant in soleus muscle. However, the fast-to-slow conversion of EDL was accompanied by an early translocation of cytosolic micro-calpain, suggesting that myofibrillar and microsomal micro-calpain was responsible for the twofold increase in activity and thus involved in controlled proteolysis during fiber transformation. This is in contrast to muscle regeneration where m-calpain translocation predominated. Taken together, we suggest that translocation is an important step in the control of calpain activity in skeletal muscle in vivo.

Topics: Animals; Calcium-Binding Proteins; Calpain; Electric Stimulation; Hindlimb; Male; Microsomes; Muscle Fibers, Fast-Twitch; Muscle Fibers, Slow-Twitch; Muscle, Skeletal; Myofibrils; Protein Isoforms; Rats; Rats, Wistar; Regeneration

The interaction between the cysteine proteases calpain and caspases during renal ischemia-reperfusion (I/R) was investigated. An increase in the activity of calpain, as determined by 1) the appearance of calpain-mediated spectrin breakdown products and 2) the conversion of procalpain to active calpain, was demonstrated. Because intracellular calpain activity is regulated by calpastatin, the effect of I/R on calpastatin was determined. On immunoblot of renal cortex, there was a 50-100% decrease of a low molecular weight (LMW) form of calpastatin (41 kDa) after I/R. Calpastatin activity was also significantly decreased after I/R compared with sham-operated rats, indicating that the decreased protein expression had functional significance. In rats treated with the caspase inhibitor, z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-D-DCB), the decrease in both calpastatin activity and protein expression was normalized, suggesting that caspases may be proteolyzing calpastatin. Caspase 3 activity increased significantly after I/R and was attenuated in ischemic kidneys from rats treated with the caspase inhibitor. In summary, during renal I/R injury, there is 1) calpain activation associated with downregulation of calpastatin protein and decreased calpastatin activity and 2) activation of caspase 3. In addition, in vivo caspase inhibition reverses the decrease in calpastatin activity. In conclusion, proteolysis of calpastatin by caspase 3 may regulate calpain activity during I/R injury. Although the protective effect of cysteine protease inhibition against hypoxic necrosis of proximal tubules has previously been demonstrated, the functional significance in ischemic acute renal failure in vivo merits further study.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Blotting, Western; Calcium-Binding Proteins; Calpain; Caspase 3; Caspases; Cell Fractionation; Enzyme Activation; Kidney; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Spectrin

The present study was conducted to study the stability of autolyzed mu-calpain activity and determine whether measurement of mu-calpain activity after anion exchange chromatography accurately reflects its activity in postmortem muscle. Ionic strength and pH affected the stability of partially autolyzed mu-calpain. Complete loss of activity was observed as a result of binding of autolyzed mu-calpain to DEAE-Sephacel when the large subunit of mu-calpain was autolyzed from 80 to 76 kDa. Therefore, determination of mu-calpain by standard anion exchange chromatography may underestimate mu-calpain activity in postmortem muscle. The activity of autolyzed mu-calpain was stabilized by inclusion of glycerol in the buffers, and this permitted us to investigate whether the apparent loss of mu-calpain activity in postmortem muscle is an artifact of the methodology. Despite the inclusion of glycerol in the buffers, a decrease in mu-calpain activity was observed during postmortem storage of muscle, even though the autolyzed enzyme was readily detectable by Western blotting in muscle extracts and column eluates. This result indicates that instability of autolyzed mu-calpain is a major cause for the decline in mu-calpain activity in postmortem muscle.

Topics: Animals; Calcium-Binding Proteins; Calpain; Chromatography, Ion Exchange; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Muscles; Osmolar Concentration; Postmortem Changes; Swine

The aim of this study was to investigate the role of secondary free radicals and calpain, a calcium-activated cysteine protease, in the development of reperfusion injury in the heart. The time course of radical generation was assessed directly by Electron Paramagnetic Resonance (EPR) and spin trapping with N-ter butyl-alpha-phenylnitrone (PBN), in isolated perfused rat heart subjected to 30 minutes of global ischemia and 30 minutes of reperfusion. The effect of leupeptin, a calpain inhibitor, was assessed on postischemic dysfunction. The antioxidant properties of leupeptin were also investigated by using allophycocyanin, a fluorescent protein sensitive to oxidative stress generated by the H2O2 + Cu++ system. Moreover, we measured the capacities of leupeptin to scavenge hydroxyl (.OH) and superoxide (O2-.) radicals using EPR technique. Our results show that myocardial reperfusion is associated with an increase of alkyl, alkoxyl free radicals release; the administration of catalase 5.10(5) UI/L significantly reduces this release, but didn't improve the postischemic contractile function of the heart. In our study leupeptin 50 microM possess, in vitro, antioxidant properties and scavenging abilities against .OH and O2-., in return leupeptin does not influence the cardiac functions during reperfusion period. In conclusion, our results confirm that myocardial reperfusion induces an important production of secondary free radicals associated with contractile dysfunction. The role of calpain in myocardial ischemia-reperfusion injury remains to be clarified 1) by assessing the activities of calpain and calpastain, its main endogenous inhibitor, during these periods, 2) by measuring the ability of leupeptin in inhibiting the calpain dependent proteolysis.

Topics: Animals; Antioxidants; Calcium-Binding Proteins; Calpain; Catalase; Cathepsins; Cyclic N-Oxides; Cysteine Proteinase Inhibitors; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Free Radicals; Hydroxyl Radical; Leupeptins; Magnetic Resonance Spectroscopy; Male; Myocardial Contraction; Myocardial Ischemia; Myocardial Reperfusion Injury; Nitrogen Oxides; Oxidative Stress; Phycocyanin; Rats; Rats, Wistar; Spin Labels; Superoxides; Time Factors

Calpain (Ca(2+)-dependent intracellular protease)-induced proteolysis has been considered to play a role in myoblast fusion to myotubes. We found previously that calpastatin (the endogenous inhibitor of calpain) diminishes transiently during myoblast differentiation. To gain information about the regulation of calpain and calpastatin in differentiating myoblasts, we evaluated the stability and synthesis of calpain and calpastatin, and measured their mRNA levels in L8 myoblasts. We show here that mu-calpain and m-calpain are stable, long-lived proteins in both dividing and differentiating L8 myoblasts. Calpain is synthesized in differentiating myoblasts, and calpain mRNA levels do not change during differentiation. In contrast, calpastatin (though also a long-lived protein in myoblasts), is less stable in differentiating myoblasts than in the dividing cells, and its synthesis is inhibited upon initiation of differentiation. Inhibition of calpastatin synthesis is followed by a diminution in calpastatin mRNA levels. A similar calpastatin mRNA diminution is observed upon drug-induced inhibition of protein translation. On the other hand, transforming growth factor beta (which inhibits differentiation) allows calpastatin synthesis and prevents the diminution in calpastatin mRNA. The overall results suggest that at the onset of myoblast differentiation, calpastatin is regulated mainly at the level of translation and that an inhibition of calpastatin synthesis leads to the decrease in its mRNA stability. The existing calpastatin then diminishes, resulting in decreased calpastatin activity in the fusing myoblasts, allowing calpain activation and protein degradation required for fusion.

Topics: Animals; Blotting, Northern; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cell Division; Cell Line; Cycloheximide; DNA, Complementary; Immunoblotting; Myocardium; Precipitin Tests; Protein Synthesis Inhibitors; Puromycin; Rats; RNA, Messenger; Time Factors; Transforming Growth Factor beta

1. The role of the sequence 1572-1651 in the C-terminal tail of the alpha1C subunit in run-down of Ca2+ channels was studied by comparing functional properties of the conventional alpha1C,77 channel with those of three isoforms carrying alterations in this motif. 2. The pore-forming alpha1C subunits were co-expressed with alpha2delta and beta2a subunits in HEK-tsA201 cells, a subclone of the human embryonic kidney cell line, and studied by whole-cell and single-channel patch-clamp techniques. 3. Replacement of amino acids 1572-1651 in alpha1C,77 with 81 different amino acids leading to alpha1C,86 significantly altered run-down behaviour. Run-down of Ba2+ currents was rapid with alpha1C,77 channels, but was slow with alpha1C,86. 4. Transfer of the alpha1C,86 segments L (amino acids 1572-1598) or K (amino acids 1595-1652) into the alpha1C,77 channel yielded alpha1C,77L and alpha1C,77K channels, respectively, the run-down of which resembled more that of alpha1C,77. These results demonstrate that a large stretch of sequence between residues 1572 and 1652 of alpha1C,86 renders Ca2+ channels markedly resistant to run-down. 5. The protease inhibitor calpastatin added together with ATP was able to reverse the run-down of alpha1C,77 channels. Calpastatin expression was demonstrated in the HEK-tsA cells by Western blot analysis. 6. These results indicate a significant role of the C-terminal sequence 1572-1651 of the alpha1C subunit in run-down of L-type Ca2+ channels and suggest this sequence as a target site for a modulatory effect by endogenous calpastatin.

Topics: Amino Acid Sequence; Amino Acid Substitution; Barium; Blotting, Western; Calcium Channels, L-Type; Calcium-Binding Proteins; Calpain; Cell Line; Cysteine Proteinase Inhibitors; Electrophysiology; Green Fluorescent Proteins; Humans; Isomerism; Kidney; Luminescent Proteins; Molecular Sequence Data; Patch-Clamp Techniques; Plasmids

The effects of a calpain-like proteinase (CaDP) isolated from the arm muscle of Octopus vulgaris on the myofibrils and myofibrillar proteins isolated from the same tissue were examined. Our studies clearly showed that treatment of intact myofibrils with CaDP in the presence of 5 mM Ca2+ results in the degradation of the major myofibrillar proteins myosin, paramyosin, and actin. From the isolated alpha- and beta-paramyosins only beta-paramyosin is degraded by CaDP in the presence of 5 mM Ca2+ producing three groups of polypeptides of 80, 75, and 60 kDa, respectively. The degradation rate depends on the proteinase to substrate ratio, temperature, and time of proteolysis and is inhibited by the endogenous CaDP inhibitory factor (CIF), as well as by various known cysteine proteinase inhibitors (E-64, leupeptin, and antipain). From the other myofibrillar proteins examined myosin, but not actin, is degraded by CaDP; myosin heavy chain (MHC, 200 kDa) is degraded by CaDP producing four groups of polypeptides of lower molecular masses (155, 125, 115, and 102 kDa, respectively); the degradation rate depends on the incubation time and the proteinase to substrate ratio. Furthermore, CaDP undergoes limited autolysis in the presence of both the exogenous casein and the endogenous beta-paramyosin producing two large active fragments of 52 and 50.6 kDa, respectively; CIF reversibly inhibits this CaDP autolysis.

Topics: Actins; Animals; Autolysis; Calcium; Calcium-Binding Proteins; Calpain; Caseins; Enzyme Activation; Muscle Proteins; Muscles; Myofibrils; Myosin Heavy Chains; Octopodiformes; Substrate Specificity; Tropomyosin

The purpose of this report is to present a method which can be used to parameterize patterns of immunofluorescent staining in cultured neural cells. The algorithm is based on the observation that the variance in pixel intensity of the image is a power function of the magnitude of the area in immunofluorescently stained PC12 cells. This property is used to derive the fractal dimension (D) of the region of interest (ROI), and corresponds to the complexity of the pixel intensity associated with the ROI, which is analogous to a fractal surface. We show that the measure is useful in characterizing immunofluorescent staining patterns, and apply this measure to study the effects of ethanol exposure on mu-calpain and calpastatin-associated immunoreactivity. Exposure of PC12 cells to ethanol (80 mM)x48 h resulted in alterations in immunofluorescent signal (Control vs ethanol) associated with actin, calpastatin and mu-calpain: 2289+/-166 vs 1709+/-69, P<0.01; 1681+/-38 vs 2224+/-95, P<0.001; 1823+/-39 vs 2841+/-68, P<0.0001 respectively, magnitudes being pixel intensity units on a scale of 0-4095. D-values for the three proteins in the same order were: 2.32+/-0.01 vs 2.31+/-0.03, NS; 2.31+/-0.01 vs 2.32+/-0.01, NS; 2.16+/-0.03 vs 2. 24+/-0.02, P<0.01, with a possible D-value range of 2-3.

Topics: Actins; Algorithms; Animals; Calcium-Binding Proteins; Calpain; Ethanol; Fluorescent Antibody Technique; Fractals; Image Processing, Computer-Assisted; Linear Models; Microscopy, Fluorescence; Models, Biological; PC12 Cells; Rats

Many studies have demonstrated that m-calpain was implicated in cell membrane reorganization-related phenomena during fusion via a regulation by calpastatin, the specific Ca2+-dependent proteolytic inhibitor. However, the real biological role of this protease is unclear because many targeted proteins are still unknown. Using different digestion experiments we have demonstrated that desmin, vimentin, talin, and fibronectin represent very good substrates for this proteinase capable of cleaving them in fragments which are immediately degraded by other enzymatic systems. Concerning intermediate filaments, we showed that during the phenomenon of fusion, the amount of desmin was significantly reduced while the concentration of vimentin presented a steady level. On the other hand, we have conducted biological assays on cultured myoblasts supplemented by exogenous factors such as calpain inhibitors or antisense oligonucleotides capable of stimulating or inhibiting m-calpain activity. The effect of such factors on fusion and concomitantly on the targeted substrates was analyzed and quantified. When m-calpain activity and myoblast fusion were prevented by addition of calpain inhibitors entering the cells, the amounts of desmin, talin, and fibronectin were increased, whereas the amount of vimentin was unchanged. Using antisense strategy, similar results were obtained. In addition, when the phenomenon of fusion was enhanced by preventing calpastatin synthesis, the amounts of desmin, talin, and fibronectin were significantly reduced. Taken together, these results support the hypothesis that m-calpain is involved in myoblast fusion by cleaving certain proteins identified here. This cleavage could modify membrane and cytoskeleton organization for the myoblasts to fuse.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Extracts; Cell Fusion; Cells, Cultured; Cysteine Proteinase Inhibitors; Desmin; Dipeptides; Octoxynol; Oligonucleotides, Antisense; Rats; Rats, Wistar; Substrate Specificity; Vimentin

Pathological activation of the intracellular Ca2+-dependent proteases calpains may be responsible for the neuronal pathology associated with neurodegenerative diseases and acute traumas to the central nervous system. Though calpain activation has been shown definitively in traumatic brain injury (TBI), no studies have investigated calpastatin (CAST), the calpains' endogenous and specific inhibitor, after TBI. The present study examined temporal changes in CAST protein following controlled cortical impact injury in the rat. Western blot analyses of CAST in cortex and hippocampus detected two bands corresponding to molecular weights of 130 kDa [high-molecular-weight (HMW)] and 80 kDa [low-molecular-weight (LMW)]. A modest decrease in the HMW band in conjunction with a significant increase in the LMW band was observed in cortex ipsilateral to the site of impact following TBI. Examination of ipsilateral hippocampus revealed an increasing trend in the LMW band after injury, while no changes were observed in the HMW band. Thus, observable changes in CAST levels appear to occur several hours after reported calpain activation and cleavage of other substrates. In addition, a new analysis was performed on previously published data examining calpain activity in the same tissue samples used in the present study. These data suggest an association between decreases in calpain activity and accumulation of LMW CAST in the ipsilateral cortex following TBI. The present study cannot exclude proteolytic processing of CAST to LMW forms. However, the absence of reciprocity between changes in LMW and HMW bands in consistent with other data suggesting that rat brain could contain different CAST isoforms.

Topics: Animals; Blotting, Western; Brain Injuries; Calcium-Binding Proteins; Calpain; Cerebral Cortex; Cysteine Proteinase Inhibitors; Disease Models, Animal; Functional Laterality; Hippocampus; Male; Rats; Rats, Sprague-Dawley; Time Factors

The average polymorphonuclear neutrophil (PMN) lives only a day and then dies by apoptosis. We previously found that the calcium-dependent protease calpain is required for apoptosis in several mouse models of cell death. Here we identify calpain, and its endogenous inhibitor calpastatin, as regulators of human neutrophil apoptosis. Cell death triggered by the translation inhibitor cycloheximide is calpain-dependent, as evidenced using either a calpain active site inhibitor (N-acetyl-leucyl-leucyl-norleucinal) or agents that target calpain's calcium binding sites (PD150606, PD151746). No significant effect on cycloheximide-triggered apoptosis was found by using inhibitors of the proteasome or of other papain-like cysteine proteases, providing further evidence that the active site calpain inhibitor prevents apoptosis via its action on calpain. In addition, we find that potentiation of calpain activity by depleting its endogenous inhibitor, calpastatin, is sufficient to cause apoptosis of neutrophils. Nevertheless, apoptosis signalled via the Fas antigen proceeds regardless of the presence of calpain inhibitor. These experiments support a growing body of work, indicating an upstream regulatory role for calpain in many, but not all, forms of apoptotic cell death. They also identify calpastatin as a participant in apoptotic cell death and suggest that for at least one cell type, a decrease in calpastatin is a sufficient stimulus to initiate calpain-dependent apoptosis.

Topics: Acrylates; Adult; Animals; Apoptosis; Calcium-Binding Proteins; Calpain; Cell Survival; Cells, Cultured; Cycloheximide; Cysteine Proteinase Inhibitors; Humans; Kinetics; Mice; Neutrophils; Oligodeoxyribonucleotides, Antisense; Protein Biosynthesis; RNA, Messenger; Thionucleotides; Transcription, Genetic

Evidence is presented that the calcium-activated protease, calpain, is required for differentiation of 3T3-L1 preadipocytes into adipocytes induced by methylisobutylxanthine (a cAMP phosphodiesterase inhibitor), dexamethasone, and insulin. Calpain is expressed by preadipocytes and its level falls during differentiation. Exposure of preadipocytes to the calpain inhibitor N-acetyl-Leu-Leu-norleucinal or overexpression of calpastatin, a specific endogenous inhibitor of calpain, blocks expression of adipocyte-specific genes, notably the CCAAT/enhancer-binding protein (C/EBP)alpha gene, and acquisition of the adipocyte phenotype. The inhibitor disrupts the differentiation-inducing effect of methylisobutylxanthine (by means of the cAMP-signaling pathway), but is without effect on differentiation induced by dexamethasone or insulin. N-acetyl-Leu-Leu-norleucinal, or overexpression of calpastatin, inhibits reporter gene expression mediated by the C/EBPalpha gene promoter by preventing C/EBPbeta, a transcriptional activator of the C/EBPalpha gene, from binding to the promoter. These findings implicate calpain in the transcriptional activation of the C/EBPalpha gene, a process required for terminal adipocyte differentiation.

Topics: 1-Methyl-3-isobutylxanthine; 3T3 Cells; Adipocytes; Animals; Calcium-Binding Proteins; Calpain; CCAAT-Enhancer-Binding Proteins; Cell Differentiation; Dexamethasone; DNA-Binding Proteins; Insulin; Leupeptins; Mice; Nuclear Proteins; Recombinant Proteins; Transcription Factors; Transfection

In a model of cerebral hypoxia-ischemia in the immature rat, widespread brain injury is produced in the ipsilateral hemisphere, whereas the contralateral hemisphere is left undamaged. Previously, we found that calpains were equally translocated to cellular membranes (a prerequisite for protease activation) in the ipsilateral and contralateral hemispheres. However, activation, as judged by degradation of fodrin, occurred only in the ipsilateral hemisphere. In this study we demonstrate that calpastatin, the specific, endogenous inhibitor protein to calpain, is up-regulated in response to hypoxia and may be responsible for the halted calpain activation in the contralateral hemisphere. Concomitantly, extensive degradation of calpastatin occurred in the ipsilateral hemisphere, as demonstrated by the appearance of a membrane-bound 50-kDa calpastatin breakdown product. The calpastatin breakdown product accumulated in the synaptosomal fraction, displaying a peak 24 h post-insult, but was not detectable in the cytosolic fraction. The degradation of calpastatin was blocked by administration of CX295, a calpain inhibitor, indicating that calpastatin acts as a suicide substrate to calpain during hypoxia-ischemia. In summary, calpastatin was up-regulated in areas that remain undamaged and degraded in areas where excessive activation of calpains and infarction occurs.

Topics: Animals; Animals, Newborn; Brain; Brain Ischemia; Calcium-Binding Proteins; Calpain; Cell Membrane; Cysteine Proteinase Inhibitors; Dipeptides; Enzyme Activation; Female; Functional Laterality; Hypoxia; Male; Rats; Rats, Wistar; Up-Regulation

We developed a ribonuclease protection assay (RPA) to quantify the mRNA mass of calpastatin and the catalytic subunits of calpains I and II in ovine and bovine tissues. The method is based on constructing standard curves using predetermined amounts of in vitro synthesized sense cRNA of the calpain system, hybridized with excess radiolabeled antisense counterprobes. This is possible because the vectors used for riboprobe preparation can be used to transcribe the sense cRNA required to generate the standard curves to quantify absolute calpain I, calpain II, and calpastatin mRNA levels. We used the RPA to study calpain I, calpain II, and calpastatin gene expression in ovine liver, heart, and skeletal muscle. The results revealed that calpain II gene expression was similar in the three tissues. However, the expression of calpain I and calpastatin genes indicates that each tissue has its unique pattern. We also analyzed the activity of calpain I, calpain II, and calpastatin by the conventional DEAE chromatographic method for comparison. The results indicated that the RPA is more repeatable than the DEAE method. Special features of the RPA as compared with DEAE chromatography are as follows; 1) the RPA is a reliable method for quantifying the expression of calpains in all tissues because it is not affected by the presence of inhibitors or activators, 2) the RPA method can be expanded to analyze the expression of the tissue-specific calpains simply by designing specific probes for them, and 3) the RPA requires a small amount of tissue. The described method will facilitate future studies on the gene expression of calpains and will contribute to determining their physiological functions.

Topics: Animals; Base Sequence; Calcium-Binding Proteins; Calpain; Catalytic Domain; Cattle; Cysteine Proteinase Inhibitors; Liver; Molecular Sequence Data; Muscle, Skeletal; Myocardium; Ribonucleases; RNA Probes; RNA, Messenger; Sheep

Calpain, a calcium (Ca2+)-activated cysteine protease presents in several somatic mammalian cells, has been demonstrated to mediate specific Ca2+-dependent reactions including cell fusion. Because spermatozoa cells have an absolute Ca2+ requirement for penetration of oocytes, we have postulated that calpain would also be found in mammalian spermatozoa. Here we show that whole sperm homogenate and cell fractions prepared from ejaculated human spermatozoa contain calpain activity. Specific calpain inhibitors impaired this proteolytic activity. Unlike the enzyme described in somatic cells, sperm calpain was mostly particulate in nature and its activity was maximal at pH 9.0. Presence of sperm calpain was confirmed by immunoblot analysis using specific anti-calpain I and anti-calpain II antibodies. A 67 kDa calpain II protein and a 75 kDa calpain I protein were detected. Also spermatozoa contain the endogenous calpain inhibitor, calpastatin. We detected 158.8 +/- 24.5 (mean +/- SD) fmol calpastatin/mg sperm protein. Immunoblot analysis using specific antibodies showed a 68 kDa calpastatin protein located in the cytosolic fraction. This is the first demonstration that a complete calpain-calpastatin system exists in mammalian spermatozoa. Because calpain is a unique effector system for calcium-dependent processes, our data reveals a novel mechanism by which calcium exerts its regulatory functions in spermatozoa.

Topics: Calcium; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Cytosol; Humans; Hydrogen-Ion Concentration; Immunoblotting; Leucine; Male; Semen; Spermatozoa; Time Factors

Calpastatin, the natural inhibitor of calpain, is present in rat brain in multiple forms, having different molecular masses, due to the presence of one (low Mr form) or four (high Mr form) repetitive inhibitory domains. Recombinant and native calpastatin forms are substrates of protein kinase C, which phosphorylates a single serine residue at their N-terminus. Furthermore, both low and high Mr calpastatins are phosphorylated by protein kinase C at the same site. These calpastatin forms are phosphorylated also by protein kinase A, although with a lower efficiency. The incorporation of a phosphate group determines an increase in the concentration of Ca2+ required to induce the formation of the calpain-calpastatin complex. This effect results in a large decrease of the inhibitory efficiency of calpastatins. We suggest that phosphorylation of calpastatin represents a mechanism capable to balance the actual amount of active calpastatin to the level of calpain to be activated.

Topics: Animals; Brain; Calcium-Binding Proteins; Calpain; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Isoenzymes; Phosphopeptides; Phosphorylation; Protein Kinase C; Rats

A negative correlation exists between calpastatin activity and meat tenderness. Therefore, it is important to determine the mechanism of calpastatin inactivation in postmortem skeletal muscle. Western immunoblot analysis was performed to determine the protease(s) responsible for degradation of muscle calpastatin during postmortem storage. To accomplish this, purified calpastatin was digested with different proteases in vitro, and their pattern of calpastatin degradation was compared with that of calpastatin degradation in postmortem muscle. Polyclonal antibodies raised in mice against recombinant bovine skeletal muscle calpastatin were used to monitor calpastatin degradation. Lamb longissimus was stored at 4 degrees C and sampled at 0, 6, 12, 24, 72, 168, and 336 h postmortem. Postmortem storage produced a discrete pattern of calpastatin degradation products that included immunoreactive bands at approximately 100, 80, 65, 54, 32, and 29 kDa. Undegraded calpastatin (130 kDa) was barely detectable after 72 h of postmortem storage at 4 degrees C, and no immunoreactive calpastatin was observed by 336 h postmortem. For in vitro proteolysis, lamb longissimus calpastatin (0 h postmortem) was purified using Affi-Gel Blue chromatography. Calpastatin was digested with m-calpain, mu-calpain, cathepsin B, proteasome, trypsin, or chymotrypsin. Each of these enzymes degraded calpastatin. Immunoreactive fragments resulting from digestion of calpastatin with m- and mu-calpain were similar to each other and closely resembled those observed during postmortem aging of lamb longissimus at 4 degrees C. Digestion of calpastatin with mu-calpain reduced calpastatin activity. Degradation of calpastatin by other proteases resulted in unique patterns of immunoreactive fragments, distinct from that observed in longissimus. Thus, m- and(or) mu-calpain seem to be responsible for calpastatin degradation during postmortem storage of meat.

Topics: Animals; Blotting, Western; Calcium-Binding Proteins; Calpain; Cattle; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Meat; Mice; Molecular Weight; Muscles; Postmortem Changes

The present experiment was conducted to determine whether calpastatin inhibits only the rate, or both the rate and extent, of calpain-induced postmortem proteolysis. Biceps femoris from normal (n = 6) and callipyge (n = 6) lamb was stored for 56 d at 4 degrees C. Calpastatin activity was higher (P < .05) in the callipyge muscle at 0 and 14 d postmortem, but not at 56 d postmortem. The activity of mu-calpain did not differ between normal and callipyge biceps femoris at 0 and 56 d postmortem (P > .05), but was higher at 14 d postmortem in the callipyge muscle (P < 0.05). The activity of m-calpain was higher in the callipyge muscle (P < 0.05). Western blot analyses of titin, nebulin, dystrophin, myosin heavy chain, vinculin, alpha-actinin, desmin, and troponin-T indicated that postmortem proteolysis was less extensive in callipyge than in normal biceps femoris at all postmortem times. The results of this experiment indicate that calpastatin inhibits both the rate and extent of postmortem proteolysis.

Topics: Actinin; Animals; Blotting, Western; Calcium-Binding Proteins; Calpain; Connectin; Cysteine Proteinase Inhibitors; Desmin; Dystrophin; Electrophoresis, Polyacrylamide Gel; Muscle Proteins; Muscle, Skeletal; Myosin Heavy Chains; Postmortem Changes; Protein Kinases; Sheep; Troponin T; Vinculin

The purpose of the present investigation was to test three calpain inhibitors (recombinant calpastatin domain I, E64, and SJA6017) against Lp82 calpain in rat lenses. Lp82 is a lens-specific isoenzyme from the calpain super family of calcium-activated, cysteine proteases (EC 34.22.17). Lp82 and m-calpain proteolytic activities and protein levels were measured by casein zymography and immunoblotting. Activity of endogenous Lp82 against vimentin was also tested by in vitro incubation of rat lens soluble and insoluble fractions with calcium. Most of the Lp82 activity could be inhibited by irreversible inhibitor E64 and reversible inhibitor SJA6017. However, a major finding of the present investigation was that Lp82 in the soluble and insoluble fractions of the lens was less sensitive to inhibition by recombinant domain I from the endogenous tissue inhibitor of ubiquitous calpains, calpastatin, than m-calpain. By using recombinant calpastatin to inhibit endogenous lens m-calpain, we were able to demonstrate the first example of a substrate for Lp82, vimentin. These data suggest that Lp82-induced proteolysis in rodent lenses may occur even in the presence of calpastatin.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Immunoblotting; Lens, Crystalline; Rats; Rats, Sprague-Dawley; Substrate Specificity

Calpain isozymes (intracellular, Ca(2+)-dependent thiol proteases) are present in the cytoplasm of many cells, along with their endogenous specific inhibitor, calpastatin. Previously, we found that the levels of mu-calpain and m-calpain (activated by microM and mM Ca(2+), respectively) remain about the same during myoblast differentiation and fusion. By contrast, the calpastatin level, which is high during the initial stages of differentiation, diminishes markedly before myoblast fusion, allowing the proteolysis that is required for myotube formation. In the present study, we used immunoprecipitation to investigate the molecular association between calpain and calpastatin in dividing myoblasts and in the initial stages of myoblast differentiation. Immunoprecipitation (IP) was performed in two ways: (1) IP of calpain, using an anti-calpain antibody that recognized both isozymes; and (2) IP of calpastatin (using anti-calpastatin). Calpastatin was co-precipitated when calpain was immunoprecipitated; calpain was co-precipitated when calpastatin was immunoprecipitated. The results indicate that calpastatin is associated with calpain in dividing myoblasts and in myoblasts during the initial stages of differentiation, thereby preventing calpain activation at this stage. Prior studies carried out in vitro have shown a Ca(2+)-dependent interaction of calpain with calpastatin. The results described here suggest that an association between calpain and calpastatin could occur within cells in the presence of physiological Ca(2+)levels. It is proposed that the status of cellular calpain-calpastatin association is modulated by cell constituents, for which some possibilities are suggested.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Cell Line; Cysteine Proteinase Inhibitors; Microscopy, Fluorescence; Muscles; Precipitin Tests; Rats

The objective of the study was to improve the understanding of the relationship between the effect of epinephrine plus exercise and meat tenderness. The calpain, calpastatin, and cathepsin B + L activities and postmortem proteolysis in porcine longissimus muscle were studied. The muscle glycogen stores were depleted in five pigs by s.c. injection of epinephrine (.3 mg/kg) at 15 h antemortem and exercise on a treadmill (5 min, 3.8 km/h) immediately before slaughter. Antemortem injection of epinephrine and treadmill exercise resulted in higher ultimate pH (6.32 vs 5.66 in control) and decreased (P < .05) thaw loss, cooking loss, and shear force values. The muscle energy depletion treatment increased (P < .05) the muscle mu-calpain activity measured 42 min postmortem, and at 24 h mu-calpain activity was still approximately 50% greater in the high ultimate pH group. Also, as the ratio of mu-calpain to calpastatin increased (P < .01), the overall proteolytic potential of the calpain system were greater. These observations suggest that the muscle energy level may influence the activity of the calpain system in the living animal. The high ultimate pH group showed lower (P < .001) cathepsin B + L activity in the myofibrillar and the soluble fractions after 8 d of storage, suggesting that the increased ultimate pH increased the stability of the lysosomal membrane and thereby reduced the release of cathepsins from the lysosomes during storage. The SDS-PAGE showed increased (P < .001) degradation of a 39-kDa band in the epinephrine and exercise-treated samples. Degradation products at 30, 31, and 32 kDa were labeled by troponin-T antibody in western blot. An appearing 24-kDa band was identified as a troponin-I degradation product in western blot. The proteolytic degradation pattern of myofibrillar proteins during storage differed in control and treated samples, supporting the hypothesis that calpain-mediated proteolysis was affected after treatment, resulting in meat with high ultimate pH.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cathepsin B; Cathepsin L; Cathepsins; Cysteine Endopeptidases; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Energy Metabolism; Epinephrine; Female; Glycogen; Meat; Muscle, Skeletal; Physical Conditioning, Animal; Swine

Since calcium-activated neutral proteinase (calpain) activity and expression are significantly increased in activated glial/inflammatory cells in the central nervous system of animals with autoimmune demyelinating diseases, this enzyme may also play a role in peripheral organ systems in these diseases. In this study, the activity and expression of calpain and the endogenous inhibitor, calpastatin, were evaluated at transcriptional and translational levels in spleens of Lewis rats with acute experimental allergic encephalomyelitis (EAE) prior to the onset of clinical symptoms. Calpain activity and translational expression were increased by 475.5% and 44.3% respectively, on day 4 post-induction in adjuvant controls and animals with EAE. These levels remained elevated compared to normal controls on days 8 and 12. Calpastatin translational expression was similarly increased at these time points although transcriptional expression was not significantly altered at any time following induction of EAE. Likewise, transcriptional expression of mu-calpain was unchanged following induction, while small increases in m-calpain transcriptional expression were observed on days 2 and 8. Most calpain expression was observed in activated splenic macrophages at day 8 post-induction even though activated T cells were also calpain positive. In spinal cords of animals with EAE, calpain expression was significantly increased in rats with severe disease compared to those exhibiting only mild symptoms at day 12 post-induction. Thus, prior to symptomatic EAE, increased calpain activity and expression in peripheral lymphoid organs may play an important role in T cell migration and subsequent disease progression.

Topics: Adjuvants, Immunologic; Animals; Autoimmune Diseases; Calcium-Binding Proteins; Calpain; Encephalomyelitis, Autoimmune, Experimental; Enzyme Induction; Lymphocyte Activation; Lymphocytes; Macrophage Activation; Macrophages; Male; Protein Biosynthesis; Rats; Rats, Inbred Lew; Spinal Cord; Spleen; Time Factors; Transcription, Genetic

In autoimmune demyelinating diseases such as multiple sclerosis (MS), the degradation of myelin proteins results in destabilization of the myelin sheath. Thus, proteases have been implicated in myelin protein degradation, and recent studies have demonstrated increased expression and activity of a calcium-activated neutral proteinase (calpain) in experimental allergic encephalomyelitis, the corresponding animal model of MS. In the present study, calpain activity and expression (at translational and transcriptional levels) were evaluated in white matter from human patients with MS and Parkinson's and Alzheimer's diseases and compared with that of white matter from normal controls. Western blot analysis revealed that levels of the active form of calpain and calpain-specific degradation products (fodrin) were increased by 90.1% and 52.7%, respectively, in MS plaques compared with normal white matter. Calpain translational expression was up-regulated by 462.5% in MS plaques compared with controls, although levels of the specific endogenous inhibitor, calpastatin, were not altered significantly. At the transcriptional level, no significant changes in calpain or calpastatin expression were detected by reverse transcription-PCR. Using double immunofluorescent labeling, increased calpain expression was observed in reactive astrocytes, activated T cells, and activated mononuclear phagocytes in and adjacent to demyelinating lesions. Calpain activity and translational expression were not increased significantly in white matter from patients with Parkinson's or Alzheimer's diseases compared with that of normal controls. Because calpain degrades all major myelin proteins, the increased activity and expression of this proteinase may play a critical role in myelinolysis in autoimmune demyelinating diseases such as MS.

Topics: Calcium; Calcium-Binding Proteins; Calpain; Demyelinating Diseases; Fluorescent Antibody Technique; Humans; Multiple Sclerosis; Protein Biosynthesis; Transcription, Genetic

Localization of the two main components of the Ca(2+)-dependent proteolytic system has been investigated in human neuroblastoma LAN-5 cells. Using a monoclonal antibody which recognizes the N-terminal calpastatin domain, it has been shown that this inhibitory protein is almost completely confined in two granule-like structures not surrounded by membranes. Similar calpastatin distribution has been found in other human and in murine cell types, indicating that aggregation of calpastatin is a general property and not an exclusive characteristic of neuronal-like cells. The existence of such calpastatin aggregates is confirmed by the kinetics of calpastatin-activity release during rat liver homogenization, which does not correspond to the rate of appearance of cytosolic proteins or to the disruption of membrane-surrounded organelles. Calpastatin distribution is affected by the intracellular increase in free Ca(2+), which results in calpastatin progressively becoming a soluble protein. However, calpain is distributed in the soluble cell fraction and, in activating conditions, partially accumulates on the plasma membrane. Similar behaviour has been observed in calpastatin localization in LAN-5 cells induced with retinoic acid, suggesting that the proteolytic system is activated during the differentiation process of these cells. The involvement of calpastatin in controlling calpain activity, rather than its activation process, and the utilization of changes in calpastatin localization as a marker of activation of the system is discussed.

Topics: Animals; Calcimycin; Calcium; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cell Membrane; Cell Membrane Permeability; Cytosol; Diffusion; Endopeptidases; Enzyme Activation; Humans; Liver; Mice; Neurons; Rats; Solubility; Tretinoin; Tumor Cells, Cultured

To improve our understanding of the regulation of mu-calpain activity in situ during postmortem storage of muscle, the effect of different calpastatin levels on proteolysis of myofibrillar proteins by mu-calpain in a system closely mimicking postmortem conditions was studied. Increasing the amount of calpastatin in the incubations limited both the rate and extent of proteolysis of myofibrillar proteins and autolysis of mu-calpain. Excess calpastatin (i.e., a mu-calpain:calpastatin ratio of 1:4) did not inhibit proteolysis completely. Western blot analysis revealed that proteolysis of myofibrillar proteins virtually ceased after 7 d of incubation, despite the presence of partly autolyzed, therefore seemingly active, mu-calpain. A series of incubations of autolyzed mu-calpain revealed that the autolyzed form of this enzyme is unstable at an ionic strength observed in postmortem muscle. The possible significance of these results in terms of the regulation of mu-calpain activity in postmortem muscle is discussed.

Topics: Animals; Blotting, Western; Calcium-Binding Proteins; Calpain; Cattle; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Isoenzymes; Muscle Proteins; Myofibrils; Postmortem Changes

p107 protein, a member of the retinoblastoma family protein, suppresses growth promotion in cancer cells. We have already reported evidence that calpain, a calcium dependent protease is involved in the cleavage of p107 protein. We show here that p107 protein can also be a substrate for ubiquitination. A negative growth regulator, the HMG-CoA reductase inhibitor lovastatin was found to induce loss of p107 protein which was reversible by a specific protease inhibitor lactacystin as well as calpain inhibitor. Following treatment with lovastatin higher molecular weight ubiquitinated forms of p107 were detected by anti-p107 immunoprecipitation and anti-ubiquitin Western blotting. These forms further increased when lactacystin was added to culture medium. These results indicate that ubiquitin-proteasome pathway plays a potential role in the degradation as well as calpain. The data presented here suggest a model in which calpain and ubiquitin-proteasome system possibly play a cooperative role in targeting the protein under certain conditions.

Topics: Acetylcysteine; Antineoplastic Agents; Blotting, Western; Calcium-Binding Proteins; Calpain; CDC2-CDC28 Kinases; Cell Cycle; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Cysteine Proteinase Inhibitors; Humans; Lovastatin; Male; Nuclear Proteins; Proliferating Cell Nuclear Antigen; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Retinoblastoma Protein; Retinoblastoma-Like Protein p107; Sarcoma, Ewing; Transfection; Tumor Cells, Cultured; Ubiquitins

Previous studies have indicated that the Ca(2+)-dependent protease, calpain, is activated in platelets within 30-60 s of thrombin stimulation, but specific roles of calpain in platelets remain to be identified. To directly test the functions of calpain during platelet activation, a novel strategy was developed for introducing calpain's specific biological inhibitor, calpastatin, into platelets prior to activation. This method involves treatment of platelets with a fusion peptide, calpastat, consisting of the cell-penetrating signal sequence from Kaposi's fibroblast growth factor connected to a calpain-inhibiting consensus sequence derived from calpastatin. Calpastat specifically inhibits thrombin peptide (SFLLR)-induced alpha-granule secretion (IC(50) = 20 microM) during the first 30 s of activation, thrombin-induced platelet aggregation (IC(50) = 50 microM), and platelet spreading on glass surfaces (IC(50) = 34 microM). Calpastat-Ala, a mutant peptide in which alanine is substituted at conserved calpastatin residues, lacks calpain inhibitory activity and fails to inhibit secretion, aggregation, or spreading. The peptidyl calpain inhibitors calpeptin, MDL 28,170 (MDL) and E64d also inhibit secretion, aggregation and spreading, but require 3-10-fold higher concentrations than calpastat for biological activity. Together, these findings demonstrate that calpain regulates platelet secretion, aggregation, and spreading and indicate that calpain plays an earlier role in platelet activation following thrombin receptor stimulation than had been previously detected.

Topics: Amino Acid Sequence; Blood Platelets; Calcium-Binding Proteins; Calpain; Humans; Molecular Sequence Data; Mutation; Peptide Fragments; Platelet Adhesiveness; Platelet Aggregation

Our previous reports revealed that calpain has proteoglycanase activity and exists in synovial fluid in osteoarthritis and rheumatoid arthritis. We examined the effects of cytokines on expression of the calpain-calpastatin system in fibroblastic synoviocytes (FLS). Primary cultures of human FLS from osteoarthritis (OA) and rheumatoid arthritis (RA) patients were stimulated with inflammatory cytokines and the amounts of m-calpain and calpastatin mRNAs expressed were determined by Northern blotting. Northern blots were subjected to computerized densitometer and band intensities were determined. Interleukin-1 (IL-1) down-regulated m-calpain and tissue-type calpastatin mRNA expression in OA and RA FLS. In RA FLS, although IL-6 did not alter m-calpain mRNA expression, IL-1 + tumor necrosis factor (TNF) and IL-1 + transforming growth factor (TGF) down-regulated m-calpain mRNA expression. These results provide new information about the effects of inflammatory cytokines on calpain and calpastatin system in OA and RA pathology.

Topics: Arthritis, Rheumatoid; Calcium-Binding Proteins; Calpain; Cells, Cultured; Cytokines; Down-Regulation; Gene Expression Regulation, Enzymologic; Humans; Interleukin-1; Kinetics; Osteoarthritis; RNA, Messenger; Synovial Fluid; Transforming Growth Factors; Tumor Necrosis Factor-alpha

Stepwise and continuous gradient ion-exchange chromatography were compared for yield of calpains and calpastatin from ovine muscle in a study designed to quantify their activities for comparative purposes. In Exp. 1, a continuous (25 to 400 mM NaCl) gradient and a two-step gradient method (200 mM NaCl to coelute mu-calpain and calpastatin together and then 400 mM NaCl to elute mu-calpain) were compared. For the two-step method, mu-calpain activities were determined by subtracting calpastatin activities before and after heat inactivation of mu-calpain. Both the two-step and the continuous gradient method yielded similar results over a broad range of activities. The stepwise gradient method does not require the use of fraction collectors and pumps, and it can be completed in a fraction of the time required for the continuous gradient method. In Exp. 2, the two-step method was compared with a three-step method (100 mM NaCl to elute calpastatin, then 200 mM NaCl to elute mu-calpain, and then 400 mM NaCl to elute m-calpain). Unlike the continuous gradient method, calpastatin and mu-calpain could not be completely separated using the three-step chromatography method. Thus, the three-step gradient method should not be used to quantify the components of the calpain proteolytic system. The present results indicate that the two-step gradient method is a fast and inexpensive method to determine calpain and calpastatin activities in studies designed to quantify the components of the calpain proteolytic system in skeletal muscle.

