leupeptins and calpastatin

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

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

In HEK293 cells, exposure to various NAD(P)H oxidants, including phenazine methosulfate (PMS), that non-enzymatically oxidize intracellular NAD(P)H to NAD(P), decreased hypoxia-induced hypoxia-inducible factor 1 (HIF-1α) accumulation. RT-PCR and cycloheximide inhibition experiments indicated that PMS-induced HIF-1α decrease is involved in post-translational degradation during hypoxia. The decrease in HIF-1α caused by PMS was not eliminated by proteasome inhibitor MG132. Moreover, the increase in HIF-1α induced by exposure to MG132 alone in normoxia was diminished by PMS. In contrast, calpastatin peptide, a calpain inhibitor, fully prevented PMS-induced reduction in HIF-1α in hypoxic cells. These data suggest that the decreased stability of HIF-1α induced by PMS is due to the activation by PMS of a protein degradation system that is independent of the ubiquitin-proteasome pathway.

Topics: Calcium-Binding Proteins; Cell Hypoxia; Cycloheximide; Cysteine Proteinase Inhibitors; HEK293 Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Leupeptins; Methylphenazonium Methosulfate; Oxidants; Oxygen; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Processing, Post-Translational; Protein Stability; Protein Synthesis Inhibitors; Proteolysis; Signal Transduction

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

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

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 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

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

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

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

Ca(2+)-activated neutral protease calpain is ubiquitously expressed and may have pleiotropic biological functions. We have previously reported that repeated treatment of NIH3T3 mouse fibroblasts with the calpain inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN) resulted in the induction of transformed foci [T. Hiwasa, T. Sawada, and S. Sakiyama (1990) Carcinogenesis 11, 75-80]. To elucidate further the effects of calpain in malignant transformation of NIH3T3 cells, calpastatin, an endogenous specific inhibitor of calpain, was expressed in NIH3T3 cells by transfection with cDNA. G418-selected calpastatin-expressing clones showed a significant increase in the anchorage-independent growth ability. A similar increase in cloning efficiency in soft agar medium was also observed in calpain small-subunit-transfected clones. On the other hand, reduced expression of calpastatin achieved by transfection with calpastatin antisense cDNA in Ha-ras-transformed NIH3T3 (ras-NIH) cells caused morphological reversion as well as a decrease in anchorage-independent growth. When NIH3T3 cells were treated with ALLN for 3 days, cell growth was stimulated by approximately 10%. This growth stimulation by ALLN was not observed in ras-NIH cells, but recovered by expression of a dominant negative form of protein kinase C (PKC)epsilon but not by that of PKCalpha. Western blotting analysis showed that an increase in PKCepsilon was much more prominent than that of PKCalpha in NIH3T3 cells after treatment with ALLN. These results are concordant with the notion that calpain suppresses malignant transformation by predominant degradation of PKCepsilon.

Topics: 3T3 Cells; Animals; Blotting, Western; Calcium; Calcium-Binding Proteins; Cell Division; Cell Line; Cysteine Proteinase Inhibitors; DNA, Complementary; Gene Expression Regulation; Genes, Dominant; Humans; Isoenzymes; Leupeptins; Mice; Phenotype; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-epsilon; ras Proteins; Time Factors; Transfection

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

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

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

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

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

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

We studied the effect of protease inhibitors at a high concentration on connectin and nebulin filaments in myofibrils. Calpastatin domain I at 0.1 mM bound to connectin and nebulin filaments, and deteriorated their physico-chemical properties; the calcium-binding ability of connectin and nebulin filaments was suppressed, the susceptibility of both filaments to trypsin was markedly decreased, and the resting tension of mechanically skinned fibers was increased by 2.5 times that of the control at a sarcomere length of 3.6 microns. This indicates that the connectin filaments were made more rigid. The same phenomenon was observed from the treatment of skinned fibers with 1 mM leupeptin whose resting tension was increased to 2 times the control value. Microscopically, both protease inhibitors induced dense aggregation and disappearance of the regular striation of myofibrils due to their non-specific binding to many myofibrillar proteins. The use of excess calpastatin domain I and leupeptin should therefore be avoided in physiological and biochemical studies on connectin and nebulin filaments, as well as on myofibrils.

Topics: Animals; Calcium-Binding Proteins; Connectin; Cysteine Proteinase Inhibitors; Leupeptins; Muscle Proteins; Myofibrils; Protein Kinases; Rabbits

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

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

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

Sympathetic neurons undergo programmed cell death (PCD) upon deprivation of nerve growth factor (NGF). PCD of neurons is blocked by inhibitors of the interleukin-1beta converting enzyme (ICE)/Ced-3-like cysteine protease, indicating involvement of this class of proteases in the cell death programme. Here we demonstrate that the proteolytic activities of the proteasome are also essential in PCD of neurons. Nanomolar concentrations of several proteasome inhibitors, including the highly selective inhibitor lactacystin, not only prolonged survival of NGF-deprived neurons but also prevented processing of poly(ADP-ribose) polymerase which is known to be cleaved by an ICE/Ced-3 family member during PCD. These results demonstrate that the proteasome is a key regulator of neuronal PCD and that, within this process, it is involved upstream of proteases of the ICE/Ced-3 family. This order of events was confirmed in macrophages where lactacystin inhibited the proteolytic activation of precursor ICE and the subsequent generation of active interleukin-1beta.

Topics: Acetylcysteine; Animals; Apoptosis; Calcium-Binding Proteins; Caspase 1; Cell Survival; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA Ligases; Electrophoresis, Polyacrylamide Gel; Leupeptins; Multienzyme Complexes; Nerve Growth Factors; Neurons; Poly(ADP-ribose) Polymerases; Proteasome Endopeptidase Complex; Rats; Sympathetic Nervous System

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

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

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 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

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

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

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