leupeptins has been researched along with calpeptin* in 19 studies
19 other study(ies) available for leupeptins and calpeptin
Article | Year |
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Identification of Phosphorylated Calpain 3 in Rat Brain Mitochondria under mPTP Opening.
The protein phosphorylation of the membrane-bound mitochondrial proteins has become of interest from the point of view of its regulatory role of the function of the respiratory chain, opening of the mitochondrial permeability transition pore (mPTP), and initiation of apoptosis. Earlier, we noticed that upon phosphorylation of proteins in some proteins, the degree of their phosphorylation increases with the opening of mPTP. Two isoforms of myelin basic protein and cyclic nucleotide phosphodiesterase were identified in rat brain non-synaptic mitochondria and it was concluded that they are involved in mPTP regulation. In the present study, using the mass spectrometry method, the phosphorylated protein was identified as Calpain 3 in rat brain non-synaptic mitochondria. In the present study, the phosphoprotein Calpain-3 (p94) (CAPN3) was identified in the rat brain mitochondria as a phosphorylated truncated form of p60-62 kDa by two-dimensional electrophoresis and mass spectrometry. We showed that the calpain inhibitor, calpeptin, was able to suppress the Ca Topics: Animals; Apoptosis; Brain; Calcium; Calcium Signaling; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Isoenzymes; Leupeptins; Male; Mitochondria; Mitochondrial Membranes; Mitochondrial Permeability Transition Pore; Molecular Weight; Muscle Proteins; Phosphorylation; Protein Transport; Rats | 2021 |
Protective effect of S-allyl-L-cysteine against endoplasmic reticulum stress-induced neuronal death is mediated by inhibition of calpain.
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 | 2014 |
T-cell receptor ligation causes Wiskott-Aldrich syndrome protein degradation and F-actin assembly downregulation.
Wiskott-Aldrich syndrome protein (WASP) links T-cell receptor (TCR) signaling to the actin cytoskeleton. WASP is normally protected from degradation by the Ca(++)-dependent protease calpain and by the proteasome because of its interaction with the WASP-interacting protein.. We investigated whether WASP is degraded after TCR ligation and whether its degradation downregulates F-actin assembly caused by TCR ligation.. Primary T cells, Jurkat T cells, and transfected 293T cells were used in immunoprecipitation experiments. Intracellular F-actin content was measured in splenic T cells from wild-type, WASP-deficient, and c-Casitas B-lineage lymphoma (Cbl)-b-deficient mice by using flow cytometry. Calpeptin and MG-132 were used to inhibit calpain and the proteasome, respectively.. A fraction of WASP in T cells was degraded by calpain and by the ubiquitin-proteasome pathway after TCR ligation. The Cbl-b and c-Cbl E3 ubiquitin ligases associated with WASP after TCR signaling and caused its ubiquitination. Inhibition of calpain and lack of Cbl-b resulted in a significantly more sustained increase in F-actin content after TCR ligation in wild-type T cells but not in WASP-deficient T cells.. TCR ligation causes WASP to be degraded by calpain and to be ubiquitinated by Cbl family E3 ligases, which targets it for destruction by the proteasome. WASP degradation might provide a mechanism for regulating WASP-dependent TCR-driven assembly of F-actin. Topics: Actins; Adaptor Proteins, Signal Transducing; Animals; Calpain; Cell Line; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; Down-Regulation; Leupeptins; Mice; Mice, Knockout; Proto-Oncogene Proteins c-cbl; Receptors, Antigen, T-Cell; T-Lymphocytes; Ubiquitination; Wiskott-Aldrich Syndrome Protein | 2013 |
Calpain modulates capacitation and acrosome reaction through cleavage of the spectrin cytoskeleton.
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 | 2010 |
An ezrin/calpain/PI3K/AMPK/eNOSs1179 signaling cascade mediating VEGF-dependent endothelial nitric oxide production.