Topics: Animals; Calcium-Binding Proteins; Calpain; Chromatography, Ion Exchange; Isoenzymes; Muscles; Sheep

Topics: Animals; Animals, Newborn; Calcium-Binding Proteins; Calpain; Hypoxia-Ischemia, Brain; Rats; Up-Regulation

We have found previously that run-down of cardiac Ca2+ channels in cell-free patches is reversed by cytoplasm plus adenosine triphosphate (ATP). Characterization of the factor in cytoplasm revealed that it is likely to be calpastatin (CS), an endogenous inhibitor of calpain (Ca2+-activated neutral protease). We therefore investigated the possible restoring effect of CS obtained from various tissues (activity 1.3-23 U/ml) on Ca2+ channel activity after run-down in inside-out patches. Although CS from porcine erythrocytes (plus 3 mM ATP) had only a minimal effect in restoring channel activity (to 4% of the control level recorded before the run-down), CS from porcine heart restored channel activity to 19% of control. The product of recombinant complementary deoxyribonucleic acid (cDNA) of human heart CS, a membrane-bound CS partially purified from bovine heart and CS from rabbit skeletal muscle (Sigma) restored channel activity to 28%, 23% and 10% of control levels, respectively. These results suggest that tissue-type CS, but not erythrocyte-type (truncated) CS, seems to have an effect on the cardiac Ca2+ channel to maintain its activity. Purified CS had relatively small effects compared to that of crude cytoplasm, implying that some other factor(s) might contribute also to the regulation of Ca2+ channel activity.

Topics: Adenosine Triphosphate; Animals; Calcium Channels; Calcium-Binding Proteins; Calpain; Cattle; Cytoplasm; Erythrocytes; Guinea Pigs; Heart Ventricles; Humans; Molecular Weight; Muscle, Skeletal; Myocardium; Organ Specificity; Patch-Clamp Techniques; Rabbits; Recombinant Proteins; Species Specificity; Swine

The calcium-activated neutral proteases (CANP, calpains) have been implicated in both acute and chronic neurodegenerative processes. In the present study, we analyzed the in situ mRNA expression of calpain I and II and their endogenous inhibitor, calpastatin, in the motor neuron degeneration (Mnd) mutant mouse, which exhibits progressive dysfunction of the spinal cord and brain. As the disease progresses, the mutants show increasingly pronounced motor abnormalities which coincide with swelling of the spinal motor neurons, neocortex, hippocampal CA regions and cerebellar Purkinje cells. In situ hybridization studies show that the Mnd mice have a significantly higher level of calpain I, calpain II and calpastatin than the congenic controls in the following brain regions and cell types: hippocampal CA3 region, pyramidal cells, cerebellar Purkinje cells and spinal cord motor neurons. However, no differences in calpain or calpastatin mRNA levels are observed in glial and cerebellar granule cells of Mnd and control mice. Western blots and competitive RT-PCR analyses of brain and spinal cord homogenates are confirmative. Such altered gene expression in specific cell types of brain and spinal cord suggests the involvement of the calpain/calpastatin system.

Topics: Animals; Brain; Calcium-Binding Proteins; Calpain; DNA Primers; Female; Gene Expression Regulation; In Situ Hybridization; Male; Mice; Mice, Mutant Strains; Motor Neurons; Nerve Degeneration; Neuroglia; Neurons; Organ Specificity; Polymerase Chain Reaction; Purkinje Cells; Spinal Cord

Effects of ischemia on the content of a ulinastatin (UT)-like substance in the murine cerebral cortex and hippocampus were studied. At 24 h post-ischemia, a significant (p < 0.05) decrease in the content of UT-like substance in the hippocampus but not the cerebral cortex and a concurrent increase in the activity of micro-calpain were observed. In in vitro experiments, a decrease was registered in the content of UT-like substance in the hippocampus in the presence of calcium. This decrease was inhibited by both EDTA and calpastatin treatments. These results implicate the destruction of UT-like substance by micro-calpain in the ischemic hippocampus.

Topics: Animals; Calcium Chloride; Calcium-Binding Proteins; Calpain; Cerebral Cortex; Edetic Acid; Glycoproteins; Hippocampus; Ischemic Attack, Transient; Kinetics; Male; Mice; Mice, Inbred ICR; Trypsin Inhibitors

A study was conducted to examine the effects of bird age and muscle tissue type on calpain and calpastatin activities in turkey skeletal muscle. Enzymatic activities of calpains and calpastatin were found to vary with bird age and muscle type. Breast muscle from younger birds (age 5 wk) had higher mu-calpain, m-calpain, and calpastatin activities (P < 0.05) than breast muscle from older birds (9, 13, and 17 wk of age). Thigh muscle calpain activities were not affected by bird age, but thigh calpastatin activity was found to increase with age, with muscle from 17-wk-old birds having 35% higher activity than muscle from 13-wk-old birds. When extracted from 9-wk-old turkeys, breast muscle mu-calpain activity was 30% higher than thigh muscle mu-calpain. By 13 wk of age, breast muscle mu-calpain activity was 20% less than thigh mu-calpain. Thigh muscle m-calpain and calpastatin activities were found to be significantly higher (P < 0.05) than that found in breast muscle, with some values more than double in older birds (17 wk of age).

Topics: Aging; Animals; Body Weight; Calcium-Binding Proteins; Calpain; Cohort Studies; Hydrogen-Ion Concentration; Muscle, Skeletal; Time Factors; Turkeys

Calpastatin, an endogeneous inhibitor protein acting on calpain (Ca(2+)-dependent cysteine proteinase), is widely distributed in animal tissues and cells. Two different expression systems, baculovirus-infected Spodoptera frugiperda (Sf9) insect cells and Escherichia coli, were used for overexpression of the human calpastatin tagged with N-terminal hexahistidine peptide. Recombinant calpastatin was purified to homogeneity by nickel ion affinity chromatography and gel filtration separation. Purified recombinant proteins from both systems have similar inhibitory activity for calpain.

Topics: Amino Acid Sequence; Animals; Baculoviridae; Base Sequence; Calcium-Binding Proteins; Calpain; Cell Line; Cloning, Molecular; Cysteine Proteinase Inhibitors; Escherichia coli; Genetic Vectors; Histidine; Humans; Molecular Sequence Data; Recombinant Fusion Proteins; Spodoptera

Vascular smooth muscle cell (VSMC) proliferation still remains a poorly understood process, although it is believed to play a critical role in pathological states, including atherosclerosis and hypertension. Several reports have suggested that proteases may be directly involved in this process; however, it was still unclear which protease is responsible for VSMC proliferation. In this study, by use of a cell-permeable calpain inhibitor (calpeptin; benzyloxycarbonyl-Leu-nLeu-H), its analogue (benzyloxycarbonyl-Leu-Met-H), the cell-impermeable serine protease inhibitor leupeptin, and antisense oligonucleotide against m-calpain to inhibit proliferation of primarily cultured human VSMCs, we investigated whether calcium-activated neutral protease (calpain) is involved in VSMC proliferation. Calpeptin and its analogue, more specific for m-calpain, equally inhibited the proliferation of VSMCs in a dose-related manner, whereas a more limited antiproliferative effect was observed in leupeptin-treated VSMCs. Antisense oligonucleotide against m-calpain, but not scrambled antisense, dose-dependently inhibited m-calpain expression and proliferation of VSMCs. Maximal inhibition was an approximately 50% reduction of cell number and m-calpain antigen observed at 50 micromol/L of antisense oligonucleotide. Calpeptin or antisense oligonucleotide against m-calpain increased the expression of the endogenous calpain substrate pp125FAK (focal adhesion kinase), whereas the expression of the endogenous calpain inhibitor calpastatin was not affected. These results suggest that the proliferation of VSMCs requires protease activity, some of which is due to m-calpain.

Topics: Calcium-Binding Proteins; Calpain; Cell Adhesion Molecules; Cell Division; Dipeptides; DNA; Flow Cytometry; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Isomerism; Muscle, Smooth, Vascular; Oligonucleotides, Antisense; Protease Inhibitors; Protein-Tyrosine Kinases

Calpain (Ca(2+)-activated cysteine protease) induced proteolysis has been suggested to play a role in myoblast fusion. We previously found that calpastatin (the endogenous inhibitor of calpain) diminishes markedly in myoblasts during myoblast differentiation just prior to the start of fusion, allowing Ca(2+)-induced calpain activation at that stage. Here, we show that a limited degradation of some proteins occurs within the myoblasts undergoing fusion, but not in proliferating myoblasts. The protein degradation is observed at the stage when calpastatin is low. Protein degradation within the myoblasts and myoblast fusion are inhibited by EGTA, by the cysteine protease inhibitors calpeptin and E-64d and by calpastatin. The degradation appears to be selective for certain myoblast proteins. Integrin beta 1 subunit, talin and beta-tropomyosin are degraded in the fusing myoblasts, whereas alpha-actinin, beta-tubulin and alpha-tropomyosin are not. A similar pattern of degradation is observed in lysates of proliferating myoblasts when Ca2+ and excess calpain are added, a degradation that is inhibited by calpastatin. The results support the notion that degradation of certain proteins is required for myoblast fusion and that calpain participates in the fusion-associated protein degradation. Participation of calpain is made possible by a change in calpain/calpastatin ratio, i.e., by a diminution in calpastatin level from a high level in the proliferating myoblasts to a low level in the differentiating myoblasts. Degradation of certain proteins, known to be responsible for the stability of the membrane-skeleton organization and for the interaction of the cell with the extracellular matrix, would allow destabilization of the membrane and the creation of membrane fusion-potent regions.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cell Division; Cell Fusion; Cell Line; Cysteine Proteinase Inhibitors; Egtazic Acid; Enzyme Activation; Muscle, Skeletal; Rats; Tropomyosin

Previous studies suggest that the Ca2+-dependent proteases, calpains, participate in remodeling of the actin cytoskeleton during wound healing and are active during cell migration. To directly test the role that calpains play in cell spreading, several NIH-3T3- derived clonal cell lines were isolated that overexpress the biological inhibitor of calpains, calpastatin. These cells stably overexpress calpastatin two- to eightfold relative to controls and differ from both parental and control cell lines in morphology, spreading, cytoskeletal structure, and biochemical characteristics. Morphologic characteristics of the mutant cells include failure to extend lamellipodia, as well as abnormal filopodia, extensions, and retractions. Whereas wild-type cells extend lamellae within 30 min after plating, all of the calpastatin-overexpressing cell lines fail to spread and assemble actin-rich processes. The cells genetically altered to overexpress calpastatin display decreased calpain activity as measured in situ or in vitro. The ERM protein ezrin, but not radixin or moesin, is markedly increased due to calpain inhibition. To confirm that inhibition of calpain activity is related to the defect in spreading, pharmacological inhibitors of calpain were also analyzed. The cell permeant inhibitors calpeptin and MDL 28, 170 cause immediate inhibition of spreading. Failure of the intimately related processes of filopodia formation and lamellar extension indicate that calpain is intimately involved in actin remodeling and cell spreading.

Topics: 3T3 Cells; Actins; Animals; Calcium-Binding Proteins; Calpain; Cell Movement; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Down-Regulation; Humans; Mice; Phosphoproteins; Substrate Specificity; Up-Regulation

Previous studies have demonstrated a role for m-calpain in myoblast fusion. Moreover, the presence, in differentiated cells, of a highly specific endogenous inhibitor of calpain, calpastatin, has led to the hypothesis that a regulation of or a protection against m-calpain activity by calpastatin could also occur during the earlier stages of muscle cell differentiation. In order to verify this hypothesis, we have investigated, in myoblast culture, the appearance of calpastatin-mRNA and its corresponding protein. Our results provide evidence that calpastatin is already present at the earlier stages of myoblast differentiation and that a significant decrease of the levels of calpastatin mRNA and its protein precedes myoblast fusion. In addition, the induction of an artificial decrease in calpastatin level, via an appropriate antisense oligodeoxyribonucleotide methodology, leads to earlier and faster myoblast fusion. Together with previous studies, these results indicate that m-calpain and calpastatin are functionally involved in myoblast fusion. Our findings also demonstrate that an acute "hyperactivity" of m-calpain resulting from the decrease of calpastatin synthesis is necessary during the early stages of this step of differentiation.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Fusion; Cells, Cultured; Muscle, Skeletal; Oligonucleotides, Antisense; Rats; Rats, Wistar; RNA, Messenger

Recombinant human mu-calpain whose active site Cys-115 was substituted with Ser was expressed in insect cells using baculovirus system. The mutant mu-calpain, purified using an affinity-column of calpastatin oligopeptides, had no proteolytic activities of autolysis and caseinolysis. The large subunit of the mutant mu-calpain was processed from the 80 kDa form to the 76 kDa form by the wild type calpain, supporting the intermolecular cleavage mechanism of procalpain during activation. Fluorescence polarization analysis revealed that the mutant mu-calpain retained high affinity toward fluorescein-labeled calpastatin domain 1. Fragmentation of the full-length calpastatin by the wild type calpain was enhanced by pre-incubating the inhibitor with the mutant calpain. The recombinant mutant calpain was suggested to retain the integrity of the high ordered structure of the wild type calpain.

Topics: Animals; Autolysis; Baculoviridae; Binding Sites; Calcium; Calcium-Binding Proteins; Calpain; Caseins; Chromatography, Affinity; Cysteine Proteinase Inhibitors; Enzyme Precursors; Humans; Mutation; Protein Binding; Protein Processing, Post-Translational; Recombinant Proteins; Spodoptera

The degradation of myelin proteins has been implicated in destabilization of the myelin sheath in autoimmune demyelinating diseases such as multiple sclerosis (MS). In order to investigate the role of calcium-activated neutral proteinase (calpain), which degrades myelin proteins, the activity and expression (translational and transcriptional) of this enzyme were examined in spinal cords of Lewis rats with experimental allergic encephalomyelitis (EAE), an animal model of MS. In addition to calpain, the translational expression of calpastatin (endogenous inhibitor of calpain) and extent of neurofilament (NFP) and myelin protein degradation were evaluated via Western blotting in controls and rats with EAE. The transcriptional expression of millicalpain, microcalpain, and calpastatin as examined by RT-PCR was not significantly increased in EAE. However, calpain translational expression was increased by 206. 5% while the levels of 68 kDa NFP and myelin-associated glycoprotein were decreased by 42.9 and 39.7%, respectively, in animals with EAE compared to controls. Calpastatin isoforms (180, 110, 80, and 68 kDa) were significantly increased in EAE as well. The findings of increased activity and translational expression of calpain in EAE suggest a major role for this enzyme in myelinolysis associated with autoimmune demyelinating diseases.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Encephalomyelitis, Autoimmune, Experimental; Gene Expression Regulation, Enzymologic; Male; Molecular Weight; Myelin Sheath; Neurofilament Proteins; Polymerase Chain Reaction; Protein Biosynthesis; Rats; Rats, Inbred Lew; Transcription, Genetic; Up-Regulation

Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the attachment of myristate onto the amino terminal glycine residue of select polypeptides. Cardiac tissue expresses high levels of cAMP-dependent protein kinase whose catalytic subunit is myristoylated; however, cardiac muscle extracts were found to contain low NMT activities. Northern blot analysis of bovine heart poly(A)+ RNA probed with bovine spleen NMT cDNA revealed a 1.7-kb mRNA. Western blot analysis of cardiac muscle extracts with human NMT antibody indicated a prominent immunoreactive band with a molecular mass of 50 kDa. The expression of mRNA and protein levels in cardiac muscle is not correlated with NMT activities, suggesting the presence of regulators of the enzyme activity. We have isolated the cDNA encoding bovine cardiac muscle NMT (cNMT) by reverse transcription polymerase chain reaction. The single long open reading frame of 1248 bp of bovine cNMT specifies a protein of 416 amino acids with a predicted mass of 46,686 Da. The cDNA clone expressed in Escherichia coli resulted in the production of functionally active 50-kDa NMT. Ultrastructural and immunolocalization of NMT utilizing the immunogold labeling technique demonstrated cytoplasmic distribution with occasional mitochondrial and myofilaments localization of the NMT antibody. Cardiac muscle NMT has a higher affinity for myristoyl-CoA than toward palmitoyl-CoA. Substrate specificity indicated that cNMT has a higher affinity toward pp60src and M2 gene segment of reovirus type 3-derived peptide substrates than toward cAMP-dependent protein kinase-derived peptide. Primary translational product of cNMT sequence contained several regions rich in proline, glutamic acid, serine, and threonine, which are known as "PEST" regions. PEST-FIND analysis of the amino acid sequences indicated eight PEST regions were present in the cNMT. These PEST regions are suggested to be recognized by specific proteases, particularly Ca(2+)-dependent neutral proteases, calpains, which are responsible for the degradation of PEST-containing proteins. We have demonstrated the abolishment of NMT activity and NMT protein degradation in vitro by m-calpain. The proteolysis of cNMT by m-calpain and the abolishment of NMT activity was prevented by the calpain inhibitor, calpastatin. These observations indicate that calpains may regulate NMT activity.

Topics: Acyltransferases; Amino Acid Sequence; Animals; Base Sequence; Calcium-Binding Proteins; Calpain; Cattle; Cloning, Molecular; Cysteine Proteinase Inhibitors; DNA, Complementary; Gene Expression; Hydrolysis; Immunohistochemistry; Kinetics; Microscopy, Electron; Molecular Sequence Data; Myocardium; RNA, Messenger; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Spleen

Calpains, Ca(2+)-dependent neutral proteinases (microM and mM Ca(2+)-sensitive), and their endogenous inhibitor calpastatin were examined in rat brain. Specific activity of m-calpain exceeded almost 10 times that of mu-calpain, and the both isoforms of calpain together with calpastatin were mainly located in the soluble fraction of homogenate. Acute postdecapitative ischemia of 15 min duration resulted in a gradual, time-dependent decrease of total mu-calpain activity (to 60% of control values) and in the moderate elevation of calpastatin activity (by 28%). The decrease of total mu-calpain activity coincided with its remarkable increase (above 300% of control values) in particulate fraction. In the case of m-calpain, the only observed effect of ischemia was its redistribution and, as a consequence, the elevation of activity in particulate fraction. The accumulation of breakdown products, resulting from calpain-catalyzed proteolysis of fodrin (as revealed by Western blotting) indicated activation of calpain under ischemia. The findings suggest that this rapid activation involves partial enzyme translocation toward membranes, and is followed (at least in acute phase) by mu-calpain downregulation and increased calpastatin activity.

Topics: Animals; Blotting, Western; Brain; Brain Ischemia; Calcium-Binding Proteins; Calpain; Carrier Proteins; Down-Regulation; Male; Membrane Proteins; Microfilament Proteins; Rats; Rats, Wistar

Four recombinant calpastatin forms, deduced from rat brain mRNAs and differing in the number of inhibitory repetitive domains from zero to four, were expressed and characterized for their inhibitory efficiency on mu- and m-calpain. Although the most effective one is a truncated calpastatin form composed of the N-terminal region (domain L) and a single inhibitory domain, all inhibitors are more active against mu-calpain, but are preferentially degraded and inactivated by m-calpain. The protein form composed exclusively of a domain L is deprived of any inhibitory activity but prevents inhibition of calpain by the other calpastatin forms, indicating that this calpastatin region could be relevant in the recognition of the proteinase. A calpastatin form having molecular properties similar to those of the recombinant truncated calpastatin, has also been found in rat brain. It does not derive from proteolysis of a higher molecular mass precursor. The expression of multiple calpastatin forms may be relevant for the specific modulation of the different calpain isozymes normally present in a single cell type.

Topics: Animals; Brain; Calcium-Binding Proteins; Calpain; Cloning, Molecular; Enzyme Inhibitors; Escherichia coli; Rats; Recombinant Fusion Proteins; RNA, Messenger

Global brain ischemia and reperfusion result in the degradation of the eukaryotic initiation factor (eIF) 4G, which plays a critical role in the attachment of the mRNA to the ribosome. Because eIF-4G is a substrate of calpain, these studies were undertaken to examine whether calpain I activation during global brain ischemia contributes to the degradation of eIF-4G in vivo. Immunoblots with antibodies against calpain I and eIF-4G were prepared from rat brain postmitochondrial supernatant incubated at 37 degrees C with and without the addition of calcium and the calpain inhibitors calpastatin or MDL-28,170. Addition of calcium alone resulted in calpain I activation (as measured by autolysis of the 80-kDa subunit) and degradation of eIF-4G; this effect was blocked by either 1 micromol/L calpastatin or 10 micromol/L MDL-28,170. In rabbits subjected to 20 minutes of cardiac arrest, immunoblots of brain postmitochondrial supernatants showed that the percentage of autolyzed calpain I increased from 1.9% +/- 1.1% to 15.8% +/- 5.0% and that this was accompanied by a 68% loss of eIF-4G. MDL-28,170 pretreatment (30 mg/kg) decreased ischemia-induced calpain I autolysis 40% and almost completely blocked eIF-4G degradation. We conclude that calpain I degrades eIF-4G during global brain ischemia.

Topics: Animals; Brain; Calcium; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Eukaryotic Initiation Factor-4G; Female; Ischemic Attack, Transient; Kinetics; Male; Peptide Initiation Factors; Rabbits; Rats; Reperfusion; Subcellular Fractions

Two cysteine protease families (caspase and calpain) participate in apoptosis. Here we report that the endogenous calpain inhibitor calpastatin is fragmented by caspase(s) to various extents during early apoptosis in two cell types. In anti-fas or staurosporine-treated Jurkat T-cells, the high-molecular-weight form (HMW) of calpastatin (apparent Mr 110 K) was extensively degraded to immunoreactive fragments of Mr 75 K and 30 K In apoptotic SH-SY5Y human neuroblastoma cells, HMW calpastatin was degraded to a major immunoreactive fragment of 75 K. In both cell types, fragmentation of HMW calpastatin was blocked by a caspase-specific inhibitor carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene. In vitro translated HMW calpastatin was sensitive to proteolysis by recombinant caspase-1, -3, and -7. By contrast, in vitro translated LMW calpastatin (which lacks domains L and I) was cleaved into multiple fragments only by caspase-1 and was relatively resistant to caspase-3, -7, and other caspases tested. Consistently with that, purified erythroid LMW calpastatin was also highly susceptible to caspase-1 digestion. Recombinant human calpastatin spanning domain I through III (CAST(DI-III)) was found cleaved by caspase-1 at at least three sites, located in either the A or the C helix of domains I and III (ALDD137*L, LSSD203*F and ALAD404*S), while only a single site (ALDD137*L) was cleaved by caspase-3. These findings suggest that both HMW and LMW calpastatins are more vulnerable to caspase-1 than to caspase-3. Surprisingly, both erythroid LMW calpastatin and recombinant CAST(DI-III) fragmented by caspase-1 suffered only a less than twofold reduction of inhibitory activity toward calpain. We propose that the proteolysis of calpastatin in early apoptosis might have yet unidentified effects on the cross-talk between the two protease systems.

Topics: Apoptosis; Binding Sites; Calcium-Binding Proteins; Calpain; Caspase 1; Caspase 3; Caspases; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Humans; Hydrolysis; Jurkat Cells; Molecular Weight; Neuroblastoma; Recombinant Proteins; Tumor Cells, Cultured

Calcium-activated neutral proteinase (calpain) is a ubiquitous, cytosolic endopeptidase which is believed to play a role in many neural functions. In the present study, we examined the transcriptional and translational expression of microcalpain (microcalpain) and millicalpain (mcalpain) isoforms and the endogenous inhibitor calpastatin in rat and bovine spinal cord, brain stem, cerebellum, and cerebral cortex tissues using reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. In rat central nervous system (CNS) samples, the microcalpain and mcalpain transcriptional expression was highest in white matter-enriched areas. Calpastatin mRNA expression demonstrated no significant differences among the CNS areas. Calpain and calpastatin translational expression levels were greatest in the spinal cord. In bovine CNS, microcalpain transcriptional expression was greatest in the spinal cord, while other CNS regions showed no significant differences. Bovine mcalpain transcriptional expression was similar among various CNS regions but marginally greater in the cortex. Translational expression of bovine calpain was greatest in the brain stem, while that of calpastatin was highest in the cerebral cortex. These results indicate that calpain expression varies among different CNS regions and is often highest in white matter-enriched areas.

Topics: Animals; Brain; Brain Stem; Calcium-Binding Proteins; Calpain; Cattle; Enzyme Precursors; Gene Expression Regulation; Organ Specificity; Protein Biosynthesis; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spinal Cord; Transcription, Genetic

Rat brain contains a calpain activator specific for the mu-form of the proteinase. We now report that this protein factor binds to the catalytic 80 kDa calpain subunit, promoting the dissociation of the heterodimer structure of the proteinase. The successive steps of the activation process, namely the two autoproteolytic steps producing the 78 kDa and the 75 kDa calpain forms, result in a 100 times faster rate. The activator competes with calpastatin and associates with the inner surface of plasma membranes. Based on its properties, the calpain activator can be visualised as the molecule indicating the sites for calpain activation at which the proteinase can also elude the negative control exerted by calpastatin.

Topics: Animals; Binding Sites; Binding, Competitive; Brain; Calcium; Calcium-Binding Proteins; Calpain; Cell Membrane; Cysteine Proteinase Inhibitors; Enzyme Activation; Erythrocyte Membrane; Humans; In Vitro Techniques; Molecular Weight; Protein Conformation; Rats

The calpains (E.C. 3.4.22.17) and calpastatin constitute an ubiquitous, intracellular, Ca2+-dependent protease/inhibitor system. This system has been implicated as a principal regulator of myofibrillar protein degradation in both ante-mortem and postmortem muscle. Although proteolytic activity of the calpains is primarily controlled through interaction of calpain and calpastatin, evidence for an activator(s) has been limited and the reported characteristics varied. The function of the activator has not been elucidated. A putative calpain activator has been isolated from the Pectoralis muscle of broiler breeders (Cobb x Cobb). The activator elutes from an ion-exchange column at approximately 200 mM NaCl. Addition of activator increased apparent m-calpain activity to a level demonstrating a fourfold increase in proteolysis. The activator/calpain complex maintains a requirement for Ca2+ for proteolytic activity. Under physiological conditions, presence of the activator negates the ability of calpastatin to inhibit m-calpain. Additionally, the activator alone does not demonstrate proteolytic activity. Effect of the activator is pH-dependent; in a physiological pH range, the activator enhances m-calpain proteolytic activity but at pH less than 6.75 the effect is to inhibit m-calpain. The activator's ability to modulate m-calpain activity and eliminate calpastatin's effect provides a further means of regulating this important enzyme system.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Chickens; Chromatography, Ion Exchange; Cysteine Proteinase Inhibitors; Enzyme Activation; Female; Hydrogen-Ion Concentration; Molecular Weight; Muscle Proteins; Muscle, Skeletal

Topics: Animals; Calcium-Binding Proteins; Calpain; Gene Expression Regulation; Muscle Fibers, Fast-Twitch; Muscle Fibers, Slow-Twitch; Muscle, Skeletal; Organ Specificity; Swine

Changes in activity and protein status of micro-calpain, m-calpain, and calpastatin in bovine semimembranosus muscle during the first 7d of postmortem storage were monitored by using assays of proteolytic activity, SDS-polyacrylamide gel electrophoresis, and Western blot analysis. Extractable m-calpain activity changed slightly during the first 7d after death (decreased to 63% of at-death activity after 7d), whereas extractable calpastatin activity decreased substantially (to 60% of at-death activity after 1d and to 30% of at-death activity after 7d of postmortem storage) during this period. Extractable micro-calpain activity also decreased rapidly (to 20% of at-death activity at 1d and to less than 4% of its at-death activity at 7d after death) during postmortem storage. Western blot analysis showed that the 80-kDa subunit of m-calpain remained undegraded during the first 7d after death but that the 125- to 130-kDa calpastatin polypeptide was gone entirely at 7d after death. Hence, the calpastatin activity remaining at 7d originates from calpastatin polypeptides that are 42 kDa or smaller. The 80-kDa micro-calpain subunit was almost entirely in the 76-kDa autolyzed form at 7d after death; this form is proteolytically active in in vitro systems, and it is unclear why the postmortem, autolyzed micro-calpain is not active. Over 50% of total muscle micro-calpain is tightly bound to myofibrils 7d after death; this micro-calpain is also nearly inactive proteolytically. Unless postmortem muscle contains some factor that enables micro-calpain in this muscle to be proteolytically active, it is not clear whether micro-calpain could be responsible for any appreciable postmortem myofibrillar proteolysis.

Topics: Animals; Blotting, Western; Calcium-Binding Proteins; Calpain; Caseins; Cattle; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Female; Food Preservation; Hydrogen-Ion Concentration; Male; Meat; Muscle Proteins; Muscle, Skeletal; Myofibrils; Postmortem Changes

To detect immunoglobulin isotype-specific autoantibodies to native human calpastatin in patients with rheumatic diseases, we performed immunoblot analysis using the heated HeLa cell extracts to enrich heat-resistant calpastatin. The calpastatin molecule that was apparently migrated to 110 kD by SDS-PAGE was confirmed to react with monoclonal anti-human calpastatin antibody in immunoblotting. IgG antibodies to calpastatin were detected in 22 of 48 sera (46%) from patients with RA, whereas only 20% (5/25), 11% (2/19) and 13% (2/15) of sera from SLE, SSc and PM/DM had IgG anti-calpastatin antibodies, respectively. IgM antibodies were also found in 40% (19/48) of RA and 12% (3/25) of SLE patients but not detected in sera from patients with other rheumatic diseases. IgA antibodies were found in only one RA and one SLE serum. In RA, 7 of 48 sera (15%) had IgM antibodies alone, but all SLE sera with IgM antibodies had IgG antibodies. Thus, anti-calpastatin autoantibodies were detected by using the native human calpastatin. Although these autoantibodies were found in patients with various rheumatic diseases, they were present in RA patients at the highest frequency. In particular, the presence of IgM antibodies appeared to be more specific in RA patients.

Topics: Arthritis, Rheumatoid; Autoantibodies; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; HeLa Cells; Hot Temperature; Humans; Immunoblotting; Immunoglobulin Isotypes; Tissue Extracts

Since myelin proteins are degraded in autoimmune demyelinating diseases such as optic neuritis, proteinases are believed to participate in myelinolysis. Calpain (calcium activated neutral proteinase) degrades myelin proteins at physiological pH and is found in glial and inflammatory cells involved in demyelination. To examine the putative role of calpain in myelinolysis, the activity and expression (translational and transcriptional) of this enzyme and endogenous inhibitor, calpastatin were examined in optic nerves of Lewis rats with experimental allergic encephalomyelitis (EAE), an animal model of optic neuritis. Calpain activity was examined via Western blotting by measuring the extent of myelin protein degradation and calpain-specific fodrin proteolysis in optic nerves from controls versus rats with experimental optic neuritis. RT-PCR studies demonstrated no significant change in millicalpain, microcalpain, or calpastatin expression at the mRNA level in optic nerves from animals with experimental optic neuritis compared to controls. However, myelin associated glycoprotein (MAG) levels were decreased by 25.5% while calpain translational expression and calpain-autolyzed fodrin levels were increased by 72.1% and 462.8% respectively, in experimental optic neuritis compared to controls. Translational expression of calpastatin isoforms (80, 68 and 55 KD) was not significantly different in rats with experimental optic neuritis compared to controls. Thus, increased activity and translational expression of calpain in experimental optic neuritis suggests this proteinase may participate in the degradation of myelin and cytoskeletal proteins in demyelinating diseases such as optic neuritis.

Topics: Animals; Autoimmune Diseases; Blotting, Western; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Encephalomyelitis, Autoimmune, Experimental; Male; Myelin Proteins; Optic Neuritis; Protein Biosynthesis; Rats; Rats, Inbred Lew; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic

Previous studies have shown that transcription of the gene encoding bovine calpastatin, an inhibitor of the calcium-activated cysteine protease calpain, is upregulated following activation of cAMP-dependent signaling pathways. In this study, deletion and site-directed mutagenesis experiments were performed to identify cis elements conferring cAMP responsiveness. Heterologous promoter assays demonstrated that all cAMP-responsive cis elements were located within -102 nucleotides (nt) of transcription initiation. Deletion of an element (GTCA) at nt +13 that is identical to half of the palindromic cAMP-responsive element (TGACGTCA) identified in other cAMP-responsive gene promoters had no effect on the response of the calpastatin promoter to dibutyryl-cAMP, although a 67% reduction in basal promoter activity was observed. In contrast, two point mutations in a cis element at nt -76 (GTCA to aTCt) abolished cAMP responsiveness. These results demonstrate that the calpastatin promoter sequence between nt -1653 and +130 contains a single cAMP-responsive element (GTCA) located at nt -76, and suggest a direct molecular pathway by which activation of cAMP signaling could lead to increased calpastatin gene transcription and reduction in calpain-mediated proteolysis.

Topics: 3T3 Cells; Animals; Base Sequence; Bucladesine; Calcium-Binding Proteins; Calpain; Cattle; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Mice; Molecular Sequence Data; Promoter Regions, Genetic; Transcription, Genetic; Transfection

Calpain activity is thought to be essential for the execution of apoptotic cell death in certain experimental models. In the present study, the physiological inhibitor of calpain, calpastatin, was found to be cleaved in three different apoptotic systems. The 110-120 kDa calpastatin protein of Jurkat T-lymphocytes and U937 monocytic leukemia cells was cleaved to a 65-70 kDa form after the induction of apoptosis with anti-CD95 monoclonal antibody, staurosporine or TNF. Cleavage of calpastatin in apoptotic cells occurred simultaneously with the cleavage of the DNA repair enzyme, poly(ADP-ribose) polymerase. The caspase inhibitors VAD-cmk and IETD-fmk prevented calpastatin cleavage in all three systems. Calpain inhibitor I, however, suppressed calpastatin cleavage only during TNF-induced apoptosis. Other protease inhibitors, such as lactacystin and pepstatin A, did not confer any significant protection against apoptotic calpastatin cleavage. The results from in vitro incubations with cell lysates and purified enzymes showed that calpain I, calpain II and recombinant caspase-3, all cleaved calpastatin, with varying efficiency. In conclusion, the results of the present study suggest that caspases may cleave calpastatin and thus, regulate calpain activity during apoptotic cell death.

Topics: Acetylcysteine; Apoptosis; Calcium-Binding Proteins; Calpain; Caspase 3; Caspases; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; fas Receptor; Flow Cytometry; Humans; Jurkat Cells; Pepstatins; Protease Inhibitors; Staurosporine; Tumor Necrosis Factor-alpha; U937 Cells

A rapid stepwise measurement for the activities of calpastatin and mu- and m-calpains was developed by using 2-stage elution at pH 8.5 and then 7.0. The activities of calpastatin, mu-calpain and m-calpain can be rapidly assayed following the separation on DEAE-Sephacel chromatography by a 2 stage elution with 90 mM NaCl (pH 8.5), and then by 200 and 300 mM NaCl in elution buffer (pH 7.0). No significant differences in the recovery of these proteinases and inhibitor was observed between stepwise gradient and linear gradient methods.

Topics: Buffers; Calcium-Binding Proteins; Calpain; Chromatography, Ion Exchange; Cysteine Proteinase Inhibitors; Hydrogen-Ion Concentration; Isoenzymes

A simplified methodology for assaying the caseinolysis by calpains was developed. This methodology, including the incubation of calpain with casein and direct measurement of the absorbance at 500 nm, is based on the turbidity of the reaction mixture caused by the aggregation of hydrolysates during the reaction. Unlike the typical caseinolysis assay, this novel assay does not need to separate the substrate from hydrolysates and can be continuously monitored in visible wavelength range. The activity of calpain is expressed by the maximum reaction velocity (delta A500/min) at 25 degrees C).

Topics: Animals; Calcium-Binding Proteins; Calpain; Caseins; Cattle; Enzyme Activation; Fluorescamine; Isoenzymes; Kinetics; Rosaniline Dyes; Solutions; Spectrophotometry, Atomic; Trichloroacetic Acid

The effect of protein depletion and refeeding with a normal diet on calpain activity was examined in mouse kidney soluble homogenate. In terms of units per gram of protein, it increased 2.9 times with depletion and decreased upon refeeding. After a DEAE-Sephacel chromatography, the homogenate yielded three enzymatic activities. Their sum, assessed as total calpain activity, was higher than the activity measured before fractionation and did not appreciably change during protein depletion and refeeding. Because the proportion of total activity displayed by the complete homogenate increased with depletion and decreased with refeeding, a low calpastatin content in depleted kidney was envisaged. This was confirmed by direct estimations: depleted kidney had 6 times less calpastatin compared to both normal and 16 h refed tissue. We concluded that a decrease in calpastatin content contributes to an increased calpain activity related to degradable protein in protein depleted kidney. In view of this, it seems not unlikely that the in vivo rate of protein breakdown depicted by kidney during protein depletion and refeeding is in part effected through modulation of the calpain proteolytic system.

Topics: Animals; Calcium-Binding Proteins; Calpain; Female; Kidney; Mice; Nutritional Physiological Phenomena; Proteins

Myocardial stunning is characterized by decreased myofilament Ca2+ responsiveness. To investigate the molecular basis of stunned myocardium, we performed PAGE and Western immunoblot analysis of the contractile proteins. Isolated rat hearts were retrogradely perfused at 37 degrees C for either 50 minutes (control group) or for 10 minutes, followed by 20-minute global ischemia and 20-minute reperfusion (stunned group), or for 20-minute ischemia without reflow. Another group consisted of hearts subjected to 20-minute ischemia in which stunning was mitigated by 10-minute reperfusion with low Ca2+/low pH solution. Myocardial tissue samples subjected to PAGE revealed no obvious differences among groups. Western immunoblots for actin, tropomyosin, troponin C, troponin T, myosin light chain-1, and myosin light chain-2 showed highly selective recognition of the appropriate full-length molecular weight bands in all groups. Troponin I (TnI) Western blots revealed an additional band (approximately 26 kD, compared with 32 kD for the full-length protein) in stunned myocardial samples only. In parallel experiments, skinned trabeculae were treated with calpain I for 20 minutes; Western blots showed a TnI degradation pattern similar to that observed in stunned myocardium. Such TnI degradation was prevented by calpastatin, a naturally occurring calpain inhibitor. The results show that (1) TnI is partially and selectively degraded in stunned myocardium; (2) this degradation could be prevented by low Ca2+/low pH reperfusion, which also prevented the contractile dysfunction of stunning; and (3) calpain I could similarly degrade TnI, supporting the idea that Ca(2+)-dependent myofilament proteolysis underlies myocardial stunning.

Topics: Animals; Blotting, Western; Calcium; Calcium-Binding Proteins; Calpain; Female; Heart; In Vitro Techniques; Male; Myocardial Reperfusion; Myocardial Reperfusion Injury; Myocardial Stunning; Rats; Rats, Sprague-Dawley; Troponin I; Ventricular Pressure

A crude fraction that contains ubiquitin-protein ligases contains also a proteolytic activity of approximately 100 kDa that cleaves p53 to several fragments. The protease does not require ATP and is inhibited in the crude extract by an endogenous approximately 250 kDa inhibitor. The proteinase can be inhibited by chelating the Ca2+ ions, by specific cysteine proteinase inhibitors and by peptide aldehyde derivatives that inhibit calpains. Purified calpain demonstrates an identical activity that can be inhibited by calpastatin, the specific protein inhibitor of the enzyme. Thus, it appears that the activity we have identified in the extract is catalyzed by calpain. The calpain in the extract degrades also N-myc, c-Fos and c-Jun, but not lysozyme. In crude extract, the calpain activity can be demonstrated only when the molar ratio of the calpain exceeds that of its native inhibitor. Recent experimental evidence implicates both the ubiquitin proteasome pathway and calpain in the degradation of the tumor suppressor, and it was proposed that the two pathways may play a role in targeting the protein under various conditions. The potential role of the two systems in this important metabolic process is discussed.

Topics: Adenosine Triphosphate; Calcium-Binding Proteins; Calpain; Chromatography, Gel; Cysteine Proteinase Inhibitors; Humans; Hydrolysis; Signal Transduction; Tumor Suppressor Protein p53

This experiment was conducted to investigate the effects of feeding a protein-free diet on mRNA levels of the calpain system in skeletal muscle of growing pigs during a 15-d feeding trial. Twenty crossbred barrows were divided into two dietary treatments: control or protein-free diet (mean initial weight for both groups: 38.3 kg). Daily diets were provided at 2.5 times energy for maintenance (twice a day). On d 0, 3, and 14, biopsies were taken from longissimus muscle between the third and fourth ribs (d 0 and 3) and between the fourth and fifth rib (d 14). On d 15, animals were slaughtered and longissimus muscles were dissected and analyzed for calpastatin, and mu- and m-calpain activity. From biopsies, mRNA level of skeletal muscle calpain, mu- and m-calpain, and calpastatin were measured using reversed transcription PCR. Subsequently, PCR products were quantified using ELISA. Feeding the protein-free diet lowered growth rate to almost zero. Only total level of mRNA of mu-calpain on d 14 was influenced by dietary treatments, being lower for the protein-free group than for the control group (P < .05). However, proteolytic activities were not different between treatments. Total RNA concentration in longissimus muscle decreased during the experiment for both treatments, but on d 14 this was more pronounced for the protein-free than for the control group (P < .05). If mRNA levels were corrected for this change, specific mRNA level on d 14 of skeletal muscle calpain and mu-calpain were lower (P < .05) for the protein-free than for the control group. These data suggest that activity of the components of the calpain system are differentially regulated.

Topics: Aging; Animals; Base Sequence; Biopsy; Body Weight; Calcium-Binding Proteins; Calpain; Diet; Diet, Protein-Restricted; DNA Primers; DNA Probes; DNA, Complementary; Eating; Enzyme-Linked Immunosorbent Assay; Male; Muscle, Skeletal; Polymerase Chain Reaction; RNA, Messenger; Swine; Weight Gain

Two groups of Brangus steers produced by nuclear transplantation cloning were used in parallel studies investigating the impact of calf- and yearling-feeding. The first group (n = 8) were fed as calves (CF; n = 4) or yearlings (YF; n = 4) to a constant age end point of 16 mo. The second group (n = 10) were fed as calves (CF; n = 5) or yearlings (YF; n = 5) to a constant live weight end point (530 kg). When slaughtered at the same age, CF and YF steers did not differ (P > .05) in feedlot ADG, but the CF steers were heavier and had higher dressing percentages, numeric yield grades, and quality grades (P < .05). Top loin steaks from the groups of steers did not differ (P > .05) in palatability traits. When fed to a constant live weight, the YF steers gained more rapidly (P < .05) and had lower (P < .05) numeric yield grades than did CF steers. Again CF steers had higher (P < .05) dressing percentages. There was no difference (P > .05) between the treatments in carcass quality grade or meat palatability characteristics. Thus, when finished to a constant weight end point, YF steers gained more rapidly, with no adverse effects on carcass quality grade or palatability traits; however, CF steers consistently produced higher dressing percentages, largely due to greater external fatness.

Topics: Aging; Animals; Birth Weight; Body Composition; Calcium-Binding Proteins; Calpain; Cattle; Cloning, Molecular; Diet; Food Technology; Male; Meat; Muscle, Skeletal

p53 is a short-lived transcription factor that is frequently mutated in tumor cells. Work by several laboratories has already shown that the ubiquitin-proteasome pathway can largely account for p53 destruction, at least under specific experimental conditions. We report here that, in vitro, wild-type p53 is a sensitive substrate for milli- and microcalpain, which are abundant and ubiquitous cytoplasmic proteases. Degradation was dependent on p53 protein conformation. Mutants of p53 with altered tertiary structure displayed a wide range of susceptibility to calpains, some of them being largely resistant to degradation and others being more sensitive. This result suggests that the different mutants tested here adopt slightly different conformations to which calpains are sensitive but that cannot be discriminated by using monoclonal antibodies such as PAb1620 and PAb240. Inhibition of calpains by using the physiological inhibitor calpastatin leads to an elevation of p53 steady-state levels in cells expressing wild-type p53. Conversely, activation of calpains by calcium ionophore led to a reduction of p53 in mammalian cells, and the effect was blocked by cell-permeant calpain inhibitors. Cotransfection of p53-null cell lines with p53 and calpastatin expression vectors resulted in an increase in p53-dependent transcription activity. Taken together, these data support the idea that calpains may also contribute to the regulation of wild-type p53 protein levels in vivo.

