calpain and pepstatin

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

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

The effects of in situ postrigor injection (24 h postmortem) of exogenous aspartic, serine, and cysteine proteinase effectors into cylindrical beef longissimus samples on tenderness and myofibrillar protein degradation and integrity were studied. Injection of phenylmethanesulphonylfluoride (PMSF) and pepstatin did not influence shear force or protein degradation measured 8 d postmortem, confirming that neither serine nor aspartic proteinases affect tenderization. Injection of leupeptin, an epoxysuccinyl peptide (E-64), or N-acetyl-Leu-Leu-norleucinal (calpain inhibitor I) blocked tenderization completely, as observed by higher (P < .05) shear force values. A causal relationship between increased toughness and prevented action of the cysteine proteinases was suggested by a concomitant reduction of myofibrillar protein degradation, generally reflected in higher (P < .05) remaining troponin-T and titin amounts and lower (P < .05) levels of 30-kDa peptide, as evaluated by semiquantitative SDS-PAGE. Moreover, parallel to these changes, amounts of salt-soluble myofibrillar protein and semiquantitative concentrations of individual salt-soluble proteins (SDS-PAGE) were also reduced (P < .05). Injection of Triton-X-100 and Ca2+ increased (P < .05) tenderness, as well as myofibrillar protein degradation and solubility, and free Ca2+, whereas EDTA induced the opposite results, indicating an important role for calpains in tenderization. Because cathepsin B, D, H, and L inhibitors did not affect texture or proteolysis, our results suggest that calpains are the main proteases involved in beef tenderization.

Topics: Animals; Calcimycin; Calcium; Calpain; Cathepsins; Cattle; Cysteine Proteinase Inhibitors; Diazomethane; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Glycoproteins; Leucine; Leupeptins; Male; Meat; Muscle Proteins; Muscles; Octoxynol; Pepstatins; Phenylmethylsulfonyl Fluoride; Postmortem Changes; Solubility

17 different proteinase inhibitors were screened for their effect on the erythrocyte invasion by the malaria parasite Plasmodium falciparum. The effect was tested when the inhibitors were present in the culture medium and when they were trapped into erythrocyte ghosts. A very strong inhibition of invasion was observed in the presence of calpain inhibitors, with IC50 in the order of 10(-7) M. Chymostatin, leupeptin, pepstatin A and bestatin also caused inhibition of the invasion, but with IC50 in the order of 10(-5) M. The results suggest that participation of various proteinases in the process and point to the possibility of a calpain-mediated proteolytic event. This study may explain previous observations on the role of calcium in the invasion of the human erythrocyte by Plasmodium falciparum.

Topics: Animals; Calpain; Cysteine Proteinase Inhibitors; Erythrocytes; Humans; Leucine; Leupeptins; Oligopeptides; Pepstatins; Plasmodium falciparum

Four Ca2+-dependent proteinase activities in lobster claw and abdominal muscle have been resolved by high-performance liquid chromatography on gel filtration and ion-exchange columns. These activities, which do not appear to be generated by autolytic or other degradative processes, differed from each other in molecular weight (peak I, Mr = 310,000; peak IIa, Mr = 125,000; peak IIb, Mr = 195,000; peak III, Mr = 59,000) and net charge, as indicated by elution from an ion-exchange column with a NaCl gradient. Although optimum activity occurred at 5-10 mM Ca2+ at pH 6.8, the enzymes differed in activation at lower Ca2+ concentrations. The concentrations required for half-maximal activation were 0.6 mM for peak III, 1 mM for peak I, 1.5 mM for peak IIa, and 2 mM for peak IIb. Only the peak III proteinase was active at 100 microM Ca2+; none were active at 10 microM and below. Although the lobster Ca2+-dependent proteinases were all inhibited, from 75 to 98%, by the cysteine proteinase inhibitors leupeptin, N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine, and iodoacetamide, they showed differential responses to the aspartic proteinase inhibitor pepstatin and the serine proteinase inhibitor phenylmethanesulfonyl fluoride. Peak I was moderately (26%) inhibited by phenylmethanesulfonyl fluoride, whereas peaks IIb and III were inhibited 26 and 90%, respectively, by pepstatin. This is the first description of multiple forms of Ca2+-dependent proteinase that require Ca2+ at millimolar levels in any tissue, either vertebrate or invertebrate.

Topics: Animals; Calcium; Calcium Channel Blockers; Calpain; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Iodoacetamide; Leupeptins; Molecular Weight; Muscles; Nephropidae; Pepstatins; Phenylmethylsulfonyl Fluoride