Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.22.56 (caspase-3)
 35,750 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have previously demonstrated cleavage of alpha-spectrin by caspase-3 and calpain during apoptosis in SH-SY5Y neuroblastoma cells (Nath, R., Raser, K. J., Stafford, D., Hajimohammadreza, I., Posner, A., Allen, H., Talanian, R. V., Yuen, P., Gilbertsen, R. B., and Wang, K. K. (1996) Biochem. J. 319, 683-690). We demonstrate here that calcium/calmodulin-dependent protein kinase IV (CaMK IV) is cleaved during apoptosis by caspase-3 and calpain. We challenged SH-SY5Y cells with the pro-apoptotic agent thapsigargin. Western blot analysis revealed major CaMK IV breakdown products of 40, 38, and 33 kDa. Digestion of control SH-SY5Y lysate with purified caspase-3 produced a 38-kDa CaMK IV fragment; digestion with purified calpain produced a major fragment of 40 kDa. Pretreatment with carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene or Z-Val-Ala-Asp-fluoromethylketone was able to block the caspase-3-mediated production of the 38-kDa fragment both in situ and in vitro. Calpain inhibitor II similarly blocked formation of the calpain-mediated 40-kDa fragment both in situ and in vitro. Digestion of recombinant CaMK IV by other caspase family members revealed that only caspase-3 produces a fragmentation pattern consistent to that seen in situ. The major caspase-3 and calpain cleavage sites are respectively identified as PAPD176*A and CG201*A, both within the CaMK IV catalytic domain. Furthermore, calmodulin-stimulated protein kinase activity decreases within 6 h in thapsigargin-treated SH-SY5Y. The loss of activity precedes cell death.
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PMID:Calcium/calmodulin-dependent protein kinase IV is cleaved by caspase-3 and calpain in SH-SY5Y human neuroblastoma cells undergoing apoptosis. 968 36

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.
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PMID:Caspase-mediated fragmentation of calpain inhibitor protein calpastatin during apoptosis. 970 9

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.
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PMID:Cleavage of the calpain inhibitor, calpastatin, during apoptosis. 989 9

We have previously demonstrated that calpain is responsible for the cleavage of Bax, a proapoptotic protein, during drug-induced apoptosis of HL-60 cells (Wood, D. E., Thomas, A., Devi, L. A., Berman, Y., Beavis, R. C., Reed, J. C., and Newcomb, E. W. (1998) Oncogene 17, 1069-1078). Here we show the sequential activation of caspases and calpain during drug-induced apoptosis of HL-60 cells. Time course experiments using the topoisomerase I inhibitor 9-amino-20(S)-camptothecin revealed that cleavage of caspase-3 substrates poly(ADP-ribose) polymerase (PARP) and the retinoblastoma protein as well as DNA fragmentation occurred several hours before calpain activation and Bax cleavage. Pretreatment with the calpain inhibitor calpeptin blocked calpain activation and Bax cleavage but did not inhibit PARP cleavage, DNA fragmentation, or 9-amino-20(S)-camptothecin-induced morphological changes and cell death. Pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) inhibited PARP cleavage, DNA fragmentation, calpain activation, and Bax cleavage and increased cell survival by 40%. Interestingly, Z-VAD-fmk-treated cells died in a caspase- and calpain-independent manner that appeared morphologically distinct from apoptosis. Our results suggest that excessive or uncontrolled calpain activity may play a role downstream of and distinct from caspases in the degradation phase of apoptosis.
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PMID:Caspase-dependent activation of calpain during drug-induced apoptosis. 1007 37

Maitotoxin is a potent toxin that activates voltage and receptor-mediated Ca2+ channels, resulting in Ca2+ overload and rapid cell death. We report that maitotoxin-induced cell death is associated with activation of calpain but not caspase-3 proteases in septo-hippocampal cell cultures. Calpain and caspase-3 activation were examined by accumulation of protease-specific breakdown products to alpha-spectrin. Cell death manifested exclusively necrotic-like characteristics including round, shrunken nuclei, even distribution of chromatin, absence of DNA fragmentation and failure of protein synthesis inhibition to reduce cell death. Necrotic cell death was observed in neurons and astroglia. Calpain inhibitor II inhibited calpain-specific processing of alpha-spectrin and significantly reduced cell death. The pan-caspase inhibitor, Z-D-DCB, nominally attenuated cell death. Results suggest that: (1) calpain, but not caspase-3, is activated as a result of maitotoxin-induced Ca2+ influx; (2) necrotic cell death caused by maitotoxin exposure is partially mediated by calpain activation; (3) maitotoxin is a useful tool to investigate pathological mechanisms of necrosis.
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PMID:Maitotoxin induces calpain but not caspase-3 activation and necrotic cell death in primary septo-hippocampal cultures. 1021 11