Topics: Animals; Calcimycin; Calcium-Binding Proteins; Calpain; Cattle; Cricetinae; Cysteine Endopeptidases; Humans; Jurkat Cells; Mice; Multienzyme Complexes; Mutagenesis; Proteasome Endopeptidase Complex; Protein Conformation; Transcription, Genetic; Transfection; Tumor Suppressor Protein p53; Ubiquitins

Proteolysis at neutral pH in the soluble fraction of cultured pig thyroid epithelial cells was examined using a synthetic calpain substrate, succinyl-Leu-Tyr-7-amino-4-methylcoumarin. The Ca2+-independent proteolytic activity was largely inhibited by substances known to affect cysteine- and metalloproteases, whereas no or little effects were obtained with inhibitors affecting serine- and aspartic proteases. Addition of Ca2+ did not significantly alter the rate of substrate degradation. Biochemical separation via hydrophobic interaction chromatography and Western blotting demonstrated the presence of both m-calpain (40% of total calpain) and mu-calpain (60%) in confluent thyrocytes. Determination of calpastatin activity indicated a 30 times higher level of the inhibitor as compared to total calpain activity. Western blotting showed the presence of a 110 kD calpastatin form with additional low mol wt forms possibly representing fragmentation products. In immunofluorescent stainings, m-calpain had a diffuse cytoplasmic distribution whereas mu-calpain was located both in the cytoplasm and at the cell-cell contacts. Calpastatin immunoreactivity was mainly granular and located close to the nucleus, although a fibrillar distribution was also observed. The results show the presence of all components of the calpain/calpastatin system and indicate a strict control of calpain activity in cultured thyrocytes. The different subcellular distributions of calpains and calpastatin suggests that they are compartmentalized and require mobilization to interact.

Topics: Animals; Blotting, Western; Calcium-Binding Proteins; Calpain; Cells, Cultured; Cysteine Proteinase Inhibitors; Cytoplasm; Cytosol; Epithelium; Fluorescent Antibody Technique; Glycoproteins; Peptide Hydrolases; Swine; Thyroid Gland

Calpains have importance in human neurodegenerative disease pathogenesis, but these mechanisms are difficult to study in postmortem tissues. To establish a cellular model of the human calpain and calpastatin system, we characterized calpain I, calpain II, and calpastatin in SH-SY5Y human neuroblastoma cells in relation to their counterparts in human brain and investigated their expression and activity after inducing cellular differentiation with retinoic acid (RA), a physiological effector of normal brain development. Calpain I in both SH-SY5Y cells and human brain existed in the cytosolic and particulate fractions as three isoforms (80, 78, and 76 kDa) and exhibited atypical isoelectric focusing behavior. Calpain II in SH-SY5Y cells, as in human brain, migrated as a single predominantly cytosolic 76-kDa protein with an isoelectric point ranging from 5.9 to 6.3. Calpastatin from both sources was also 90% cytosolic. In the cells it was composed of four discrete bands, ranging in molecular weight from 110 to 127 kDa. Levels of activated (76 and 78 kDa) and precursor (80 kDa) calpain I isoforms rose 54% (P < 0.0001) in the particulate fraction and 26% (P < 0.0001) in the soluble fraction after 3 days of RA exposure. Because levels and activity of calpastatin remain unchanged during the first 7 days of RA exposure, the increased abundance of calpain I implies a net activation of the calpain system during differentiation. Calpain I activation may contribute to the remodeling of cell shape and neurite extension/retraction associated with neuronal differentiation.

Topics: Brain; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cysteine Proteinase Inhibitors; Humans; Isomerism; Molecular Weight; Neurites; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

Both silica and lipopolysaccharide (LPS) induce a rapid degradation of I kappa B alpha, an intracellular inhibitor of the nuclear factor (NF)-kappa B transcription factor. In this report, we demonstrate that MG132, a relatively specific proteasome inhibitor, is capable of suppressing LPS-induced I kappa B alpha degradation and NF-kappa B activation in mouse macrophage line RAW 264.7 cells, but is unable to influence the same induction produced by silica. In contrast, the lysosome inhibitor chloroquine has little effect on I kappa B alpha degradation induced by either silica or LPS. In fact, chloroquine enhances the signal-induced nuclear expression of NF-kappa B p50/p65 heterodimer by inhibiting the resynthesis of I kappa B alpha. With the use of transient transfection of a plasmid that expresses calpastatin, a natural inhibitor for calpain, the silica-induced degradation of I kappa B alpha and NF-kappa B activation was attenuated. In contrast, no inhibition of LPS-induced I kappa B alpha degradation and NF-kappa B activation was observed by the overexpression of calpastatin. This suggests that calpain contributes to silica-induced I kappa B alpha degradation and NF-kappa B activation but not to LPS-induced I kappa B alpha degradation and NF-kappa B activation.

Topics: Animals; Blotting, Western; Calcium-Binding Proteins; Calpain; Cell Extracts; Cell Line; Chloroquine; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; DNA, Complementary; I-kappa B Proteins; Leupeptins; Lipopolysaccharides; Macrophages; Mice; NF-kappa B; NF-KappaB Inhibitor alpha; Silicon Dioxide; Transfection

The calpain-calpastatin system, which consists of calpains I and II (two ubiquitously distributed calcium-activated papain-like cysteine proteases), as well as calpastatin (the endogenous calpain inhibitor), plays an important role in cell proliferation and differentiation in many tissues. However, its contribution to the regulation of osteoprogenitor or pluripotent stem cell proliferation and differentiation into osteoblasts remains poorly defined. In these studies, rat pluripotent mesodermal cells (ROB-C26) and mouse MC3T3-E1 preosteoblasts were induced to differentiate into osteoblasts by long-term culture or in response to bone morphogenetic protein (BMP). The occurrence and distribution of calpain-calpastatin system proteins were determined by immunofluorescent microscopy, measurement of calcium-dependent proteolytic activity, and Western blotting. Treatment of intact MC3T3-E1 cells with an irreversible, membrane-permeable cysteine protease inhibitor attenuated proliferation and alkaline phosphatase upregulation under differentiation-enhancing conditions. Calpain II activity increased during differentiation of MC3T3-E1 cells in postconfluent culture. When ROB-C26 cells were maintained in long-term culture, neutral protease, calpain I, and calpain II activities increased 2- to 3-fold in the absence of BMP. In the presence of partially purified native BMP, neutral protease and calpain I activities also increased similarly, but calpain II activity increased by 10-fold in 3 days. The maximal increase in alkaline phosphatase occurred 4 to 11 days after the calpain II activity had peaked. Induction of differentiation in long-term MC3T3-E1 cultures was associated with higher calpain II and 70- and 110-kDa calpastatin protein levels and lower 17-kDa calpastatin degradation product levels. In conclusion, cysteine protease activity is essential for preosteoblastic proliferation and differentiation. The calpain-calpastatin system is regulated during osteoprogenitor proliferation and differentiation, as it is in other cells, and bone morphogenetic protein is a specific regulator of calpain II.

Topics: Alkaline Phosphatase; Animals; Bone Morphogenetic Proteins; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cell Division; Cell Line; Cysteine Proteinase Inhibitors; Mice; Mice, Inbred C57BL; Osteoblasts; Rats; Skull; Time Factors

Cerebral deposition of amyloid beta-protein (Abeta) as senile plaques is a pathological hallmark of Alzheimer's disease (AD). Abeta falls into two major subspecies defined by their C-termini, Abeta40 and Abeta42, ending in Val-40 and Ala-42, respectively. Although Abeta42 accounts for only approximately 10% of secreted Abeta, Abeta42 is the predominant species accumulated in senile plaques in AD brain and appears to be the initially deposited species. Its secretion level has recently been reported to be increased in the plasma or culture media of fibroblasts from patients affected by any of early-onset familial AD (FAD). Thus, inhibition of Abeta42 production would be one of the therapeutic targets for AD. However, there is little information about the cleavage mechanism via which Abeta40 and Abeta42 are generated and its relationship to intracellular protease activity. Here, we examined by well-characterized enzyme immunoassay the effects of calpain and proteasome inhibitors on the levels of Abeta40 and Abeta42 secretion by cultured cells. A calpastatin peptide homologous to the inhibitory domain of calpastatin, an endogenous calpain specific inhibitor, induced a specific increase in secreted Abeta42 relative to the total secreted Abeta level, a characteristic of the cultured cells transfected with FAD-linked mutated genes, while a proteasome specific inhibitor, lactacystin, showed no such effect. These findings suggest that the Abeta42 secretion ratio is modulated by the calpain-calpastatin system and may point to the possibility of exploring particular compounds that inhibit Abeta42 secretion through this pathway.

Topics: Acetylcysteine; Alzheimer Disease; Amyloid beta-Peptides; Calcimycin; Calcium-Binding Proteins; Calpain; Cell Line; Cysteine Proteinase Inhibitors; Dipeptides; Embryo, Mammalian; Humans; Kidney; Peptide Fragments; Transfection

The differentiation of keratinocytes involves numerous steps including the formation of the cornified envelope and the aggregation of keratin filaments by filaggrin monomer molecules. In this study, we investigated whether mu-calpain is involved in the processing of profilaggrin to filaggrin monomers by using both an active mu-calpain specific antibody and a 27-mer synthetic calpastatin peptide, a cell-permeable calpain-specific inhibitor. Upon incubation of cultured keratinocytes with Ca2+ for 96 hours, active mu-calpain with a molecular mass of 76 kDa appeared preceded by an increase in mu-calpain mRNA. In synchrony with the appearance of active mu-calpain, the processing of profilaggrin occurred. Furthermore, the Ca2+-induced activation of mu-calpain and the processing of profilaggrin were affected by the addition of the synthetic calpastatin inhibitor. These results indicate that the activation of mu-calpain plays a major role in the profilaggrin processing.

Topics: Amino Acid Sequence; Calcium; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cells, Cultured; Cysteine Proteinase Inhibitors; Cytosol; Filaggrin Proteins; Humans; Infant, Newborn; Intermediate Filament Proteins; Keratinocytes; Kinetics; Male; Molecular Sequence Data; Peptide Fragments; Protein Precursors; Protein Processing, Post-Translational; Skin

The presence of the calpain-calpastatin system in human umbilical vein endothelial cells (HUVEC) was investigated by means of ion exchange chromatography, Western blot analysis, and Northern blot analysis. On DEAE anion exchange chromatography, calpain and calpastatin activities were eluted at approximately 0.30 M and 0.15-0.25 M NaCl, respectively. For half-maximal activity, the protease required 800 microM Ca2+, comparable to the Ca2+ requirement of m-calpain. By Western blot analysis, the large subunit of mu-calpain (80 kDa) was found to be eluted with calpastatin (110 kDa). Both the large subunit of m-calpain (80 kDa) and calpastatin were detected in the respective active fractions. By Northern blot analysis, mRNAs for large subunits of mu- and m-calpains were detected in single bands, each corresponding to approximately 3.5 Kb. Calpastatin mRNA was observed in two bands corresponding to approximately 3.8 and 2.6 Kb. Furthermore, the activation of mu-calpain in HUVEC by a calcium ionophore was examined, using an antibody specifically recognizing an autolytic intermediate form of mu-calpain large subunit (78 kDa). Both talin and filamin of HUVEC were proteolyzed in a calcium-dependent manner, and the reactions were inhibited by calpeptin, a cell-permeable calpain specific inhibitor. Proteolysis of the cytoskeleton was preceded by the appearance of the autolytic intermediate form of mu-calpain, while the fully autolyzed postautolysis form of mu-calpain (76 kDa) remained below detectable levels at all time points examined. These results indicate that the calpain-calpastatin system is present in human endothelial cells and that mu-calpain may be involved in endothelial cell function mediated by Ca2+ via the limited proteolysis of various proteins.

Topics: Antibody Specificity; Blotting, Western; Calcimycin; Calcium; Calcium-Binding Proteins; Calpain; Cells, Cultured; Chromatography, DEAE-Cellulose; Cytoskeletal Proteins; Dipeptides; Endothelium, Vascular; Enzyme Activation; Enzyme Precursors; Extracellular Space; Humans; Hydrolysis; RNA, Messenger; Umbilical Veins

Topics: Adult; Biomarkers; Calcium-Binding Proteins; Calpain; Female; Hippocampus; Humans; Male; Middle Aged; Neural Pathways; Obsessive-Compulsive Disorder; Reference Values; Temporal Lobe

In this study, we examined calcium-mediated degradation of a neurofilament protein (NFP), and autolytic activation of calpain in Lewis rat optic nerve in vitro. After incubation with calcium, homogenized optic nerve samples were analysed by SDS-PAGE in association with ECL immunoblot techniques. 68 kD NFP, calpain, and calpastatin antibodies were used for identification of the respective proteins. The extent of calcium-mediated 68 kD NFP degradation compared to EGTA controls, served to quantify calpain activity, while the extent of calpain autolysis measured the activation of the enzyme. A progressive loss of 68 kD NFP was observed at 15 min (42.1%), 1 hr (52.7%) and 6 hr (73.4%) incubation periods compared to EGTA controls. The immunoreactive calpain bands showed progressive autolysis after 15 min (26.6%), 1 hr (31.4%) and 6 hr (43.4%) incubations. We also found degradation of low molecular weight isoforms of calpastatin (43 kD and 27 kD) in the presence of calcium compared to controls. These results indicate that calpain is present in optic nerve in its inactive form but when calcium is added, it undergoes autolysis and becomes active. Thus, active calpain is capable of degrading endogenous substrates (e.g. cytoskeletal and myelin proteins) and may promote the degeneration of optic nerve in optic neuritis.

Topics: Animals; Blotting, Western; Calcium; Calcium-Binding Proteins; Calpain; Enzyme Activation; Enzyme Precursors; Neurofilament Proteins; Optic Nerve; Rats; Rats, Inbred Lew

Calpains, the thiol proteinases of the calcium-dependent proteolytic system, are regulated by a natural inhibitor, calpastatin, which is present in brain tissue in two forms. Although both calpastatins are highly active on human erythrocyte calpain, only one form shows a high inhibitory efficiency with both rat brain calpain isozymes. The second calpastatin form is almost completely inactive against homologous proteinases and can be converted into an active one by exposure to a phosphoprotein phosphatase, also isolated from rat brain. Phosphorylation of the active calpastatin by protein kinase C and protein kinase A promotes a decrease in its inhibitory efficiency. The interconversion between the two inhibitor forms seems involved in the adjustment of the level of intracellular calpastatin activity on specific cell requirements.

Topics: Animals; Brain; Calcium-Binding Proteins; Calpain; Phosphoprotein Phosphatases; Phosphorylation; Protein Processing, Post-Translational; Rats

Parathyroid hormone (PTH) activates calpains I and II (calcium-activated papain-like proteases) and stimulates the synthesis and secretion of cathepsin B (a lysosomal cysteine protease) in osteoblastic cells. Anabolic doses of PTH also stimulate osteoprogenitor cell proliferation and differentiation into mature, fully functional osteoblasts capable of elaborating bone matrix, whereas catabolic doses of PTH stimulate calcium mobilization and matrix turnover. Previous investigations in other cell types have demonstrated that calcium-activated calpains play a major role in regulating proliferation and differentiation by catalyzing limited regulatory proteolysis of nuclear proteins, transcription factors, and enzymes. We tested the hypothesis that inhibition of intracellular cysteine proteases such as the calpains will ablate PTH-mediated osteoblast proliferation and differentiation, two fundamental indices of bone anabolism. A brief preincubation with the membrane-permeable, irreversible cysteine protease inhibitor E64d (10 micrograms/mL) before short-term PTH treatment blunted PTH-induced cell proliferation in subconfluent cultures and also attenuated proliferation and inhibited differentiation in longer-term confluent cultures. This confirms the hypothesis that cysteine proteases such as the calpains are important in mediating the proliferative and prodifferentiating or anabolic effects of PTH on MC3T3-E1 cells in culture. Immunofluorescent localization demonstrated that calpain I, calpain II, and calpastatin (the endogenous calpain inhibitor) are abundant and widely distributed within actively proliferating MC3T3-E1 preosteoblasts. Since the calpains are active and stable at neutral intracellular pH levels in osteoblasts, whereas cathepsins are not, our results support a role for these calcium-activated regulatory proteases in mediating the anabolic effects of PTH in bone.

Topics: Alkaline Phosphatase; Animals; Calcium-Binding Proteins; Calpain; Cell Count; Cell Division; Cell Line; Cell Membrane Permeability; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Leucine; Mice; Osteoblasts; Parathyroid Hormone; Rats; Tumor Cells, Cultured

Myoblast differentiation and fusion to multinucleated muscle cells can be studied in myoblasts grown in culture. Calpain (Ca(2+)-activated thiol protease) induced proteolysis has been suggested to play a role in myoblast fusion. We previously showed that calpastatin (the endogenous inhibitor of calpain) plays a role in cell membrane fusion. Using the red cell as a model, we found that red cell fusion required calpain activation and that fusibility depended on the ratio of cell calpain to calpastatin. We found recently that calpastatin diminishes markedly in myoblasts during myoblast differentiation just prior to the start of fusion, allowing calpain activation at that stage; calpastatin reappears at a later stage (myotube formation). In the present study, the myoblast fusion inhibitors TGF-beta, EGTA and calpeptin (an inhibitor of cysteine proteases) were used to probe the relation of calpastatin to myoblast fusion. Rat L8 myoblasts were induced to differentiate and fuse in serum-poor medium containing insulin. TGF-beta and EGTA prevented the diminution of calpastatin. Calpeptin inhibited fusion without preventing diminution of calpastatin, by inhibiting calpain activity directly. Protein levels of mu-calpain and m-calpain did not change significantly in fusing myoblasts, nor in the inhibited, non-fusing myoblasts. The results indicate that calpastatin level is modulated by certain growth and differentiation factors and that its continuous presence results in the inhibition of myoblast fusion.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cell Fusion; Cell Line; Cysteine Proteinase Inhibitors; Muscles; Rats

Cell-permeant peptidyl aldehydes and diazomethylketones are frequently utilized as inhibitors of regulatory intracellular proteases. In the present study the specificities of several peptidyl inhibitors for purified human mu-calpain and 20 S proteasome were investigated. Acetyl-LLnL aldehyde, acetyl-LLM aldehyde, carbobenzyloxy-LLnV aldehyde (ZLLnVal), and carbobenzyloxy-LLY-diazomethyl ketone produced half-maximum inhibition of the caseinolytic activity of mu-calpain at concentrations of 1-5 x 10(-7) M. In contrast, only ZLLnVal was a reasonably potent inhibitor of the caseinolytic activity of 20 S proteasome, producing 50% inhibition at 10(-5) M. The other inhibitors were at least 10-fold less potent, producing substantial inhibition only at near saturating concentrations in the assay buffer. Further studies with ZLLnVal demonstrated that its inhibition of the proteasome was independent of casein concentration over a 25-fold range. Proteolysis of calpastatin or lysozyme by the proteasome was half-maximally inhibited by 4 and 22 microM ZLLnVal, respectively. Thus, while other studies have shown that ZLLnVal is a potent inhibitor of the hydrophobic peptidase activity of the proteasome, it appears to be a much weaker inhibitor of its proteinase activity. The ability of the cell permeant peptidyl inhibitors to inhibit growth of the yeast Saccharomyces cerevisiae was studied because this organism expresses proteasome but not calpains. Concentrations of ZLLnVal as high as 200 microM had no detectable effect on growth rates of overnight cultures. However, yeast cell lysates prepared from these cultures contained 2 microM ZLLnVal, an amount which should have been sufficient to fully inhibit hydrophobic peptidase activity of yeast proteasome. Degradation of ubiquitinylated proteins in yeast extracts by endogenous proteasome was likewise sensitive only to high concentrations of ZLLnVal. The higher sensitivity of the proteinase activity of calpains to inhibition by the cell permeant inhibitors suggests that calpain-like activities may be targets of these inhibitors in animal cells.

Topics: Acetylcysteine; Calcium-Binding Proteins; Calpain; Catalysis; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Diazomethane; Enzyme Precursors; Humans; Kinetics; Leupeptins; Multienzyme Complexes; Muramidase; Oligopeptides; Plant Proteins; Protease Inhibitors; Proteasome Endopeptidase Complex; Saccharomyces cerevisiae; Serine Proteinase Inhibitors; Ubiquitins

Neonatal rats were subjected to transient cerebral hypoxic-ischemia (HI, unilateral occlusion of the common carotid artery +7.70% O2 for 100 min) and allowed to recover for up to 14 days. Calpain caseinolytic activity was found to increase in both hemispheres for at least 20 hr. Hypoxic exposure per se increased the activity of calpains, more pronounced in a membrane-associated fraction, probably through interaction with cellular components, whereas HI introduced a loss of activity, most likely through consumption and loss of proteases. Consecutive tissue sections were stained with antibodies against calpastatin, alpha-fodrin, the 150-kDa breakdown product of alpha-fodrin (FBDP, marker of calpain proteolysis) or microtubule-associated protein 2 (MAP-2, marker of dendrosomatic neuronal injury). Areas with brain injury displayed a distinct loss of MAP-2, which clearly delineated the infarct. FBDP accumulated in injured and borderline regions ipsilaterally, and a less conspicuous, transient increase in FBDP also occurred in the contralateral hemisphere, especially in the white matter. The cytosolic fraction (CF) and the membrane and microsomal fraction (MMF) of cortical tissue were subjected to Western blotting and stained with antibodies against calpain, calpastatin and the 150-kDa breakdown product of alpha-fodrin (FBDP). Calpain immunoreactivity decreased bilaterally in the CF during the insult (62-68% of controls) and remained significantly lower during early recovery, whereas the MMF showed no significant changes. This translocation of calpains coincided with the appearance of FBDP in the ipsilateral, HI hemisphere, displaying a significantly higher level of FBDP from immediately after the insult until at least 1 day of recovery (204-292% of controls). No significant changes in FBDP were found in the contralateral, undamaged hemisphere, despite translocation of calpains in both hemispheres, a prerequisite for calpain activation. This discrepancy may be related to changes in the endogenous inhibitor, calpastatin. Calpastatin protein was found to decrease during and shortly after HI in the ipsilateral, but not the contralateral, hemisphere. The inhibitory activity of calpastatin also tended to decrease after HI, indicating that a reduction of calpastatin may be necessary for extensive calpain activation to occur. The mRNA of m-calpain increased in the HI hemisphere 48 hr after the insult (167%, p < 0.001), a time point when the protein was also increased.

Topics: Animals; Animals, Newborn; Calcium-Binding Proteins; Calpain; Carrier Proteins; Cerebral Cortex; Cysteine Proteinase Inhibitors; Cytosol; DNA Primers; Female; Functional Laterality; Hypoxia, Brain; Ischemic Attack, Transient; Male; Microfilament Proteins; Nerve Tissue Proteins; Rats; Rats, Wistar; Transcription, Genetic

We used 69 steers of varying percentage Brahman (B) breeding (0% B, n = 11; 25% B, n = 13; 37% B, n = 10; 50% B, n = 12; 75% B, n = 12; 100% B, n = 11) to study the relationship between carcass traits, the calpain proteinase system, and aged meat tenderness in intermediate B crosses. Calpains and calpastatin activities were determined on fresh longissimus muscle samples using anion-exchange chromatography. The USDA yield and quality grade data (24 h) were collected for each carcass. Longissimus steaks were removed and aged for 5 or 14 d for determination of shear force and 5 d for sensory panel evaluation. Even though some yield grade factors were affected by the percentage of B breeding, USDA yield grades did not differ (P > .15) between breed types. Marbling score and USDA quality grade decreased linearly (P < .01) with increasing percentage of B breeding. Shear force after 5 and 14 d of aging was higher (P < .05) in the 100% B steers than in all other breed types, which were not significantly different. Sensory panel tenderness and connective tissue scores decreased linearly (P < .05) with increasing B breeding. A quadratic effect was also noted for tenderness and connective tissue scores; 37% B steers received the highest scores. A similar response was found in mu-calpain activities; the 37% B steers had the highest activities. Conversely, calpastatin activity increased linearly (P < .01) with increasing percentage B breeding. These data show strong linear relationships between calpastatin activity (positive), marbling score (negative), and percentage B breeding, suggesting a possible combined effect of these traits on aged tenderness of intermediate Brahman crosses.

Topics: Animals; Body Composition; Breeding; Calcium-Binding Proteins; Calpain; Cattle; Chromatography, Ion Exchange; Female; Food Technology; Male; Meat; Muscle, Skeletal; Time Factors; United States; United States Department of Agriculture

The activities of calpain and its endogenous inhibitor, calpastatin, were measured in the soluble fraction of perfused rat heart after ischemia for 5-20 min and reperfusion for up to 30 min. The method for m-calpain measurement was modified: washing of the DEAE-cellulose column with 0.18 M NaCl instead of 0.15 M NaCl increased the m-calpain activity 12.5-fold. Ischemia for 20 min followed by reperfusion for 30 min did not affect the m-calpain activity but decreased the calpastatin activity. m-Calpain was enriched in the nucleus-myofibril fraction but was not further translocated on ischemia-reperfusion. Mu-calpain was below the limit of detection on immunoblotting or casein zymography, but its mRNA was substantially expressed, as detected on Northern blotting. Casein zymography also revealed a novel Ca2+-dependent protease without the typical characteristics of mu- or m-calpain. The immunoblotting of myocardial fractions showed that calpastatin was proteolyzed on ischemia-reperfusion. The calpastatin proteolysis was suppressed by a calpain inhibitor, Ac-Leu-Leu-norleucinal. Calpastatin may sequester calpain from its substrates in the normal myocardium, but may be proteolyzed by calpain in the presence of an unidentified activator in the early phase of calpain activation during ischemia-reperfusion, resulting in the proteolysis of calpastatin and then other calpain substrates.

Topics: Animals; Blotting, Northern; Blotting, Western; Calcium-Binding Proteins; Calpain; Chromatography, DEAE-Cellulose; Cysteine Proteinase Inhibitors; Down-Regulation; Hydrolysis; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Rats; Rats, Wistar; RNA, Messenger

Using red cells as an experimental model, we previously showed that a limited degradation of certain membrane proteins by calpain (Ca2+-activated thiol protease) was a necessary prerequisite for cell fusion and that fusibility depended on the ratio of calpain to its endogenous inhibitor calpastatin. Here we show that fusion of rat L8 line myoblasts is accompanied by a dramatic change in the calpain/calpastatin ratio. The protein levels of mu-calpain and m-calpain increased only slightly during myoblast differentiation. In contrast, calpastatin diminished by a factor of 10 at the stages of myoblast alignment and start of fusion, allowing calpain activity to become apparent. Calpastatin reappeared at a late stage of myoblast fusion (myotube formation). The results indicate that calpastatin is regulated during myoblast differentiation, and that its diminution is important in determining the activity of the calpain required for myoblast fusion.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cell Fusion; Cell Line; Cysteine Proteinase Inhibitors; Dipeptides; Egtazic Acid; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Kinetics; Molecular Weight; Muscle Fibers, Skeletal; Rats

Nuclear factor kappaB (NF-kappaB) is a transcription factor that is critical for the inducible expression of multiple cellular and viral genes. Using the electrophoretic mobility shift assay, we demonstrated that DNA binding activity of NF-kappaB was abolished by proteolysis with mu- and m-calpains in vitro. The proteolysis of NF-kappaB by calpains and hence the abolition of its DNA binding was prevented by calpastatin, calpain inhibitor I and proteasome inhibitor. We also provided evidence that calpains degrade the C-terminal domain of NF-kappaB by Western blot using anti-NF-kappaB (p65) C-terminal antibody. These observations indicate that calpains regulate gene expression through processing of NF-kappaB.

Topics: Base Sequence; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; DNA; Glycoproteins; Humans; Leupeptins; Molecular Sequence Data; NF-kappa B; Protein Processing, Post-Translational

Although hepatocellular carcinoma (HCC) cells are more resistant to anoxic injury than normal hepatocytes, the mechanisms responsible for this differential sensitivity remain obscure. Because enhanced calpain protease activity contributes to hepatocyte necrosis, we tested the hypothesis that HCC cells resist anoxia by preventing calpain activation. Cell viability in two rat HCC cell lines (N1S1 and McA-RH7777 cells) was fourfold greater compared to rat hepatocytes after 4 h of anoxia. Although calpain activity increased twofold in rat hepatocytes during anoxia, no increase in calpain activity occurred in HCC cells. Western and Northern blot analysis revealed greater or equivalent expression of calpains and calpastatin in HCC cells compared to hepatocytes. Because increases in cytosolic free Ca++ (Cai++) and phospholipid degradation products regulate calpains in vitro, we measured Cai++ and phospholipid degradation. Ca++i did not change in any cell types during 60 min of anoxia. In contrast, phospholipid degradation was fourfold greater in hepatocytes compared to HCC cells. Melittin, a phospholipase A2 activator, increased calpain activity and cell necrosis in all cell types; melittin-induced cell necrosis was ameliorated by a calpain protease inhibitor. In summary, these data demonstrate for the first time 1) calpain activation without a measureable increase in Ca++i, 2) phospholipase-mediated calpain activation in hepatocytes and HCC cells, and 3) the adaptive mechanism responsible for the resistance of HCC cells to anoxia-an inhibition of phospholipid-mediated calpain activation. Interruption of phospholipase-mediated calpain activation may be a therapeutic strategy for preventing anoxic cell injury.

Topics: Adenosine Triphosphate; Animals; Calcium; Calcium-Binding Proteins; Calpain; Cell Hypoxia; Cell Survival; Cells, Cultured; Cysteine Proteinase Inhibitors; Enzyme Activation; Gene Expression Regulation, Neoplastic; Liver; Liver Neoplasms, Experimental; Male; Melitten; Necrosis; Phospholipases; Rats; Rats, Sprague-Dawley; Tumor Cells, Cultured

A Chinese hamster ovary cell line (CHOp) was cultured in the presence of benzyloxycarbonyl-Leu-Leu-Tyr diazomethyl ketone (ZLLY-CHN2), to select for resistance to this cell-permeant calpain inhibitor. A clone isolated after several courses of exposure (SHI cells) demonstrated decreased sensitivity to ZLLY-CHN2 toxicity and a decreased growth rate. SHI cells also possessed less mu-calpain isozyme relative to CHOp cells, as determined by activity measurement or by protein immunoblotting. Activities of m-calpain, calpastatin, cathepsin B, cathepsin L, and glycogen phosphorylase were not altered. SHI mu-calpain was partially purified by sequential chromatography on Bio-Gel A-1.5m and DEAE-Sepharose. Its chromatographic behavior in either system was the same as for CHOp mu-calpain. Further studies with the partially purified SHI and CHOp mu-calpain fractions failed to distinguish any difference in Ca2+ requirement or in sensitivity to inhibition by calpastatin or ZLLY-CHN2 for these enzymes. These experiments suggest that SHI cells underproduce a form of mu-calpain which is very similar to, if not identical with, CHOp mu-calpain. SHI cells displayed a population doubling time of 29 h compared with 19 h for CHOp cells. The decreased growth rate of SHI cells was the result of a prolonged G1 phase. Introduction of purified human mu-calpain into SHI cells by electroporation transiently restored the growth rate and also increased toxicity associated with exposure to ZLLY-CHN2. SHI cells should be a valuable model in further studies to delineate the function of mu-calpain in cell proliferative growth.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cathepsin L; Cathepsins; Cell Division; Cell Survival; CHO Cells; Cricetinae; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Diazomethane; Endopeptidases; Isoenzymes; Mutation; Oligopeptides; Phosphorylases

We studied the activity of plasma membrane (Ca+Mg)ATPase from erythrocytes of Milan hypertensive rat strain (MHS) and Milan low calpastatin rat strain (MLCS), that show an activity level of the specific calpain inhibitor, calpastatin, about five fold reduced in comparison with the Milan normotensive rat strain (MNS), while the protease activity level is similar. This imbalance of calpain:calpastatin ratio leads to a decrease of the erythrocyte plasma membrane (Ca+Mg)ATPase activity and to the appearance of 124 kDa fragments, which are the typical products of proteolytic calpain action on the 136 kDa (Ca+Mg)ATPase native form.

Topics: Animals; Blood Pressure; Blotting, Western; Ca(2+) Mg(2+)-ATPase; Calcium-Binding Proteins; Calpain; Enzyme Inhibitors; Erythrocyte Membrane; Hypertension; Male; Molecular Weight; Rats; Rats, Mutant Strains

The relationship between plasma epinephrine and postmortem activity of the calpain system in porcine longissimus muscle was investigated. Two groups of Large White pigs were continuously infused intravenously with either placebo (control) or epinephrine (treated) at a rate of .15 micrograms.kg-1.min-1 for a period of 1 wk before slaughter. Samples of longissimus muscle were taken at 0, 1, 2, 4, 8, 24, 48, and 192 h (t0 to t192) after slaughter and immediately snap-frozen in liquid nitrogen for subsequent analysis of mu-calpain, m-calpain, and calpastatin activity. Epinephrine infusion had no effect on the activities of mu- and m-calpain at t0. Calpastatin activity at t0 was increased (P < .01) in treated pigs by 97%. The ratio of total calpain:calpastatin activity at t0 was reduced (P < .01) in treated pigs. The activity of mu-calpain decreased rapidly after slaughter, irrespective of treatment, dropping to less than 10% of the initial activity within 8 h. The activity of m-calpain also decreased over the first 8 h, although the rate of decrease was less (P < .05) in treated pigs. Consequently, m-calpain activity remained greater in treated pigs compared with controls throughout the period normally associated with tenderization. Postmortem values for calpastatin activity tended to be highly variable, with activities being similar between control and treated pigs within 1 h after slaughter. Over the entire 192 h sampling period, calpastatin activity decreased (P < .001), although the effect was independent of treatment. In general the results imply that variations in plasma epinephrine concentrations, which may naturally occur as part of the stress response, perturb the calpain enzyme system.

Topics: Animals; Calcium-Binding Proteins; Calpain; Epinephrine; Female; Hydrogen-Ion Concentration; Infusions, Intravenous; Muscle, Skeletal; Swine; Time Factors

Calpains are intracellular Ca2+-dependent proteases that are thought to participate in Ca2+-associated signal transduction pathways. It has been proposed that calpains are activated by an autoproteolytic mechanism. If this is true one would expect a relatively short half-life for calpain protein in cells. To test this hypothesis, WI-38 human diploid fibroblasts were pulse-labeled with [35S]methionine, and calpain was immunoprecipitated at various times after chasing with nonradioactive methionine to determine residual radioactivity. The results demonstrated that the two major calpain isozymes, m-calpain and micro-calpain, had metabolic half-lives of approximately 5 days. Calpains were long-lived proteins in several human cell lines, A-431, HeLa, VA-13, C-33A, and TE2 cells. In addition, calpastatin, the calpain-specific inhibitor protein, also had a long metabolic half-life. These observations suggest that the model for calpain activation by autoproteolysis requires re-investigation. Based on a knowledge of calpain metabolic stability, a protocol was devised for chronic exposure of WI-38 cells and HeLa cells to a calpain small subunit antisense oligodeoxyribonucleotide. Depletion of calpain small subunit after 5 or more days of treatment led to inhibition of cell proliferation that could be reversed by removal of antisense oligodeoxyribonucleotide from the culture medium. Together with previous studies, these results indicate a requirement for calpains in mammalian cell proliferation.

Topics: Amino Acid Sequence; Base Sequence; Calcium-Binding Proteins; Calpain; Cell Division; Cell Line; Cysteine Proteinase Inhibitors; Diazomethane; Fibroblasts; Half-Life; HeLa Cells; Humans; Isoenzymes; Molecular Sequence Data; Molecular Structure; Oligonucleotides, Antisense; Oligopeptides; Protein Conformation

Calpains are Ca-activated neutral proteases present in all cells together with an endogenous inhibitor, calpastatin. Proposed substrates are; cytoskeletal proteins like microtubules and actin, protein kinases such as PKC and membrane-bound enzymes like Ca-ATPase and the Ca-channel. In lenses from different species calpains have been detected in decreasing amounts from the epithelium to the cortex to the nucleus. Several substrates for calpain in the lens have been demonstrated: crystallins, vimentin, actin, beaded filaments and MP26 among others. Both studies on animal models and capsulorhexis indicate that calpains are mainly involved in cortical cataract.

Topics: Calcium; Calcium-Binding Proteins; Calpain; Cataract; Epithelium; Humans; Lens, Crystalline; Substrate Specificity

Calpains, calcium activated neutral proteases (CANP), and calpastatin (CAST), their specific inhibitor, are involved in the proteolysis of amyloid precursor protein (APP), which is thought to be abnormal in Alzheimer's disease (AD). We studied the CANP/CAST system in erythrocytes of 14 clinically probable AD patients, 11 young and 14 old controls. CANP and CAST activities in the control subjects significantly correlated with increasing age; old controls showed a significant increase in CANP and CAST activities compared to young controls. Values of CANP and CAST activities in AD patients were similar to those of young controls. The physiological gage-related increase in proteolysis seems to be lost in AD patients, and this could play a role in the pathogenesis of the disease. However, due to the overlap of results between patients and controls, we could not reliably differentiate the healthy from the disease state on the basis of erythrocytic CANP/CAST activity.

Topics: Aged; Alzheimer Disease; Analysis of Variance; Biomarkers; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Erythrocytes; Female; Humans; Male; Middle Aged

The family of calpains (CANP or calcium activated neutral proteases) and their endogenous inhibitor calpastatin have been implicated in many neural functions; however, functional distinctions between the major calpain isoforms, calpain I and II, have not been clearly established. In the present study we analyzed the gene expression patterns for calpain I and II and calpastatin in mouse brain and spinal cord by measuring both their mRNA and protein levels. Our results show that the overall mRNA level measured by competitive reverse transcription polymerase chain reaction for calpain II is 15-fold higher and for calpastatin is three-fold higher than that for calpain I. Overall, both mRNA and protein expression levels for the calpains and calpastatin showed no significant difference between the spinal cord and the brain. The cellular distributions of mRNA for calpain I or calpastatin, measured by in situ hybridization, are relatively uniform throughout the brain. In contrast, calpain II gene expression is selectively higher in certain neuron populations including pyramidal neurons of the hippocampus and the deep neocortical layers, Purkinje cells of cerebellum, and motor neurons of the spinal cord. The motor neurons were the most enriched in calpain message. Motor neurons possessed 10-fold more calpain II mRNA than any other spinal cord cell type. The differential distribution of the two proteases in the brain and the spinal cord at the mRNA level indicates that the two calpain genes are differentially regulated, suggesting that they play different physiological roles in neuronal activities and that they may participate in the pathogenesis of certain regional neurological degenerative diseases.

Topics: Animals; Base Sequence; Blotting, Western; Brain; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Female; Gene Expression Regulation, Enzymologic; In Situ Hybridization; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Messenger; Spinal Cord; Transcription, Genetic

Satellite cells are the source of new muscle fibers in postnatal skeletal muscle growth and regeneration. Regulation of satellite cell survival is of fundamental importance in maintaining normal muscle function. Here we describe and characterize a tissue culture model of satellite cell apoptosis. Kinetic studies indicate that serum deprivation triggers a set of sequential events: early cell death, transient cell cycle traverse, and delayed cell death. The satellite cell death occurs by apoptosis based on the internucleosomal DNA laddering, in situ DNA end-labeling, and the requirements for de novo protein synthesis and extracellular calcium influx. The transient period of cell cycle progression (7-11 h after serum withdrawal) is accompanied by temporal induction of members of the immediate early gene family, such as c-myc, c-fos, and SRF, and appears to precede the delayed phase of cell death. Satellite cell apoptosis can be suppressed by several growth factors and by blocking the activity of calpain, a calcium-regulated protease. The late phase of apoptosis is marked by selective activation of ubiquitin-mediated protein conjugation and degradation. This study defines several control points where satellite cell apoptosis may be genetically or pharmacologically intervened.

Topics: Animals; Apoptosis; Blood Physiological Phenomena; Calcium-Binding Proteins; Calpain; Cell Cycle; Cell Differentiation; Cell Division; Cell Line; DNA; DNA-Binding Proteins; Enzyme Inhibitors; Gene Expression Regulation; Genes, Immediate-Early; Growth Substances; L-Lactate Dehydrogenase; Mice; Mice, Inbred C3H; Muscle Proteins; Muscle, Skeletal; Nuclear Proteins; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-myc; RNA, Messenger; Serum Response Factor; Ubiquitins

The proteinase inhibitor set in skeletal muscle is poorly characterized at present. This study was aimed to investigate in mouse skeletal muscle 1) the tissue-associated counterpart, if any, of serum protease inhibitors (which may also play antiproteolytic functions in tissues) and 2) calpastatin, a tissue inhibitor of calcium-activated neutral proteases (calpains). Triton-extracts were prepared from muscle homogenates of mice, which had been perfused extensively with phosphate buffered saline (PBS) (under deep anesthesia) to remove blood inhibitors. Among various inhibitors tested, the following muscle-associated inhibitors were identified by western-blotting: alpha-2-macroglobulin (185, 165, 35 kDa), alpha-1-antitrypsin (52 kDa), inter-alpha-trypsin inhibitor (220, 180 kDa) and calpastatin (70 kDa). Combined light microscope and confocal immunohistochemical experiments revealed that, in all muscles examined (soleus, plantaris, extensor digitorum longus) the above specific immunoreactivities were localized outside the muscle fibers (in periendomysium, blood vessel wall) as well as within them. Inter-alpha-trypsin inhibitor, however, completely lacked the intracellular localization. This wide distribution of proteinase inhibitors suggests that numerous muscular structures may be normally protected from unwanted proteolysis, thus providing an essential background for further studies on pathological models with altered proteolysis (m. dystrophy, denervation atrophy, etc.).

Topics: Animals; Blotting, Western; Calcium-Binding Proteins; Calpain; Immunoenzyme Techniques; Mice; Mice, Inbred C57BL; Muscle, Skeletal; Protease Inhibitors

Calpain, a Ca(2+)-dependent neutral protease was examined to investigate its involvement in postmortem proteolysis in the rat brain. Western blotting analysis showed that the 240 kDa alpha-subunit of fodrin, a well-known substrate for calpain, was degraded to generate 150 kDa and 145 kDa fragments in the postmortem interval (0-24 h) at 25 +/- 3 degrees C. Postmortem proteolysis was dependent on ambient temperature. In in vitro experiments, the 150 kDa and 145 kDa fragments appeared in the homogenate with addition of Ca2+ (1 microM-1 mM) or in the microsomal fraction by incubation with purified calpain. Both calpain inhibitor-1 and leupeptin suppressed in vitro proteolysis. During the initial 0-24 h postmortem, the activity of m-calpain in the brain remained unaltered, while that of its endogenous inhibitor, calpastatin, decreased with the postmortem interval. These results indicate that calpain is involved in fodrin proteolysis in the postmortem rat brain. The ratio of the amount of the 150 kDa proteolytic product to that of the 240 kDa fodrin alpha-subunit was correlated significantly with the postmortem interval (0-16 h; r = 0.745).

Topics: Animals; Blotting, Western; Brain Chemistry; Calcium-Binding Proteins; Calpain; Carrier Proteins; Forensic Medicine; Male; Microfilament Proteins; Nerve Tissue Proteins; Postmortem Changes; Rats; Rats, Wistar; Temperature; Time Factors

Most purification schemes of calpain (CANP) involve a number of chromatographic steps. The final preparations often contain impurities, including degradation fragments. Two peptide-affinity columns were developed, using peptides of 27 amino acids and 30 amino acids, corresponding to the products of exons 1B and 1C, respectively, of the natural inhibitor (calpastatin) gene, coupled to CNBr-activated Sepharose 4B. Crude preparations of calpain, isolated by anion-exchange chromatography on a DEAE-Sepharose column, were incubated with a reversible or an irreversible synthetic inhibitor which blocks the catalytic subunit of the enzyme in the inactive 80-kDa form. The crude preparation was then loaded onto the peptide column in the presence of calcium. Calpain was eluted with an EGTA-containing buffer. Using the two peptide-affinity columns connected in tandem, calpain was isolated with a high degree of purity, suitable for structural and mechanistic studies, i.e. as an 80/30-kDa heterodimer or in the form of dissociated monomers.