Calpain was recently reported to mediate vascular endothelial growth factor (VEGF)-induced angiogenesis. In the present study, we investigated detailed molecular mechanisms. VEGF (100 ng/mL) induced a marked increase in endothelial cell production of NO(*), specifically detected by electron spin resonance. This response was abolished by inhibition of calpain with N-acetyl-leucyl-leucyl-norleucinal (ALLN) or Calpeptin. Both also diminished membrane-specific calpain activation by VEGF, which was intriguingly attenuated by silencing ezrin with RNA interference. A rapid membrane colocalization of calpain and ezrin occurred as short as 10 minutes after VEGF stimulation. AKT, AMP-dependent kinase (AMPK), and endothelial nitric oxide synthase (eNOS)(s1179) phosphorylations in VEGF-stimulated endothelial cells were markedly enhanced, which were however significantly attenuated by either ALLN, Calpeptin, or ezrin small interfering RNA, as well as by Wortmannin or compound C (respectively for phosphatidylinositol 3-kinase [PI3K] or AMPK). The latter 3 also abolished VEGF induction of NO(*). These data indicate that AMPK and AKT are both downstream of PI3K and that AKT activation is partially dependent on AMPK. The interrelationship between AMPK and AKT, although known to be individually important in mediating VEGF activation of eNOS, is clearly characterized. Furthermore, AMPK/AKT/eNOS(s1179) was found downstream of a calpain/ezrin membrane interaction. These data no doubt provide new insights into the long mystified signaling gap between VEGF receptors and PI3K/AKT or AMPK-dependent eNOS activation. In view of the well-established significance of VEGF-dependent angiogenesis, these findings might have broad and important implications in cardiovascular pathophysiology. Topics: AMP-Activated Protein Kinases; Animals; Aorta; Calpain; Cattle; Cells, Cultured; Cytoskeletal Proteins; Dipeptides; Endothelial Cells; Endothelium, Vascular; Enzyme Activation; Hydrogen Peroxide; Leupeptins; Membrane Proteins; Nitric Oxide; Nitric Oxide Synthase Type III; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Interaction Mapping; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction; Vascular Endothelial Growth Factor A | 2009 |
Down-regulation of brain-pancreas relative protein in diabetic rats and by high glucose in PC12 cells: prevention by calpain inhibitors.
Brain-pancreas relative protein (BPRP) is a novel protein that we found in our laboratory. Previously we demonstrated that it is involved in ischemia and depression. In light of the putative association between diabetes and clinical depression, and the selective expression of BPRP in brain and pancreas, the present study examined whether BPRP levels are affected by induction of diabetes by alloxan injection in rats and exposure to high glucose levels in PC12 cells. Western blot and immunohistochemical analyses revealed that BPRP levels were decreased in the hippocampal CA1 neurons of diabetic rats 4 and 8 weeks post-alloxan injection and in PC12 cells 48 h after exposure to high concentrations of glucose. BPRP protein levels were not affected by osmolarity control treatments with mannitol. Follow-up pharmacological experiments in PC12 cells revealed that glucose-induced BPRP down-regulation was markedly attenuated by the calpain inhibitors N-acetyl-Leu-Leu-norleucinal (ALLN) or calpeptin, but not the proteasome-specific inhibitor carbobenzoxy-Leu-Leu-leucinal (MG132). The ability of calpain inhibitors to specifically counter the effects of high glucose exposure on BPRP levels further suggests that BPRP and calpain activity may contribute to diabetes complications in the central nervous system. Topics: Animals; Calpain; Cysteine Proteinase Inhibitors; Diabetes Mellitus, Experimental; Dipeptides; Down-Regulation; Glucose; Half-Life; Hippocampus; Insulin; Leupeptins; Male; Nerve Tissue Proteins; Neurons; PC12 Cells; Rats; Rats, Sprague-Dawley; Time Factors | 2008 |
Complex effects of IL1A polymorphism and calpain inhibitors on interleukin 1 alpha (IL-1 alpha) mRNA levels and secretion of IL-1 alpha protein.