GRP94 is a 94-kDa chaperone glycoprotein with Ca(2+)-binding properties. We report here that during apoptosis induced by the topoisomerase II inhibitor etoposide, a fraction of GRP94 associated with the endoplasmic reticulum membrane undergoes specific proteolytic cleavage, coinciding with the activation of the caspase CPP32 and initiation of DNA fragmentation. In vivo, inhibitors of caspases able to block etoposide-induced apoptosis can only partially protect GRP94 from proteolytic cleavage, whereas complete inhibition is observed with calpain inhibitor I but not with the proteasome inhibitor. In vitro, GRP94 is not a substrate for CPP32; rather, it can be completely cleaved by calpain, a Ca(2+)-regulated protease. The cleavage of GRP94 by calpain is Ca(2+)-dependent and generates a discrete polypeptide of 80 kDa. In contrast, calpain has no effect on other stress proteins such as GRP78 or HSP70. Further, immunohistochemical staining reveals specific co-localization of GRP94 with calpain in the perinuclear region following etoposide treatment. We further showed that reduction of GRP94 by antisense decreased cell viability in etoposide-treated Jurkat cells. Our studies provide new evidence that the cytoprotective GRP94, as in the case of the antiapoptotic protein Bcl-2, can be targets of proteolytic cleavage themselves during the apoptotic process.
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PMID:The endoplasmic reticulum chaperone glycoprotein GRP94 with Ca(2+)-binding and antiapoptotic properties is a novel proteolytic target of calpain during etoposide-induced apoptosis. 1049 10

Studies examined the phenotypic characteristics of glutamate-induced cell death and their relationship to calpain and caspase-3 activation. Cell viability was assessed by fluorescein diacetate and propidium iodide staining and lactate dehydrogenase release. Calpain and caspase-3 activity was inferred from signature proteolytic fragmentation of alpha-spectrin. Characterization of cell death phenotypes was assessed by Hoechst 33258 and DNA fragmentation assays. Exposure of septohippocampal cultures to 1.0, 2.0, and 4.0 mmol/L glutamate induced a dose-dependent cell death with an LD50 of 2.0 mmol/L glutamate after 24 hours of incubation. Glutamate treatment induced cell death in neurons and astroglia and produced morphological alterations that differed from necrotic or apoptotic changes observed after maitotoxin or staurosporine exposure, respectively. After glutamate treatment, cell nuclei were enlarged and eccentrically shaped, and aggregated chromatin appeared in a diffusely speckled pattern. Furthermore, no dose of glutamate produced evidence of internucleosomal DNA fragmentation. Incubation with varying doses of glutamate produced calpain and caspase-3 activation. Calpain inhibitor II (N-acetyl-Leu-Leu-methionyl) provided protection only with a narrow dose range, whereas carbobenzoxy-Asp-CH2-OC(O)-2,6-dichlorobenzene (Z-D-DCB; pan-caspase inhibitor) and MK-801 (N-methyl-D-aspartate receptor antagonist) were potently effective across a wider dose range. Cycloheximide did not reduce cell death or protease activation.
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PMID:Novel characteristics of glutamate-induced cell death in primary septohippocampal cultures: relationship to calpain and caspase-3 protease activation. 1072 20