Topics: Amino Acid Sequence; Calcium; Calcium-Binding Proteins; Calpain; Chromatography, Affinity; Chromatography, Gel; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Erythrocytes; Humans; Molecular Sequence Data; Peptide Fragments; Sepharose

The Ca-induced autoproteolysis calpain proceeds through the sequential formation of two forms of active enzyme with molecular masses of 78 kD and 75 kD, respectively. The autolysed calpains are produced by the cleavage of the peptide bond between Ser15-Ala16 and then between Gly27-Leu28. Calpastatin reduces with high efficiency the transition from 78 kD to 75 kD calpain forms. At higher concentration also the first autolytic event is blocked. The data are consistent with the presence of two calpain forms with different susceptibility to calpastatin. Furthermore, calpain, once bound to phospholipid vesicles, undergoes autoproteolysis which preferentially accumulates the 78 kD species. These data provide new information on the activation process of calpain, indicating that a Ca-induced conformational change is the triggering event, followed by the appearance of the active 78 kD calpain which can be considered the preferential form of calpain at the membrane level.

Topics: Amino Acid Sequence; Autolysis; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Erythrocytes; Humans; Liposomes; Models, Biological; Molecular Sequence Data; Phospholipids

Vertebrate m-calpain, calpastatin, constitutive nitric oxide synthase, myelin basic protein, and dynamin I are substrates of protein kinase C (PKC). The presence/absence of similar/related protein in nonvertebrate was investigated by immunological methods, including (1) affinity chromatography on agarose-secondary antibodies and agarose IgG for removal of nonspecific immunoreactivities from crude extracts; (2) omitting beta-mercaptoethanol treatment and boiling prior to SDS-PAGE to increase the immunoreactivity; (3) immunoreactivity comparisons of nonspecific IgG as controls with specific anti-(vertebrate PKC-substrates/related proteins) in Western blots. It was found that (a) m-calpain and dynamin I were absent in baker's yeast, wheat germ and lobster tail muscle, (b) m-calpain, nitric oxide synthase, myelin basic protein and dynamin II were present in all three samples, and (c) calpastatin was present in baker's yeast and lobster tail muscle. The presence and absence of these proteins suggest evolutionary conservation and divergence, respectively, of these PKC substrates.

Topics: Animals; Blotting, Western; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Dynamin I; Dynamins; Electrophoresis, Polyacrylamide Gel; GTP Phosphohydrolases; Microtubules; Muscles; Myelin Basic Protein; Nephropidae; Nitric Oxide Synthase; Saccharomyces cerevisiae; Triticum

The increasing effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was characterized. The proteolytic activity was markedly elevated by the addition of regucalcin (0.1-0.5 microM) in the absence of Ca2+. This increase was not significantly altered by the presence of diisopropylfluorophosphate (DPF; 2.5 mM)--although DFP caused a significant decrease in the proteolytic activity. Regucalcin (0.25 microM) additively enhanced the dithiothreitol (DTT; 1.0 mM)--increased proteolytic activity, while the regucalcin or DTT effect was completely abolished by NEM (5 mM), indicating that regucalcin may act on the SH group in proteases. Also, regucalcin (0.25 microM) enhanced the effect of Ca2+ (10 microM) increasing liver proteolytic activity, suggesting that regucalcin does not influence on the active sites for Ca2+ in proteases. Moreover, the proteolytic activity of regucalcin (0.25 microM) was significantly decreased by the presence of calpastatin (24 micrograms/ml), an inhibitor of Ca(2+)-activated neutral protease (calpain). Now, regucalcin (0.25 microM) increased about 7-fold the activity of m-calpain isolated from rabbit skeletal muscle. These observations demonstrate that regucalcin directly activates cysteinyl-proteases. Regucalcin may have a role as a potent proteolytic activator in the cytoplasm of liver cells.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Carboxylic Ester Hydrolases; Cysteine Endopeptidases; Cytosol; Dithiothreitol; Drug Synergism; Edetic Acid; Enzyme Activation; Ethylmaleimide; Intracellular Signaling Peptides and Proteins; Isoflurophate; Liver; Male; Muscle, Skeletal; Rabbits; Rats; Rats, Wistar; Sulfotransferases

We have examined the mechanism for the selective down-regulation of protein kinase C epsilon (nPKC epsilon) in rat pituitary GH4C1 cells responding to thyrotropin-releasing hormone (TRH) stimulation. Among various low molecular weight protease inhibitors examined, only a cysteine protease inhibitor (calpain inhibitor I, N-acetyl-Leu-Leu-norleucinal) blocked the down-regulation of nPKC epsilon. Furthermore, the introduction of a synthetic calpastatin peptide, an exclusively specific inhibitor of calpain, into the cells also reduced the down-regulation, suggesting the involvement of calpain among all the intracellular cysteine proteases in this process. In accordance, we observed TRH-induced translocation of m-calpain from the cytosol to the membrane and the concomitant up-regulation of calpastatin isoforms; presumably, the former represents activation of the protease initiating the kinase degradation, while the latter constitutes a negative feedback system protecting the cells from activated calpain. These results suggest that in GH4C1 cells, TRH mobilizes both protease (m-calpain) and inhibitor (calpastatin) as a strictly regulating system for the nPKC epsilon pathway mediating TRH signals.

Topics: Amino Acid Sequence; Animals; Calcium-Binding Proteins; Calpain; Cell Line; Down-Regulation; Isoenzymes; Molecular Sequence Data; Pituitary Gland; Protease Inhibitors; Protein Kinase C; Rats; Thyrotropin-Releasing Hormone

m-Calpain, a Ca(2+)-dependent neutral cysteine proteinase (EC 3.4.22.17), has been demonstrated to be present in the lower hypertrophic zone of the rat growth plate. Using the pelleted culture system as an in vitro model of rat epiphyseal chondrocyte differentiation, we studied m-calpain contents and activities in pelleted cultures during chondrocyte differentiation and the role of m-calpain in the mineralization process. m-Calpain was demonstrated immunohistochemically in epiphyseal chondrocytes, and immunoreactive m-calpain content in cells increased with terminal differentiation into hypertrophic cells. Immunoblotting also showed the association of the increase in m-calpain in cell pellets and in cell culture medium with development of the culture. Ca(2+)-dependent caseinolytic activities of m-calpain extracted from cell pellets and from the medium increased with chondrocyte differentiation, coincident with the increase in enzyme content. The inhibition of m-calpain by the addition of calpastatin, a specific inhibitor of calpain, caused suppression of matrix mineralization in pelleted cultures; the addition of E-64c, a specific inhibitor of cysteine proteinases, during the mineralization stage also caused a significant inhibition of the matrix mineralization. The addition of E-64c resulted in altered composition of proteoglycan monomers and aggregates in cell pellets and in suppression of mineral growth. These findings support an important role of cysteine proteinases, especially m-calpain, in the regulation of the cartilage mineralization process through proteoglycan degradation.

Topics: Alkaline Phosphatase; Animals; Calcification, Physiologic; Calcium; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cells, Cultured; Cysteine Proteinase Inhibitors; DNA; Extracellular Matrix; Growth Plate; Leucine; Male; Proteoglycans; Rats; Rats, Wistar; Uronic Acids

Activity of L-type Ca2+ channels in a membrane patch disappears rapidly when the patch is excised from the cell into an artificial solution. This channel run-down observed in isolated membrane patches can however, be prevented by application of calpastatin, an endogenous protease inhibitor, and ATP. The high specificity of calpastatin for the protease calpain would clearly point to a participation of calpain activity in the run-down of Ca2+ channels. In an attempt to examine a possible involvement of calpain, three synthetic and rather specific calpain inhibitors were substituted for calpastatin. One of these inhibitors chosen for its membrane permeability in addition allowed calpain activity to be inhibited even before patch excision. The potency of these compounds in inhibiting calpain, specifically mu- and m-calpain, was first determined in a biochemical assay and then compared with their efficacy in preventing Ca2+ channel run-down. Surprisingly, calpastatin was least effective in calpain inhibition but by far the most potent in prevention of Ca2+ channel run-down. In addition run-down of Ca2+ channel activity was examined for its reversibility, which would not be expected upon involvement of a proteolytic process. However, Ca2+ channel activity clearly recovered after run-down by application of calpastatin. In contrast, synthetic calpain inhibitors were unable to reverse Ca2+ channel run-down. These results indicate that proteolysis might only be partially responsible for channel run-down and suggest an as yet unidentified function for calpastatin beyond its inhibitory action on calpain in the regulation of Ca2+ channel activity.

Topics: Adenosine Triphosphate; Animals; Barium; Calcium; Calcium Channels; Calcium-Binding Proteins; Calpain; Guinea Pigs; Heart; Time Factors

A calcium-dependent proteinase (calpain) has been suggested to play an important role in muscle degradation in Duchenne muscular dystrophy (DMD). In immunohistochemical studies, calpain and its endogenous inhibitor (calpastatin) were located exclusively in the cytoplasm in normal human muscles. The intensity of the staining was stronger in type 1 than in type 2 fibers. Quantitative immunohistochemical study showed an increase of calpain in biopsied muscles from the patients with DMD and Becker muscular dystrophy. Abnormal increases in calpain and calpastatin were demonstrated mainly in atrophic fibers, whereas necrotic fibers showed moderate or weak immunoreactions for the enzymes. Opaque fibers and hypertrophic fibers were negative. Not all dystrophin-deficient muscle fibers necessarily showed a strong reaction for calpain. We suggest that calpain may play an important role in muscle fiber degradation, especially in the early stage of muscle degradation in muscular dystrophy.

Topics: Adolescent; Adult; Calcium-Binding Proteins; Calpain; Child; Cysteine Proteinase Inhibitors; Humans; Immunohistochemistry; Male; Muscle, Skeletal; Muscular Dystrophies

We identified an autoantibody that reacts with calpastatin [an inhibitor protein of the calcium-dependent neutral protease calpain (EC 3.4.22.17)]. In early immunoblot studies, sera from patients with rheumatoid arthritis (RA) recognized unidentified 60-, 45-, and 75-kDa proteins in HeLa cell extracts. To identify these autoantigens, we used patient sera to clone cDNAs from a lambda gt11 expression library. We isolated clones of four genes that expressed fusion proteins recognized by RA sera. The 1.2-kb cDNA insert (termed RA-6) appeared to encode a polypeptide corresponding to the 60-kDa antigen from HeLa cells, since antibodies bound to the RA-6 fusion protein also reacted with a 60-kDa HeLa protein. The deduced amino acid sequence of the RA-6 cDNA was completely identical with the C-terminal 178 amino acids of human calpastatin except for one amino acid substitution. Patient sera that reacted with the RA-6 also bound pig muscle calpastatin, and a monoclonal antibody to human calpastatin recognized the RA-6 fusion protein, confirming the identity of RA-6 with calpastatin. Moreover, the purified RA-6 fusion protein inhibited the proteolytic activity of calpain, and IgG from a serum containing anti-calpastatin antibodies blocked the calpastatin activity of the RA-6 fusion protein. Immunoblots of the RA-6 product detected autoantibodies to calpastatin in 57% of RA patients; this incidence was significantly higher than that observed in other systemic rheumatic diseases, including systemic lupus erythematosus (27%), polymyositis/dermatomyositis (24%), systemic sclerosis (38%), and overlap syndrome (29%). Thus, anti-calpastatin antibodies are present most frequently in patients with RA and may participate in pathogenic mechanisms of rheumatic diseases.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Arthritis, Rheumatoid; Autoantibodies; Base Sequence; Binding, Competitive; Calcium-Binding Proteins; Calpain; DNA, Complementary; HeLa Cells; Humans; Immunoglobulin G; Molecular Sequence Data; Muscles; Recombinant Fusion Proteins; Rheumatic Diseases; Species Specificity; Swine

Calpastatin molecule contains four repeated inhibition domains, each having highly conserved internal regions A, B and C. The synthetic oligopeptides of regions A and C had no calpain inhibition activity while region B oligopeptide showed weak inhibition activity. Real-time biomolecular interaction analysis using a BIAcore instrument revealed that the bacterially expressed calmodulin-like domain of the calpain large subunit (L-CaMLD) and that of the small subunit (S-CaMLD) interacted, in a Ca(2+)-dependent fashion, preferentially with the immobilized synthetic oligopeptide of region A and that of region C, respectively. Calmodulin showed no specific binding to these oligopeptides. The tripartite structure of the calpastatin functional domain may confer the specific interactions with the protease domain and the two CaMLDs of calpain.

Topics: Amino Acid Sequence; Calcium; Calcium-Binding Proteins; Calpain; Humans; Molecular Sequence Data; Oligopeptides; Recombinant Proteins

The Milan hypertensive strain (MHS) of rats, in addition to having hypertension, is also characterized by a genetic deficiency in calpastatin, the endogenous inhibitor of calpain. Since this protease has been implicated in long-term potentiation (LTP), we have investigated whether induction of this form of plasticity was altered in this strain of rats as compared to control animals (Milan normotensive strain, MNS). Progressive induction of LTP by increasing numbers of high frequency trains resulted in a greater degree of potentiation measured with all inducing protocols in MHS as compared with MNS animals. This difference was not related to the hypertension, since another hypertensive strain (the SHR strain) and a segregated Milan hypertensive strain, expressing only the hypertension but not the calpastatin deficiency (the MHNE strain), exhibited an LTP indistinguishable from control rats. Treatment of MHNE rats for 2 months with isovalerylcarnitine, a compound that increases calpain activity, also resulted in a greater amount of LTP induced by high frequency trains. These effects were not related to an enhancement of the NMDA receptor dependent component of responses to burst stimulation. These results are consistent with the idea that conditions under which calpain activation is facilitated are associated with a greater degree of synaptic potentiation.

Topics: Animals; Calcium-Binding Proteins; Calpain; Carnitine; Hippocampus; Hypertension; Long-Term Potentiation; Rats; Rats, Inbred SHR; Rats, Inbred Strains; Rats, Wistar

Death of dystrophin-deficient muscle purportedly results from increases in [Ca]in that cause the activation of calpains. We have tested whether calpains play a role in this process by assaying for changes in calpain concentration and activation in peak necrotic mdx mice (4 weeks of age) and in completely regenerated mdx mice (14 weeks of age). Biochemical fractionation and immunoblotting with epitope-specific antisera allowed measurement of the concentrations of m- and mu-calpains and the extent of autoproteolytic modification. Our findings show that total calpain concentration is elevated in both 4-week and 14-week mdx mice. This increase in concentration was shown to result primarily from a significant increase in m-calpain concentration at 4 weeks. Northern analysis demonstrated that neither m- nor mu-calpain mRNA concentrations differed between mdx and controls suggesting that the increased calpain concentration results from post-translational regulation. Immunoblotting with antibodies directed against amino-terminal peptides revealed an increase in autoproteolysis of mu-calpain, indicative of increased activation. The extent of autoproteolysis of mu-calpain returns to control levels during regeneration. This is not a consequence of increased calpastatin mRNA or protein. The findings reported here support a role for calpains in both the degenerative and regenerative aspects of mdx dystrophy.

Topics: Age Factors; Animals; Calcium-Binding Proteins; Calpain; Dystrophin; Enzyme Activation; Gene Expression; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Muscular Dystrophy, Animal; Necrosis; RNA, Messenger

Calpain activity was measured in the various subfractions of rat myocardia after global ischemia for 60 min or after ischemia followed by 30 min of reperfusion after the chromatographic separation of mu- and m-calpains. The activity of m-calpain after ischemia and that of mu-calpain after reperfusion were both higher than that in the control. The activity of the endogenous calpain inhibitor calpastatin in 10,000 x g supernatant was decreased after both ischemia and ischemia-reperfusion. The increase in m- and mu-calpain activities was suppressed by pre-ischemic perfusion with a synthetic calpain inhibitor, transepoxysuccinyl-L-leucylamido (4-guanidino) butane (E64d, 100 micrograms/ml). After reperfusion, the calpain activity in the 10,000 x g pellet was also increased, which was inhibited by pre-ischemic perfusion with E64d or dimethylsulfoxide (a solvent for E64d) or by reperfusion with 1 mmol/L ethyleneglycol bis (beta-aminoethylether)-N, N, N', N'-tetraacetic acid. SDS-polyacrylamide gel electrophoresis revealed the proteolysis of several proteins, including fodrin, in the 10,000 x g and 100,000 x g pellet fractions after ischemia and reperfusion, some of which were confirmed to be in vitro substrates of calpain. The creatine kinase release during the reperfusion was also partially inhibited by E64d or dimethylsulfoxide. Thus, calpain activity in the soluble or particulate fractions was altered during ischemia or reperfusion, and appeared to be implicated in the proteolysis of the membrane proteins, which may contribute to myocardial injury.

Topics: Animals; Calcium-Binding Proteins; Calpain; Creatine Kinase; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Leucine; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Rats; Rats, Wistar

The theory that net muscle growth is, at least partly, regulated by catabolic factors has been tested in order to set up an animal model to study meat aging and post-mortem tenderization. Male and female chickens of a layer strain (White Leghorn), a commercial broiler strain (Ross), and two experimental broiler lines (designated GL and FC) were used to estimate differences in proteolytic enzyme activities in the breast muscles. The GL and the FC lines were selected for high body weight gain and high feed efficiency, respectively. At 6 wk of age the birds were slaughtered and the activities of endogenous proteinases and their specific inhibitors in breast muscles measured. The Leghorns showed significant differences in all traits compared with the three broiler genotypes. Within the broiler types, FC birds tended in the direction of the Leghorns and GL birds in the opposite direction. Ross birds were intermediate between FC and GL birds. All types and sexes differed significantly in slaughtering weight. Feed conversion ratio and protein conversion ratio were highest for Leghorns. The FC birds showed the lowest feed conversion. Ross and GL birds showed intermediate values. The Leghorns showed higher calpain activities and lower calpastatin activity than the three broiler genotypes. The FC broilers showed intermediate calpain and calpastatin activities but higher cathepsin H and total cystatin values. The GL broilers showed lower cathepsin B, D, and H activities. In all cases the Ross broilers showed intermediate values. From these figures it is concluded that the strains of birds used in this study can be used as a natural source of variability to study the mechanisms involved in post-mortem proteolytic degradation and thus in the study of muscle tenderization and meat aging. It is also concluded that it could be very interesting to study the behavior of the different proteolytic systems more carefully in relation to muscular growth characteristics and compare them to anabolic factors involved in muscle growth.

Topics: Animals; Body Weight; Calcium-Binding Proteins; Calpain; Cathepsins; Chickens; Female; Male; Muscle, Skeletal; Sex Factors; Species Specificity

Calcium-activated neutral proteases (calpains) are regulated by specific endogenous protein inhibitors, the calpastatins, which are widely distributed in mammalian tissues. Calpastatins from different species or in various tissues from the same species exhibit considerable size heterogeneity on sodium dodecyl sulfate (SDS) gels, reflecting both transcriptional and posttranslational regulation. This heterogeneity has complicated previous biochemical characterizations. In this study, we purified bovine brain calpastatin to homogeneity. The inhibitor was purified 2,463-fold from a cytosolic fraction of fresh bovine cerebral cortex by chromatographies on diethylaminoethyl cellulose, Ultrogel AcA44, phenyl-Sepharose, concanavalin A-Sepharose, and Q-Sepharose. The major calpastatin displayed a native molecular mass of 250-300 kDa by gel filtration and was composed of 125-kDa polypeptide chains by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Small amounts of a 68-kDa calpastatin fragment were detected particularly in molecules exhibiting smaller native molecular mass (250 kDa). When electroeluted from SDS gels, the 125- and 68-kDa polypeptides each inhibited calpain. The purified protein was strongly immunoreactive toward antibodies raised against a synthetic peptide, CEKLGEKEETIPPDYR, shown to be a conserved repetitive motif in the calpastatin gene or a recombinant polypeptide corresponding to domains L and 1 of human calpastatin. Calpastatins purified from bovine and human erythrocytes exhibited molecular masses of 78 and 68 kDa, respectively, by SDS-PAGE. Both erythrocyte calpastatins reacted strongly with antibodies against the conserved sequence but not with antibodies raised against domains L and 1 of human calpastatin, indicating that the erythrocyte inhibitors lack these two domains.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Brain Chemistry; Calcium-Binding Proteins; Calpain; Cattle; Molecular Weight; Protease Inhibitors

A recombinant rat strain--Milan low-calpastatin strain (MLCS)--was derived from Milan hypertensive (MHS/Gib) and Milan normotensive (MNS/Gib) strains. The MLCS rats have normal blood pressure and low calpastatin activity, and this strain is proposed as a model for studies of the calpain-calpastatin system, which is involved in important cellular mechanisms. Calpastatin polymorphism was observed in 10 different strains of laboratory rats and a single locus hypothesis is suggested as the mode of inheritance.

Topics: Animals; Blood Pressure; Calcium-Binding Proteins; Calpain; Crosses, Genetic; Cysteine Proteinase Inhibitors; Female; Genotype; Male; Phenotype; Polymorphism, Genetic; Rats; Rats, Inbred Strains; Recombination, Genetic

Topics: Alternative Splicing; Animals; Calcium-Binding Proteins; Calpain; Gene Expression; In Vitro Techniques; Muscle, Skeletal; Myocardium; Organ Specificity; RNA, Messenger; Swine

This experiment was conducted to determine the relationship between 3-methylhistidine (3MH) production and proteinase activity in skeletal muscles of growing barrows. Barrows at 13 wk of age were randomly assigned to either control diet available on an ad libitum basis (21% of ME consisted of protein; control group), control diet fed restricted (pair-fed with barrows in protein-free group; intake-restricted group), or protein-free diet available on an ad libitum basis (protein-free group) for 14 d. During the last 3 d, blood samples were collected for determination of 3MH production rate, which is a measure of myofibrillar protein breakdown. At slaughter, two muscles were taken: masseter (M) and longissimus (L) muscles. The muscle samples were analyzed for calpastatin, mu-calpain, m-calpain, multicatalytic proteinase (MCP), cathepsin B, cathepsins B+L, and cystatins activities. Both muscles were also analyzed for amounts of DNA, RNA, total protein, and myofibrillar and sarcoplasmic proteins. Growth rate (kilograms/day) was influenced by dietary treatments (P < .05). Fractional breakdown rate (FBR, percentage/day) of skeletal muscle, as calculated from 3MH production rate (micromoles.kilogram-1.day-1), was 27% higher for the protein-free group than for the control group. However, no differences in proteinase activities were observed, except for lower MCP activity in the M muscle of the protein-free group than in that of the other groups (P < .05). In the present study, no direct relation was observed between myofibrillar protein degradation rate and proteinase activities in skeletal muscle during a protein-free feeding strategy.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cathepsins; Cystatins; Diet, Protein-Restricted; Dietary Proteins; DNA; Endopeptidases; Liver; Male; Methylhistidines; Muscle Proteins; Muscle, Skeletal; Organ Size; Protein Deficiency; Random Allocation; RNA; Swine; Swine Diseases

Calpain secreted by lymphoid (MOLT-3, M.R.) or monocytic (U-937, THP-1) cell lines activated with PMA and A23187 degraded myelin antigens. The degradative effect of enzymes released in the extracellular medium was tested on purified myelin basic protein and rat central nervous system myelin in vitro. The extent of protein degradation was determined by SDS-PAGE and densitometric analysis. Various proteinase inhibitors were used to determine to what extent protein degradation was mediated by calpain and/or other enzymes. Lysosomal and serine proteinase inhibitors inhibited 20-40% of the myelin-degradative activity found in the incubation media of cell lines, whereas the calcium chelator (EGTA), the calpain-specific inhibitor (calpastatin), and a monoclonal antibody to m calpain blocked myelin degradation by 60-80%. Since breakdown products of MBP generated by calpain may include fragments with antigenic epitopes, this enzyme may play an important role in the initiation of immune-mediated demyelination.

Topics: Animals; Antibodies, Monoclonal; Calcimycin; Calcium; Calcium-Binding Proteins; Calpain; Chelating Agents; Culture Media, Conditioned; Demyelinating Diseases; Egtazic Acid; Humans; Leukemia-Lymphoma, Adult T-Cell; Lymphoma, Large B-Cell, Diffuse; Monocytes; Myelin Basic Protein; Myelin Sheath; Neoplasm Proteins; Protease Inhibitors; Rabbits; Rats; T-Lymphocytes; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The present experiment was conducted to determine the effect of the callipyge phenotype on traits affecting muscle growth and meat tenderness. Dorset wethers (N = 40) that were either carriers or non-carriers were fed grain and slaughtered at 169 d of age. Callipyge phenotype did not affect (P > .05) slaughter weight, hot carcass weight, or weights of the heart, spleen, viscera, kidney-pelvic fat, head, and pelt; however, callipyge lambs had a higher dressing percentage and lighter lungs, liver, and kidneys (P < .01). Callipyge lambs had reduced fat thickness and marbling score and higher leg scores and longissimus area (34%). Adductor (30%), biceps femoris (42%), gluteus group (31%), longissimus (32%), psoas group (20%), quadriceps femoris (18%), semimembranosus (38%), and semitendinosus (26%) weights were higher in the callipyge phenotype (P < .01); however, phenotype did not affect (P > .05) weights of infraspinatus or supraspinatus. Longissimus pH and temperature declines were not affected (P > .05) by phenotype. Longissimus myofibril fragmentation index was lower at 1 (27%), 7 (35%), and 21 (37%) d postmortem and Warner-Bratzler shear force was higher at 1, 7, and 21 d postmortem in the callipyge phenotype (P < .01). Shear force values of callipyge lambs at 21 d postmortem tended to be greater (P = .12) than shear force values of non-carriers at 1 d postmortem . Activities of calpastatin (83%) and m-calpain (45%) were higher in the callipyge (P < .01); however mu-calpain activity was not affected (P > .05). Longissimus and semitendinosus RNA concentration, DNA content, RNA content, protein content, and the RNA:DNA ratio were higher (P < .05), but DNA concentration, protein concentration, and protein:DNA were not affected in the callipyge phenotype. The higher calpastatin activity associated with callipyge suggests that protein degradation may be reduced in the live animal. Additionally, the increased muscle DNA content associated with the callipyge phenotype suggests an increase in satellite cell proliferation, and results in an increased capacity of skeletal muscle to accumulate and maintain myofibrillar protein. These results suggests that both reduced rate of protein degradation and higher capacity for protein synthesis are consequences of the callipyge condition.

Topics: Actinin; Animals; Blotting, Western; Body Weight; Calcium-Binding Proteins; Calpain; Connectin; Desmin; Hydrogen-Ion Concentration; Hypertrophy; Male; Meat; Muscle Development; Muscle Fibers, Skeletal; Muscle Proteins; Muscle, Skeletal; Nucleic Acids; Phenotype; Protein Kinases; Sheep; Sheep Diseases; Temperature; Troponin

The calpains-calpastatin system (calcium-activated neutral proteases and endogenous inhibitor) seems to be, in the skeletal muscle, a fine enzymatic system of myofibrillar turnover regulation, in normal as well as pathological conditions (for ex., dystrophic muscle). The purpose of the research is to establish in qualitative and quantitative terms whether the level of calpastatin can evidence differences between a muscle in normal activity conditions and one having dysfunctional alterations experimentally induced. So the masseter muscle of rabbit in normal conditions and with experimentally modified occlusal plane has been used. Our results confirm the presence of the 68 KDa calpastatin in the masseter muscle. The presence of the inhibitor in the same subcellular structures in which the calpains have been detected (myofibrillars, sarcolemma, endomysial connective) has been confirmed. Finally, variations in calpastatin amount in the muscle of animals experimentally treated with respect to the controls have been found. Thus, calpastatin seems to act as a marker of muscle dysfunctions connected to occlusal plane alteration and to loss of vertical dimention.

Topics: Animals; Blotting, Western; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Enzyme-Linked Immunosorbent Assay; Immunochemistry; Immunohistochemistry; Malocclusion; Masseter Muscle; Rabbits; Tooth Abrasion; Vertical Dimension

Forty-eight crossbred rabbits were used in three replications of a 2 x 4 factorial arrangement to investigate the short-term responses of tissue accretion, calpains and calpastatin activity, and nucleic acid and protein concentrations to beta-adrenergic agonist (BAA) feeding. Rabbits were fed a 17% CP diet with or without 7 ppm of L644,969 and slaughtered after 1, 4, 8, or 16 d of treatment. Empty body dressing percentage and biceps femoris weight (as a percentage of empty body weight [EBW]) were significantly higher in the treated rabbits than in the controls after 16 d of treatment. Heart and liver weights (as a percentage of EBW) were higher (P < .05) after 1 d and liver weight (as a percentage of EBW) was lower (P < .05) after 16 d in treated vs controls. Except for an elevation of skeletal muscle m-calpain after 16 d, BAA-supplementation did not affect the calpain-calpastatin system. Muscle RNA concentrations and RNA:DNA ratios were higher (P < .05) in treated rabbits after 1 d and remained higher thereafter. Protein:RNA ratios were lower (P < .01) in treated than in control rabbits after 4 d and remained lower throughout the trial. Muscle DNA content was lower after 4 d and higher after 16 d; RNA content was higher after 4, 8, and 16 d; and protein content was higher after 16 d in treated vs control rabbits. Liver nucleic acid and protein concentrations were not affected by BAA treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adrenergic beta-Agonists; Animals; Blood Urea Nitrogen; Calcium-Binding Proteins; Calpain; DNA; Female; Heart; Liver; Muscle Development; Muscle Proteins; Muscles; Myocardium; Organ Size; Pyridines; Rabbits; RNA

The mode(s) of skeletal muscle protein turnover as well as muscle and animal growth may be studied by using lean and obese animals as models. The objectives of this study were to look at proteolytic variables implicated in these processes. A lean and obese strain of swine from similar genetic lineage (Duroc x Yorkshire, 50:50) have been well established and may prove ideal for this purpose. This study was done in two phases. Phase I included eight lean and eight obese pigs at 2.5 months of age, and phase II was identical, but the pigs were 7 months old. Longissimus muscle samples were processed immediately after euthanasia for activity measurements of mu-calpain, m-calpain, calpastatin, and lysosomal cathepsins B and B + L. Additional samples were taken for DNA, RNA, and total protein determinations. In phase I, total calpastatin activity, total and specific cathepsin B+L activity, and total protein/g muscle were greater in the obese pigs than in the lean pigs. In contrast, DNA and RNA/g muscle were greater in the lean pigs. No other differences were observed in phase I. In phase II, total calpastatin activity and total cathepsin B activity were greater in the obese pigs than in the lean pigs. No other differences were observed in phase II. From phase I to phase II, mu-calpain total activity increased in the lean pigs but not in the obese pigs and calpastatin activity decreased in both lean and obese pigs; however, the phase-II-obese and phase-I-lean total calpastatin concentrations were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aging; Animals; Calcium-Binding Proteins; Calpain; Cathepsin B; Cathepsin L; Cathepsins; Cysteine Endopeptidases; DNA; Endopeptidases; Muscles; Obesity; RNA; Swine

The objectives were to investigate the mechanisms by which glucocorticoids control proteolysis in muscle cells and the relationship between the calpain:calpastatin system and proteolysis in muscle. Female rabbits were treated with 1 mg dexamethasone (Dex)/kg body weight per day for 0, 1, 2 or 4 days after which animals were killed and muscle samples taken for analyses. Dex reduced urinary N tau-methylhistidine (NMH) 48% (day 4 versus day 1 of Dex treatment) and muscle NMH concentrations by 49% (day 1) to 40% (day 2) respectively, suggesting that protein degradation was reduced. To investigate whether the changes in apparent proteolysis were related to calpains, we examined the effects of Dex on muscle calpain and calpastatin activities. These were unaffected by Dex. This implies that Dex-dependent changes in degradation are not mediated by changes in muscle calpain or calpastatin activities. We studied the effects of Dex on calpain and calpastatin gene expression as a means of clarifying the relationships between proteinase gene expression and proteinase activities. mu-Calpain mRNA concentration was unaffected by Dex but m-calpain mRNA and calpastatin mRNA concentrations were reduced by 42-55% and 40% respectively. Dex had a similar effect on beta-actin mRNA. Although calpain and calpastatin genes behaved as house-keeping genes, changes in their expression mimicked apparent changes in proteolysis. The observation that calpain and calpastatin activities were unchanged indicates that additional regulation of the calpain:calpastatin system exists at other sites in muscle cells. To determine whether Dex-dependent changes in proteolysis were mediated indirectly, we assayed the effects of Dex on plasma thyroid hormone concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Blotting, Northern; Calcium-Binding Proteins; Calpain; Dexamethasone; Female; Gene Expression; Homeostasis; Methylhistidines; Muscles; Rabbits; RNA; Thyroxine; Triiodothyronine

Our previous studies demonstrated that fibronectin could be proteolyzed by m-calpain during muscle cell differentiation. Recent results indicated also that m-calpain could be exteriorized and more particularly associated to extracellular matrix components. To clarify one of the possible physiological functions of this proteinase during myogenesis, we have analyzed the incidence of added purified m-calpain and calpain inhibitors on the fusion kinetics of cultured myoblasts. Our results provided evidence that at low concentration (0.01 microgram/ml), added m-calpain induces precocious fusion and increases myoblast fusion by 78%. At high concentrations (10 micrograms/ml), the viability of the cells was not affected but the myoblasts were unable to fuse. Leupeptin and calpastatin--potent m-calpain inhibitors--added to the culture medium reduced myoblast fusion by 70%. On the other hand, the addition of monospecific m-calpain polyclonal antibodies to the culture medium induced a 76% decrease of myoblast fusion. In order to trap exteriorized m-calpain, myoblasts were incubated for 24 h with m-calpain antibodies. Following this treatment, nonpermeabilized myoblasts exposed to labeled secondary antibodies showed fluorescent spots scattered at the cell surface. These results strongly support that m-calpain which was involved in myoblast fusion was exteriorized and suggest therefore that this enzyme may play an important role extracellularly.

Topics: Animals; Biological Assay; Calcium-Binding Proteins; Calpain; Cell Fusion; Cells, Cultured; Culture Media; Immunoglobulin G; Immunohistochemistry; Kinetics; Leupeptins; Muscle Fibers, Skeletal; Rats; Rats, Wistar

Calpain has been identified as the intracellular proteinase that catalyzes the selective down-regulation of protein kinase C (PKC) isoforms, occurring in the early stages of commitment to terminal erythroid differentiation of murine erythroleukemia (MEL) cells induced by hexamethylenebisacetamide. This conclusion has been reached through direct experiments performed with two MEL cell clones, one characterized by a high and the other by a low rate of differentiation. In both cell types, introduction of an anti-calpain antibody resulted in a significant delay in the onset of down-regulation of PKC isoforms, and in an increase in the latent period that precedes differentiation. Both cell lines also displayed reduced rates of PKC decay and accumulation of mature erythroid cells. Furthermore, in the fast-responding clone, calpastatin, the natural calpain-inhibitor protein, was found to be almost completely absent, resulting in activation and expression of proteolytic activity of calpain even at micromolar concentrations of Ca2+, a condition not sufficient to trigger calpain activation in the slowly responding clone which contains high levels of calpastatin. The fast-responding MEL cell clone, enriched with calpastatin, displayed a lower rate of cell differentiation, with a kinetics almost identical to that observed following introduction of the anti-calpain antibody. It is proposed that Ca(2+)-dependent proteolysis plays a crucial role for the progress of MEL cell differentiation through the specific degradation of PKC isozymes.

Topics: Acetamides; Antibodies; Antineoplastic Agents; Calcium; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cell Line; Erythrocytes; Humans; Isoenzymes; Kinetics; Leukemia, Erythroblastic, Acute; Protein Kinase C; Tumor Cells, Cultured

Calpastatin is a widely distributed endogenous inhibitor protein specifically acting on calpain (Ca(2+)-dependent proteinase) and is known to interact with the calmodulin-like domain (CaMLD) of the proteinase in a Ca(2+)-dependent fashion. The calpastatin molecule consists of four inhibitory domains (domains 1-4) with mutually homologous sequences in three regions designated as A, B, and C. Acidic amphiphilic alpha-helical motifs are found in both regions A and C. We investigated the correlation between the calpain inhibition potency and the ability of calpastatin to bind to recombinant CaMLD of the mu-calpain large subunit using various mutant proteins of pig calpastatin domain 1 expressed in Escherichia coli. Substitution of conserved Leu-161 with Pro in region A caused a reduction in activity of both calpain inhibition and CaMLD binding. Additional substitution of Leu-236 with Pro in region C further decreased the calpain inhibitory activity and caused a loss of CaMLD binding ability. The effects of mutation in region C alone on the above activities were smaller than those in region A. Although a mutant of deletion in the entire region B had no calpain inhibitory activity, it retained the CaMLD binding ability. On the other hand, although a region B oligopeptide had a moderate inhibitory activity, it had no CaMLD binding ability. These results suggest that region A has a role in potentiating the inhibitory activity of calpastatin by interacting with CaMLD of calpain to form a tighter complex where region B exerts the inhibitory function.

Topics: Amino Acid Sequence; Animals; Calcium-Binding Proteins; Calmodulin; Calpain; Conserved Sequence; Cysteine Proteinase Inhibitors; Molecular Sequence Data; Mutation; Sequence Alignment; Swine

The peptide EKLGERDDTIPPEYRELLEKKTGV was synthesized to mimic the central consensus sequence of calpastatin, the specific, endogenous inhibitor of the calpains (EC 3.4.22.17). The peptide competitively inhibits hydrolysis of casein by either micro- or milli-calpain but does not affect the activity of other proteases. This inhibitory peptide was preferentially cross-linked to milli-calpain in the presence of calcium using the heterobifunctional cross-linking reagent m-maleimidobenzoyl-N-hydroxysuccinimide ester. Cross-linking of the peptide was blocked by calpastatin. The site of cross-linking for the peptide within milli-calpain was localized using random chemical cleavage of the enzyme-peptide complex at cysteine residues. Calpain fragments were identified as amino-terminal fragments through reactivity with a peptide-specific antiserum or as non-amino-terminal fragments through incorporation of 14C from 14CN. Analysis of the control and cross-linked fragments, from experiments using both milli-calpain and micro-calpain, maps the chemical cross-linking site to cysteine-497 and localizes the binding site for the calpastatin-like peptide to this highly conserved region of domain III of calpains catalytic subunit.

Topics: Amino Acid Sequence; Animals; Antibodies; Binding Sites; Binding, Competitive; Calcium-Binding Proteins; Calpain; Caseins; Cattle; Immunoblotting; Molecular Sequence Data; Peptides

Calpains are Ca(2+)-requiring, nonlysosomal proteases which are thought to participate in some aspects of intracellular Ca(2+)-signal transduction. However, their exact physiologic function has not yet been established. Addition of the cell-permeant, irreversible calpain inhibitor, ZLLY-CHN2, to human TE2 or C-33A cells inhibited growth, as assessed either by mitochondrial MTT reductase activity or by direct cell counting. Inhibition of growth produced by a 24-h exposure to 50 microM ZLLY-CHN2 was reversed upon substituting growth medium without inhibitor. Homogenates produced from cells cultured in the presence of ZLLY-CHN2 displayed decreased calpain and Ca(2+)-independent proteolytic activities. Protein immunoblot analysis showed that cell cultures which had lost 80% of their calpain activity still retained full calpain immunoreactivity. Therefore, inhibition by ZLLY-CHN2 appeared to result in accumulation of irreversibly inactivated calpain within the cells. Homogenates from cells cultured in the presence of 20 or 50 microM ZLVG-CHN2, a cell-permeant inhibitor with little activity against calpains, had decreased Ca(2+)-independent proteolytic activity, but demonstrated no decrease in calpain activity. ZLVG-CHN2 did not inhibit cell growth under these conditions. Growth of Saccharomyces cerevisiae cells, which do not appear to express calpain-like proteases, was not inhibited by including 50 microM ZLLY-CHN2 in the culture medium. These results indicate that calpains participate in the social regulation of cell growth in multicellular organisms.

Topics: Calcium-Binding Proteins; Calpain; Cell Division; Cell Line, Transformed; Cell Membrane Permeability; Cell Transformation, Viral; Cysteine Endopeptidases; Diazomethane; Female; Humans; Kinetics; Molecular Weight; Multienzyme Complexes; Oligopeptides; Protease Inhibitors; Proteasome Endopeptidase Complex; Saccharomyces cerevisiae; Simian virus 40; Time Factors; Uterine Cervical Neoplasms

Changes in expression of calpains and calpastatin during differentiation in GOTO cells were examined using antibodies specific to calpains and calpastatin. Neuronal differentiation induced by dibutyryl cyclic AMP elicited a remarkable decrease in m-calpain. No marked changes in the levels of calpains were found in bromodeoxyuridine-induced Schwannian differentiation. Calpastatin was detected as a single band of 110k in undifferentiated and in neuronally differentiated cells by Western blot analysis. However, the appearance of a 120k species was detected in Schwannian differentiation associated with morphological change. The data show that marked changes in m-calpain and calpastatin occur in a differentiation-specific manner.

Topics: Bromodeoxyuridine; Bucladesine; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cytosol; Humans; Neuroblastoma; Neurons; Schwann Cells; Tumor Cells, Cultured

To study nuclear transport of purified calpains in an in vitro system, A431 cells were permeabilized with digitonin, and fluorescein-labeled calpains were introduced under conditions known to facilitate energy-dependent nuclear transport of proteins. Fluorescein-mu-calpain was transported into nuclei in an ATP-dependent fashion. The calpain-specific inhibitor protein, calpastatin, could not block mu-calpain translocation. Fluorescein-calpastatin and fluorescein-m-calpain were poorly transported at best. In the presence of rat liver cytosolic factors, accumulation of nuclear mu-calpain was maximum at approximately 1 microM Ca2+, and no transport was observed at 0.3 microM Ca2+. Rat erythrocyte and HeLa cell extracts supported transport in the absence of Ca2+.

Topics: Amino Acid Sequence; Animals; Biological Transport; Calcium; Calcium-Binding Proteins; Calpain; Cattle; Cell Nucleus; Digitonin; Fluorescein; Fluoresceins; Humans; Molecular Sequence Data; Tumor Cells, Cultured

We examined the activation of mu-calpain in human epidermoid carcinoma KB cells following a rise in intracellular calcium concentration using antibodies specifically recognizing different activation states of mu-calpain. KB cells possess calpastatin activity in large excess of calpain activity as analyzed by ion exchange HPLC. Stimulation of the cells with a calcium ionophore, ionomycin, caused production of the autolytic intermediate form (M(r) = 78 k) of mu-calpain derived from the preautolysis form (80 k), while the fully autolyzed postautolysis form (76 k) remained below detectable levels at all times. The appearance of the autolytic intermediate paralleled limited proteolysis of the membrane-associated calpastatin fractions (110 k and 106 k); the resulting fragments (68 k and 45 k) were released into the cytosol. Both the production of the autolytic mu-calpain intermediate and the limited proteolysis of calpastatin in cell lysates in the presence of calcium were inhibited by a synthetic calpastatin peptide, indicating that proteolysis of calpastatin was indeed catalyzed by calpain and that the autolytic intermediate may have exerted the proteolytic activity. Furthermore, mu-calpain autolysis and calpastatin degradation, upon ionomycin treatment, were both augmented by epidermal growth factor (EGF). These results suggest that calpastatin serves not only as an inhibitor but also as a substrate for calpain at cell membranes and that intracellular conditions associated with the cell cycle may affect the activation of mu-calpain.