Alleles of IL1A-889(C>T) and IL1A+4845(G>T) are in linkage disequilibrium. Interleukin 1alpha (IL-1alpha) is produced as a precursor protein and cleaved at positions 117-118 by calpain, generating a mature protein for export. IL1A+4845 affects amino acids expressed at position 114 and hence may modulate calpain-mediated cleavage. We sought evidence for this mechanism in intact cells. Blood leukocytes from heterozygous donors released more IL-1alpha protein than cells from IL1A(1,1) donors, while release from IL1A(2,2) cells was variable. Genotype did not affect levels of IL-1alpha mRNA, so differential cleavage of the precursor is a feasible mechanism. However, genotype also had no effect on inhibition of IL-1alpha release by pretreatment with calpain inhibitors, and calpain inhibitors reduced IL-1alpha and tumor necrosis factor alpha mRNA levels. Hence, calpain inhibitors probably affect inhibition of signal transduction pathway rather than cleavage of IL-1alpha protein. As ratios of mu-calpain/calpastatin were lowest in heterozygous donors, genetically determined IL-1alpha levels may modulate transcription of calpain and calpastatin. This could reduce the impact of IL1A genotype on IL-1alpha secretion and amplify individual variation in levels generated in culture. Topics: Acrylates; Calpain; Cells, Cultured; Dipeptides; Glycoproteins; Humans; Interleukin-1alpha; Leupeptins; Polymorphism, Genetic; RNA, Messenger | 2008 |
Regulation of plasma membrane Ca2+-ATPase in human platelets by calpain.
The plasma membrane Ca(2+)-ATPase (PMCA) plays an essential role in maintaining low cytosolic Ca(2+) in resting human platelets by extruding Ca(2+) from the cytoplasm across the plasma membrane. Since PMCA is the main agent of Ca(2+) efflux in platelets, it is a key point for regulation of platelet Ca(2+) metabolism. PMCA has been shown to be an excellent substrate for the Ca(2+)-activated cysteine protease calpain, a major platelet protein that is turned on during platelet activation. The objectives of the present work were to determine if PMCA is degraded during thrombin- and collagen-mediated platelet activation, and if calpain is responsible. The kinetics of PMCA degradation during platelet activation were analysed using SDS polyacrylamide gel electrophoresis and immunoblotting. The role of calpain was tested using the calpain inhibitors calpeptin and ALLN. Platelet activation mediated by both collagen and thrombin resulted in degradation of 60% of platelet PMCA within 18 minutes. Calpeptin and ALLN significantly inhibited the rate and extent of PMCA degradation. We conclude that calpain-mediated degradation of PMCA during platelet activation likely contributes significantly to Ca(2+) regulation and, therefore, to platelet function. Topics: Blood Platelets; Calcium; Calcium-Transporting ATPases; Calpain; Cell Membrane; Collagen; Dipeptides; Enzyme Activation; Humans; Leupeptins; Platelet Activation; Thrombin | 2007 |
Myoblast migration is regulated by calpain through its involvement in cell attachment and cytoskeletal organization.
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 | 2004 |
Inhibition of calpain is a component of nitric oxide-induced down-regulation of human mast cell adhesion.
Nitric oxide is an important messenger that regulates mast cell activity by modifications to gene expression and intracellular pathways associated with exocytosis and adhesion. Integrin interactions with extracellular matrix components modulate an array of cell activities, including mediator production and secretion. To investigate the molecular mechanisms underlying NO regulation of mast cell function, we studied its effects on adhesion of a human mast cell line (HMC-1) to fibronectin (FN). The NO donors S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine strongly down-regulated the adhesion of HMC-1 to FN. Inhibitors of soluble guanylate cyclase and protein kinase G did not alter the response of cells to NO. A peroxynitrite scavenger did not affect modulation of adhesion by NO, nor could the effect of NO be mimicked by the peroxynitrite-producing compound 3-morpholinosydnonimine. NO donors inhibited the cysteine protease, calpain, while calpain inhibitors mimicked the effect of NO and led to a decrease in the ability of HMC-1 cells to adhere to FN. Thus, NO is an effective down-regulator of human mast cell adhesion. The mechanism for this action does not involve peroxynitrite or activation of soluble guanylate cyclase. Instead, a portion of NO-induced down-regulation of adhesion may be attributed to inhibition of the cysteine protease, calpain, an enzyme that has been associated with control of integrin activation in other cell types. The inhibition of calpain is most likely mediated via nitrosylation of its active site thiol group. Calpain may represent a novel therapeutic target for the regulation of mast cell activity in inflammatory disorders. Topics: Calpain; Cell Adhesion; Cyclic GMP; Dipeptides; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Fibronectins; Humans; Leupeptins; Mast Cells; Nitric Oxide; Nitric Oxide Donors; Peroxynitrous Acid; Protein Binding; S-Nitroso-N-Acetylpenicillamine; S-Nitrosoglutathione; Tumor Cells, Cultured | 2003 |
Protein kinase C inhibition induces DNA fragmentation in COLO 205 cells which is blocked by cysteine protease inhibition but not mediated through caspase-3.