There are at least three types of inositol 1,4,5-trisphosphate receptor (IP(3)R) [IP(3)-gated Ca(2+)channels], which are expressed in different cell types and mammalian tissues. In this study, we have identified three IP(3)R subtypes in human Jurkat T-lymphoma cells. All three subtypes have a molecular mass of about 260 kDa, and display Ca(2+)channel properties in an IP(3)-dependent manner. We have also demonstrated that TNFalpha promotes the activity of different proteases (e.g. caspase-8, caspase-3 and calpain), alters the TCR-mediated Ca(2+)response and subsequently induces apoptosis in Jurkat cells. During the first 6 h of incubation with TNFalpha, several IP(3)R subtype-related changes occur (e.g. proteolysis of IP(3)R subtypes, inhibition of IP(3)binding and impairment of IP(3)-mediated Ca(2+)flux) concomitantly with an elevation of protease (caspase-8, caspase-3 and calpain) activity. Furthermore, the caspase inhibitor, Z-VAD-fmk, significantly reduces TNFalpha-mediated perturbation of IP(3)R1 and IP(3)R2 (but not IP(3)R3) function; whereas the calpain inhibitor I, ALLN, is capable of blocking the inhibitory effect of TNFalpha on IP(3)R3 function. These findings suggest that IP(3)R1 and IP(3)R2 serve as cellular substrates for caspases, and IP(3)R3 is a substrate for calpain. We propose that the selective down-regulation of IP(3)R subtype-mediated Ca(2+)function by caspase-dependent and calpain-sensitive mechanisms may be responsible for the early onset of the apoptotic signal by TNFalpha in human T-cells.
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PMID:Selective down-regulation of IP(3)receptor subtypes by caspases and calpain during TNF alpha -induced apoptosis of human T-lymphoma cells. 1101 62

Upon apoptosis induction, the proapoptotic protein Bax is translocated from the cytosol to mitochondria, where it promotes release of cytochrome c, a caspase-activating protein. However, the molecular mechanisms by which Bax triggers cytochrome c release are unknown. Here we report that before the initiation of apoptotic execution by etoposide or staurosporin, an active calpain activity cleaves Bax at its N-terminus, generating a potent proapoptotic 18-kDa fragment (Bax/p18). Both the calpain-mediated Bax cleavage activity and the Bax/p18 fragment were found in the mitochondrial membrane-enriched fraction. Cleavage of Bax was followed by release of mitochondrial cytochrome c, activation of caspase-3, cleavage of poly(ADP-ribose) polymerase, and fragmentation of DNA. Unlike the full-length Bax, Bax/p18 did not interact with the antiapoptotic Bcl-2 protein in the mitochondrial fraction of drug-treated cells. Pretreatment with a specific calpain inhibitor calpeptin inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and caspase-3 activation. In contrast, transfection of a cloned Bax/p18 cDNA into multiple human cancer cell lines targeted Bax/p18 to mitochondria, which was accompanied by release of cytochrome c and induction of caspase-3-mediated apoptosis that was not blocked by overexpression of Bcl-2 protein. Therefore, Bax/p18 has a cytochrome c-releasing activity that promotes cell death independent of Bcl-2. Finally, Bcl-2 overexpression inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and apoptosis. Our results suggest that the mitochondrial calpain plays an essential role in apoptotic commitment by cleaving Bax and generating the Bax/p18 fragment, which in turn mediates cytochrome c release and initiates the apoptotic execution.
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PMID:N-terminal cleavage of bax by calpain generates a potent proapoptotic 18-kDa fragment that promotes bcl-2-independent cytochrome C release and apoptotic cell death. 1102 54

Testicular torsion requires emergent release of the twisted spermatic cord. Ischemia/reperfusion (I/R) plays an important role in its pathogenesis, and recent data suggest that germ cells undergo apoptosis during I/R. In a model of torsion/detorsion (i.e., I/R) of the rat testis, involvement of calpain and caspase in necrotic and apoptotic cell death was examined. After 1 h of ischemia followed by 0, 0.5, 1, 6, or 24 h of reperfusion, the germ cells positively stained with in situ TUNEL, and DNA fragmentation, activation of caspase-3, and proteolysis of caspase substrates increased with time of reperfusion, demonstrating apoptosis. In addition, m-calpain activation and proteolysis of alpha-fodrin were increased during reperfusion, and its activation is thought to be involved in the necrosis. A calpain inhibitor, acety-leucyl-leucyl-norleucinal, inhibited the phenomena associated with apoptosis and necrosis induced by I/R, although a caspase inhibitor, Z-Val-Ala-Asp-fluoromethlyketone, only inhibited apoptotic changes. The inhibition of calpain but not caspase ameliorated the injury after 60 days of reperfusion following 1 h of ischemia. The calpain inhibitor injected just before reperfusion effectively suppressed alpha-fodrin proteolysis, suggesting its usefulness in the treatment of testicular torsion.
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PMID:Inhibition of calpain but not caspase protects the testis against injury after experimental testicular torsion of rat. 1105 63


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