Topics: Amino Acid Sequence; Antibody Specificity; Autolysis; Binding Sites; Calcium; Calcium-Binding Proteins; Calpain; Carcinoma, Squamous Cell; Cysteine Proteinase Inhibitors; Enzyme Activation; Humans; Molecular Sequence Data; Tumor Cells, Cultured

To gain knowledge about the behaviour of calpastatin (the specific inhibitor of the Ca(2+)-dependent thiol protease calpain) in the intact cell, we analysed the inhibitor by specific antibodies and determined its activity in erythrocytes from individuals 20-34 years old (young) and 70-93 years old (old). Differences between old and young in the behaviour of erythrocyte calpastatin were observed. Erythrocytes of old individuals had lower amounts of calpastatin and less calpastatin activity than those of young ones. A difference between old and young was also found in the molecular-mass distribution of calpastatin subunits. Increasing the erythrocyte Ca2+ induced changes in calpastatin in young individuals, rendering it similar to calpastatin in cells of old individuals. When calpastatin (isolated from erythrocytes of a young individual) was added to erythrocyte membranes, the initial binding and subsequent association of calpastatin with the membrane were lower in old than in young individuals. We had previously found that calpain binding and activation were enhanced in erythrocyte membranes from old individuals, along with enhanced degradation of band 3 (a major erythrocyte transmembrane anion-transport protein). The overall results indicate an interaction of calpain with calpastatin in the intact cell. Enhanced activation of erythrocyte calpain and degradation of calpastatin occur under conditions of increased cellular Ca2+ and in cells of the aged.

Topics: Adult; Aged; Aged, 80 and over; Aging; Calcium; Calcium-Binding Proteins; Calpain; Cytosol; Erythrocyte Membrane; Erythrocytes; Ethylmaleimide; Humans; Molecular Weight

To estimate the heritability (h2) of postrigor calpastatin activity (CA), 555 steers were reared and processed conventionally. Breed-types included purebreds (Angus [A], Braunvieh [B], Charolais [C], Gelbvieh [G], Hereford [H], Limousin [L], Pinzgauer [P], Red Poll [RP], and Simmental [S]), composite populations (MARC I [1/4 C, 1/4 B, 1/4 L, 1/8 H, 1/8 A], MARC II [1/4 S, 1/4 G, 1/4 H, 1/4 A], and MARC III [1/4 RP, 1/4 H, 1/4 P, 1/4 A]), and F1 crosses (H, A, C, G, P, Shorthorn, Galloway, Longhorn, Nellore, Piedmontese, or Salers x H or A). Steers were serially slaughtered on an age-constant (across breed groups) basis. Heritability estimates for CA, i.m. fat content (IMF), Warner-Bratzler shear (WBS) force, retail product yield (RPY), and ADG were .65 +/- .19, .93 +/- .02, .53 +/- .15, .45 +/- .18, and .32 +/- .26, respectively. The genetic correlations (rg) of CA with WBS, RPY, and ADG were .50 +/- .22, .44 +/- .25, and -.52 +/- .37, respectively. The rg of IMF with WBS, RPY, and ADG were -.57 +/- .16, -.63 +/- .15, and -.04 +/- .11, respectively. These h2 and rg estimates indicate that it should be possible to select for improvements in CA, IMF, and WBS. However, selection against CA may be a more suitable approach for improving meat tenderness than selection for increased IMF because the level of genetic antagonism between CA and RPY was not as great as that between IMF and RPY.

Topics: Adipose Tissue; Animals; Body Composition; Breeding; Calcium-Binding Proteins; Calpain; Cattle; Least-Squares Analysis; Male; Meat; Muscles; Phenotype; Postmortem Changes; Weight Gain

The detailed localization of mRNAs for calpain II and calpastatin was examined in adult rat brain by in situ hybridization histochemistry. The expression patterns of the two mRNAs were similar to each other throughout the brain in terms of relative expression intensity, and almost all neurons expressed both mRNAs more or less. Among them, neurons in cranial nerve nuclei and some others in the brain stem expressed at relatively high levels, suggesting the high involvement of the non-lysosomal proteolytic system in the function of these neurons. On the other hand, the expression levels of the two mRNAs in non-neuronal cells including glia were basically low with the choroid plexuses expressing calpastatin mRNA relatively highly.

Topics: Animals; Base Sequence; Brain; Brain Chemistry; Calcium-Binding Proteins; Calpain; Gene Expression; In Situ Hybridization; Male; Molecular Sequence Data; Nerve Tissue Proteins; Rats; Rats, Wistar; RNA, Messenger

Calpain requires Ca2+ both for proteolysis of its substrates and for interaction with its endogenous inhibitor, calpastatin. Although calmodulin-like domains (CaMLDs) of large and small subunits of calpain have been suggested to be the sites for Ca(2+)-dependent interaction with calpastatin, specificity and molecular basis of the interaction have remained unclear. We investigated the interaction between the CaMLD of human mu-calpain large subunit expressed in Escherichia coli and a 19-residue synthetic oligopeptide corresponding to the region A (the amino-terminal conserved acidic region) of one of the four repetitive functional domains of calpastatin. The recombinant CaMLD bound to the oligopeptide immobilized on Sepharose beads in a Ca(2+)-dependent manner. The CaMLD failed in binding to a mutant oligopeptide with one amino acid substitution. Enhancement of fluorescence intensity of a hydrophobic probe, 2-(p-toluidino)naphthalene-6-sulfonate, was observed upon incubating with the CaMLD and further increased by Ca2+. The Ca(2+)-dependent enhancement of fluorescence intensity was strongly suppressed by the wild type oligopeptide, but not by the mutant one. Kinetic experiments were performed with BIAcore where binding of the CaMLD to the oligopeptide immobilized on a biosensor chip was detected as real time signals of surface plasmon resonance. The determined dissociation constant (KD) was 3.1 x 10(-9) M. These results suggest that the region A of calpastatin binds to the CaMLD in a specific manner similar to interactions between calmodulin-binding peptides and calmodulin where hydrophobic properties are known to be important.

Topics: Amino Acid Sequence; Calcium; Calcium-Binding Proteins; Calmodulin; Calpain; Humans; Kinetics; Molecular Sequence Data; Protein Binding; Recombinant Proteins; Spectrometry, Fluorescence

Micromolar and millimolar Ca(2+)-requiring neutral protease (calpain I and calpain II) along with their endogenous inhibitor calpastatin were isolated and partially purified from the same preparation of rat intestinal epithelial cells. Calpain I and II were partially purified by 1300 and 900-fold with 57 and 53 per cent yield, respectively. The optimum assay conditions revealed pH 7.5, 20 min incubation at 25 degrees C and 0.24% casein substrate for both calpains. The optimum calcium concentration obtained for calpain I and II were 25 microM and 4 mM, respectively. Distribution of rat intestinal epithelial cells calpain I and II along with calpastatin during cell differentiation stages in weanling to senescence age were studied. Calpain I in weanling rats was in an increasing order from villus to crypt regions. Adult rats indicated well expressed consistent calpain I throughout the differentiation stages. Whereas, significant lowering towards crypt region cells were evident in old rats. Calpain II in weanling and adult rats was found to be consistent throughout the differentiation stages. Old animals revealed an increasing trend from villus to crypt region with insignificant activity present in upper villus cells. Concomitantly, different concentrations of calpastatin were observed throughout the differentiation stages in all the age groups. Moreover, the levels of calpains exceeded that of calpastatin in most of the epithelial cell populations during developmental stages. In addition to casein, intestinal epithelial cell membranes were found to be equally good substrates for calpains. Proteolytic susceptibility of weanling, adult and old rat membrane proteins varied significantly all along the ageing process in rats. Simultaneous age-dependent calpastatin response were also evident. Taken together the results obtained provided strong evidence that calpain plays significant role in rat intestinal cell differentiation and ageing process with calpastatin as its specific regulatory protein.

Topics: Aging; Animals; Calcium; Calcium-Binding Proteins; Calpain; Caseins; Cell Differentiation; Cell Membrane; Cysteine Proteinase Inhibitors; Epithelial Cells; Epithelium; Hydrogen-Ion Concentration; Intestine, Small; Isoenzymes; Male; Rats; Rats, Wistar; Substrate Specificity

Rabbits were subjected to hypoxia (5% O2) for up to 90 min and allowed to recover for a maximum of 4 days. Hippocampus homogenate was assayed for fodrin breakdown product (BDP). After separation into a nuclear and mitochondrial fraction (NMF), a membrane and microsomal fraction (MMF), and a cytosolic fraction (CF), samples were assayed for mu-calpain, m-calpain, and calpastatin immunoreactivity. Calpain and calpastatin immunoreactivity decreased in the NMF and CF but increased in the MMF during hypoxia and short-term recovery. This translocation occurred in parallel with the increase in fodrin BDP. Because the increase in the MMF was not large enough to explain the decrease in the other two fractions, it was assumed that the translocation and activation was accompanied by a reduction in the total amounts of calpains and calpastatin. Glucocorticoid pretreatment (beta-methasone, 0.4 mg x kg-1 x day-1) for 7 days produced a decrease in the ratio of activated mu-calpain in all three fractions in nearly all samples before, during, and after hypoxia, compared with untreated animals. Glucocorticoid pretreatment also prevented the increase in fodrin BDP that occurred in untreated animals during hypoxia and short-term recovery, indicating impairment of calpain activation.

Topics: Animals; Calcium-Binding Proteins; Calpain; Carrier Proteins; Cell Membrane; Cell Nucleus; Cytosol; Glucocorticoids; Hippocampus; Hypoxia; Microfilament Proteins; Microsomes; Mitochondria; Rabbits; Subcellular Fractions

The effect of protein depletion and refeeding with a normal diet on mouse liver soluble homogenate calpain activity was studied. It was unchanged when expressed in terms of whole liver (units/liver). However, when expressed in terms of degradable protein (units/mg protein) it increased with depletion and decreased with refeeding. DEAE Sephacel chromatography of soluble homogenate yielded three calpain activities which were eluted at 0.04, 0.16 and 0.23 M NaCl, respectively. On the basis of whole liver, they decreased with depletion and increased with refeeding. Immunochemical analysis revealed similar changes in the mass of the calpain eluted with 0.16 M NaCl. The sum of these three activities (total liver calpain activity) was higher than the activity displayed by the soluble homogenate, indicating that they were separated from calpastatin. Furthermore, the percentage of total calpain activity displayed by soluble homogenate increased with depletion and decreased with refeeding, suggesting that depleted liver had the lowest calpastatin content. This was confirmed by direct measurements which indicated that depleted homogenate had in average 5.5 and 3.1 times less calpastatin compared to normal and 16 hours refed liver, respectively. It is concluded that a remarkable decrease in calpastatin content maintained unchanged whole liver soluble homogenate calpain activity during protein depletion and refeeding and contributes to an increased calpain activity related to degradable protein in depleted livers. This increase is in accordance with the high in vivo rate of protein breakdown depicted by these livers.

Topics: Animals; Calcium-Binding Proteins; Calpain; Chromatography, DEAE-Cellulose; Diet; Dietary Proteins; Electrophoresis, Polyacrylamide Gel; Female; Immunodiffusion; Kidney; Liver; Mice; Protein Deficiency

The chronology of appearance of calpain I and calpain II was analyzed during myogenesis of embryonic myoblasts in culture. The influence of the hormones insulin and corticosterone, and insulin growth factor-1 (IGF-1) and transforming growth factor-beta (TGF-beta) on the modulation of calpain-calpastatin levels during myogenesis was also analyzed. Immunodetection assays using specific antibodies and enzymic activities showed that during muscle cell differentiation in vitro, calpain II is present from the beginning of myoblast fusion (2nd day) increasing until the 6th day and then reaching a plateau. These observations were confirmed by an analysis of the expression of total calpain mRNAs which followed the same time profile, thereby providing evidence for a transcriptional regulation in the expression of calpains. Even if an increase in calpain II activity occurs at approximately the same time as an increase of fusion, calpain II activity and rate of fusion are not closely correlated. The involvement of calpain II in some event that follows myoblast fusion is suggested. On the other hand, calpain I and calpastatin were detected only on the 6th day of cell culture growth; these results enable us to argue that if calpain I has any biological role (which remains to be established), this role occurs during the final stages of muscle cell differentiation. The presence of exogenous factors which are known to affect muscle cell differentiation by altering either the rate of protein synthesis, or degradation or both, significantly affects the modulation of calpain-calpastatin levels. Such a regulation at the transcriptional level suggests that calpains do not act as housekeeping enzymes during myogenesis.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cells, Cultured; Cortisone; Gene Expression Regulation; Insulin; Insulin-Like Growth Factor I; Isoenzymes; Muscles; Rats; Rats, Wistar; RNA, Messenger

The calcium-activated neutral protease, calpain, was tested for its proteolytic effects to degrade the cell membrane spectrin-like protein, fodrin, during hypoxia. Cardiac myocytes, isolated from neonatal rat hearts, were incubated under hypoxic conditions for 6 hours. The cell death during hypoxia rose to 80% after 6 hours. Extracellular protease activity was much more elevated during hypoxia, than in aerobic states at 6 hours. Intracellular protease activity was also elevated in hypoxia. These protease activities were markedly inhibited by the cysteine protease inhibitor E-64 and the calpain specific inhibitor, calpastatin. Hypoxic cell death was also suppressed by E-64. Cell membrane proteins prepared from hypoxic myocytes were examined with electroblots stained for fodrin by the peroxidase method. A 125 kilodalton immunoreactive degradation product of fodrin was found under hypoxic conditions. Treatment with E-64 inhibited both the appearance of this degradation band and the decrease in the content of fodrin. These observations indicate that calpain is activated during hypoxia and that it is related to cell membrane protein degradation, especially in fodrin. The data also suggest that protease inhibitor E-64 treatment may be beneficial in protection against hypoxic myocyte injury.

Topics: Animals; Animals, Newborn; Calcium; Calcium-Binding Proteins; Calpain; Cell Death; Cell Hypoxia; Cysteine Proteinase Inhibitors; Heart; Hydrogen-Ion Concentration; Leucine; Myocardium; Rats; Rats, Wistar

Two calpastatins, with Mr 110 KD and named calpastatin I and II, have been isolated from rat heart and kidney and displayed distinct inhibitory efficiency with mu- and m-calpain, respectively, as those isolated from rat skeletal muscle. Whereas the level of calpastatin I always exceeds that of mu-calpain, the level of calpastatin II appears to be more closely correlated to the level of m-calpain. As previously shown for skeletal muscle, the two inhibitor proteins can be interconverted by a phosphorylation-dephosphorylation reaction; the enzyme responsible for phosphate incorporation in calpastatin I is now identified in c-AMP dependent protein kinase A. In rat erythrocytes, containing a single calpain form, the single low Mr calpastatin form does not undergo reversible phosphorylation and is equally efficient in respect to typical mu- and m-calpain. The presence of two interconvertible calpastatin forms provides the cells with a highly sensitive mechanism of regulation of the Ca(2+)-dependent proteolytic system.

Topics: Animals; Calcium-Binding Proteins; Calpain; Chromatography, Affinity; Chromatography, DEAE-Cellulose; Cyclic AMP-Dependent Protein Kinases; Cysteine Proteinase Inhibitors; Erythrocytes; Globins; Humans; Kidney; Kinetics; Myocardium; Phosphorylation; Protein Kinase C; Protein Kinases; Rats

The activation of calpain in normal human erythrocytes incubated in the presence of Ca2+ and the Ca2+ ionophore A23187 led to the decline of the Ca(2+)-dependent ATPase activity of the cells. Preloading of the erythrocyte with an anticalpain antibody prevented the decline. The pump was also inactivated by applied to isolated erythrocyte plasma membranes. The decline of the pump activity corresponded to the degradation of the pump protein and was inversely correlated to the amount of the natural inhibitor of calpain, calpastatin, present in the cells. In erythrocytes containing only 50% of the normal level the degradation started at a concentration of Ca2+ significantly lower than in normal cells. A comparison of the concentrations of Ca2+ required for the degradation of a number of erythrocyte membrane proteins showed that the Ca2+ pump and band 3 were the most sensitive. All other membrane proteins tested were attacked at higher levels of intracellular Ca2+. Thus, the degradation of the Ca2+ pump protein may be a simple and sensitive means to monitor calpain activation in vivo. Furthermore, the results have shown that the calpastatin level correlated directly with the amount of activable calpain and with the concentration of Ca2+ required to trigger the activation process.

Topics: Calcium; Calcium-Binding Proteins; Calcium-Transporting ATPases; Calpain; Erythrocytes; Humans

The integrin beta 4 subunit often undergoes proteolytic cleavage within its long cytoplasmic tail to yield a characteristic protein pattern of 205, 165, and 125 kDa. The results in this study suggest that beta 4 cleavage often occurs during or after cell lysis, where it was readily inhibitable by calcium chelators (EDTA, EGTA) and inhibitors of cysteine proteases (E64c, leupeptin). The cleavage of beta 4 is catalyzed by a calpain-like enzyme because (i) it requires calcium, (ii) it is mimicked by purified milli-calpain, and (iii) it is inhibited by several calpain inhibitors including the calpain-specific inhibitor calpastatin. Within intact cells, cleavage of beta 4 was cell type-specific and observed only when the cells were made permeable to calcium. Substantial cell viability was retained during beta 4 cleavage induced by ionomycin plus calcium, indicating that cleavage within intact cells was not necessarily a consequence of cell death. However, manipulations of cells including suspension, synchronization, and stimulation with serum, phorbol esters, or other agents all failed to induce cleavage, suggesting that if cleavage is physiologically relevant, it is not easily duplicated in vitro. Analysis of multiple cell types showed a wide variation in beta 4 sensitivity to proteolytic cleavage, suggesting that this process might be differentially regulated depending on the cellular environment.

Topics: Antigens, Surface; Artifacts; Calcium; Calcium-Binding Proteins; Calpain; Cell Adhesion; Cell Survival; Digitonin; Egtazic Acid; Endopeptidases; Humans; Integrin alpha6beta4; Integrin beta4; Integrins; Ionomycin; Peptide Fragments; Tumor Cells, Cultured

Calpastatin is a specific inhibitor of the calpains. Calpains play a key role in postmortem tenderization of meat and have been hypothesized to be involved in muscle protein degradation in living tissue. Isolation, cloning of complementary DNA, and nucleotide sequencing of bovine calpastatin from the longissimus muscle have been completed. Two clones were identified that encompass the entire coding sequence. Clone pCR41, derived by reverse transcription-PCR, covers domains L and 1; clone pBSA1, obtained from cDNA library screening, covers domains 2 through 4 in addition to the 3'-nontranslated region. Nucleotide sequence analysis of the cDNA for bovine calpastatin revealed an average nucleotide sequence identity of approximately 70 to 80% compared with published calpastatin nucleotide sequences of human, rabbit, and pig. Exon 3, corresponding to a highly conserved 22-amino acid region, was deleted from bovine calpastatin domain L. The calculated molecular weight of bovine skeletal muscle calpastatin of 706 amino acid residues (M(r) 75,842) corresponds to the value of purified bovine skeletal muscle calpastatin as determined by SDS-PAGE (M(r) 68,000). Northern blot analysis revealed the presence of multiple calpastatin mRNA transcripts having estimated sizes of 3.8, 3.0, and 1.5 kb in beef and 3.8, 3.0, 2.5, and 1.5 kb in sheep. Calpastatin mRNA expression was increased with beta-adrenergic agonist-induced muscle hypertrophy.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Calcium-Binding Proteins; Calpain; Cattle; Cloning, Molecular; Consensus Sequence; DNA; DNA Primers; Gene Expression Regulation; Gene Library; Male; Molecular Sequence Data; Muscles; Polymerase Chain Reaction; RNA, Messenger; Sequence Analysis, DNA

An experiment was conducted to determine whether prostaglandin F2 alpha (PGF2 alpha)-induced secretion of oxytocin (OT) by the bovine corpus luteum (CL) was associated with changes in the activities of protein kinase-C (PKC), calpains, and calpastatin. On day 8 of the estrous cycle (estrus = day 0), beef heifers were restrained and given a 500-micrograms iv injection of cloprostenol, a PGF2 alpha analog. Corpora lutea were surgically removed from beef heifers 0, 2, 7.5, or 30 min (n = 4 animals/time period) after cloprostenol injection. Blood samples were collected before injection and at frequent intervals after injection. Distribution of PKC activity in cytosol and membrane fractions and activities of microcalpain, millicalpain, and calpastatin were determined for all CL. OT was measured in plasma and tissue by RIA. Relative to mean plasma levels of OT at time zero (85 +/- 7 pg/ml), peak plasma levels occurred between 1.5-10 min (270 +/- 36 pg/ml) for all animals. The mean luteal concentration of OT was greater at 0, 2, and 7.5 min (145 +/- 27, 232 +/- 82 and 269 +/- 115 ng/g, respectively) than at 30 min (93 +/- 33), but differences in tissue OT over time were not significant (P > 0.05). PKC activities (percentage over nonactivated control values) in the membrane or cytosolic fractions did not differ significantly among the times of CL removal; however, membrane PKC activity was positively correlated with the plasma OT level at the time of CL removal (r = 0.82; P < 0.0025). Luteal millicalpain activity was approximately twice that of microcalpain at each time point (P < 0.001), although the activities of the individual calpains over time after PGF2 alpha injection did not change. Calpastatin activity was significantly higher at 30 min (515 +/- 28 U/g tissue) than at 0, 2, or 7.5 min (373 +/- 26, 423 +/- 26, and 426 +/- 24 U/g tissue, respectively). PKC activity in the membrane appears to be positively correlated with OT secretion from the bovine CL, and increased calpastatin activity after PGF2 alpha injection may inhibit calpains present in the CL, thereby maintaining an active pool of PKC.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Cell Membrane; Corpus Luteum; Cytosol; Dinoprost; Female; Oxytocin; Protein Kinase C; Radioimmunoassay; Time Factors

The effect of castration on endogenous proteinase activity and myofibrillar protein turnover was investigated in cattle. Six each of MARC III composite bulls and steers weighing approximately 210 kg were given ad libitum access to a typical growing diet. At 0, 42, 84, 126, and 168 d, two consecutive 24-h urine samples were obtained. Urine was analyzed for N tau-methylhistidine (N tau MH) and creatinine. Following slaughter after 170 d on feed, a longissimus muscle sample was removed immediately from each carcass for quantification of mu-calpain, m-calpain, calpastatin, cystatin(s), cathepsin B, and cathepsin B + L activities. Bulls were heavier (P < .05) at 126 and 168 d and more efficient (P < .05) in conversion of feed to gain at 84 and 168 d than were steers. Compared with steers, bulls excreted less (P < .05) N tau MH at 84, 126, and 168 d and displayed lower (P < .05) fractional degradation rates (FDR) at all sample times. No differences (P > .05) in calpain or cathepsin activities were observed between bulls and steers. However, muscle from bulls had greater (P < .05) activities of calpastatin and cystatin(s) than that from steers. A negative relationship existed between d-168 FDR and calpastatin (r = -.72; P < .05) and cystatin (r = -.62; P < .05) activities. These results indicate that decreased FDR of skeletal muscle from growing bulls contributes to their greater efficiency of growth and could be related partially to cystatin-mediated cathepsin activity and(or) calpastatin-mediated calpain activity.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cathepsins; Cattle; Creatinine; Cystatins; DNA; Endopeptidases; Male; Methylhistidines; Muscle Development; Muscle Proteins; Muscles; Orchiectomy; Random Allocation; RNA; Weight Gain

Forty wether lambs were used in a 2 x 4 factorial arrangement to determine the response of animal performance, muscle growth, proteinase activity, and meat tenderness to beta-adrenergic agonist (BAA) supplementation. Lambs were fed a finishing diet with or without 4 ppm of L644,969 and slaughtered after 0, 2, 4, and 6 wk of treatment. The ADG was higher (P < .05) in the treated than in the control lambs after 2 wk and returned to control levels thereafter. Semitendinosus weight and calpastatin activity were higher and mu-calpain activity was lower in the treated than in the control lambs after 2, 4, and 6 wk. Cathepsin B activity was higher (P < .01) and cystatin-like activity was lower (P < .05) after 2 wk in treated than in control lambs but returned to control levels thereafter. Longissimus protein:DNA was higher after 4 (P < .05) and 6 (P < .01) wk in the treated lambs than in the controls. The concentration of RNA and RNA:DNA ratio were higher (P < .01) in the longissimus and semitendinosus muscles in the treated lambs after 2 wk and remained higher throughout the study. Semitendinosus protein and RNA content were higher after 2, 4, and 6 wk and DNA content was higher after 2 and 6 wk in the treated than in the control lambs. Longissimus shear-force values were higher (P < .001) in the treated than in the control lambs at all slaughter end points. These data indicate a rapid alteration of muscle growth, activity of the calpain-calpastatin system, and meat tenderness during BAA treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adrenergic beta-Agonists; Aging; Animal Feed; Animals; Calcium-Binding Proteins; Calpain; Cathepsins; Cystatins; DNA; Endopeptidases; Male; Meat; Muscle Development; Muscle Proteins; Muscles; Pyridines; Random Allocation; RNA; Sheep; Weight Gain

Calpain, a calcium-activated neutral proteinase, is ubiquitously present in human tissues. To determine if lymphoid cells implicated in pathogenesis of demyelination may harbor calpain in a functionally active form, we determined both muCalpain and mCalpain activities in human lymphoid cell lines. DEAE-cellulose and phenylsepharose column chromatography were used to isolate the enzyme from the natural inhibitor, calpastatin. Lymphocytic lines (CCRF-CEM, MOLT-3, MOLT-4, M.R.) showed predominance of muCalpain (55-80%) whereas the monocytic line (U-937) showed predominance of mCalpain (77%). Proportion and subcellular distribution of both isoforms varied among cell lines. Calpains isolated from U-937 cells degraded myelin basic protein. These results indicate that human lymphoid cells harbor functionally active calpain that can degrade myelin components in vitro. The study suggests a degradative role for calpain in demyelinating diseases.

Topics: Calcium-Binding Proteins; Calpain; Cell Membrane; Cytosol; Humans; Lymphocytes; Myelin Basic Protein; Myelin Sheath; Tumor Cells, Cultured

Prolonged dietary inclusion of beta-adrenergic agonists can induce skeletal muscle hypertrophy in meat animals, by a mechanism probably related to the calcium-dependent proteolytic enzymes, or calpains, and in particular to their specific inhibitor calpastatin. Calpain and calpastatin activities are also believed to be important factors during post-mortem tenderisation of meat. beta-Agonist treatment is generally associated with increased calpastatin activity, which may lead to meat toughness. The aim of the present study was to examine the effect of a short period of cimaterol (feeding for 8 days, followed by reversion to a normal diet for a further 24 days) on muscle growth and on calpain isoform and calpastatin activities and specific mRNA abundance in the longissimus dorsi (LD) muscle. Significant changes were detected in LD wet weight and in calpastatin activity and mRNA after only 8 days treatment with cimaterol. After 24 further days on a control diet, both LD wet weight and calpastatin activity were not significantly different (P > 0.05) from untreated controls of the same age, although calpastatin mRNA stayed surprisingly high. In contrast to several earlier studies, changes in calpain I (or mu-calpain) and calpain II (or m-calpain) activity and calpain I mRNA were not significantly different (P > 0.05) from controls in any groups. These data suggest that calpastatin activity rather than the activity of either calpain isoform is closely linked to beta-agonist-induced muscle hypertrophy. Changes in calpastatin mRNA are not directly proportional to inhibitory activity, suggesting that variable mRNA species may be transcribed, spliced or stabilised, but not necessarily translated as part of the beta-agonist response.

Topics: Adrenergic beta-Agonists; Animals; Base Sequence; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Diet; Ethanolamines; Female; Molecular Sequence Data; Muscle Development; Muscles; RNA, Messenger; Sheep

1. Activities of calpains I and II and calpastatin in two different types of porcine skeletal muscles were compared. 2. Semimembranosus muscle was composed of mixed fiber types with mainly glycolytic fibers (65%), while masseter muscle was composed exclusively of oxidative fibers. 3. Calpain I activity was lower and calpain II activity was higher in masseter muscle as compared to semimembranosus muscle. Calpastatin activities were similar in both muscles.

Topics: Animals; Calcium-Binding Proteins; Calpain; Male; Muscles; Swine

Neonatal rats were subjected to transient cerebral hypoxic-ischemia (unilateral occlusion of the common carotid artery plus 7.7% O2 for 2 h) and allowed to recover for 0 min, 30 min or 20 h. The calpain and calpastatin activities were assayed in subcellular fractions of the ipsilateral, hypoxic-ischemic and the contralateral, hypoxic hemisphere. An upregulation of calpain activity occurred in the hypoxic hemisphere, both in the major, cytosolic fraction and in the hypotonic, membrane associated fraction (110% and 133% of controls, respectively). The hypoxic-ischemic hemisphere displayed a decrease in calpain activity in the cytosolic fraction but an increase in the hypotonic fraction (90% and 111% of controls, respectively). The changes in calpastatin activity were less pronounced. This indicates that an upregulation of calpain activity occurs in parallel with development of hypoxic-ischemic damage. However, this upregulation is not necessarily coupled to development of injury as lesions are not seen in the hypoxic hemisphere.

Topics: Animals; Brain; Calcium-Binding Proteins; Calpain; Humans; Hypoxia, Brain; Infant, Newborn; Ischemic Attack, Transient; Rats; Rats, Sprague-Dawley; Subcellular Fractions; Up-Regulation

1. The objective of this study was to estimate the difference between broiler and layer chicks in the activities of calpain and calpastatin (inhibitor of calpain) in breast muscle. Differences between broilers and layers in body weight, daily gain at 3 weeks of age and fractional growth rate (FGR) during 2 and 3 weeks of age were statistically significant (P < 0.01). 2. Calpain and calpastatin activities were measured at three weeks of age with alkali-denatured casein as a substrate. The m-calpain (calpain activated by millimolar calcium concentration) activities in units/g muscle and units/mg extractable muscle protein were 0.779 and 0.353 for broilers, and 1.042 and 0.440 for layers, respectively. The calpastatin activities in units/g muscle and units/mg extractable muscle protein were 0.332 and 0.153 for broilers, and 0.262 and 0.112 for layers, respectively. 3. Broilers with high FGR showed low m-calpain and high calpastatin activities. In contrast, layers with low FGR showed high m-calpain and low calpastatin activities. 4. These results suggest that m-calpain and calpastatin activities in skeletal muscle vary between breeds which have different rates of muscle production.

Topics: Animals; Body Weight; Calcium-Binding Proteins; Calpain; Chickens; Muscles; Substrate Specificity

Calpain autolyses in the presence of Ca2+. In the case of m-calpain (80 + 30 kDa) the first product is an 80 + 18 kDa species which has an intact large subunit and the C-terminal Ca(2+)-binding domain of the small subunit. It was possible to bind E64 into the active site of calpain in the presence of Ca2+ before cleavage of either calpain subunit. This suggests that the active site is functional before any autolysis has occurred and that calpain is not a proenzyme. Prolonged autolysis generates several fragments including a 42 kDa active-site domain fragment that showed no proteolytic activity and Ca(2+)-binding domain fragments. Some of the Ca(2+)-binding domain fragments were found to exist as heterodimers (23 + 18 kDa and 22 + 18 kDa), with the Ca(2+)-binding domain of the large subunit interacting with the Ca(2+)-binding domain of the small subunit. These species were true heterodimers, as they showed co-elution of the two Ca(2+)-binding domains on ion-exchange and gel-filtration chromatography, and immunoprecipitation of both polypeptides with an antiserum specific for the small-subunit Ca(2+)-binding domain. The generation of the dimer species after only 15 min autolysis suggests that the interaction between the Ca(2+)-binding domains is present in the native calpain structure. The interaction of calpain with calpastatin was investigated using an assay based on binding to calpastatin-Sepharose and a competitive binding assay. Calpain, active-site-blocked calpain and calpain fragments generated by autolysis were studied. Calpain bound to calpastatin in the presence of Ca2+; however, the isolated active-site-containing 80 kDa large subunit (proteolytically inactive), a 42 kDa active-site-containing fragment (proteolytically inactive) and Ca(2+)-binding domain fragments of calpain did not. Active-site-blocked calpain bound to calpastatin, but with an affinity reduced by approximately two orders of magnitude when compared with native calpain.

Topics: Amino Acid Sequence; Animals; Binding Sites; Blotting, Western; Calcium-Binding Proteins; Calpain; Chromatography, Affinity; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Kidney; Macromolecular Substances; Mass Spectrometry; Molecular Sequence Data; Molecular Weight; Myocardium; Peptide Fragments; Swine

The ultrastructural localization of calpastatin, the endogenous inhibitor of the neutral calcium-dependent proteases (calpains), was investigated in rabbit skeletal muscle fibers using a polyclonal antibody against the 34 kDa form of the inhibitor isolated from rabbit. Quantitative studies by pre- and postembedding immunogold techniques revealed that the distribution pattern of the specific immunoreactivity included: 1) the sarcolemma with the adjacent cytoplasm (about 1 micron wide); 2) the myofibrils; 3) the mitochondria and 4) the nuclei (condensed as well as extended chromatin). Other cell substructures, such as lysosomes and the intermyofibrillar cytoplasm, were substantially devoid of immunoreactivity. Furthermore, in accordance to previous light microscope immunohistochemical experiments, an extracellular (endomysial) localization of specific immunoreactivity was confirmed. These results favour the view, which is also supported by a series of biochemical evidences, that calpastatin in rabbit skeletal muscle is present in cell structures also containing calpains and/or their putative substrates. The above multiple patterns of distribution also suggest that the muscular calpain-calpastatin system in skeletal muscle fibers may play different physiological roles in the various subcellular compartments.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Nucleus; Immunohistochemistry; Microscopy, Immunoelectron; Mitochondria; Muscles; Myofibrils; Rabbits; Sarcolemma

Topics: Animals; Calcium-Binding Proteins; Calpain; Cathepsins; Male; Myocardium; Sheep

Using a rapid FPLC procedure for the separation of calpain I, calpain II and the inhibitor calpastatin from pork meat, results of proteolytic and inhibitory activity were obtained. Duplicate measurements showed variation coefficients less than 0.13. Compared to others we found lower calpain I activity 1 h post-mortem. Three groups of pigs were randomly selected. One group was exposed to work immediately before slaughter, another group was exposed to work but allowed 30 min rest before slaughter, while the third group was a control group. Samples were taken from m longissimus dorsi 1 h and 6 h post-mortem. Work had no influence on the calpain/calpastatin activity, but a significant decrease in the calpain and calpastatin activity 6 h post-mortem was found. It should be noted that the results are preliminary and are part of an ongoing study. The method described allows separation of three extracts per instrument per day followed by activity determinations of calpains and calpastatin by the casein assay.

Topics: Animals; Autolysis; Calcium-Binding Proteins; Calpain; Chemical Fractionation; Chromatography, High Pressure Liquid; Cysteine Proteinase Inhibitors; Muscles; Reproducibility of Results; Swine

The objectives of this study were to examine the effects of castration on the calpain proteinase system (mu-calpain, m-calpain, and calpastatin) activities and meat tenderness. Six each, MARC III bulls and steers were slaughtered at approximately 12 mo of age. Longissimus muscle samples were obtained for determining myofibril fragmentation index, Warner-Bratzler shear force, and sensory panel evaluation at 1, 7, and 14 d postmortem, and mu- and m-calpain and calpastatin activities at 24 h postmortem. Bulls produced leaner carcasses with lower (P < .05) quality grades than did steers. Meat from bulls had higher (P < .05) shear force values than meat from steers; however, sensory panelists were unable (P > .05) to detect differences in tenderness or other sensory traits between bulls and steers. Activities of mu- and m-calpain were not affected (P > .05) by castration; however, calpastatin was higher (P < .05) in muscles from the bull carcasses. Lower (P < .05) myofibril fragmentation index values indicate that less proteolysis occurred in muscle from bulls than in muscle from steers during the first 7 d postmortem. Greater calpastatin 24-h activity may be associated with the increased shear force of meat from bulls.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Male; Meat; Muscles; Orchiectomy; Postmortem Changes; Random Allocation; Taste

The localization of the two Ca-activated extralysosomal proteases m-calpain and mu-calpain in the eye of the adult rabbit was examined by immunohistochemistry, using poly- and monoclonal antibodies against the corresponding rabbit antigens. Immunoreactivity against the two forms of calpains was observed in the epithelial cells on the external and internal surface of the cornea as well as in the epithelial cells covering the iris and ciliary body. The sclera and choroid layers showed a relatively weak immunoreactivity. Using anti m-calpain antibodies, the pigment epithelium in the retina was heavily labelled as well as the outer and inner plexiform layers. The other and inner borders of the Müller cells were clearly labelled. The outer segments of the receptor cells showed a strong immunoreactivity for both mu-calpain and m-calpain. Labelling was also observed in the retinal ganglion cells and in the nerve fiber layer. The immunohistochemical localization of calpastatin, an endogenous inhibitor of both m- and mu-calpain was also examined. A high level of calpastatin immunoreactivity was observed in the outer segments of the receptor cells. The results may be compatible with a role for calpains, especially m-calpain, in the secretory/phagocytic process and as modulators of the cytoskeleton in cell processes.

Topics: Animals; Calcium-Binding Proteins; Calpain; Ciliary Body; Cornea; Eye; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique; Immunohistochemistry; Iris; Organ Specificity; Rabbits; Retina

Calpain requires Ca2+ for both proteolysis of its substrates and interaction with its endogenous inhibitor, calpastatin. The mechanism of inhibition of calpain by calpastatin has remained unsolved, although Nishimura and Goll [J. Biol. Chem. 266, 11842-11850 (1991)] reported that autolyzed calpain fragments containing calmodulin-like domains (CaMLDs) bound to an immobilized calpastatin column. We investigated the correlation between CaMLD-binding and calpain inhibition using immobilized columns of gene-engineered CaMLDs derived from the human mu-calpain large subunit and various recombinant calpastatin mutants. Among the four internally repetitive inhibitory domains of calpastatin, each having conserved regions A, B, and C, only domains 1 and 4 showed the binding activity. The region B deletion mutant of domain 1, retaining the CaMLD-binding ability, no longer had the calpain inhibition activity, and became susceptible to proteolysis. In contrast, a synthetic oligopeptide of region B with moderate calpain inhibition activity did not bind to the column. Domain 3 acquired the binding ability on substitution of region A with that of domain 1. These results suggest that calpain inhibition and binding to the CaMLDs are not correlated or mediated by different subdomains of calpastatin.

Topics: Amino Acid Sequence; Base Sequence; Binding Sites; Calcium; Calcium-Binding Proteins; Calmodulin; Calpain; Electrophoresis, Polyacrylamide Gel; Glutathione Transferase; Magnesium; Molecular Sequence Data; Recombinant Fusion Proteins; Sulfonamides

Rat intoxication with a single dose of the hepatotoxin carbon tetrachloride induces a significant modification of liver protein kinase C total activity which depends on the degree of the intrahepatocyte oxidative unbalance provoked by various concentrations of the haloalkane. Low carbon tetrachloride amounts stimulate total protein kinase C activity, while one order of magnitude higher amounts exert strong enzyme inhibition. The latter effect is due to an early inactivation followed with progress of time by a proteolytic degradation of the enzyme. A pathological recruitment of the calcium-dependent protein kinase C regulatory enzymes calpain and calpastatin appears responsible for protein kinase C loss. The prolonged excess of cytosolic calcium which characterizes the single high dose carbon tetrachloride poisoning also leads to inactivation of calpain II and calpastatin in a time-dependent manner.

Topics: Animals; Calcium-Binding Proteins; Calpain; Carbon Tetrachloride; Carbon Tetrachloride Poisoning; Dose-Response Relationship, Drug; Immunoblotting; Isoenzymes; Kinetics; Liver; Male; Oxygen Consumption; Protein Kinase C; Rats; Rats, Wistar; Time Factors

Calpain and its endogenous inhibitor, calpastatin, were isolated from erythrocytes of various mammals and their properties were compared. It has been widely believed that mammalian erythrocytes contain only mu-calpain. However, rat and human erythrocytes were found to contain two species of calpain, identified as mu-calpain and m-calpain from their elution positions on DEAE-cellulose column chromatography and their Ca(2+)-requirements. Thus, it is apparent that rat and human erythrocytes contain not only mu-calpain, but m-calpain as well. On the other hand, rabbit erythrocytes contain only mu-calpain. Western blot analysis showed that human and rabbit erythrocytes contain predominantly 70-kDa calpastatin (erythrocyte-type), but unnegligible amounts of 110-kDa calpastatin (tissue-type) are also present. Rat erythrocytes were shown to contain a calpastatin with a molecular mass of approx. 100 kDa almost exclusively; this molecular mass was in perfect coincidence with the mass of the calpastatin in rat lung. These results strongly suggest that rat erythrocytes contain a tissue-type calpastatin. No essential change in the calpain/calpastatin system during maturation of rabbit reticulocytes into mature erythrocytes was observed.

Topics: Animals; Blotting, Western; Calcium-Binding Proteins; Calpain; Cell Differentiation; Chromatography, DEAE-Cellulose; Erythrocytes; Humans; Rabbits; Rats; Rats, Wistar; Reticulocytes; Species Specificity

Intracellular localization of calpain (calcium dependent cysteine proteinase) was studied in resting or activated human platelets. When stimulated with 2 U/ml thrombin, approximately 40% of total cellular calpain activity and 25% of antigen translocated mainly to the intracellular membrane fractions with autolytic activation. Translocation of calpain was completely abolished by the addition of EDTA to the sonication medium. However an endogenous calpain inhibitor (calpastatin) activity was not detected in the membrane fractions both in resting and in thrombin stimulated platelets. Translocation of calpain was also observed in the platelets stimulated with ionomycin, collagen or phorbor myristate acetate (PMA). These data suggest that cytosolic calpain reversibly translocates to the intracellular membranes during platelet activation without an interference by calpastatin.

Topics: Blood Platelets; Calcium-Binding Proteins; Calpain; Cell Membrane; Collagen; Cysteine Proteinase Inhibitors; Edetic Acid; Humans; Immunoblotting; Intracellular Membranes; Ionomycin; Tetradecanoylphorbol Acetate; Thrombin

Calpain, a ubiquitously distributed intracellular cysteine protease in animals, is thought to be activated at the cell membrane in the presence of micromolar levels of calcium ions. Calpastatin, the sole specific proteinaceous inhibitor for calpain, inhibits not only proteolytic activity, but also the binding of calpain to cell membranes. The inhibition of calpain binding to membranes by calpastatin fragments was analyzed. A calpastatin peptide containing the inhibitory sequence did not inhibit the binding of calpain to membranes. On the other hand, binding was prevented by a fragment without the activity to inhibit proteolytic activity. These data indicate that calpain binds to cell membranes through a site (regulatory site) other than the active site and that calpastatin inhibits the binding of calpain to cell membranes via a site (regulatory inhibition site) other than the inhibitory sequence. Calpain and calpastatin can undergo independent interactions: interaction between the catalytic site of calpain and the inhibitory sequence of calpastatin, and interaction between the regulatory site of calpain and the regulatory inhibition site of calpastatin. The interaction between the regulatory site of calpain and the regulatory inhibition site of calpastatin is essential for the regulation of calpain activity.