Enhancing apoptosis to remove abnormal cells has potential in reversing cancerous processes. Caspase-3 activation generally accompanies apoptosis and its substrates include enzymes responsible for DNA fragmentation and isozymes of protein kinase C (PKC). Recent data, however, question its obligatory role in apoptosis. We have examined whether modulation of PKC activity induces apoptosis in COLO 205 cells and the role of caspase-3. Proliferation ([3H]thymidine) and apoptosis (DNA fragmentation and FACS) of COLO 205 cells were measured in response to PKC activation and inhibition. Caspase-3 activity was assayed and the effects of its inhibition with Ac-DEVD-cmk, and the effect of other protease inhibitors, on apoptosis were determined. PKC activation and inhibition both reduced DNA synthesis and induced DNA fragmentation. As PKC inhibitors induced DNA fragmentation more rapidly than PKC activators and failed to block activator effects, we conclude that it is PKC down-regulation (i.e., inhibition) after activator exposure that mediates apoptosis. Increases in caspase-3 activity occurred during apoptosis but apoptosis was not blocked by caspase inhibition. By contrast, the cysteine protease inhibitor, E-64d, blocked apoptosis. Cysteine proteases not of the caspase family may either act more closely to the apoptotic process than caspases or lie on an alternative, more active pathway. Topics: Aged; Alkaloids; Amino Acid Chloromethyl Ketones; Aprotinin; Benzophenanthridines; Benzyl Compounds; Caspase 3; Caspases; Cell Division; Cell Transformation, Neoplastic; Colonic Neoplasms; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dipeptides; DNA; DNA Fragmentation; Down-Regulation; Humans; Hydrocarbons, Fluorinated; Leucine; Leupeptins; Male; Pepstatins; Phenanthridines; Protein Kinase C; Pyridines; Tumor Cells, Cultured | 2003 |
Enhanced proteolysis of IkappaBalpha and IkappaBbeta proteins in astrocytes by Moloney murine leukemia virus (MoMuLV)-ts1 infection: a potential mechanism of NF-kappaB activation.
Moloney murine leukemia virus (MoMuLV)-ts1-mediated neuronal degeneration in mice is likely due to loss of glial support and release of inflammatory cytokines and neurotoxins from surrounding ts1-infected glial cells including astrocytes. NF-kappaB is a transcription factor that participates in the transcriptional activation of a variety of immune and inflammatory genes. We investigated whether ts1 activates NF-kappaB in astrocytes and examined the mechanism(s) responsible for the activation of NF-kappaB by ts1 infection in vitro. Here we present evidence that ts1 infection of astrocytes in vitro activates NF-kappaB by enhanced proteolysis of the NF-kappaB inhibitors, IkappaBalpha and IkappaBbeta. In in vitro studies using protease inhibitors, IkappaBalpha proteolysis in ts1-infected astrocytes was significantly blocked by a specific calpain inhibitor calpeptin but not by MG-132, a specific proteasome inhibitor, whereas rapid IkappaBbeta proteolysis was blocked by MG-132. Furthermore, treatment with MG-132 increased levels of multiubiquitinated IkappaBbeta protein in ts1-infected astrocytes. These results indicate that the calpain proteolysis is a major mechanism of IkappaBalpha proteolysis in ts1-infected astrocytes. Additionally, ts1 infection of astrocytes in vitro increased expression of inducible nitric oxide synthase (iNOS), a NF-kappaB-dependent gene product. Our results suggest that NF-kappaB activation in ts1-infected astrocytes is mediated by enhanced proteolysis of IkappaBalpha and IkappaBbeta through two different proteolytic pathways, the calpain and ubiquitin-proteasome pathways, resulting in increased expression of iNOS, a NF-kappaB-dependent gene. Topics: Active Transport, Cell Nucleus; Animals; Astrocytes; Calpain; Cells, Cultured; Cysteine Endopeptidases; Dipeptides; DNA-Binding Proteins; Enzyme Induction; Gene Expression Regulation, Viral; I-kappa B Proteins; Leupeptins; Ligases; Mice; Moloney murine leukemia virus; Multienzyme Complexes; Nerve Tissue Proteins; NF-kappa B; NF-KappaB Inhibitor alpha; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Protease Inhibitors; Proteasome Endopeptidase Complex; Ubiquitin | 2001 |
Differential toxicity of protease inhibitors in cultures of cerebellar granule neurons.