Topics: Amino Acid Sequence; Animals; Calcium; Calcium-Binding Proteins; Calpain; Enzyme Activation; Erythrocyte Membrane; In Vitro Techniques; Molecular Sequence Data; Peptide Fragments; Protein Binding; Rabbits

Vasospasm was produced in the canine basilar artery by a two-hemorrhage method, while contraction was induced in the normal canine basilar artery by a local application of KCl or serotonin after transclival exposure. The control animals were injected with saline instead of fresh blood. The activation of mu-calpain, a Ca(++)-dependent neutral protease, in the basilar artery was studied by evaluating the conversion from its inactivated into its activated form on immunoblots. In addition, the activity of calpastatin, an intrinsic inhibitor of calpain, in the basilar artery was determined by assay. The majority of the mu-calpain was inactivated in the control group. In the spastic group, mu-calpain was generally activated markedly in the early stage of vasospasm and moderately thereafter. The contraction induced by KCl or serotonin application was classified into the early phasic and the later tonic stages; mu-calpain was usually activated in the phasic stage and inactivated in the tonic stage. Calpastatin activity was significantly decreased during vasospasm, whereas it was not significantly changed in KCl- or serotonin-induced contraction. The final activity of mu-calpain results from the balance of mu-calpain and calpastatin. This suggests that mu-calpain activity was enhanced continuously in the spastic group and transiently in the KCl or serotonin group, and that the continuous activation of mu-calpain during vasospasm probably induced more proteolytic changes compared to those in the KCl or serotonin group.

Topics: Animals; Basilar Artery; Calcium-Binding Proteins; Calpain; Dogs; Immunoblotting; In Vitro Techniques; Ischemic Attack, Transient; Potassium Chloride; Serotonin

The effects of bovine skeletal muscle m-calpain and calpastatin on the degradation of casein and isolated bovine myofibrils were characterized under various pH values (7.0, 6.2, 5.7) and ionic strengths (32 to 400 mM KCl) at 25 degrees C. Caseinolytic assays indicated that m-calpain activity increased with increasing pH (P < .01) but decreased with increasing ionic strength (P < .01). Regardless of the presence of m-calpain, SDS-PAGE of myofibrils showed increased solubilization of myofibrillar proteins as pH and ionic strength increased. However, only in the presence of m-calpain were changes normally observed during postmortem storage reproduced. Protein release attributed to m-calpain activity increased with pH, but the effects of elevated ionic strength on the ability of m-calpain to hydrolyze myofibrillar proteins were not evident from SDS-PAGE, except for the decreased troponin-T degradation by m-calpain at the higher ionic strengths. A pH x ionic strength interaction was observed for calpastatin activity determined by caseinolytic assays (P < .01). No changes in m-calpain inhibition were detected at pH 7.0 and 6.2 at different ionic strengths. However, at pH 5.7 the ability of calpastatin to inhibit m-calpain decreased with increasing ionic strength. No changes in m-calpain inhibition could be detected with SDS-PAGE. Based on these results, it can be concluded that although m-calpain and calpastatin activities decrease with increasing ionic strength, their activities in the presence of myofibrils were not affected by ionic strengths typically found in postmortem muscle.

Topics: Animals; Calcium-Binding Proteins; Calpain; Caseins; Cattle; Culture Techniques; Hydrogen-Ion Concentration; Muscle Proteins; Muscles; Osmolar Concentration

The objective of this study was to examine the effects of clenbuterol administration on meat quality traits of veal. Sixteen Holstein-Friesian veal calves (male) were randomly assigned to one of four treatment groups; either control (n = 4) or clenbuterol-treated (1.6 micrograms.kg [corrected] BW-1.d-1, 42 d) with a withdrawal period between clenbuterol treatment and slaughter of 8 d (n = 4), 4 d (n = 4), or 2 d (n = 4). All animals were slaughtered at the same day at a commercial slaughterplant. At 30 min postmortem the carcasses were split and the right carcass side was electrically stimulated. After 24 h of cooling the longissimus, semimembranosus, triceps brachii, and psoas major muscles were excised and vacuum-packaged. After 1, 7, and 13 d of vacuum storage at 2 +/- 2 degrees C the muscles were sampled to determine tenderness, water-holding capacity, and color characteristics. Clenbuterol treatment resulted in a slower rate of pH decline in the unstimulated longissimus muscle but did not affect the ultimate pH. Clenbuterol treatment resulted in toughening of the longissimus, semimembranosus, and triceps brachii muscles after 1 and(or) 7 d of storage (P < .05). It is suggested that this resulted from a decrease in postmortem proteolysis because both the intensity of a 30-kDa peptide and the myofibril fragmentation index were lower in clenbuterol-treated muscles. Clenbuterol treatment resulted in increased lightness (L*-value) of longissimus and semimembranosus muscles (P < .05), coincident with a lower water-holding capacity. In a following experiment, the effect of clenbuterol administration (0 [n = 5] and 1.0 [n = 5] mg/kg of feed for 27 d) on calpain and calpastatin levels at 1 d postmortem in longissimus muscles of Friesian Pie Noire veal calves was investigated. Clenbuterol administration resulted in an increase in calpastatin levels (P < .05) and a trend (P < 0.1) toward a decrease in mu-calpain activity at 1 d postmortem.

Topics: Animals; Body Composition; Calcium-Binding Proteins; Calpain; Cattle; Clenbuterol; Electric Stimulation; Food Preservation; Hydrogen-Ion Concentration; Male; Meat; Muscle Proteins; Muscles; Pigmentation; Postmortem Changes; Random Allocation; Sarcomeres; Temperature

A new artificial cell adhesive protein was engineered by grafting the Arg-Gly-Asp (RGD) sequence, the minimal recognition signal of fibronectin for interaction with integrins, to a calpastatin segment by in vitro mutagenesis. The mutagenized protein showed cell adhesive activity in addition to calpain inhibitory activity. The RGD signal grafted to the calpastatin segment was recognized by the vitronectin receptor but not by the fibronectin receptor.

Topics: Amino Acid Sequence; Base Sequence; Calcium-Binding Proteins; Calpain; Cell Adhesion; Cell Adhesion Molecules; Cell Communication; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligopeptides; Receptors, Cytoadhesin; Receptors, Fibronectin; Receptors, Vitronectin; Recombinant Fusion Proteins; Signal Transduction

The aim of this study was to investigate the correlation between hypoxic myocardial cell injury and intracellular protease activity. Cardiac myocytes were isolated from neonatal rat hearts and cultured in Eagle's modified minimum essential medium. Myocytes were incubated in hypoxic conditions for 6 hours. The cell death rate during hypoxia rose to 80% after 6 hours. Extracellular protease activity was elevated to 4 units during hypoxia, much higher than the 0.7 units in aerobic states at 6 hours. This extracellular protease activity in hypoxic conditions was markedly inhibited by leupeptin and EDTA, and weakly inhibited by the cysteine protease inhibitor, NCO-700, but phenylmethyl sulfonyl fluoride did not inhibit the protease activity. To identify the protease activated during hypoxia, calpain-specific inhibitors were added to the incubation mixture. Calpain inhibitor 1 and calpastatin, an endogenous selective calpain inhibitor, markedly inhibited extracellular protease activity during hypoxia. NCO-700 also inhibited intracellular protease activity. NCO-700 reduced hypoxic cell death to 30% after 6 hours of hypoxygenation. These observations indicate that calpain is activated during hypoxia and leads to irreversible cell membrane degradation after 6 hours of hypoxygenation.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Hypoxia; Cells, Cultured; Glycoproteins; In Vitro Techniques; Myocardium; Piperazines; Protease Inhibitors; Rats

The Ca2+ activated neutral protease calpain II in a concentration-dependent manner sequentially degrades the junctional foot protein (JFP) of rabbit skeletal muscle triad junctions in either the triad membrane or as the pure protein. This progression is inhibited by calmodulin. Calpain initially cleaves the 565 kDa JFP monomer into peptides of 160 and 410 kDa, which is subsequently cleaved to 70 and 340 kDa. The 340 kDa peptide is finally cleaved to 140 and 200 kDa or its further products. When the JFP was labeled in the triad membrane with the hydrophobic probe 3-(trifuoromethyl) 3-(m)[125I]iodophenyl) diazirine and then isolated and proteolysed with calpain II, the [125I] was traced from the 565 kDa parent to Mr 410 kDa and then to 340 kDa, implying that these large fragments contain the majority of the transmembrane segments. A 70-kDa fragment was also labeled with the hydrophobic probe, although weakly suggesting an additional transmembrane segment in the middle of the molecule. These transmembrane segments have been predicted to be in the C-terminal region of the JFP. Using an ALOM program, we also predict that transmembrane segments may exist in the 70 kDa fragment. The JFP has eight PEDST sequences; this finding together with the calmodulin inhibition of calpain imply that the JFP is a PEDST-type calpain substrate. Calpain usually cleaves such substrates at or near calmodulin binding sites. Assuming such sites for proteolysis, we propose that the fragments of the JFP correspond to the monomer sequence in the following order from the N-terminus: 160, 70, 140 and 200 kDa. For this model, new calmodulin sequences are predicted to exist near 160 and 225 kDa from the N-terminus. When the intact JFP was labeled with azidoATP, label appeared in the 160 and 140 kDa fragments, which according to the above model contain the GXGXXG sequences postulated as ATP binding sites. This transmembrane segment was predicted by the ALOM program. In addition, calpain and calpastatin activities remained associated with triad component organelles throughout their isolation. These findings and the existence of PEDST sequences suggest that the JFP is normally degraded by calpain in vivo and that degradation is regulated by calpastatin and calmodulin.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Animals; Calcium Channels; Calcium-Binding Proteins; Calmodulin; Calpain; In Vitro Techniques; Molecular Sequence Data; Muscle Proteins; Muscles; Protein Binding; Rabbits; Receptors, Cholinergic; Ryanodine Receptor Calcium Release Channel

Administration of beta-adrenergic agonists to domestic species can lead to skeletal muscle hypertrophy, probably by reducing the rate of myofibrillar protein breakdown. Myofibrillar breakdown is associated with the calcium-dependent proteinase system (calpains I,II and calpastatin) whose activity also changes during beta-agonist treatment. A number of growth trials using the agonists cimaterol and clenbuterol with cattle, sheep, chicken and rat are reported which suggest a general mechanism whereby beta-agonists reduce calpain I activity, but increase calpain II and calpastatin activity in skeletal muscle. Parallel changes in specific mRNAs indicate that changes in gene expression or stabilisation of mRNA could in part explain the changes in activity.

Topics: Adrenergic beta-Agonists; Animals; Calcium-Binding Proteins; Calpain; Cattle; Chickens; Ethanolamines; Female; Gene Expression; Male; Muscles; Rats; Rats, Inbred Strains; RNA, Messenger; Sheep

Administration of beta-adrenergic agonists to mammals can produce skeletal muscle hypertrophy in some species and muscle types. The growth-promoting effect appears to be due to suppression of protein breakdown rather than stimulation of synthesis, although evidence from turnover studies is equivocal. In ovine muscle, changes in the activity of the calcium dependent neutral proteinases (calpains I and II) and their specific inhibitor (calpastatin) accompany beta-agonist-induced hypertrophy. These observations suggest that the calpain system is involved in myofibrillar protein turnover in some way. Alternatively, the relationship with hypertrophy may be indirect, since the calpains also interact with hormone and growth-factor receptors, protein kinase C and transcription factors, in addition to a range of membrane, cytoskeletal and nuclear proteins. In the present study, attempts have been made to determine if the beta-agonist-induced effects on the calpain system are associated with corresponding changes in specific mRNA. The activity of both calpain isoforms and calpastatin was measured in bovine longissimus dorsi samples from trials in which test animals were treated with the beta agonist cimaterol. Total RNA was extracted from the muscle samples. A cDNA probe for calpastatin mRNA was generated from bovine RNA by the polymerase chain reaction. This cDNA and a human calpain-II large-subunit cDNA were used to detect specific mRNA by Northern-blot analysis. beta-agonist treatment of Friesian steers caused significant longissimus dorsi hypertrophy. Increases in muscle mass (+37%, P less than 0.005), calpain-II specific activity (+27%, P less than 0.05) and calpastatin-specific activity (+76%, P less than 0.05) were found in treated animals. Total RNA was unchanged, but there was a 96% overall increase in calpastatin mRNA and a 30% increase in calpain-II large-subunit mRNA in muscle from treated animals. The mRNA changes are similar in direction and degree to the activity changes. Both calpain-II large subunit and inhibitor expression may therefore be stimulated by agonist action at the level of transcription or mRNA stabilisation. Multiple calpastatin mRNA species were detected in steers, as reported for other species. Differential changes in these messages, induced by the beta agonist, suggest that expression or stability of alternative mRNA species may be a factor in calpastatin regulation.

Topics: Adrenergic beta-Agonists; Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Calcium-Binding Proteins; Calpain; Cattle; DNA Probes; Ethanolamines; Male; Molecular Sequence Data; Muscles; Polymerase Chain Reaction; Receptors, Adrenergic, beta; RNA, Messenger

Eight MARC III composite (1/4 Hereford, 1/4 Angus, 1/4 Pinzgauer, and 1/4 Red Poll) steers weighing approximately 350 kg were fed 0 or 3 ppm of the beta-adrenergic agonist L644,969 (Merck Sharp and Dohme Laboratories, Rahway, NJ) for 6 wk in a high-concentrate diet. Feed efficiency was higher (P less than .05) in beta-adrenergic agonist-fed steers at 1, 3, 5, and 6 wk on trial. Average daily gain was greater (P less than .05) in beta-adrenergic agonist-fed steers at 3, 5, and 6 wk on treatment. Fractional degradation rate (percentage/day) of skeletal muscle myofibrillar protein was 27.1% lower (P less than .05) in beta-adrenergic agonist-fed steers at 3 wk on trial. Fractional accretion rate (percentage/day) of skeletal muscle myofibrillar protein in beta-adrenergic agonist-fed steers was higher (P less than .05) at 1, 3, 5, and 6 wk on trial. The beta-adrenergic agonist-fed steers had heavier (P less than .05) carcasses (9.6%), larger (P less than .05) longissimus muscle areas (24.3%), and lower (P less than .05) USDA yield grades (43.8%). Marbling degree, USDA quality grade, kidney, pelvic, and heart fat percentage, and 12th rib fat thickness were not different (P greater than .05). Calpastatin activity was higher (P less than .05) in muscle from the beta-adrenergic agonist-fed steers at 0 and 7 d postmortem. There were no differences (P greater than .05) in mu- or m-calpain or in cathepsins B or B+L or cystatin(s) between beta-adrenergic agonist-fed and control steers.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adrenergic beta-Agonists; Animals; Calcium-Binding Proteins; Calpain; Cathepsins; Cattle; Crosses, Genetic; Endopeptidases; Male; Meat; Muscle Development; Muscle Proteins; Muscles; Pyridines; Random Allocation; Weight Gain

The presence of low levels of calpastatin activity in erythrocytes of hypertensive rats affects regulation of calpain activity so it is highly susceptible to activation within physiological fluctuations in [Ca2+]. Under identical conditions, in red cells of normotensive rats, calpain activation is efficiently controlled by the high levels of calpastatin activity, and a progressive increase in proteinase activity can only be observed in parallel with a decrease in the level of calpastatin. In intact erythrocytes from hypertensive rats exposed to small variations in [Ca2+], degradation of anion transport protein (band 3) and Ca(2+)-ATPase appears as a primary event indicating that these two transmembrane proteins are probably early recognized as targets of intracellular calpain activity. Furthermore, band 3 protein seems to be structurally modified in erythrocytes from hypertensive rats, as indicated by its increased susceptibility to degradation in the presence of 10-50 microM Ca2+. In addition, when exposed to progressive and limited increases in [Ca2+], erythrocytes from hypertensive rats, but not those from normotensive rats, show a high degree of fragility that can be restored to normal values by inhibition of calpain. These results indicate that, within fluctuations in [Ca2+] close to physiological values, regulation of calpain activity is efficiently accomplished in normal erythrocytes but is completely lost in cells from hypertensive animals. Regulation is of critical importance in maintaining normal structural and functional properties of selective red cell membrane and cytoskeletal proteins, among which band 3 and Ca(2+)-ATPase appear to be the substrates with highest susceptibility to digestion by calpain.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calcium-Transporting ATPases; Calpain; Enzyme Activation; Erythrocyte Membrane; Hypertension; Membrane Proteins; Rats

To investigate the role of calpains in myofibrillar protein degradation in skeletal muscle and the regulation of their activity in vivo, we studied the effects of fasting on gene expression of calpains and calpastatin in the skeletal muscle of rabbits. In response to fasting, myofibrillar protein degradation increased 2-fold and mRNA levels of calpain I, calpain II and calpastatin were also increased. However, calpain and calpastatin activities remained unchanged. To investigate this discrepancy, we analysed polysomal calpain mRNA. Results indicated that fasting caused a 2-fold increase in the loading of calpain I and II mRNAs on ribosomes. Thus transcription of genes encoding calpain may be increased during fasting to ensure adequate synthesis of the proteinases needed to mobilize muscle protein reserves. The effect of fasting on calpain and calpastatin mRNA expression is shared by cathepsin D and proteasome C2 but not by beta-actin, implying that fasting invokes control of several proteolytic systems in skeletal muscle and underscores the possibility that each proteolytic system plays a role in the adaptation of skeletal muscle to the fasted state.

Topics: Animals; Blotting, Western; Body Weight; Calcium-Binding Proteins; Calpain; Cathepsin D; Cysteine Endopeptidases; Fasting; Gene Expression; Multienzyme Complexes; Muscles; Polyribosomes; Proteasome Endopeptidase Complex; Rabbits; RNA, Messenger

Little is known about the relative intracellular localizations of the calcium-dependent proteases, calpains, and their naturally occurring inhibitor, calpastatin. In the present study, the intracellular localization of mu-calpain, m-calpain, and calpastatin was studied at the light microscopic level in proliferating A431 cells. Highly specific antibodies against the three antigens revealed distinct staining patterns in interphase and mitotic cells. Most notably, calpastatin in interphase cells was localized near the nucleus in tube-like, or large granular structures, while the calpains were more uniformly distributed through the cytoplasm in either a fibrillar form (mu-calpain) or a diffuse or fine granular form (m-calpain). The distribution patterns of the two calpain isozymes were distinctly different during mitosis. m-Calpain was concentrated at the mitotic spindle poles and midbody, while mu-calpain appeared to accumulate at the cell membrane and the spindles. Four other human cell lines as well as normal human monocytes were examined to determine if the calpains-calpastatin segregation patterns are common to other cells or are unique to the A431 line. With the exception of abundant nuclear mu-calpain in the C-33A cervical carcinoma, the segregation of the proteins was similar to that of A431. These studies indicate that calpains may be localized at regions which are relatively poor in calpastatin content. Proteins at these sites may be susceptible to calpain-catalyzed cleavage.

Topics: Antibodies, Monoclonal; Blotting, Western; Calcium-Binding Proteins; Calpain; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cytoskeleton; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Interphase; Subcellular Fractions; Tumor Cells, Cultured

To improve our understanding of the regulation of calpain activity in situ during postmortem storage, the effects of pH, temperature, and inhibitors on the autolysis and subsequent proteolytic activity of mu-calpain were studied. Calpains (mu- and m-calpain) and calpastatin were purified from bovine skeletal muscle. All autolysis experiments were conducted in the absence of substrate at different pH (7.0, 6.2, and 5.8) and temperatures (25 and 5 degrees C). Autolysis of mu-calpain generated polypeptides with estimated masses of 61, 55, 40, 27, 23, and 18 kDa. The rate of autolysis was significantly increased with decreasing pH. The rate of degradation of the 80-kDa subunit was significantly decreased with decreasing temperature. However, degradation of the 30-kDa subunit was not affected by decreasing temperature. By conducting autolysis experiments at 5 degrees C and immunoblotting of autolytic fragments with anti-80 kDa, it was demonstrated that with the exception of 18 kDa, which originates from 30 kDa, all other fragments probably originate from degradation of the 80-kDa subunit. Calpastatin, leupeptin, and E-64 did not inhibit the initial step of autolysis, but they did inhibit further breakdown of these fragments. However, zinc, which also inhibits the proteolytic activity of calpain, only reduced the rate of autolysis, but did not inhibit it. The possible significance of these results in terms of the regulation of calpain in postmortem muscle is discussed.

Topics: Animals; Autolysis; Calcium-Binding Proteins; Calpain; Cattle; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Muscles; Postmortem Changes; Temperature

Because freezing samples decreases calpastatin activity and the application of exogenous calcium activates the calpain proteolytic system, thereby improving tenderness, the objective of this study was to determine whether freezing would enhance the effects of CaCl2 marination on the tenderness of beef steaks. Longissimus steaks were obtained from 10 beef steers 6 d postmortem. One-half of the steaks were frozen at -30 degrees C for 6 wk. The remaining steaks were treated fresh; one-half were subjected to a 150 mM CaCl2 marinade for 48 h. Frozen steaks were thawed and subjected to the same treatment. Treatments consisted of 1) fresh control, 2) fresh marinated, 3) frozen control, and 4) frozen marinated. Samples were taken before and after treatment (6 and 8 d) for calpastatin activity determination and d 8 for SDS-PAGE. Warner-Bratzler shear force values were measured 8 d postmortem. Data were analyzed using a paired comparison t-test procedure. Results showed that freezing and marination significantly decreased calpastatin activity. A .35-kg improvement (P = .07) in Warner-Bratzler shear force was observed with freezing, whereas a .78-kg improvement (P less than .01) in tenderness was observed with marination. However, prior freezing enhanced the effects of marination. Therefore, the decrease in calpastatin activity seemed to allow greater proteolysis by the calpains with the application of Ca2+. The SDS-PAGE of myofibril preparations indicated that more small polypeptide fragments (28 to 32 kDa) appeared and a 95-kDa fragment was more intense in the marinated samples than in control samples, indicating that proteolysis was enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcium Chloride; Calcium-Binding Proteins; Calpain; Cattle; Cryopreservation; Electrophoresis, Polyacrylamide Gel; Food Preservation; Hydrolysis; Male; Meat; Muscle Proteins; Muscles; Postmortem Changes

1. In millimolar Ca2+, smooth muscle calpains I and II were inhibited by aluminum ion. 2. At sub-millimolar Ca2+, calpain II, but not calpain I, was activated by low millimolar aluminum ion. 3. Calpastatin inhibited aluminum ion-activated calpain II. 4. Aluminum ion-activated and Ca(2+)-activated calpain II gave almost identical patterns of desmin cleavage. 5. Aluminum-activated calpain II, unlike the Ca(2+)-activated enzyme, did not autolyze and retained its proteolytic activity over extended periods of time.

Topics: Aluminum; Animals; Calcium; Calcium-Binding Proteins; Calpain; Chickens; Muscle, Smooth; Substrate Specificity

We examined the distribution of calpains I and II in human hematopoietic system cell lines by Western and Northern blot analyses and enzyme activity assay. Expression of calpain I, a low Ca(2+)-requiring cysteine protease, was observed in all human T-cell lines tested. By contrast, expression of calpain II, a high Ca(2+)-requiring form, in human T-cells was closely correlated with human T-cell leukemia virus type I (HTLV-I) infection, which is known to result in the expression of adult T-cell leukemia-associated antigens, interleukin-2 (IL-2) receptor alpha, and Ca(2+)-dependent cell proliferation. Specific expression of calpain II in HTLV-I-infected cells occurred at the mRNA level. Furthermore, expression of calpain II in human natural killer-like cells was augmented by HTLV-I pX gene transfection. In HTLV-I-infected cells, the trans-acting transcriptional activation of the long terminal repeat and control elements for the IL-2 receptor alpha, c-fos, and granulocyte-macrophage colony-stimulating factor genes by the Tax from the pX gene is already known. Our results suggest that the similar trans-activation occurs to the calpain II gene in HTLV-I-infected hematopoietic system cells.

Topics: Blotting, Western; Calcium-Binding Proteins; Calpain; Gene Expression; Genes, pX; Hematopoietic Stem Cells; HTLV-I Infections; Humans; In Vitro Techniques; RNA, Messenger; T-Lymphocytes; Transfection; Tumor Cells, Cultured

Rat skeletal muscle calpastatin form is markedly modified in its inhibitory properties by means of a reverse reaction which involves both phosphorylation and dephosphorylation. Dephospho-calpastatin shows greater inhibitory efficiency versus mu-calpain, whereas phospho-calpastatin shows maximal inhibition versus m-calpain. Both forms are present in fresh rat muscle. Phosphorylation has been reproduced "in vitro" using a homologous Ca2+ independent protein kinase and found to result in the incorporation of approximately one mole of 32P per mole of protein. Dephosphorylation was induced by treatment with alkaline phosphatase and 32P release shown found to correlate with modifications of the inhibitory properties. This reversible covalent modification of calpastatin is considered an important advancement in the understanding of how different calpain isoforms can be more efficiently controlled by a single inhibitor isozyme form.

Topics: Alkaline Phosphatase; Animals; Calcium-Binding Proteins; Calpain; Humans; Muscles; Phosphorylation; Protein Kinases; Rats; Structure-Activity Relationship

Of a total of three Friesian cows, two of which had been treated with adrenalin before slaughter, Mm longissimus (LO), supraspinatus (SS), triceps brachii (TB) and rectus abdominis (RA) were sampled at different times post mortem (pm). pH, calpain/calpastatin activities and degradation of myofibrillar proteins, as evidenced by SDS-PAGE, were assessed. Contraction characteristics were measured by determining myofibrillar ATPase activities. Adrenalin treatment resulted in a high ultimate pH (6.48 +/- 0.40) and a faster decline pm of calpain I activity. The effect was similar in all four investigated muscles (72.4 +/- 5.4% decline at 24 h pm). The decline in calpain I activity in the control muscles was muscle-dependent and ranged from 22.8-74.3% at 24 h pm. Differences in ultimate pH did not lead to distinct rates of breakdown of proteins with molecular weights lower than that of myosin heavy chain. Calpastatin levels were muscle-dependent and correlated with myofibrillar ATPase activity (r = -0.99). In a second experiment Mm rectus abdominis (RA) and psoas major (PM) of adrenalin-treated (n = 6) and control (n = 6) Friesian-Holstein calves were sampled at 1 and 29 h pm for assessment of calpain activities. At seven days pm the M longissimus (LO) was sampled for tenderness evaluation. pH values were measured at 30 min, 4 h and 29 h pm. Adrenalin treatment resulted in a higher ultimate pH in the three muscles. Higher ultimate pH resulted in lower calpain activities in the RA at 29 h pm (P less than or equal to 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphatases; Animals; Calcium-Binding Proteins; Calpain; Cattle; Epinephrine; Hydrogen-Ion Concentration; Muscle Proteins; Muscles; Myofibrils; Postmortem Changes

Immunofluorescence and immunogold localization studies show that the two Ca(2+)-dependent proteinases (mu-calpain for the micromolar Ca(2+)-requiring proteinase and m-calpain for the millimolar Ca(2+)-requiring proteinase) and their protein inhibitor (calpastatin) are located exclusively intracellularly in normal rat soleus muscle. Quantitative immunogold studies indicate that binding of antibodies to both calpains and to calpastatin is approximately two times greater at the Z-disk of myofibrils than it is at the I-band or A-band regions. Mitochondria and nuclei in muscle cells contain both calpains and calpastatin at concentrations approximately one-tenth and one-fifth, respectively, of the concentration at the Z-disk, as estimated by antibody binding. Very little calpain or calpastatin was seen in the cytoplasmic intermyofibrillar spaces, and most of the calpain and calpastatin in muscle cells is associated with intracellular structures. Immunofluorescence results suggest that concentration of m-calpain but not mu-calpain or calpastatin is, in some instances, slightly higher near the intracellular surface of the plasma membrane than elsewhere in the muscle cell. Most m-calpain, however, is distributed throughout the interior of mature rat skeletal muscle cells. Denervation, or fasting and refeeding increases the concentration of the calpains and calpastatin in the muscle cell but does not change their distribution. Some mu- and m-calpain and calpastatin is found extracellularly in denervated soleus muscle or soleus muscle from fasting rats, but the extracellular calpains and calpastatin seem to originate from "leakage" of these proteins out of the cell because serum creatine kinase levels are much higher than normal in denervated or fasting rats.

Topics: Animals; Calcium-Binding Proteins; Calpain; Creatine Kinase; Fasting; Fluorescent Antibody Technique; Microscopy, Immunoelectron; Muscle Denervation; Muscles; Rats; Rats, Inbred Strains

The objectives of this study were to examine the amount of postmortem proteolysis in three different lamb muscles at different ages and to determine whether a relationship exists between the extent of myofibrillar degradation and certain endogenous proteinase activities. Wether lambs were slaughtered at 8 (n = 6) and 26 (n = 6) wk of age. Samples were taken from the longissimus (LM), gluteus medius (GM), and supraspinatus (SS) muscles for determining myofibrillar fragmentation index (MFI) at 1 and 7 d postmortem, cathepsins B and B + L, cystatin(s), mu- and m-calpain and calpastatin 24-h activities, and muscle fiber type and area. Muscle samples were removed for SDS-PAGE analysis at 0, 1, and 7 d postmortem. The SS muscle consisted of more (P less than .05) oxidative fibers, whereas no age effects (P greater than .10) were observed for fiber type. The younger lambs had higher (P less than .01) cathepsins B and B + L, cystatin, and calpastatin 24-h activities but less (P less than .01) m-calpain activity than did the 26-wk-old lambs. Within each age, the SS muscle had the highest (P less than .05) mu-calpain, m-calpain, and calpastatin specific activities. The MFI values and SDS-PAGE results indicate that less proteolysis occurred in the SS muscle. Samples from 26-wk-old lambs tended to have greater MFI values at 1 and 7 d postmortem, especially for the LM and GM muscles. From these results, it seems that less postmortem proteolysis occurs in younger and more oxidative muscles and this may be attributed to the greater calpastatin 24-h activity.

Topics: Age Factors; Animals; Calcium-Binding Proteins; Calpain; Cathepsins; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Glycolysis; Male; Muscle Proteins; Muscles; Oxidation-Reduction; Postmortem Changes; Sheep

All mammalian cells contain a calcium-dependent proteolytic system, composed by a proteinase, calpain, and an inhibitor, calpastatin. In some cell types an activator protein has also been identified. Moreover, two calpain isoforms, distinguishable on the basis of a different calcium requirement, can be present in a single cell. Both calpain forms are heterodimers composed of a heavy subunit (80 kDa) that contains the catalytic site and a smaller (regulatory?) subunit (30 kDa). Calpain I expresses full activity at 10-50 microM Ca2+, whereas calpain II requires calcium concentrations in the millimolar range. The removal by autoproteolysis of a fragment from the N-terminus of both calpain subunits generates a proteinase form that can express catalytic activity at concentrations of Ca2+ close to the physiological range. This process is significantly accelerated in the presence of cell membranes or phospholipid vesicles. Calpastatin, the specific inhibitor of calpain, prevents activation and the expression of catalytic activity of calpain. It is in itself a substrate of the proteinase and undergoes a degradation process which correlates with the general mechanism of regulation of the intracellular proteolytic system. The natural calpain activator specifically acts on calpain II isoform, by reducing the Ca2+ required for the autoproteolytic activation process. Based on the general properties of the calpain-calpastatin system and on the substrate specificity, its role in the expression of specific cell functions can be postulated.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Enzyme Activation; Erythrocytes; Hypertension; Macromolecular Substances; Molecular Weight; Muscles; Rats

The effect of the treatment of HeLa cells with a tumor-promoting phorbol ester, 12-o-tetradecanoyl-phorbol-13-acetate (TPA) on the expression of the genes for the calpain family has been examined. Among the mRNAs for the calpain family, only the mRNA for the large subunit of human m-calpain (calpain mL) was specifically induced by treatment of cells with TPA, suggesting its specific function in response to cellular stimuli. The effect of TPA on the expression of the calpain mL gene was further examined using fusion genes containing the promoter/enhancer region of the calpain mL gene fused upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene, showing that the promoter/enhancer sequence of the calpain mL gene contains a cis-acting element which responds to TPA and activates transcription of the downstream sequence.

Topics: Calcium-Binding Proteins; Calpain; Gene Expression Regulation; HeLa Cells; Humans; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transfection

Purified calpains are capable of proteolyzing several high Mr nuclear proteins and solubilizing a histone H1 kinase activity from rat liver nuclei upon exposure to 10(-6) - 10(-5) M Ca2+. Major nuclear substrates displayed apparent molecular masses of 200, 130, 120, and 60 kDa on Coomassie Blue-stained SDS-PAGE gels. The nuclear proteins and the H1 kinase were released from Triton-treated nuclei following incubation with buffer containing 0.5 M NaCl. They therefore appeared to be internal nuclear matrix proteins. The nuclear H1 kinase activity solubilized by incubation with m-calpain was eluted in the void volume of a Bio-Gel A-1.5m column, indicating an apparent mass greater than 1,500 kDa. Treatment of the calpain-solubilized kinase with 0.5 M NaCl dissociated it to a form having an apparent mass of 300 kDa (Stokes radius = 5.6 nm), suggesting that the 300-kDa (Stokes radius = 5.6 nm), nuclei by calpain treatment as a large complex containing other internal matrix proteins. Purified human erythrocyte mu-calpain was capable of proteolyzing the nuclear matrix proteins at 10(-6) M Ca2+. In contrast, human erythrocyte multicatalytic protease complex produced little cleavage of the nuclear proteins. Proteolysis of nuclear proteins by either mu-calpain or m-calpain was inhibited by calpastatin. These experiments suggest a physiologic role for the calpains in the turnover of nuclear proteins.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Cysteine Endopeptidases; In Vitro Techniques; Liver; Molecular Weight; Multienzyme Complexes; Nuclear Proteins; Peptide Mapping; Protamine Kinase; Proteasome Endopeptidase Complex; Rats; Solubility

The objectives of this experiment were to assess effects of animal age and castration on activities of calpain I, calpain II, and calpastatin in sheep skeletal muscle. Ten newborn male lambs (2.9 kg), six weaned wethers (23.2 kg), six weaned rams (22.2 kg), six market wethers (55.4 kg), and six market rams (60.2 kg) were slaughtered and samples of biceps femoris were taken for assay of calpain I (micromolar calcium-dependent proteinase), calpain II (millimolar calcium-dependent proteinase), and calpastatin. Preweaning weight gain was similar for rams and wethers; however, postweaning ram growth exceeded (P less than .05) that of wethers. Ram biceps femoris weights at market were greater than those of wethers (P less than .05). Irrespective of age or gender, activity of calpain II was two- to threefold greater than that of calpain I. Muscle calpastatin activity was severa fold higher than calpain I and II activities. Activities of calpains and calpastatin declined (P less than .05) between birth and weaning. A portion of these losses were due to a dilution effect caused by accumulation of muscle proteins. Neonatal attenuation of calpain activities may underlie age-related attenuation of fractional rates of muscle protein degradation. Although ram muscle growth exceeded that of wethers, no differences (P greater than .05) in activities of muscle calpains or calpastatin were detected between these two groups at weaning or at market weight. Hence, castration did not influence lamb muscle growth by altering muscle calpain or calpastatin activities.

Topics: Aging; Animals; Calcium-Binding Proteins; Calpain; Male; Muscle Development; Muscle Proteins; Muscles; Orchiectomy; RNA; Sheep; Weight Gain

Marchi-positive bodies are structures present paranodally in large myelinated nerve fibers. They have morphological and biochemical characteristics closely resembling the partially degraded myelin fragments formed during the early phases of Wallerian degeneration. Levels of calcium-activated neutral proteases (calpains) and their endogenous specific inhibitor calpastatin were measured in highly purified rabbit myelin and a spinal cord subcellular light ('floating') fraction heavily enriched in Marchi-positive bodies. Calpain levels were found to be significantly higher in the floating fraction as compared to myelin. No calpastatin was detectable in either fraction.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Central Nervous System; Myelin Basic Protein; Myelin Sheath; Proteolipids; Rabbits; Subcellular Fractions

To examine the effect of a beta-adrenergic agonist (BAA) on muscle growth, proteinase activities, and postmortem proteolysis, 16 wether lambs were randomly assigned to receive 0 or 4 ppm of L644,969 in a completely mixed high-concentrate diet for 6 wk. Weight of the biceps femoris was 18.6% heavier in treated lambs. At 0 h after slaughter, treated lambs had higher cathepsin B (35.6%), cathepsins B + L (19.1%), calpastatin (62.8%), and m-calpain (24.6%) than control lambs, but both groups had similar mu-calpain activities. In both longissimus and biceps femoris muscles, treated lambs had higher protein and RNA and lower DNA concentrations. However, total DNA was not affected, indicating that the increase in muscle mass was probably due to muscle hypertrophy rather than to hyperplasia. The pattern of postmortem proteolysis was significantly altered by BAA feeding. In treated lambs, postmortem storage had no effect on the myofibril fragmentation index and degradation of desmin and troponin-T. These results indicate that the ability of the muscle to undergo postmortem proteolysis has been dramatically reduced with BAA feeding. Similar proteolytic systems are thought to be involved in antemortem and postmortem degradation of myofibrillar proteins, so BAA-mediated protein accretion is probably due, at least in part, to reduced protein degradation. To examine whether protein synthesis was altered with BAA feeding, the level of skeletal muscle alpha-actin mRNA was quantified. Longissimus muscle alpha-actin mRNA abundance was 30% greater in BAA-fed lambs. Collectively, these results indicate that dietary administration of BAA increases muscle mass through hypertrophy and that the increase in muscle protein accretion is due to reduced degradation and possibly to increased synthesis of muscle proteins.

Topics: Actins; Adrenergic beta-Agonists; Animals; Calcium-Binding Proteins; Calpain; Cathepsins; DNA; Endopeptidases; Hypertrophy; Least-Squares Analysis; Male; Meat; Muscle Development; Muscle Proteins; Muscles; Postmortem Changes; Pyridines; Random Allocation; RNA; RNA, Messenger; Sheep; Weight Gain

Rat skeletal muscle contains a calpain activator protein characterized by a high specificity for calpain II, the high Ca(2+)-requiring isoform of this class of proteinases. The activator protein increases the rate of intramolecular conversion of the native 80-kDa catalytic subunit of calpain into the autolysed 75-kDa forms with maximal rate at concentrations of calcium approximately 25 times lower than those required by the native proteinase. The activator protein interacts with native calpain II forming a 1:1 complex; interaction does not occur with the fully activated form, produced by autoproteolysis. Even after immobilization to membranes, the activator binds to calpain, which then undergoes sequential activation and release from its bound form. The activator is itself resistant to digestion by calpain II, whereas it increases the rate at which homologous calpastatin is degraded by the proteinase. Taken together, these results are indicative of the existence in rat skeletal muscle of an activating system specific for calpain II which is potentially involved in the regulation of the inhibitory efficiency of calpastatin, through modulation of its intracellular level.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Edetic Acid; Kinetics; Muscles; Proteins; Rats

Our objectives were to characterize events underlying changes in skeletal muscle calpain and calpastatin activities, using maturation as a model. Muscle samples were taken from rabbits of four ages (newborn and 1, 2, and 5 mo old). Concentrations of RNA and protein and activities of calpains I and II and calpastatin were determined. Steady-state concentrations of mRNAs encoding calpain I, calpain II, calpastatin, alpha- and beta-tubulin, and beta-actin were determined using Northern blot analysis. Calpain and calpastatin activities declined markedly between birth and 1 mo of age and remained unchanged thereafter. Several factors accounted for the neonatal losses of calpains and calpastatin. First, muscle protein concentration increased between birth and 1 mo of age and diluted calpain and calpastatin specific activities. Second, there was a marked reduction of muscle RNA concentration between birth and 1 mo of age, which indicates that protein synthetic capacity declined with age. Finally, calpastatin mRNA concentration declined between birth and 1 mo of age and further contributed to developmental losses of calpastatin activity. Calpain I mRNA concentration was unaffected by age, and although calpain II mRNA concentration declined with age, losses were not detected between birth and 1 mo; hence age-related changes in calpain I and II activities are not mediated at the mRNA level. The age-related reductions in calpain II and calpastatin mRNA concentrations resembled age-related changes in alpha- and beta-tubulin and beta-actin mRNA concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aging; Animals; Blotting, Northern; Calcium-Binding Proteins; Calpain; Female; Muscle Development; Muscles; Organ Size; Rabbits; RNA, Messenger

The structure of human platelets differs from that of bovine platelets in that human platelets have a surface-connected canalicular system that bovine platelets lack. Platelets are one of the richest known sources of the calpains, and the calpains have been implicated in many of the specific cleavages of cytoskeletal and surface-receptor proteins that occur during platelet activation and aggregation. Several studies have reported that human platelets are rich in mu-calpain and contain less m-calpain, whereas bovine platelets contain principally m-calpain and almost no mu-calpain. The immunolocalization studies reported here show that calpastatin is distributed throughout the cytosol of both human and bovine platelets and that calpain is located throughout the interior of human platelets. Calpain in bovine platelets is located primarily in alpha-granules, however. Because bovine platelets contain predominantly m-calpain and because alpha-granules are translocated to the platelet surface during activation, bovine m-calpain may be responsible for the specific cleavages of platelet surface proteins such as glycoprotein Ib that occur during platelet activation and at extracellular Ca2+ concentrations high enough to activate m-calpain.

Topics: Animals; Blood Platelets; Calcium-Binding Proteins; Calpain; Cattle; Cytoplasmic Granules; Cytosol; Humans; Immunohistochemistry; Microscopy, Immunoelectron; Species Specificity

According to the difference of molecular masses estimated by SDS-polyacrylamide gel electrophoresis, calpastatins are classified into two types, i.e. muscle type (110 kDa) and erythrocyte type (70 kDa). Muscle type calpastatin contains four internally repetitive sequences (Domains 1-4) and one nonhomologous sequence on the amino-terminal side (Domain L), whereas erythrocyte type lacks Domains L and 1. By immuno-blot analysis, chicken erythrocytes, nucleated cells, were found to contain muscle type calpastatin. In avian erythrocytes, diminution of the calpastatin molecule as in mammalian erythrocytes was not observed.

Topics: Animals; Calcium-Binding Proteins; Calpain; Chickens; Erythrocytes; Liver; Molecular Weight; Tissue Distribution

Polyamine synthesis is induced by various extracellular signals, and it is widely held that this biochemical response participates in cell growth and differentiation. Certain of the triggers for synthesis in brain tissues also increase the breakdown of high-molecular-weight structural proteins, apparently by activating calcium-dependent proteases (calpains). The present experiments tested the possibility that calpain activity is modulated by polyamines. Spermine, spermidine, and putrescine all increased calcium-dependent proteolysis of [14C]casein by soluble fractions of rat brain. The order of potency was spermine greater than spermidine greater than putrescine, with apparent affinities of 30, 300, and 6,000 microM, respectively. Each of the three polyamines at physiological concentrations also potentiated the calcium-dependent breakdown of two endogenous high-molecular-weight structural proteins known to be substrates of calpain, in both supernatant and membrane fractions. The thiol protease inhibitor leupeptin, a known calpain inhibitor, also inhibited calcium-dependent proteolysis in the presence and absence of polyamines. The polyamines did not increase the activity of purified calpain I or calpain II determined with either [14C]casein or purified spectrin as the substrate, nor did they interfere with the inhibitory effects of calpastatin, an endogenous inhibitor of calpain. However, polyamines potentiated the stimulation of endogenous but not purified calpain activity produced by an endogenous calpain activator. These results suggest a role for polyamines in protein degradation as well as protein synthesis.

Topics: Animals; Brain; Calcium-Binding Proteins; Calpain; Chemical Fractionation; Male; Peptide Hydrolases; Polyamines; Rats; Rats, Inbred Strains

Two forms of calpastatin, differing in their specificity for the homologous calpain isozymes I and II, have been separated from rat skeletal muscle extracts and purified to homogeneity. Calpastatin I, the first form to elute in chromatography on DE32, is more effective against calpain I, while calpastatin II is more effective as an inhibitor of calpain II. Based on their molecular mass (approximately 105 kDa) both calpastatin forms belong to the high molecular mass class found in muscles of other animal species (Murachi, T., 1989, Biochem. Int. 18, 263-294). For calpain I, which is active with low (mu-M) concentrations of Ca2+, maximum inhibition with either calpastatin form was observed over a wide range of Ca2+ concentrations. With calpain II, which requires high (mM) concentrations of Ca2+ for activity, maximum inhibition required Ca2+ concentrations above 1 mM. Both calpastatin forms were found to be highly sensitive to degradation by calpain II, but almost completely resistant to degradation by calpain I. Degradation of calpastatin by calpain II is competitively inhibited by the addition of a calpain substrate. Isovaleryl carnitine (IVC), an intermediate product of L-leucine catabolism, previously demonstrated to be a potent and specific activator of rat skeletal muscle calpain II (Pontremoli, S., Melloni, E., Viotti, P. L., Michetti, M., Di Lisa, F., and Siliprandi, N., 1990. Biochem. Biophys. Res. Commun. 167, 373-380) greatly enhances the rate of degradation of calpastatins by calpain II. IVC, which decreases the Ca2+ requirement for maximal calpain II activity, also decreases the concentration of Ca2+ required for digestion of the inhibitor. For calpain II, regulation by either calpastatins may occur only in the presence of high [Ca2+].

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Chromatography, Affinity; Chromatography, DEAE-Cellulose; Electrophoresis, Polyacrylamide Gel; Kinetics; Male; Molecular Weight; Muscles; Rats; Substrate Specificity

Calpains and calpastatin in the brain of the rabbit were examined in experimental situations that could mimic some features of brain ischemia. Incubations of bisected brains in saline at 39 degrees C for 0.5, 1, or 1.5 h resulted in a decreased calpain I activity in the cytosol and in an increased hydrophobicity of cytosolic calpain II activity. Incubation of brain homogenates at different pH levels demonstrated an almost-complete transfer of calpains from the cytoplasmic compartment to the membranes when pH was lowered from 6 to 5. At pH values lower than 5, the total calpain activity (soluble plus membrane-bound) markedly decreased. No significant changes of calpastatin activity or its subcellular distribution was found following incubation of the homogenates at different pH levels.

Topics: Animals; Brain; Brain Ischemia; Calcium-Binding Proteins; Calpain; Chromatography, High Pressure Liquid; Cytosol; Dithiothreitol; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; In Vitro Techniques; Perfusion; Rabbits; Subcellular Fractions

Vascular smooth muscle contains large amounts of the Ca(2+)-dependent protease calpain II. In this study, we compared bovine aortic muscle (muscle phenotype) to cultured bovine aortic cells of smooth muscle origin (modulated phenotype) with respect to major constituents of the calpain-calpastatin system. Bovine aortic muscle contained only calpain II by activity measurements, Western blot of tissue extracts and Northern blot of poly(A)+ RNA. On the other hand, using the same methodologies, both calpains I and II as well as the 110 kDa inhibitor protein, calpastatin, were identified in cultured bovine aortic cells of smooth muscle origin. We conclude that the phenotypic state of smooth muscle cells is associated with differential expression of major components of the calpain-calpastatin system. Moreover, bovine aortic muscle is the only tissue identified to date that contains calpain II exclusively.

Topics: Animals; Blotting, Northern; Calcium-Binding Proteins; Calpain; Cattle; Gene Expression; Muscle Proteins; Muscle, Smooth, Vascular; RNA, Messenger

We studied calpastatin activity in erythrocytes of Milan hypertensive and prehypertensive rats, in their normotensive controls, in F1 and F2 hybrids, and in two inbred strains derived from F2, one hypertensive and the other normotensive. Our results show that the decrease in calpastatin activity observed in Milan hypertensive rats was not caused by hypertension, it was transmitted in a recessive way in heterozygous, and it was not correlated to hypertension.

Topics: Animals; Blood Pressure; Calcium-Binding Proteins; Calpain; Erythrocytes; Heterozygote; Homozygote; Hypertension; Rats; Rats, Inbred Strains

Since calcium homeostasis is altered in cultured skin fibroblasts from aged and Alzheimer donors, the present study examined the degradation of spectrin, a substrate of the calcium dependent protease calpain. Spectrin proteolysis was estimated as the percentage of spectrin breakdown products (e.g., 150 + 155 kDa bands) per total spectrin immunoreactivity. In the baseline condition (e.g., unstimulated fibroblasts), spectrin breakdown was 53% greater in cells from aged donors when compared to cells from either young or Alzheimer donors. Compared to unstimulated cells, serum increased spectrin breakdown in cells from aged (22.4%) or Alzheimer (92.1%) donors but was ineffective in cells from young donors. Thus, when compared to young donors (100%), serum stimulation increased spectrin proteolysis by 183.9% (aged) or 231.7% (Alzheimer) after serum stimulation. Treatment of unstimulated cells with carbonyl cyanide 4-trifluoromethoxy-phenylhydrazone (FCCP), an uncoupler of mitochondrial function, increased spectrin degradation by 360.6% (young), 242.4% (aged) or 239.7% (Alzheimer) when compared to unstimulated cells of the same group. The combination of FCCP and serum stimulation enhanced spectrin breakdown in cells from aged (123.6%) and Alzheimer (154.0%) donors when compared to young cells (100%). Thus, changes in the regulation of calcium dependent proteases may contribute to decreased cell spreading and may play a role in the altered cytoskeletal dynamics characteristic of Alzheimer's disease.

Topics: Aged; Aging; Alzheimer Disease; Calcium-Binding Proteins; Calpain; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cells, Cultured; Cytoskeleton; Electrophoresis; Fibroblasts; Humans; Skin; Spectrin

Their cDNA-derived amino acid sequences predict that the 80-kDa subunits of the micromolar and millimolar Ca(2+)-requiring forms of the Ca(2+)-dependent proteinase (mu- and m-calpain, respectively) each consist of four domains and that the 28-kDa subunit common to both mu- and m-calpain consists of two domains. The calpains were allowed to autolyze to completion, and the autolysis products were separated and were characterized by using gel permeation chromatography, calpastatin affinity chromatography, and sequence analysis. Three major fragments were obtained after autolysis of either calpain. The largest fragment (34 kDa for mu-calpain, 35 kDa for m-calpain) contains all of domain II, the catalytic domain, plus part of domain I of the 80-kDa subunit of mu- or m-calpain. This fragment does not bind to calpastatin, a competitive inhibitor of the calpains, and has no proteolytic activity in either the absence or presence of Ca2+. The second major fragment (21 kDa for mu-calpain and 22 kDa for m-calpain) contains domain IV, the calmodulin-like domain, plus approximately 50 amino acids from domain III of the 80-kDa subunit of mu- or m-calpain. The third major fragment (18 kDa) contains domain VI, the calmodulin-like domain of the 28-kDa subunit. The second and third major fragments bind to a calpastatin affinity column in the presence of Ca2+ and are eluted with EDTA. The second and third fragments are noncovalently bound, so the 80- and 28-kDa subunits of the intact calpain molecules are noncovalently bound at domains IV and VI. After separation in 1 M NaSCN, the 28-kDa subunit binds completely to calpastatin, approximately 30-40% of the 80-kDa subunit of mu-calpain binds to calpastatin, and the 80-kDa subunit of m-calpain does not bind to calpastatin in the presence of 1 mM Ca2+.

Topics: Amino Acid Sequence; Animals; Calcium; Calcium-Binding Proteins; Calpain; Cattle; Chromatography, Affinity; Chromatography, Gel; DNA; Electrophoresis, Polyacrylamide Gel; Hydrolysis; Molecular Sequence Data; Sequence Homology, Nucleic Acid

Calcium-induced autolysis of bovine erythrocyte calpain I occurs in multiple stages. Initially, a 14 amino acid segment is cleaved from the N-terminus of the native 80 kDa catalytic subunit, yielding a 78 kDa form of the subunit. Then, an additional 12 amino acid segment is cleaved from the N-terminus, forming a 76 kDa subunit. The 76 kDa enzyme is the active form of the catalytic subunit that is able to proteolyze the 30 kDa regulatory subunit as well as exogenous substrates. While the initial autolytic step requires high calcium, the 76 kDa enzyme form is active in microM calcium and can cleave the amino termini of native 80 kDa and intermediate 78 kDa enzyme forms at low calcium. Both intramolecular and intermolecular proteolysis of the catalytic subunit appear to yield the same products.

Topics: Amino Acid Sequence; Animals; Calcium; Calcium-Binding Proteins; Calpain; Carbohydrate Sequence; Cattle; Enzyme Activation; Erythrocytes; Leupeptins; Molecular Sequence Data

Calpastatin activity, significantly reduced in erythrocytes of patients affected by essential hypertension, is restored to normal values by appropriate therapeutical treatments in a time-dependent fashion and in parallel with the decline in blood pressure. Evidence is also presented indicating that red cell calpastatin is degraded in human and rat red cells by homologous calpain, and that the rate of degradation is approx. 5-times higher in rat erythrocytes. Thus, increased proteolytic degradation catalyzed by calpain could explain both the decrease in the amount of calpastatin activity and the profound difference between the intracellular level of the calpain inhibitor observed in erythrocytes from patients with essential hypertension and the genetically hypertensive rats.

Topics: Animals; Blood Pressure; Calcium-Binding Proteins; Calpain; Erythrocytes; Humans; Hypertension; Kinetics; Rats

Recent evidence has suggested that Alzheimer's disease may result from an underlying defect of protein catabolism. In an attempt to identify such a defect, we have determined the levels of Ca(2+)-activated proteinase (principally calpain II) and endogenous inhibitor (calpastatin) activity in normal and Alzheimer's disease cases, following fractionation of parietal cortex (grey and white matter) via anion-exchange chromatography. The chromatographic elution profiles and levels of calpain II activity were found to be similar in grey and white matter in both normal and Alzheimer's disease cases. The characteristics of calpain II, including Ca2+ concentration required for optimum activity for enzymes partially purified from normal or Alzheimer's disease cortex were identical. Similarly, the chromatographic elution profiles and levels of total calpastatin activity (approximately equal to that for calpain II activity) were found to be similar in grey and white matter from normal and Alzheimer's disease cases. These data suggest that the characteristic neurodegeneration associated with Alzheimer's disease does not result from alteration in the level of activity or characteristics of the calpain/calpastatin system in the cerebral cortex of patients with this disorder.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Calcium-Binding Proteins; Calpain; Chromatography, Gel; Chromatography, Ion Exchange; Female; Humans; Male; Middle Aged; Parietal Lobe

Dietary administration of 4 ppm of the beta-agonist L-644,969 (Merck Sharpe and Dohme Research Laboratories) to finishing lambs induced a decrease (10 to 14%, P less than .05) in extractable calpain I activity in the longissimus muscle (LD) at death (d 0). At 4 d postmortem (d 4), extractable calpain I levels in the LD of both control and treated lambs were reduced (P less than .001) from those present at d 0, but the extractable activity in the LD was reduced to a greater extent in control than in treated lambs. Calpain II activity was increased 42% (P less than .005) in LD of treated lambs; however, no significant differences were observed between d 0 and d 4 calpain II activity within treated or control LD samples (P greater than .1). Calpastatin activity was higher in the LD of treated lambs (74% on d 0, P less than .001 and 430% on d 4, P less than .001) than in the LD of control lambs. Measurable cathepsin B activity was decreased (29% on d 0, P less than .05) and measurable cathepsin H activity was increased (10% on d 0, P less than .05 and 10% on d 4, P less than .05) in the LD of treated lambs compared with controls. On d 2, 4 and 6 postmortem, degradation in myofibrils isolated from the LD was lower for treated than for control lambs. Warner-Bratzler shear values for loin chops from treated lambs were higher on both d 3 (111%) and 6 (108%) postmortem than for chops from control lambs (P less than .001). L-644,969-induced decreases in muscle proteolytic capacity may limit postmortem myofibril degradation and contribute to the reduced tenderness observed. This decreased proteolytic capacity may contribute to increased muscularity of L-644,969-treated lambs.

Topics: Adrenergic beta-Agonists; Animals; Calcium-Binding Proteins; Calpain; Cathepsin B; Cathepsin H; Cathepsins; Cysteine Endopeptidases; Electrophoresis, Polyacrylamide Gel; Male; Muscle Proteins; Myofibrils; Postmortem Changes; Pyridines; Sheep; Time Factors

DEAE-cellulose chromatography of mouse brain extract demonstrated the occurrence of two calpastatin fractions, CS-0.1 and CS-0.2, with distinctly higher content of the latter. CS-0.1 emerged from the column at 0.1 M NaCl, inhibited calpain II more strongly than calpain I, and identified also immunologically with hitherto known calpastatin. CS-0.2 emerged at 0.2 M NaCl, inhibited calpain I more strongly than calpain II, and did not crossreact with anti-calpastatin antibody used. Fairly consistent amounts of CS-0.2 and calpain II were found in the brain of mice from 10 days to 10 weeks after birth, while CS-0.1 became measurable only after 4-week growth. In adult mice, CS-0.1 was highest in specific activity in brainstem, lower in cerebellum, and not detectable in cerebral hemisphere. Physiological significance of multiple forms of calpastatin and their variations found is not known.

Topics: Aging; Animals; Blotting, Western; Brain; Brain Chemistry; Calcium-Binding Proteins; Calpain; Chromatography, DEAE-Cellulose; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Mice; Mice, Inbred A; Molecular Weight

Calpains (calcium-dependent cysteine proteinases; optimum pH 7.0-7.5) have been regarded as intracellular proteinases. We examined the cell-free components of synovial fluid from 14 patients with osteoarthritis and demonstrated the existence of calpains, as the caseinolytic activities of chromatographic fractions, together with calpastatin, the specific endogenous inhibitor of calpains. The presence of these calpains and calpastatin was verified by immunoblotting with their respective specific antibodies. Calpain fractions showed proteoglycan-degrading activity. The results suggest that the calpain-calpastatin system may contribute to the turnover of cartilage matrix components.

Topics: Aged; Aged, 80 and over; Animals; Calcium-Binding Proteins; Calpain; Cartilage, Articular; Drug Stability; Endopeptidases; Hot Temperature; Humans; Immunoelectrophoresis; Metalloendopeptidases; Middle Aged; Osteoarthritis; Proteoglycans; Swine; Synovial Fluid

The levels of the neutral proteolytic enzymes calpains and their endogenous inhibitor calpastatin were determined in the retina and in the retrobulbar optic pathway in the albino rabbit. The highest level of calpains was observed in the optic nerve with decreasing levels in the optic tract and superior colliculus. The level of calpastatin in the retina was very low compared to that in the optic nerve and tract and other parts of the nervous system.

Topics: Animals; Calcium-Binding Proteins; Calpain; Optic Nerve; Peptide Hydrolases; Rabbits; Retina; Subcellular Fractions; Superior Colliculi

The human calpastatin gene (CAST) was assigned to chromosome 5 by spot-blot hybridization analysis with flow-sorted chromosomes, and it was further sublocalized to bands 5q14----q22 using in situ hybridization to metaphase chromosomes.

Topics: Calcium-Binding Proteins; Calpain; Chromosome Mapping; Chromosomes, Human, Pair 5; Flow Cytometry; Humans; Karyotyping; Nucleic Acid Hybridization

The mechanism for binding of human erythrocyte calpain I to human erythrocyte inside-out vesicles was studied by immunoelectrophoretic blot analysis. Binding of calpain I to inside-out vesicles was observed both in the absence and presence of Ca2+. Moreover, in the absence of Ca2+, acidic proteins like casein, ovalbumin and calpastatin suppressed while basic proteins like arginase and lysozyme did not affect the binding of calpain I to inside-out vesicles. Here, we propose a model for the binding of calpain to the membrane.

Topics: Arginase; Calcium; Calcium-Binding Proteins; Calpain; Caseins; Erythrocyte Membrane; Humans; Immunoelectrophoresis; Immunoglobulins; Leupeptins; Models, Biological; Muramidase; Ovalbumin; Protease Inhibitors

The purpose of this study was to provide a direct assay for calpain and its endogenous inhibitor calpastatin in normal rabbit epithelium. Corneal epithelial extracts were fractionated by DEAE (1) chromatography on HPLC. Fractions were analyzed for calpain by ELISA, immunoblotting, and caseinolytic enzyme activity with FITC-labeled casein. Results demonstrated immunoreactive peaks for calpains I and II. Calpain II from the soluble fraction of corneal epithelium eluted at a similar NaCl concentration (260 mM) as calpain II from other tissues, was inhibited by both E64 and the removal of Ca, contained an 80 kDa subunit in immunoblots, and was present at specific activity of 220 units/g protein (in a crude homogenate). Calpain antigen was also present in the EDTA/EGTA washed insoluble fraction of corneal epithelium. Calpastatin in corneal epithelium eluted at 130 - 160 mM NaCl on DEAE, coeluted with calpain I, and was present at 330 units/g protein (crude homogenate). The results demonstrated a calpain/calpastatin system in corneal epithelium, where it is speculated to play a role in epithelial cell turnover and wound healing.

Topics: Animals; Calcium-Binding Proteins; Calpain; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Cornea; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Epithelium; Immunoblotting; Rabbits

The muscle enzyme calpain II, in contrast to muscle calpain I, was markedly inhibited by millimolar concentrations of the polyamines spermine and spermidine. These compounds and the calpain inhibitor calpastatin had synergistic inhibitory effects on calpain II. These results suggest that the polyamines may have possible regulatory effects on the in vivo activity of calpain II enzymes.

Topics: Animals; Calcium-Binding Proteins; Calpain; Chickens; Cricetinae; Gizzard, Avian; Muscles; Spermidine; Spermine

We have found a novel type of Ca2(+)-activated neutral protease in rat brain cytosol which cleaves -Tyr-Arg-containing calpastatin fragments to release the neuropeptide kyotorphin. This enzyme was purified about 26,000-fold by column chromatography as follows: DE52 cellulose, Ultrogel AcA 44, thiopropyl-Sepharose 6B, second DE52 cellulose, Ultrogel AcA 34, and blue Sepharose CL-6B. The molecular mass of the enzyme was estimated to be 65-75 kDa by gel filtration. The purified enzyme gave a single band of 74 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Some properties of this enzyme were similar to those of the calpains, i.e. an absolute requirement for Ca2+, maximal activity at neutral pH, and inhibition by sulfhydryl reagents such as p-chloromercuriphenylsulfonic acid and N-ethylmaleimide. However, it differs from the calpains in that it possesses no caseinolytic activity, separates from the calpains on the first DE52 column, and is insensitive to leupeptin and E-64 (N-[N-(L-3-trans-carboxyoxrian-2-carbonyl)-L-leucyl]agmatine). Thus, the molecular mass, the substrate specificity, the chromatographic behavior, and the inhibitor spectrum all suggest that this enzyme is a novel type of Ca2(+)-activated neutral protease.

Topics: Amino Acid Sequence; Animals; Brain; Calcium; Calcium-Binding Proteins; Calpain; Chromatography; Cytosol; Electrophoresis, Polyacrylamide Gel; Endorphins; Humans; Hydrogen-Ion Concentration; Male; Molecular Sequence Data; Molecular Weight; Peptide Fragments; Rabbits; Rats; Rats, Inbred Strains; Substrate Specificity; Sulfhydryl Reagents

In previous studies, we found a significantly higher (100% or more) content of cathepsin D in the aging brain. In the present study, we determined activity of Ca2(+)-activated neutral protease requiring millimolar Ca2+ (calpain II, CANP II) and amount of its endogenous inhibitor, calpastatin, in extracts of various brain regions of 3-month-old and 24-month-old male Fischer-344 rats. Calpain II was separated from calpastatin in a single step (chromatography) and its activity was tested using as substrates [methyl-14C]alpha-casein, the cytoskeletal proteins desmin and actin, and a mixture of neurofilament triplet proteins and glial fibrillary acidic proteins (GFAP). We found no changes in calpain II activity in pons-medulla and spinal cord, but significant increases were detected in cortex (72%) and striatum (63%) of the 24-month-old rats using [methyl-14C]alpha-casein as substrate. The profile of desmin and actin breakdown showed regional variations somewhat different from those of [methyl-14C]alpha-casein. With desmin, the greatest increases with age were in the striatum (82%) and hypothalamus (46%), but there were no alterations in cortex, cerebellum, and pons-medulla. With actin, slightly enhanced activity in cortex and cerebellum was noticeable. Calpastatin content in brain regions was also increased, with the regional pattern of increase fairly similar to the pattern of enzyme activity increase. The causes and the physiological consequences of increased calpain and calpastatin content in the aged brain are being investigated. That changes with age are somewhat different with the various brain protein substrates indicates that some of the properties of the enzyme also undergo alteration with age.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Actins; Aging; Animals; Brain; Calcium-Binding Proteins; Calpain; Caseins; Cerebellum; Cerebral Cortex; Desmin; Glial Fibrillary Acidic Protein; Male; Medulla Oblongata; Pons; Rats; Rats, Inbred F344; Spinal Cord

An additional component of the regulatory system of rat skeletal muscle calpain has been identified. It exerts a potent activating effect on calpain activity and is a heat stable small molecular weight protein. Of the two calpain isozymes present in muscle, the activator is specific for calpain II, being uneffective with calpain I. It promotes activation of the proteinase by reducing 50 fold, from 1 mM to of 20 microM, the requirement of Ca2+ for maximum catalytic activity of the proteinase. However in the presence of the activator calpain II expresses a consistent fraction of the maximum activity even at significantly lower concentrations of Ca2+ (below 5 microM Ca2+). The activator effect follows kinetics that are consistent with the presence of specific binding sites on the calpain molecules. The activator not only removes in a dose dependent fashion the inhibition of calpain by calpastatin, but also prevents inhibition of the proteinase upon the addition of calpastatin. Competition experiments revealed that the proteinase contains distinct sites for the activator and the inhibitor, and that both ligands can bind to calpain with the formation of an almost fully active ternary complex.

Topics: Animals; Binding, Competitive; Biological Factors; Calcium; Calcium-Binding Proteins; Calpain; Chromatography, Gel; Enzyme Activation; Kinetics; Ligands; Muscles; Rats

Expression plasmids were constructed from the cDNA of human calpastatin to examine the contribution to the inhibition of calpain of highly conserved sequences in each of four repetitive domains. A series of deletion derivatives of domain 1 proteins, truncated at either the amino or carboxy terminus, were produced in E. coli. Deletion from the amino terminus past the amino terminal conserved sequence decreased the inhibition. When the middle conserved sequence, the M-sequence, was further deleted, no inhibition was detected, but deletion from the carboxy terminus past the carboxy terminal conserved sequence did not decrease the inhibition until the M-sequence was reached. Nuclear magnetic resonance and circular dichroism spectra showed that domain 1 has an unfolded structure. Peptides that contained the M-sequence and some neighboring sequences were synthesized to measure the minimum size of the inhibitory peptide, which was the M-sequence with the next six residues on the amino terminal side.

Topics: Amino Acid Sequence; Base Sequence; Calcium-Binding Proteins; Calpain; Circular Dichroism; Cloning, Molecular; Escherichia coli; Kinetics; Molecular Sequence Data; Peptides; Plasmids; Protein Conformation; Recombinant Proteins; Restriction Mapping; Terminator Regions, Genetic

The objectives of this study were to examine effects of a beta-adrenergic agonist (cimaterol) on growth and muscle development in rabbits and to examine cimaterol's effects on myofibrillar protein degradation (MPD) and on activities of several proteolytic enzymes including the calcium-dependent proteinases (CDP). Twelve New Zealand White rabbits were assigned to either control diets or to diets containing cimaterol for 35 d, after which they were killed and effects on performance and tissue weight gains were determined. Urine was collected from d 21 through 28 from each rabbit for assessment of N tau-methylhistidine (NMH) excretion. Cimaterol increased rates of gain, efficiency of gain and skeletal muscle weights. Enhancement in muscle weight was associated with an increase in total DNA and with a reduction in NMH. Cimaterol did not affect activities of cathepsin B, cathepsin D or neutral serine proteinase, but it reduced activities of the millimolar and micromolar forms of the CDP by 58 and 57%, respectively, and it reduced activity of the inhibitor of the CDP (calpastatin) by 52%. Cimaterol-dependent myofibrillar protein accretion was likely mediated, at least in part, by a reduction in MPD. The change in MPD was associated with a reduction in muscle CDP activities. Cimaterol-dependent muscle hypertrophy therefore may involve changes in calcium-dependent proteolysis of myofibrillar proteins. The significance of the effects of cimaterol on calpastatin activity is not known.

Topics: Animals; Calcium-Binding Proteins; Calpain; DNA; Ethanolamines; Liver; Male; Methylhistidines; Muscle Development; Muscle Proteins; Muscles; Myofibrils; Rabbits; Random Allocation; RNA; Weight Gain

The purposes of the current study were to: determine if human lenses contain calpain II (EC.34.22.17) activity, measure the effect of aging and anatomical location on lens calpain II activity, and determine if human lenses contain the endogenous calpain inhibitor calpastatin. Both enzymatic and immunologic assays indicated that human lenses contained calpain II activity. Calpain II activity was highest in the cortex of lenses from young donors, and lowest in the nucleus of aged lenses, where it was sometimes nondetectable. In some cases, calpain II activity persisted in the nucleus of lenses from donors greater than 70 years of age. Human lenses also contained endogenous calpain inhibitor (calpastatin) in excess over calpain enzymatic activity. Calpastatin activity did not decrease during aging. Although human lenses contained approximately 3% of the calpain activity found in rat lenses, calpain II may still be a major endopeptidase in human lenses. Demonstration of calpain II in human lenses suggested that calpain II could be involved in both lens maturation and cataract formation.

Topics: Aged; Aging; Calcium-Binding Proteins; Calpain; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Humans; Lens, Crystalline; Middle Aged; Tissue Distribution

Topics: Animals; Blood Platelets; Calcium-Binding Proteins; Calpain; Cattle; Chromatography, Gel; Chromatography, Ion Exchange; Humans; Indicators and Reagents; Isoenzymes; Kinetics; Substrate Specificity

Extracellular location of calpain and calpastatin was demonstrated in the cell-free synovial fluid obtained from the knee joint of healthy adult humans and several patients with rheumatoid arthritis. Calpains I and II and a few molecular species of calpastatin were identified by chromatographies on DEAE-cellulose and on Ultrogel AcA 34 columns as well as by immunoelectrophoretic blot analysis. Calpains I and II in the synovial fluid of the patients increased 6.7 times and 3.5 times, respectively, compared with those of the control subjects. With the patients, shortening of the heavy subunits of calpains was noted. Calpastatin also increased in the patients, but it showed rather extensive fragmentation.

Topics: Adult; Arthritis, Rheumatoid; Blotting, Western; Calcium-Binding Proteins; Calpain; Chromatography, DEAE-Cellulose; Extracellular Space; Female; Humans; Knee Joint; Molecular Weight; Synovial Fluid

1. The levels of Ca-independent and Ca-dependent proteolytic activity as well as the activities of calpains and calpastatin in different organs of the rabbit was examined at various developmental stages. 2. Calpain and calpastatin levels were highest in the lung and in the kidney. 3. In all organs examined except the thymus the total level of calpain was higher than that of calpastatin. 4. In the thymus the levels of calpains and calpastatin decreased markedly with age.

Topics: Aging; Animals; Brain; Calcium; Calcium-Binding Proteins; Calpain; Kidney; Lung; Myocardium; Rabbits; Spleen; Thymus Gland; Tissue Distribution

Chicken breast muscle has three Ca2+-dependent proteinases, two requiring millimolar Ca2+ (m-calpain and high m-calpain) and one requiring micromolar Ca2+ (mu-calpain). High m-calpain co-purifies with mu-calpain through successive DEAE-cellulose (steep gradient), phenyl-Sepharose, octylamine agarose, and Sephacryl S-300 columns, but elutes after mu-calpain when using a shallow KCl gradient to elute a DEAE-cellulose column. The mu- and m-calpains have 80 and 28 kDa polypeptides and are analogous to the mu- and m-calpains that have been purified from bovine, porcine and rabbit skeletal muscle. High m-calpain, which seems to be a new Ca2+-dependent proteinase, is still heterogeneous after the DEAE-cellulose column eluted with a shallow KCl gradient. Additional purification through two successive HPLC-DEAE columns and one HPLC-SW-4000 gel permeation column produces a fraction having six major polypeptides and 6-8 minor polypeptides on SDS-PAGE. A 74-76 kDa polypeptide in this fraction reacts in Western blots with monospecific, polyclonal anti-calpain antibodies that react with both the 80 kDa and the 28 kDa polypeptides of mu- or m-calpain. High m-calpain also is related to mu- and m-calpain in that it causes the same limited digestion of skeletal muscle myofibrils, has a similar pH optimum near pH 7.9-8.4, requires Ca2+ for activity, and reacts with the calpain inhibitor, calpastatin, and a variety of serine and cysteine proteinase inhibitors in a manner identical to mu- and m-calpain. High m-calpain differs from mu- and m-calpain in its elution off DEAE-cellulose columns and its requirement of 3800 microM Ca2+ for one-half maximal activity compared with 5.35 microM Ca2+ for mu-calpain and 420 microM Ca2+ for m-calpain. The physiological significance of high m-calpain in unclear. The presence of mu-calpain in chicken breast muscle suggests that all skeletal muscles contain both mu- and m-calpain, although the relative proportions of these two proteinases may vary in different species.

Topics: Actins; Animals; Blotting, Western; Calcium; Calcium-Binding Proteins; Calpain; Chickens; Chromatography; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Female; Hydrogen-Ion Concentration; Molecular Weight; Muscles; Myofibrils; Myosins; Peptide Fragments; Troponin; Troponin I; Troponin T

Autolyzed mu-calpain, unautolyzed mu-calpain, autolyzed m-calpain, and unautolyzed m-calpain (mu-calpain is the micromolar Ca2+-requiring proteinase, m-calpain is the millimolar Ca2+-requiring proteinase) were passed through a calpastatin-affinity column at different free Ca2+ concentrations, and binding of the calpains to calpastatin was compared with proteolytic activity of that calpain at each Ca2+ concentration. Unautolyzed m-calpain, autolyzed m-calpain, and autolyzed mu-calpain required less Ca2+ for half-maximal binding to calpastatin than for half-maximal activity. Unautolyzed mu-calpain, however, required slightly more Ca2+ for half-maximal binding to calpastatin than for half-maximal activity. Half-maximal binding of oxidatively inactivated mu- or m-calpain to calpastatin required approximately the same Ca2+ concentrations as half-maximal binding of unautolyzed mu- or m-calpain, respectively, to calpastatin. Binding of unautolyzed m-calpain and autolyzed mu-calpain to calpastatin occurred over a wide range of Ca2+ concentrations, and it seems likely that two or more Ca2+-binding sites with different Ca2+-binding constants are involved in binding of the calpains to calpastatin. Proteolytic activity occurs at different Ca2+ concentrations than calpastatin binding, suggesting a second set of Ca2+-binding sites associated with proteolytic activity. Third and fourth sets of Ca2+-binding sites may be involved in autolysis and in binding to phosphatidylinositol or cell membranes; these four Ca2+-dependent properties of the calpains may require the eight potential Ca2+-binding sites that amino acid sequences predict are present in the calpain molecules.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Cattle; Chromatography, Affinity; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Molecular Weight; Muscles; Protease Inhibitors; Protein Binding

The binding of a calcium-activated neutral protease (CANP) with high calcium sensitivity (muCANP) to erythrocyte membranes and its subsequent autolytic activation on the membranes were analyzed by an immunoblot technique. In the presence of calcium ions, muCANP bound to the erythrocyte membranes as a heterodimer of 79- and 28-kDa subunits and was converted quickly on the membranes to an active form with a 76-kDa large subunit. The active form was then released from the membranes to the soluble fraction. These sequential reactions, however, were not specific to inside-out vesicles, but occurred also, except for some Ca2+-independent binding, on right side-out vesicles. A rapid degradation of some membrane proteins was observed after binding of muCANP to the membranes. The binding of muCANP to erythrocyte membranes was inhibited by substrates and the endogenous CANP inhibitor, which is also a suicide substrate. These results strongly suggest that muCANP binds to membranes by recognition of membrane proteins as substrates and not at a special site for activation. Thus, a possible mechanism for muCANP activation on membranes is that muCANP first binds to substrates on membranes, is activated, and then degrades the substrates to deform the membrane structures.

Topics: Animals; Blood Proteins; Calcium; Calcium-Binding Proteins; Calpain; Caseins; Chickens; Egtazic Acid; Enzyme Activation; Erythrocyte Membrane; Immunoblotting; Kinetics; Leupeptins; Macromolecular Substances; Membrane Proteins; Mice; Molecular Weight; Rabbits; Vimentin

Calpastatin is a widely distributed endogenous inhibitor protein specifically acting on calpain (Ca2+-dependent cysteine endopeptidase). The inhibitor consists of four inhibitory domains (Domains 1-4) with mutually homologous sequences. NH2-terminal Domain L is non-homologous, and all domains have 120-140 residues each. A human calpastatin genomic DNA clone was isolated using a previously obtained human calpastatin cDNA probe. Sequence analysis has revealed that the clone contains Domain 1 and segments of neighboring domains (Domains L and 2). Each of three highly conserved, restricted regions within Domain 1 was located on separate exons, 1A, 1B, and 1C. Exon 2A, corresponding to the first exon of Domain 2, is homologous to Exon 1A and follows Exon 1D of Domain 1. A 27-residue peptide encoded by Exon 1B, including a 12-residue middle conserved sequence, was chemically synthesized and tested for protease inhibitory activities. The synthetic peptide showed strong inhibition against calpain I (low Ca2+-requiring form), and calpain II (high Ca2+-requiring form), but no inhibition against papain or trypsin. These results indicated that Exon 1B forms a self-sufficient functional subdomain of the calpastatin inhibitory domain.

Topics: Amino Acid Sequence; Animals; Base Sequence; Calcium-Binding Proteins; Calpain; DNA; Exons; Genes; Genome, Human; Humans; Molecular Sequence Data; Oligopeptides; Rabbits; Restriction Mapping; Sequence Homology, Nucleic Acid; Species Specificity; Swine

Pig brain was found to contain two calpain-specific, heat-stable inhibitory fractions which could be separated by DEAE-cellulose chromatography. CS-0.1, which was eluted from the column at 0.1 M NaCl, was identified as an ordinary, well-known calpastatin. CS-0.2, eluted at 0.2 M NaCl, was different from CS-0.1 in that it inhibited calpain 1 more strongly than calpain II and that it did not cross-react with anti-calpastatin antibodies. Partial purification indicated that CS-0.2 contained inhibitor proteins smaller than ordinary calpastatin, but whether they are the products derived from CS-0.1 or entirely different genetic products has not yet been determined.

Topics: Animals; Blotting, Western; Brain Chemistry; Calcium-Binding Proteins; Calpain; Chromatography, Ion Exchange; Cross Reactions; Cysteine Proteinase Inhibitors; Swine

PC12 cells are a nerve growth factor-responsive clone derived from a rat pheochromocytoma. Treatment with nerve growth factor causes the cells to differentiate. One of the hallmarks of this differentiation is the generation of neurites. PC12 cells contain both calpain I and calpain II; about 90% of the total calpain activity is due to calpain II. Treatment of the cells with nerve growth factor causes a time-dependent decrease in calpain activity, more than 50% being lost over a 5-day period. Both the decrease in calpain activity and the growth of neurites are reversible upon the removal of nerve growth factor from the cultures. Agents other than nerve growth factor that cause neurite outgrowth, such as fibroblast growth factor and dibutyryl cyclic AMP, also cause a decrease in calpain activity. Calpain levels, as detected with immunoblotting or immunohistochemistry, show no decrease. Removal of calpastatin, the endogenous inhibitor of the calpains, by phenyl-Sepharose chromatography increases the calpain activity of extracts from both control and nerve growth factor-treated cells and brings the activity in the extracts from treated cells up to the activity in those from controls. Calpastatin-containing fractions from extracts of nerve growth factor-treated cells inhibit more calpain activity than do comparable fractions from control cells. These studies suggest that nerve growth factor causes a decrease in the activity of calpain in morphologically differentiating PC12 cells by causing an increase in the activity of calpastatin.

Topics: Adrenal Gland Neoplasms; Animals; Calcium-Binding Proteins; Calpain; Cell Line; Fluorescent Antibody Technique; Kinetics; Nerve Growth Factors; Pheochromocytoma; Rats; Tumor Cells, Cultured

A major part of the Ca-activated proteolytic activity in the soluble fraction from rabbit brain could be due to the activity of the neutral thiol-proteases calpain I and II. The activity of calpains exceeded that of the endogenous inhibitor, calpastatin, at all developmental stages studied. The level of calpains increased rapidly from the prenatal stage to reach a peak 10-20 days postnatally. From this period the level of calpains decreased slowly to reach the adult levels. The level of calpastatin increased steadily from the prenatal stage to old age.

Topics: Aging; Animals; Brain; Calcium-Binding Proteins; Calpain; Embryonic and Fetal Development; Rabbits

1. Carnosine, anserine and L-1-methyl-histidine activated muscle calpain II assayed at 2.5 mM Ca2+. 2. At 5 microM Ca2+, none of these compounds activated calpain II sufficiently to bring its activity up to the level measured at 2.5 mM Ca2+. 3. Carnosine increased, whereas both anserine and L-1-methyl-histidine decreased the inhibitory effect of calpastatin on calpain II. 4. These results suggest that although the compounds are not potent activators of calpain II, the ratio of the dipeptides in muscle may have an effect on calpain II-calpastatin interaction.

Topics: Alanine; Animals; Anserine; Calcium-Binding Proteins; Calpain; Carnosine; Chickens; Dipeptides; Histidine; Male; Methylhistidines; Muscle, Smooth; Muscles

Five and nine-tenth kg of Elodea densa (Anacharis), a common aquarium plant, was extracted, and the extract was subjected to column chromatographic procedures that successfully purify the two Ca2(+)-dependent proteinases (calpains) and their protein inhibitor (calpastatin) from a variety of animal tissues. Although these procedures purified a protein having 55- and 16-kDa polypeptides, neither this protein nor any of the other chromatographic fractions contained detectable proteinase or calpastatin activity. Moreover, the purified 55- and 16-kDa polypeptides did not react on immunoblots with polyclonal antibodies that were monospecific for the calpains or calpastatin. We conclude that Elodea densa contains no calpain nor calpastatin at the level of 4 micrograms per g plant protein (1 part per 250,000), which was the sensitivity of our assay.

Topics: Actins; Animals; Calcium; Calcium-Binding Proteins; Calpain; Cattle; Chromatography, DEAE-Cellulose; Intermediate Filaments; Plants

The purpose of these experiments was to determine how the activity and regulation of calpain in rat lens changed during aging. Calpain II enzyme activity and immunoreactivity decreased with both chronological and anatomical age. Two pieces of data suggested that loss of soluble calpain II was a result of both autolysis and insolubilization during aging: (i) proteolytic fragments of calpain were detected in lenses with molecular weights similar to fragments produced during incubation of purified calpain II with calcium; (ii) the water-insoluble fraction of lens cortex contained increasing amounts of calpain antigen during aging both the 75-kDa calpain subunit and a unique high-molecular-weight immunoreactive protein. The regulation of calpain II also appeared to change with age. The activity of calpain II in vivo may be regulated by the relative concentrations of calpain II and its endogenous inhibitor calpastatin. Calpain II concentrations decreased in the rat lens with age, whereas levels of the endogenous inhibitor calpastatin were maintained. Assays of calpain II and calpastatin indicated that upon aging there was insufficient activity of calpain II to overcome the inhibition of calpastatin in the nucleus. These findings were confirmed by incubation of crude lens homogenates of 2-week- and 7-month-old rat lens regions with calcium. It is hypothesized that binding of calpain II to membrane may be important for calpain II activation, especially in older lens regions, because it may allow escape from the inhibitory action of calpastatin.

Topics: Aging; Animals; Calcium-Binding Proteins; Calpain; Electrophoresis, Polyacrylamide Gel; Immunoblotting; Lens Cortex, Crystalline; Lens Nucleus, Crystalline; Lens, Crystalline; Rats; Rats, Inbred Strains; Solubility

Calpain I requires low Ca2+ for activation and calpain II requires high Ca2+. It was generally accepted that erythrocytes contain calpain I and calpastatin, but no calpain II. We have recently found, however, that nucleated chicken erythrocytes contain both calpains I and II in addition to calpastatin. The finding is significant in rectifying the previous view that the chicken has only one molecular species of calpain, whereas mammals have two. Another erroneous view which prevailed previously was that polymorphonuclear (PMN) cells contain only one calpain species. We could also recently demonstrate that pig PMN cells do contain both calpains I and II. The cloning of cDNAs for calpastatin enabled us to utilize them as the probes in studying the expression of calpastatin in various hematopoietic cell-line cells. We found that several T cells infected with human retrovirus HTLV-I markedly increased the production of calpastatin, which could be measured both by calpain-inhibition assay and by Western blot analysis, but the level of mRNA for calpastatin did not significantly change when compared with noninfected T cells. The increase in calpastatin protein always parallels with the expression of interleukin 2 receptor protein by the HTLV-I-infected T cells, although the biological implication of such phenomena is almost entirely unknown yet.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Line; Chickens; Erythrocytes; Hematopoietic Stem Cells; Humans; Neutrophils

Calpain and calpastatin are known to be very widely distributed in animal cells. Although the physiological roles of calpain have not yet been clarified, the proteinase-proteinase inhibitor system has been suggested to play important roles in various cellular functions coupled with Ca2+ mobilization. By nucleotide sequencing of the cloned cDNAs, the primary structures of calpains and calpastatins have recently been disclosed. The expression of the genes for calpains and calpastatins in vitro as well as in vivo is being studied using cDNAs and their fragments. This paper reviews several topics on the recent progress of calpain research.

Topics: Amino Acid Sequence; Animals; Calcium-Binding Proteins; Calpain; DNA Probes; DNA, Recombinant; Gene Expression; Humans; Models, Chemical; Molecular Sequence Data; Rabbits; RNA, Messenger; Swine; T-Lymphocytes

DEAE-Sephacel and phenyl-Sepharose chromatography were compared as methods for separating and quantitatively isolating calpain I, calpain II, and calpastatin from lamb muscle extracts. DEAE-Sephacel chromatography gave greater than 90% recovery of all three proteins, while phenyl-Sepharose gave only 70, 66, and 48% of the DEAE recovery of calpain I, calpain II, and calpastatin, respectively. Additionally, DEAE-Sephacel chromatography was shown to effectively separate calpastatin and calpain I. Consequently DEAE-Sephacel appears to be superior to phenyl-Sepharose for quantitative isolation of the components of the calcium-dependent proteinase system from muscle extracts. Dietary administration of beta-agonist (L-644, 969; Merck Sharpe & Dohme Research Laboratories) decreases extractable calpain I activity in lamb longissimus dorsi (LD) muscle by 10-14% (P less than 0.05), increases calpain II activity by 34-42% (P less than 0.001), and increases calpastatin activity by 59-75% (P less than 0.001). Additionally, net cathepsin B activity is reduced by 30% (P less than 0.05) in the LD of beta-agonist-treated lambs. Reduced activity of the calcium-dependent or catheptic proteinase systems may contribute to the increased protein accretion in muscles of beta-agonist-treated lambs.

Topics: Adrenergic beta-Agonists; Animals; Calcium-Binding Proteins; Calpain; Cathepsin B; Cathepsin H; Cathepsins; Chromatography, Ion Exchange; Cysteine Endopeptidases; Female; Isoenzymes; Kinetics; Muscles; Pyridines; Reference Values; Sheep

cDNA of human calpastatin, an inhibitor protein specific for calpain (EC 3.4.22.17; Ca2(+)-dependent cysteine proteinase) was isolated by screening of a library prepared from human liver mRNA with pig calpastatin cDNA fragment as a probe. The primary structure of human calpastatin was deduced from the nucleotide sequence of the cDNA and compared with that of pig and rabbit calpastatins already reported. Human calpastatin consisted of 673 amino acid residues and had 78% and 77% identity to pig or rabbit calpastatins, respectively. Human calpastatin had a domain structure with four internally repetitive sequences and one N-terminal non-homologous sequence like the other calpastatins. Human calpastatin had two deletions, 22 and 13 residues long in domain L and domain 1, respectively, compared to pig or rabbit calpastatins.