Involvement of proteases has been postulated in several neurodegenerative processes. Accordingly, protease inhibition has been proposed as a potential therapeutic tool to limit damage in some neuropathological states. The timed turn-over of proteins is, however, an essential biochemical process and its prolonged block may be dangerous to the cell. We report here data on toxicity consequent to 24-h exposure of cerebellar granule neurons in culture to inhibitors of different classes of proteases. Inhibition of calpains (calcium-activated cysteine proteases) resulted in dose-dependent neuronal death which largely occurred through apoptotic process. Leupeptin, an inhibitor acting on a broad spectrum of cellular serine proteases, was less toxic but resulted in definite morphological alteration of the cells. On the contrary, inhibitors of caspases, proteases belonging to the ICE (interleukin 1-beta converting enzyme) family, did not apparently damage granule neurons upon exposure for 24 h to high concentrations (up to 200 microM) of two inhibitors specific for ICE (Ac-YAVD-CHO) and CPP-32 (Ac-DEVD-CHO), respectively. These results suggest that inhibition of proteases that are activated by stressful stimuli but are not essential for the normal functioning of healthy cells, as it is likely the case for caspases, may not be harmful to neurons. Instead, the potential risks and side effects of prolonged inhibition of proteases such as calpains, that regulate the disposal and the turn-over of key cellular proteins, should be carefully tested in the assessment of possible neuroprotective roles. Topics: Animals; Apoptosis; Cells, Cultured; Cerebellum; Cysteine Proteinase Inhibitors; Dipeptides; In Situ Nick-End Labeling; L-Lactate Dehydrogenase; Leupeptins; Neurons; Oligopeptides; Protease Inhibitors; Rats; Rats, Wistar | 1998 |
Constitutive apoptosis in human neutrophils requires synergy between calpains and the proteasome downstream of caspases.
Programmed cell death invariably requires the activation of proteolytic cascades that are not yet well defined but are initiated after apical caspase activation. We provide evidence that calpains and the proteasome function synergistically downstream of caspases to assist the constitutive apoptotic program of aging neutrophils, which plays an important role in resolution of inflammatory responses. Inhibitor studies indicated that "tethering" of preapoptotic senescent neutrophils to human macrophages required caspase activity. However, the development of morphological features characteristic of apoptosis, including nuclear morphology, PS exposure, surface protein shedding, and the capacity to be ingested by macrophages, required the downstream action of either calpains or the proteasome. Calpain activities were constitutively active in freshly isolated neutrophils and responsible for rearrangements in the protein composition and structure of the plasmalemmal cytoskeleton as they aged in culture and underwent apoptosis. This included a dissociation of protein(s) from F-actin, a candidate mechanism for increased susceptibility to cleavage, and a loss in immunodetectable alpha-actinin and ezrin, two actin-binding, membrane-anchoring proteins. These results clarify roles for different classes of proteases in a physiologically important form of constitutive apoptosis. Topics: Actinin; Actins; Apoptosis; Calpain; Caspases; Cell Communication; Cells, Cultured; Cellular Senescence; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Dipeptides; Drug Synergism; Humans; Leupeptins; Multienzyme Complexes; Neutrophils; Phosphoproteins; Proteasome Endopeptidase Complex | 1998 |
Persyn, a member of the synuclein family, influences neurofilament network integrity.
Topics: Calcium; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; Endopeptidases; gamma-Synuclein; Leupeptins; Neoplasm Proteins; Nerve Tissue Proteins; Neurofilament Proteins; Neurons, Afferent | 1998 |
Comparison of the effect of calpain inhibitors on two extralysosomal proteinases: the multicatalytic proteinase complex and m-calpain.
The potencies of three peptide aldehyde inhibitors of calpain (calpain inhibitors 1 and 2 and calpeptin) as inhibitors of four catalytic activities of the multicatalytic proteinase complex (MPC) were compared with their potencies as inhibitors of m-calpain. The chymotrypsinlike activity (cleavage after hydrophobic amino acids) and the caseinolytic activity (degradation of beta-casein) of MPC were strongly inhibited by calpain inhibitors 1 and 2 (IC50 values in the low micromolar range). Cleavage by MPC after acidic amino acids (peptidylglutamyl-peptide bond hydrolyzing activity) and basic amino acids (trypsinlike activity) was inhibited less effectively, declining moderately with increasing concentrations of calpain inhibitors 1 and 2. Calpeptin only weakly inhibited the four MPC activities, yet was the most potent inhibitor of m-calpain. These results indicate that caution must be exercised when calpain inhibitors 1 and 2 are used to infer calpain function. Calpeptin may be a better choice for such studies, although its effect on other cysteine or serine proteinases remains to be determined. Topics: Amino Acid Sequence; Animals; Calpain; Cattle; Cysteine Endopeptidases; Dipeptides; Kinetics; Leupeptins; Molecular Sequence Data; Multienzyme Complexes; Oligopeptides; Pituitary Gland; Protease Inhibitors; Proteasome Endopeptidase Complex; Substrate Specificity | 1994 |
Calcium-induced degradation of the inositol (1,4,5)-trisphosphate receptor/Ca(2+)-channel.
Ca(2+)-induced degradation of the neuronal inositol (1,4,5)-trisphosphate receptor, a protein which regulates Ca(2+)-release from intracellular stores, has been examined. The IP3-receptor, immunopurified from rat cerebellum, appeared to be an excellent substrate for purified Ca(2+)-activated neutral protease (calpain). Incubation of membranes or immunopurified IP3-receptor with Ca2+ and cerebellar cytosol also resulted in degradation of the receptor. Two main fragments with approximate molecular masses of 130 and 95 kDa were generated, both of which appeared to derive from the carboxyterminal Ca(2+)-channel-containing part of the protein. These data suggest that activation of the IP3-receptor, by causing increases in intracellular [Ca2+], might result in degradation of the N-terminal, IP3-binding part of the receptor. Topics: Animals; Astrocytes; Calcium; Calcium Channels; Calpain; Cell Membrane; Cerebellum; Chromatography, Affinity; Dipeptides; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Leupeptins; Male; Molecular Weight; Neurons; Phosphorylation; Rats; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Testis; Tumor Cells, Cultured; Vas Deferens | 1993 |
Calpain 1-protein kinase C complex: effect of calpain inhibitors after dissociation.
A calpain 1-protein kinase C (PKC) complex was isolated from rabbit skeletal muscle by hydrophobic interaction chromatography on phenyl-sepharose and by strong anion exchange chromatography on Q-Sepharose. Calpain 1 and kinase activities were then dissociated on a phenyl-Sepharose matrix using gradients of decreasing ionic strength. The purified PKC obtained corresponded to conventional PKC and was recognized by a monoclonal antibody specific for alpha and beta isotypes. Leupeptin, calpain inhibitor II, and the more selective calpain inhibitors calpeptin and MDL 28170 did not block the activation of the purified PKC by Ca2+ and phosphatidylserine. Topics: Amino Acid Sequence; Animals; Blotting, Western; Calpain; Dipeptides; Enzyme Activation; Enzyme Stability; Leupeptins; Male; Molecular Sequence Data; Muscles; Protein Kinase C; Rabbits | 1991 |
Synthesis of a new cell penetrating calpain inhibitor (calpeptin).
N-terminal of Leu-norleucinal or Leu-methioninal was modified to obtain a cell penetrative peptide inhibitor against calpain. Benzyloxycarbonyl (Z) derivatives had less active against papain than phenylbutyryl derivatives and leupeptin. Z-Leu-nLeu-H (calpeptin) was more sensitive to calpain I than Z-Leu-Met-H and leupeptin. Calpeptin was most potent among synthesized inhibitors in terms of preventing the Ca2+-ionophore induced degradation of actin binding protein and P235 in intact platelets. After 30 min incubation with intact platelets, calpeptin completely abolished calpain activity in platelets but no effect was observed in case of leupeptin. Calpeptin also inhibited 20K phosphorylation in platelets stimulated by thrombin, ionomycin or collagen. Thus calpeptin was found to be a useful cell-penetrative calpain inhibitor. Topics: Animals; Blood Platelets; Calcimycin; Calpain; Cell Membrane Permeability; Dipeptides; Leupeptins; Microfilament Proteins; Papain; Peptides; Swine | 1988 |