Topics: Amino Acid Sequence; Animals; Base Sequence; Calcium-Binding Proteins; Calpain; Cloning, Molecular; DNA; Gene Library; Humans; Liver; Molecular Sequence Data; Poly A; Rabbits; Restriction Mapping; RNA, Messenger; Sequence Homology, Nucleic Acid

Two forms of Ca++-dependent cysteine proteinases, calpain I, requiring low Ca++ (microM concentration), and calpain II, requiring high Ca++ (mM concentration), were purified from the cytosolic fraction of pig epidermis. Calpains I and II were separated on DEAE-cellulose chromatography, and thereafter they were purified by separate but almost identical procedures, which included chromatographies on Sephacryl S-300, Blue Sepharose CL-6B, and DEAE Bio-Gel A. Purified calpains I and II required 10 and 450 microM Ca++ for half-maximal activation, respectively, and had an optimal pH of 7.0 to 8.0. Both enzymes were heterodimers and composed of one heavy subunit (83 kDa for calpain I and 80 kDa for calpain II) and one light subunit (29 kDa for both enzymes). The action of calpains I and II on keratin extracted from the same tissue was studied. Both enzymes rapidly cleaved keratin into small fragments. The cleavage depends on Ca++ and could be blocked by leupeptin and calpastation, an endogenous calpain-specific inhibitor, which was also found in the cytosolic fraction of pig epidermis and partially purified.

Topics: Animals; Calcium-Binding Proteins; Calpain; Chromatography; Epidermis; In Vitro Techniques; Keratins; Swine

Thirteen cell lines of the human hematopoietic system were tested for expression of calpastatin at three different levels (mRNA, product and activity). The amount of calpastatin product and its inhibitory activity varied markedly depending upon cell types or human T-cell leukemia virus type I infection, although there were difference in the expression at the transcriptional level. Cell lines with high content of calpastatin comprised T-cell lineage infected by human T-cell leukemia virus type I, B-cell line and myelocytic leukemia cell lines. Non-infected T-cell lines and null cell line revealed themselves as poor in both calpastatin products and inhibitor activities. Two different molecular forms of calpastatin were observed: a 102-kDa form in T- and B-cell lines and a 94-kDa form in one of myelocytic cells. Three mRNA species of calpastatin (3.8, 3.0 and 2.5 kb) were identified in human hematopoietic system cells.

Topics: Calcium-Binding Proteins; Calpain; Cell Line; DNA; Hematopoietic System; Humans; RNA

Bovine myocardial calpastatin, the endogenous inhibitor of the calcium-dependent proteinases, calpains, could bind to sarcoplasmic reticulum preparations at neutral pH and low ionic strength. Even in the presence of 100 to 200 mM KCl, 4 to 5 micrograms of calpastatin was bound per mg of membrane. Although calpastatin is found associated with bovine myocardial sarcolemma, neither canine nor human erythrocyte calpastatins were present in isolated erythrocyte membrane preparations. The bovine myocardial calpastatin, but not human erythrocyte calpastatin, could associate with purified phospholipid vesicles at low ionic strength. Thus, phospholipids appear to be involved in the binding of calpastatin to membranes.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Cell Membrane; Dogs; Erythrocyte Membrane; Humans; Hydrogen-Ion Concentration; Liposomes; Myocardium; Osmolar Concentration; Phospholipids; Sarcolemma; Sarcoplasmic Reticulum

We have already determined the primary structure of the endogenous inhibitor for calcium-dependent protease (CANP inhibitor, calpastatin) from the cDNA sequence and revealed that the CANP inhibitor contains four internally repeating units which could be responsible for its multiple reactive sites (Emori, Y., Kawasaki, H., Imajoh, S., Imahori, K., and Suzuki, K. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 3590-3594). Restriction fragments of the cDNA corresponding to each of the four domains (encoding 104-156 amino acid residues of the total 718 residues) were subcloned into the multicloning site of pUC9 or pUC18 in a direction and frame matched to the lacZ' open reading frame of the vector. Under the lac operator-promoter system, we succeeded in producing truncated fragments of the CANP inhibitor in Escherichia coli. The CANP inhibitor fragments were partially purified, and the inhibitory activities toward calcium-dependent protease (CANP) were examined. All fragments containing well conserved regions of about 30 amino acid residues (domains I-IV) located in the middle of the four units exhibited the inhibitory activity. However, their inhibitory activities varied considerably. Further truncation experiments revealed that small fragments containing 30-70 amino acid residues of the CANP inhibitor still retained inhibitory activity. From these experimental results the following conclusions can be drawn: 1) each of the four repeating units of the CANP inhibitor (about 140 amino acid residues) is a real functional unit and can inhibit CANP activity independently; and 2) domains corresponding to well conserved sequences of about 30 amino acid residues containing a consensus Thr-Ile-Pro-Pro-X-Tyr-Arg sequence are essential for the inhibitory activity, and the bordering regions are important for its modulation.

Topics: Amino Acid Sequence; Calcium-Binding Proteins; Calpain; DNA; Escherichia coli; Molecular Sequence Data; Plasmids; Repetitive Sequences, Nucleic Acid

1. Two experiments are reported in which the effect of alteration in growth rate on the levels of avian skeletal muscle calcium-activated neutral proteinase (EC 3.4.22.17) (CANP or calpain) and its specific inhibitor (calpastatin), a system thought to be implicated in myofibrillar catabolism, was studied by means of manipulation of dietary protein concentration. 2. In Expt 1 broiler chicks were given free access to diets containing 105, 149, 197 and 212 g protein/kg for 20 d. In Expt 2 the four dietary treatments were 119, 141, 182 and 227 g protein/kg diet given for 16 d. Chick growth rate and total leg skeletal muscle weight significantly increased (P less than 0.001) with increasing dietary protein concentration in both experiments. Total skeletal muscle protein increased with the level of dietary protein, the effect being significant (P less than 0.01 and P less than 0.001 in Expts 1 and 2 respectively). 3. Minced leg muscle was homogenized in low-salt buffers, and the extract chromatographed on DEAE-cellulose to separate proteinase and inhibitor activity. The partially purified CANP enzyme and inhibitor proteins were present at a concentration broadly consistent with literature reports, and their elution characteristics and Ca2+ concentration dependence were not varied by dietary protein concentration. 4. Both the muscle CANP and CANP inhibitor activities (units/kg muscle) exhibited upward trends with growth rate and increased muscle weight. However, these differences were not statistically significant (P greater than 0.05) and were not present at all when the results were expressed as units/g muscle protein.

Topics: Animals; Calcium-Binding Proteins; Calpain; Chickens; Dietary Proteins; Male; Muscles; Protease Inhibitors

Mitotic spindle disassembly requires major structural alterations in the associated cytoskeletal proteins and mitosis is known to be associated with Ca2+-sequestering phenomena and calcium transients. To examine the possible involvement of a ubiquitous Ca2+-activated protease, calpain II, in the mitotic process, synchronized PtK1 cells were monitored by immunofluorescence for the relocation of calpain II. The plasma membrane was the predominant location of calpain II in interphase. However, as mitosis progressed, calpain II relocated to (i) an association with mitotic chromosomes, (ii) a perinuclear location in anaphase, and (iii) a mid-body location in telophase. Microinjection of calpain II near the nucleus of a PtK1 cell promoted the onset of metaphase. Injection of calpain II at late metaphase promoted a precocious disassembly of the mitotic spindle and the onset of anaphase. These data suggest that calpain II is involved in mitosis.

Topics: Anaphase; Animals; Calcium; Calcium-Binding Proteins; Calpain; Cell Line; Cell Membrane; Cell Nucleus; Chromosomes; Enzyme Activation; Fluorescent Antibody Technique; Fluorescent Dyes; Interphase; Metaphase; Mitosis; Muscles; Rhodamines; Spindle Apparatus; Swine

The specific inhibitor protein (calpastatin) for the calcium-dependent intracellular proteinases (calpains) is an important regulator of these enzymes. In this communication we describe a one day procedure for purifying 3 to 5 mg of calpastatin from a kilogram of bovine myocardium. This represents a substantial improvement over previously described methods, and should facilitate future studies of calpastatin structure and function. A key, novel step in the purification was dye-matrix chromatography on an Affi-Gel Blue column. Contrary to previous indications, calpastatin purified by the new method did not contain significant amounts of carbohydrate. However, the presence of covalently bound phosphate in purified bovine myocardial calpastatin was confirmed and co-migration of phosphate and calpastatin activity was demonstrated on Bio-Gel A-1.5m chromatography. Thus, it is possible that calpastatin function is regulated by phosphorylation.

Topics: Animals; Calcium-Binding Proteins; Calpain; Carbohydrates; Cattle; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Immunochemistry; Myocardium; Phosphates

The occurrence and molecular sizes of calpains and calpastatin in the lysates of human erythrocytes, platelets, lymphocytes/monocytes, and polymorphonuclear cells were studied by immunoelectrophoretic blot analysis. The basic uniformity among these cells of the 85-kDa and 83-kDa heavy subunits of low- and high-Ca2+-requiring calpains I and II, respectively, and of the 29-kDa light subunit was confirmed. Molecular diversity of calpastatin species, ranging from 70 kDa to 107 kDa, among different blood cells was also shown. The obtained data are consistent with those known for other animal tissues, thus settling hitherto uncertain or rather controversial issues on the distribution of calpains and calpastatin in human blood cells.

Topics: Blood Platelets; Calcium-Binding Proteins; Calpain; Erythrocytes; Humans; Leukocytes; Lymphocytes; Molecular Weight; Monocytes; Neutrophils; Protease Inhibitors

Structure-function relationships in pig calpastatin were investigated. Calpastatin is an endogenous inhibitor protein specifically acting on calpains (Ca2+-dependent cysteine endopeptidases). We recently cloned and sequenced the cDNA for pig heart calpastatin and determined the amino acid sequence of the molecule from the nucleotide sequence. Various deletion mutants in one of the four internally repetitive domains (Domain 3, approximately 140 amino acid residues) were created by in vitro site-directed mutagenesis of a cloned cDNA fragment and expressed in Escherichia coli. Deletion of a conserved region on either the amino-terminal or carboxyl-terminal side caused a drastic loss of inhibitory activity against calpain I (low Ca2+-requiring form) and, to a lesser degree, against calpain II (high Ca2+-requiring form). Inhibitory activities were below the detectable level in mutants deleted further toward the central region. Substitution of two amino acids in the latter region of the wild-type Domain 3 protein caused a drastic loss of activity against both calpains. The creation of lowered affinity inhibitors enabled us to perform a conventional kinetic analysis which showed the mode of inhibition to be competitive. Prediction of the secondary structure of Domain 3 suggests that both the amino- and carboxyl-terminal conserved regions form alpha-helical structures, which are largely located in the interior of the calpastatin molecule, whereas the central region does not form alpha-helix or beta-structure. The central region contains a 12-residue consensus sequence common to Domains 1, 2, and 4, and this portion is predicted to be located on the surface of the calpastatin molecule. These results suggest that the central conserved region of each domain of calpastatin is an area for direct interaction either with the active center of calpain or a region in close proximity, and the rest of the domain is a region stabilizing the functionally important tertiary structure of the domain.

Topics: Amino Acid Sequence; Animals; Base Sequence; Calcium-Binding Proteins; Calpain; Cloning, Molecular; DNA; DNA Restriction Enzymes; Escherichia coli; Genes; Kinetics; Molecular Sequence Data; Mutation; Protease Inhibitors; Structure-Activity Relationship; Swine

Calcium-activated neutral proteases (calpain, EC 3.4.22.17) bind to agarose matrices (Bio-Gel A-150m, Sepharose 4B, and Ultrogel AcA 34) with high affinity in the presence of calcium. 6-O-beta-Galactopyranosyl-D-galactose, a disaccharide which closely resembles the repeating unit of the agarose matrices, completely blocks the binding of calpains and can release agarose-bound enzymes in the presence of calcium. At least 1 microM level of free calcium is required for binding. Other calcium binding proteins, including calmodulin, calpastatin, casein, and neurofilament proteins, fail to bind under the same conditions. Both calpain I and calpain II can be readily purified from crude enzyme preparations by agarose chromatography in the presence of calcium and leupeptin. Agarose-bound enzymes are eluted with calcium-free solutions or can be released in the presence of calcium by 1% Triton X-100, but not by 1 M urea or 20% ethylene glycol. Enzymes eluted from agarose are activated, as evidenced by the appearance of faster migrating forms (76 and 78 kDa) of the 80-kDa catalytic subunit of calpain I upon electrophoresis and by the increased sensitivity of calpain II to activation by micromolar levels of calcium. The electrophoretic migration of the 30-kDa regulatory subunit is, however, unaltered in enzyme fractions eluted from an agarose column. When the enzyme subunits are dissociated in 1 M NaSCN, only the 30-kDa subunit binds to the agarose matrix. Furthermore, neither calpain I nor calpain II binds to agarose when their 30-kDa subunit is autocatalyzed to an 18-kDa fragment, indicating that the NH2-terminal of the 30-kDa subunit is important for the binding of calpains to an agarose matrix.

Topics: Calcium; Calcium-Binding Proteins; Calpain; Carbohydrate Metabolism; Chromatography; Disaccharides; Enzyme Activation; Ethylene Glycol; Ethylene Glycols; Leupeptins; Octoxynol; Polyethylene Glycols; Sepharose; Thiocyanates; Urea

Ca2+-dependent proteolytic activity was detected at pH 7.5 in head extracts of the fruit fly Drosophila melanogaster. This activity was abolished by iodoacetate, but was unaffected by phenylmethanesulphonyl fluoride. These properties resemble those of the Ca2+-dependent thiol-proteinase calpain. The activity appeared at Mr 280,000 on Sepharose CL-6B gel chromatography. DEAE-cellulose chromatography revealed two activity peaks, with elution positions corresponding to vertebrate calpains I and II. The fly head enzymes were inhibited by a heat-stable and trypsin-sensitive component of the fly head extract, which also inhibited calpains from rat kidney. The inhibitor emerged from Sepharose CL-6B columns at Mr 310,000 and from DEAE-cellulose at a position corresponding to the protein inhibitor calpastatin from other sources. It is concluded that Drosophila heads comprise the Ca2+-dependent calpain-calpastatin proteolytic system.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Chromatography, DEAE-Cellulose; Chromatography, Gel; Drosophila melanogaster; Endopeptidases; Isoenzymes; Protease Inhibitors

The decrease ('run-down') of the L-type Ca2+-current during long-lasting recordings with the whole-cell patch-clamp technique was examined in guinea pig ventricular myocytes. We have tested whether proteolysis is involved in the decay of the Ca2+-current by intracellular application of several concentrations of the Ca2+-dependent proteases calpain I and II, as well as their endogenous inhibitor calpastatin. The major finding was that calpain I and calpain II accelerated the Ca2+-current run-down in a concentration dependent manner, whereas calpastatin retarded it. These observations indicate that a proteolytic degradation of Ca2+-channels might be the reason for the run-down phenomenon.

Topics: Animals; Calcium Channels; Calcium-Binding Proteins; Calpain; Cell Membrane; Guinea Pigs; In Vitro Techniques; Membrane Potentials; Myocardium; Protein Kinase C

In erythrocytes of patients with essential hypertension the level of calpastatin activity was found to be significantly lower than in red cells of normotensive subjects (1). We now demonstrate, by Western blot analysis, that the decreased inhibitory activity is due to a corresponding decrease in the amount of the inhibitor protein. This is also supported by the observation that calpastatins isolated and purified from erythrocytes of normotensive and hypertensive patients, have identical specific activity. Data are presented indicating that the decreased level of calpastatin cannot be ascribed to an accelerated decay of the inhibitor during the erythrocyte life span. Taken together the previous and present results further emphasize that an umbalanced proteolytic system may represent one of the molecular mechanisms responsible for those membrane abnormalities underlying the development of essential hypertension and its clinical complications.

Topics: Antibodies, Monoclonal; Blotting, Western; Calcium-Binding Proteins; Calpain; Cytosol; Erythrocyte Aging; Erythrocyte Membrane; Erythrocytes; Humans; Hypertension; Protease Inhibitors

1. Lamb growth trials were designed to modify growth and protein content of muscle by diet and also by beta-agonist treatment, and to correlate any changes to the activities of calpain proteinases (EC 3.4.22.17) and their inhibitor calpastatin. 2. Wether lambs in a control group were fed on a barley-based diet designed to give a growth rate of 350 g/d; a second group was fed on the same diet but the intake was restricted to give an expected gain of 44 g/d; a third group was fed on the same diet as the first group but the diet included 2 mg clenbuterol/kg. At the end of a 6-week trial, longissimus dorsi wet weights were 635 (n6), 377 (n4) and 788 g (n6) (standard error of difference 53.0) in the three groups respectively. 3. Minced L. dorsi was extracted in low-salt buffers and analysed by a fast protein liquid-chromatographic system for calpain I (low calcium-requiring), calpain II (high Ca2+-requiring) and calpastatin activities. No significant changes in the three activities were associated with reduced muscle weight in the restricted-intake group. The inclusion of clenbuterol in the diet, however, led to highly significant increases (P less than 0.001) in calpain II and calpastatin to approximately double the control values. 4. The results did not support a direct relation between these activities and muscle growth, except when protein accretion was stimulated by a beta-agonist, suggesting a role for this enzyme system in the mechanism by which these agents exert their effect.

Topics: Animals; Calcium-Binding Proteins; Calpain; Clenbuterol; Diet; Ethanolamines; Male; Muscle Development; Muscles; Sheep

Topics: Animals; Calcium-Binding Proteins; Calpain; Chickens; Cricetinae; Enzyme Inhibitors; Gizzard, Avian; Imidazoles; Kinetics; Muscle, Smooth; Muscles

1. Two fast-twitch skeletal muscles from normal and dystrophic hamsters were analysed for their calpain and calpastatin contents. 2. Assays of wide-specificity calpain II showed that the activity levels in the two muscles were increased 1.5 and 1.6 times in dystrophic animals. 3. Analysis of calpastatin levels showed that the respective dystrophic muscles had activity levels of 2.2 and 2.8 times those of control muscles. 4. These results contrast with previous studies on denervated hamster muscles which showed that denervation causes an increase in calpain levels but a decrease in calpastatin levels.

Topics: Animals; Calcium-Binding Proteins; Calpain; Muscles; Muscular Dystrophy, Animal; Proteins

Topics: Amino Acid Sequence; Animals; Calcium; Calcium-Binding Proteins; Calpain; Crystallins; Cysteine Proteinase Inhibitors; Erythrocyte Membrane; Gene Expression Regulation, Enzymologic; Membrane Proteins; Molecular Sequence Data; Receptors, Glucocorticoid; Species Specificity; Thalassemia

1. Hamster skeletal muscle contains a wide-specificity calpain which was found to be a calpain II type and which is composed of a single Mr 80,000 polypeptide. 2. The muscle also contains a calpain I type enzyme which is specific for desmin degradation, and this enzyme consists of a single subunit of Mr 67,000. 3. Three calpastatins were detected in the tissue, one of which inhibited both calpains, whereas the other two appeared to be specific for the desmin-specific calpain. These calpastatins possessed the same inhibition properties when assayed with chicken gizzard calpains.

Topics: Animals; Calcium-Binding Proteins; Calpain; Chromatography, Gel; Chromatography, Ion Exchange; Cricetinae; Isoelectric Focusing; Male; Mesocricetus; Molecular Weight; Muscles; Protease Inhibitors

Complementary DNA portions coding for each domain (domain L and internally repetitive domains, domains 1-4, each composed of approximately 140 amino acid residues) of pig calpastatin were subcloned into E. coli plasmids to express the respective portions of the proteinase inhibitor gene in bacteria. Cell extracts of E. coli harboring recombinant plasmids were assayed for calpain inhibition. All four internally repetitive domains showed inhibitory activities, essentially similar to one another, against calpains I and II. No inhibition was observed in the case of the N-terminal non-homologous domain (domain L). These results support our previous conclusion that the repetitive region is a functional unit of the proteinase inhibitor.

Topics: Amino Acid Sequence; Animals; Base Sequence; Calcium-Binding Proteins; Calpain; Cloning, Molecular; Molecular Sequence Data; Recombinant Proteins; Structure-Activity Relationship; Swine

Topics: Calcium-Binding Proteins; Calpain; Erythrocytes; Hemoglobin E; Hemoglobins, Abnormal; Humans; Reference Values; Thalassemia

The action of calpain (EC 3.4.22.17; Ca2+-dependent cysteine proteinase) on platelet factor XIII has been studied. Calpain I activated platelet factor XIII up to 76% of the maximum level observed with thrombin. Activation was accompanied by the limited proteolysis of the a subunit of platelet factor XIII to produce a 76 kDa fragment which was comparable to the proteolytic product by thrombin. Activation of platelet factor XIII by calpain was inhibited by EDTA, leupeptin, and endogenous calpain-specific inhibitor calpastatin. These findings suggest that calpain is responsible for the intracellular activation of platelet factor XIII.

Topics: Blood Platelets; Calcium; Calcium-Binding Proteins; Calpain; Enzyme Activation; Factor XIII; Humans; In Vitro Techniques; Leupeptins; Thrombin; Transglutaminases

A cDNA encoding an endogenous inhibitor, termed calpastatin, for calcium-dependent cysteine protease (calpain, EC 3.4.22.17) was cloned by screening rabbit cDNA libraries with a synthetic oligodeoxynucleotide probe based on the partial amino acid sequence of the purified protein. The deduced amino acid sequence contains 718 amino acid residues (Mr, 76,964), and the mature protein corresponds to the deduced sequence from the 80th residue of the primary translation product (resultant Mr, 68,113). This deduced molecular weight is significantly lower than that determined by NaDodSO4/polyacrylamide gel electrophoresis, suggesting the possibility that the inhibitor is post-translationally modified. The sequence of the mature inhibitor contains four consecutive internal repeats approximately 140 amino acid residues long, each of which might be responsible for the inhibitory activity. Calpastatin is apparently different from a typical cysteine protease inhibitor (cystatin), suggesting that the mechanism of inhibition of calcium-dependent cysteine protease by the inhibitor might be different from that of other cysteine proteases by cystatin.

Topics: Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Calcium-Binding Proteins; Calpain; Cloning, Molecular; DNA; DNA Restriction Enzymes; Genes; Liver; Lung; Protein Biosynthesis; Rabbits

The antioxidant, butylated hydroxytoluene (BHT), causes lung toxicity in mice followed by regenerative repair, and can also modulate the development of carcinogen-induced lung adenomas. We are investigating changes in pulmonary biochemistry following BHT treatment in order to understand the mechanisms of BHT-induced pulmonary regenerative repair. BHT administration lowered cytosolic Ca2+-activated neutral protease (calpain) activity, increased the activity of the endogenous calpain inhibitor, calpastatin, increased the extent of photoincorporation of 8-N3-[32P]cAMP into a Mr 37,000 proteolytic product derived from cAMP-dependent protein kinase regulatory (R) subunits, and increased membrane-associated protease activity. All of these changes were dependent on the BHT dosage; the altered proteolytic activities occurred at a dose lower than that which caused observable lung toxicity as assessed by the lung weight/body weight ratio. Decreased cytosolic calpain activity was detectable within 1 day after BHT administration, was lowest at 4-7 days, and had not returned to control levels by Day 21, a time when normal lung morphology had been regained. The decrease in calpain activity cannot fully be accounted for by increased calpastatin activity; upon separation of these proteins by DEAE chromatography, the amount of calpain activity from BHT-treated mice remained lower than the corresponding peak from control mice. Increased photolabeling of the Mr 37,000 protein began at 1 day and continued to increase up to 4 days after BHT. All of the cytosolic changes preceded the increased particulate proteolytic activity by 1-2 days. R-subunits which have dissociated from their catalytic subunits are more susceptible to degradation by calpain, but BHT treatment did not enhance subunit dissociation as determined by the elution profile of 8-N3-[32P]cAMP-labeled R-subunits following DEAE chromatography. A large percentage of the particulate protease activity was inhibited by calpastatin, leupeptin, and E-64, all of which are known to inhibit calpain activity; this suggested that calpain accounted for most of this activity. Changes in the activities of proteases which catalyze limited proteolysis reactions may play an important role in the repair of acute lung injury.

Topics: Animals; Butylated Hydroxytoluene; Calcium-Binding Proteins; Calpain; Chromatography, DEAE-Cellulose; Cyclic AMP; Cytosol; Dose-Response Relationship, Drug; Female; Lung; Male; Mice; Mice, Inbred A; Photochemistry

Activity of a calcium-dependent neutral protease (calpain II) and its specific endogenous inhibitor was investigated in the myocardium of rats subjected to different stressors: cold, anaesthesia, 24 and 48 h starvation and food restriction for 7 and 14 days. Enzyme and inhibitor activities were determined in the 37,200 g supernatant of homogenates prepared from the free left ventricular wall of the heart. The specific activity of the myocardial calcium-dependent proteinase increased in all rats exposed to stressful stimuli, reaching maximum values in animals starved for 48 hours. Decrease in the specific activity of the inhibitor accompanied the changes in enzyme activity. Differences from normal control values were statistically significant in the starved animals and in animals fed a restricted diet for 7 or 14 days. These observations suggest that interaction between calpain II and its specific inhibitor plays a role in the regulation of the enzyme activity and furthermore, that stressful stimuli lead to increased calcium-dependent proteolysis in the myocardium.

Topics: Animals; Calcium-Binding Proteins; Calpain; Hot Temperature; Male; Muscle Proteins; Myocardium; Rats; Stress, Physiological

Three leg muscles (soleus, extensor digitorum longus, and plantaris) of adult male golden Syrian hamsters were denervated by bilateral transecting of the sciatic nerve. Eighteen days after denervation, wet weights, amounts of soluble protein, and activities of wide-specificity calpain II, intermediate filament protein-specific calpain I, and calpastatin were measured by protein determination and enzyme and immunological assays. In comparison with control (nondenervated) muscles and depending on the specific muscle and protein, the activities of the calpains increased 1.3 to 1.9 times the control values, whereas the calpastatin decreased to one-half and one-third of control values. The muscle which showed the greatest increase in both calpain activities and the largest decrease in calpastatin activity was the plantaris (a fast-twitch, oxidative glycolytic muscle). The extensor digitorum longus (fast-twitch glycolytic) showed increases in calpain II activity similar to those in the plantaris, but smaller changes in calpain I and calpastatin. The soleus (slow-twitch, oxidative) showed the smallest changes in calpain II and calpastatin activities, although an increase in the calpain I activity was seen after denervation. These results suggest a possible relationship between the presence of fast-twitch, oxidative glycolytic fibers in a muscle and increased potential for intracellular proteolysis following denervation.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cricetinae; Male; Mesocricetus; Muscle Denervation; Muscle Proteins; Muscles; Organ Size; Protease Inhibitors; Solubility

The immunohistochemical distribution of Ca2+-dependent cysteine proteinases (calpains I and II) and their endogenous inhibitor calpastatin in normal and adenomatous human pituitary tissue was studied using specific antibodies. The distributions of calpain and calpastatin were dissimilar in human pituitary gland, i.e. ACTH-immunoreactive cells were strongly positive for calpastatin and negative for calpains. PRL-, GH-, FSH-, and TSH-producing cells were negative for calpastatin, but moderately positive for calpains, especially for calpain II, the high Ca2+-requiring form of the enzyme. Similar results were found in pituitary adenoma tissue. These findings indicate that each type of cells producing a specific hormone is equipped with a different balance of the enzyme-inhibitor system involved in the Ca2+-dependent degradation of intracellular proteins.

Topics: Adenoma; Adrenocorticotropic Hormone; Calcium-Binding Proteins; Calpain; Histocytochemistry; Humans; Immunoenzyme Techniques; Pituitary Gland; Pituitary Neoplasms; Staining and Labeling

Recent studies have shown that soluble calcium activated proteases (calpains) in brain degrade proteins associated with the cytoskeleton and vary markedly in activity across regions and as a function of development. It was suggested that the observed differences in calpain activity reflect differences in the turnover rate of structural elements. The present study extends this analysis by measuring the properties and activity of calpain in representatives of the five classes of vertebrates with particular emphasis on the mammals. No evidence for proteolysis was found in soluble fractions of fish brains at neutral pH in the presence or absence of added calcium. A substantial calcium-independent proteolytic activity was found in amphibian brains--the effects of a variety of protease inhibitors indicated that it is also a neutral thiol (cysteine) protease. Reptilian brains exhibited both calcium-independent and calcium-dependent proteolytic activity. Virtually all proteolytic activity in birds (5 species) and mammals (9 species) measured at neutral pH was calcium-dependent. The endogenous substrates for the calcium activated proteases were very similar in several species of birds and mammals as were the effects of a variety of protease inhibitors. However, the activity of the enzyme, expressed per mg of soluble protein, was highly and negatively correlated with brain size in the mammals. The allometric expression for this relationship was similar to that found for the density of neurons in cerebral cortex as a function of absolute brain size. These results indicate that soluble proteolytic enzymes in brain are differentially expressed among classes of vertebrates and suggest that the turnover of cytoskeletal elements in birds and mammals differs in important ways from that found in fish and amphibians. The results obtained for mammals raise the possibility of a relationship between brain size and the rate at which structural elements are broken down and replaced in this vertebrate class.

Topics: Amphibians; Animals; Birds; Brain; Calcium-Binding Proteins; Calpain; Cattle; Fishes; Guinea Pigs; Horses; Hydrolysis; Mice; Opossums; Protease Inhibitors; Rabbits; Rats; Reptiles; Species Specificity; Swine

Rat erythrocytes fuse when treated with the membrane mobility agent, 2-(2-methoxyethoxy)ethyl-cis-8-(2-octylcyclopropyl)octanoate (A2C) and Ca2+, whereas human cells do not. Membrane proteolysis promoted by calpain is required for rat cell fusion [(1986) Eur. J. Biochem., in press]. Human calpain induced a selective proteolysis in both the human and rat erythrocyte ghosts (mainly band 4.1 in the human, band 4.1 and band 3 in the rat cell) and rendered them fusible. Calpastatin (calpain inhibitor) prevented A2C-induced fusion in both ghosts, via inhibition of proteolysis. The human erythrocyte has excess calpastatin and resists A2C-promoted fusion. A regulatory role of calpastatin in membrane fusion is thus indicated.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Cell Fusion; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Humans; Protease Inhibitors; Rats; Species Specificity; Stearates

Calpains I and II isolated from diverse tissues possess both Ca2+-independent, and Ca2+-dependent accessible hydrophobic regions. Possible subcellular organelle association of calpains involving these hydrophobic regions was studied. By homogenizing rat tissues directly in Ca2+ (50 microM), about 30-60% of the cytosolic calpain I and II activity reversibly associated with isolated subcellular fractions (microsomal greater than plasma membrane greater than nuclear). After binding to the particulate fraction, calpain II converted to a calpain I-like form exhibiting stronger Ca2+-independent binding to phenyl-Sepharose and a lower Ca2+ requirement for optimal activity. However, it retained its DEAE-cellulose chromatographic pattern, and precipitated with monospecific anti-calpain II antibodies. Although purified calpastatin (endogenous inhibitor) is known to form a Ca2+-dependent complex with calpains, it was not able to reverse the binding of calpains to the particulate fraction upon short incubation. It was, however, effective in blocking calpain binding when the isolated cytosolic fraction or a mixture of purified calpain and calpastatin was preincubated in the presence of Ca2+, and then added to the particulate fraction. Extraction of tissues under controlled conditions revealed that in fact calpains are already loosely associated with subcellular organelles even in the absence of Ca2+. This is the reason why in the crude homogenates with the addition of Ca2+, calpains strongly bind to the particulate fraction without interference by cytosolic calpastatin. Although calpastatin by complexing initially to calpain can prevent the association of this protease with subcellular organelles, it cannot dissociate calpains already bound to these subcellular fractions. By prior Ca2+-independent association with the hydrophobic proteins present in the subcellular fractions, calpains overcome the 3- to 30-fold inhibitory excess of calpastatin in tissues.

Topics: Animals; Brain; Calcium-Binding Proteins; Calpain; Cell Compartmentation; Chromatography, DEAE-Cellulose; Kidney; Lung; Male; Rats; Rats, Inbred Strains; Spleen; Subcellular Fractions

Two types of Ca2+-requiring cysteine proteinase (calpain, EC 3.4.22.17), which required for full activation 100 microM Ca2+ (calpain I) and 1 mM Ca2+ (calpain II) were found to exist in the cytosolic fraction of bovine lens. Since calpain may play an important role on the degradation of lens proteins during the aging process of the lens, we attempted to study the distribution of calpain I, calpain II, and calpastatin (an endogenous specific inhibitor of calpain) in bovine lens. It was found that both the capsule-epithelium and cortex fiber cells contained calpains I and II and calpastatin, although the content of calpain I was much lower than that of calpain II. Calpains I and II and calpastatin activities were not detected in the nuclear region at all.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cattle; Endopeptidases; Epithelium; Lens Capsule, Crystalline; Lens Cortex, Crystalline; Lens, Crystalline; Protease Inhibitors

Calpain (Ca2+-dependent cysteine proteinase) was purified to apparent homogeneity from carp muscle by the method of DEAE-cellulose, hydroxylapatite and Ultrogel AcA 34 column chromatographies. The purified enzyme is classified as calpain II (high-Ca2+-requiring form of calpain) from the effects of Ca2+ concentration, pH and the antibiotics on the activity. Carp muscle calpain II was inhibited by rat liver calpastatin, the specific inhibitor for calpain. It is probable that the calpain-calpastatin system may play a biologically fundamental and common role in various cells, since the inhibitory effect of calpastatin on calpain from different tissues of different species is well conserved.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Carps; Cations, Divalent; Endopeptidases; Kinetics; Liver; Molecular Weight; Muscles; Protease Inhibitors; Rats; Rats, Inbred Strains

The inhibitory effect of calpastatin (specific inhibitor for calpain) on calpain (Ca2+-dependent cysteine proteinase, EC 3.4.22.17) was examined using carp muscle, carp erythrocytes and rat liver preparations. A mutual inhibitory effect between calpains and calpastatins from different tissues and species was observed. The conservation of the inhibitory effect of calpastatin on calpain among vertebrates suggests that the calpain-calpastatin system may play a biologically fundamental and common role in various cells.

Topics: Animals; Calcium-Binding Proteins; Calpain; Carps; Erythrocytes; Kinetics; Liver; Muscles; Organ Specificity; Protease Inhibitors; Rats; Species Specificity

In order to examine the existence of calpain I, a low (micromolar)-Ca2+-requiring form of calpain, in fish tissues, carp erythrocytes were chosen as the experimental material, since only calpain I is known to exist in mammalian erythrocytes. By DEAE-cellulose chromatography, calpain and calpastatin (specific inhibitor for calpain) were separated from carp erythrocyte hemolysate. Carp erythrocyte calpain is classified as calpain II, a high (millimolar)-Ca2+-requiring form of calpain, from the result of Ca2+-requirement for the activity.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Carps; Endopeptidases; Erythrocytes; Hydrogen-Ion Concentration; Kinetics; Protease Inhibitors

A rapid and reliable method for quantitating tissue calpains (Ca2+-activated, neutral, thiol proteases) was developed using hydrophobic chromatography with phenyl-Sepharose. Calpains I and II isolated by this method are free of endogenous inhibitor(s) (calpastatin), activator(s), and nonspecific proteases. These calpains expose hydrophobic regions in the presence of Ca2+ and bind tightly to phenyl-Sepharose. Inactivation of bound calpain is prevented by the addition of leupeptin (20 microM). Calpains I and II bound initially by phenyl-Sepharose in a Ca2+-dependent manner are then eluted successively on the basis of their Ca2+-independent binding to phenyl-Sepharose. Because calpastatin may prevent binding of calpain to phenyl-Sepharose by forming a protease-inhibitor complex in the presence of Ca2+, preadsorbing the protease to a suspension of phenyl-Sepharose beads initially in the absence of Ca2+ separates most of the calpain present in tissue extracts from calpastatin. The isolated calpains obtained are assayed by casein digestion. This quantitation procedure is suitable for measuring calpain activity in various tissues and cells including erythrocytes.

Topics: Animals; Brain; Calcium; Calcium-Binding Proteins; Calmodulin; Calpain; Cattle; Chromatography; Female; Rats; Rats, Inbred Strains; Sepharose

The soluble fraction from rabbit brain was adsorbed on a column of phenyl-Sepharose. By applying a linear gradient with decreasing salt concentration and increasing pH, it was possible to separate calpain I and calpain II from each other and from the endogenous inhibitor calpastatin. Both enzymes were capable of degrading endogenously labelled neuronal proteins, including slowly axonally transported soluble proteins and rapidly transported membrane-bound proteins, as well as casein.

Topics: Calcium; Calcium-Binding Proteins; Calpain; Caseins; Chromatography, Affinity; Protease Inhibitors

Topics: Animals; Calcium-Binding Proteins; Calpain; Retina; Swine

Topics: Animals; Calcium-Binding Proteins; Calpain; Humans; Kinetics; Lens, Crystalline; Macromolecular Substances; Molecular Weight; Proteins; Substrate Specificity; Tissue Distribution

Variation of calpain I, calpain II, and calpastatin in rat liver during growth from 0 to 14 weeks was studied by chromatographic fractionation of the liver cytosol and enzyme assays on the eluted fractions. When compared in terms of units per g wet liver, high-Ca2+-requiring calpain II always exceeded low-Ca2+-requiring calpain I in male and female rats. The level of calpain II in neonatal (0 week) rat liver was 1.9-2.9 times higher than that for the adults (7 to 14 weeks). The contents of calpastatin, calpain-specific inhibitor protein, were were always higher than those of calpain II in adult rat liver, but the difference was much less, or sometimes even reversed, in neonatal and young (1 and 2 weeks) animals. In general, the variation was more pronounced in female than in male rats.

Topics: Aging; Animals; Animals, Newborn; Calcium-Binding Proteins; Calpain; Female; Liver; Male; Protease Inhibitors; Rats; Rats, Inbred Strains

Low and high Ca2+-requiring forms of Ca2+-dependent cysteine proteinase are known as calpain I and calpain II, respectively. We have obtained, for the first time, monospecific antibodies for calpain I and for calpain II. Using these antibodies and an electrophoretic blotting method, we have found that a small, but reproducible, amount of calpain I was associated with human erythrocyte membranes while the bulk of the protease was contained in the cytosol. Most of membrane-associated calpain I was extractable with 1% Triton X-100, but not with 0.1% detergent. In the presence of 0.1 mM Ca2+ and 5 mM cysteine, membrane-associated calpain I degraded the membrane protein band 4.1 preferentially and band 3 protein only slowly. The Ca2+-induced autodigestion of the membrane preparation was inhibited by leupeptin but not by a cytosolic calpain inhibitor, calpastatin, added to the incubation medium. No calpain II was detected in either erythrocyte cytosol or membranes when anti-calpain II antibody was used under the same conditions as those for the detection of calpain I.

Topics: Antibodies, Monoclonal; Calcium; Calcium-Binding Proteins; Calpain; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Erythrocyte Membrane; Humans; Immunoelectrophoresis; Immunoglobulin G; Leupeptins; Protease Inhibitors

Topics: Animals; Calcium-Binding Proteins; Calpain; Concanavalin A; Endopeptidases; Lipopolysaccharides; Lymphocyte Activation; Male; Mice; Mice, Inbred C3H; Protease Inhibitors; Spleen

Two forms of Ca2+-dependent cysteine proteinase (calpain, EC 3.4.22.17) and their specific endogenous inhibitor (calpastatin) were partially purified from porcine retina: calpain I (low-Ca2+-requiring form) was half-maximally activated at 8 microM-Ca2+, and calpain II (high-Ca2+-requiring form) at 250 microM-Ca2+. Both calpain I and calpain II were inhibited by calpastatin. Calpain I from porcine retina was shown to be composed of 83 000- and 29 000-Mr subunits, and calpain II of 80 000- and 29 000-Mr subunits, by the use of monospecific antibodies. Calpains I and II were both found to hydrolyse microtubule-associated proteins 1 and 2 rapidly.

Topics: Animals; Calcium-Binding Proteins; Calpain; Chromatography, DEAE-Cellulose; Endopeptidases; Immunoelectrophoresis; Isoenzymes; Microtubules; Protease Inhibitors; Retina; Swine

A highly sensitive sandwich enzyme immunoassay for a Ca2+-dependent cysteine proteinase (calpain I) and its specific endogenous inhibitor protein (calpastatin) was developed. The calpain I and calpastatin used as immunogens were purified from human erythrocytes. Anti-calpastatin antisera having sufficiently high titer were obtained only when the immunogen was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The assay method was principally based on the report by M. Imagawa et al. (1982, J. Appl. Biochem. 4, 41-57), using a specific antibody-coated polystyrene ball and horseradish peroxidase-conjugated Fab' fragment of the antibody. The sensitivity was 0.1 ng of calpain I or calpastatin per assay tube. Starting with 50 microliter of the hemolysate from human erythrocytes, the method permitted direct and simultaneous determination of calpain I and calpastatin, without prior separation of these two enzymatically counteracting components by chromatography. The present method as applied to the erythrocytes from 14 healthy adults gave 120-170 micrograms for calpain I and 164-211 micrograms for calpastatin per gram of hemoglobin, respectively.

Topics: Calcium-Binding Proteins; Calpain; Endopeptidases; Erythrocytes; Hemolysis; Humans; Immunoenzyme Techniques; Methods; Protease Inhibitors

Homogenously purified porcine calpain I (Mr 112000), a low-Ca2+-requiring form of Ca2+-dependent cysteine proteinase [EC 3.4.22.17], was coupled to Sepharose 4B gel as an active form. It was used as a ligand to calpastatin (Mr 70000), calpain-specific inhibitor protein, for an affinity chromatography. Only in the presence of Ca2+, calpastatin bound to calpain-Sepharose, but the interaction resulted in rather extensive fragmentation of a calpastatin molecule into several peptides of Mr 14000 to 70000, which still retain inhibitory activities against calpain. Fragmentation was demonstrated both by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by high-performance liquid chromatography in the presence of 6 M guanidine-HCl.

Topics: Animals; Calcium-Binding Proteins; Calpain; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Erythrocytes; Molecular Weight; Peptide Fragments; Protease Inhibitors; Swine

The cytosol of human erythrocytes was found to contain a Ca2+-dependent thiol protease (calpain) and its specific inhibitor (calpastatin) by DEAE-cellulose chromatography at pH 8.0, although no proteolytic activity toward casein was detected in the unfractionated hemolysate. The protease required only 40 microM Ca2+ for 50% activation, indicating that it belongs to the highly Ca2+-sensitive type of calpain, namely, calpain I. It was not inactivated by heating at 58 degrees C for 10 min at pH 7.2, the optimal pH for its action on casein. The inhibitor comprised major and minor components, calpastatin H (Mr = 280,000) and caplastatin L (Mr = 48,000). Both were heat-stable proteins which were readily inactivated by tryptic digestion. The inhibition of erythrocyte calpain by erythrocyte calpastatin H or L was not due to sequestering of Ca2+ from the reaction medium by the inhibitor protein. The calpain preparation preferentially digests bands III and IVa of human erythrocyte membrane proteins, with little or no cleavage of the bands corresponding to spectrin.

Topics: Calcium; Calcium-Binding Proteins; Calpain; Cytosol; Endopeptidases; Enzyme Activation; Erythrocyte Membrane; Erythrocytes; Humans; Molecular Weight; Protease Inhibitors

Topics: Animals; Brain; Calcium; Calcium-Binding Proteins; Calpain; Endopeptidases; Erythrocytes; Humans; Isoenzymes; Liver; Peptide Hydrolases; Protease Inhibitors; Rats; Rats, Inbred Strains