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)

The serine protease granzyme B, which is secreted by cytotoxic cells, is one of the major effectors of apoptosis in susceptible targets. To examine the apoptotic mechanism of granzyme B, we have analyzed its effect on purified proteins that are thought to be components of death pathways inherent to cells. We demonstrate that granzyme B processes interleukin 1beta-converting enzyme (ICE) and the ICE-related protease Yama (also known as CPP32 or apopain) by limited proteolysis. Processing of ICE does not lead to activation. However, processing by granzyme B leads directly to the activation of Yama, which is now able to bind inhibitors and cleave the substrate poly(ADP-ribose) polymerase whose proteolysis is a marker of apoptosis initiated by several other stimuli. Thus ICE-related proteases can be activated by serine proteases that possess the correct specificity. Activation of pro-Yama by granzyme B is within the physiologic range. Thus the cytotoxic effect of granzyme B can be explained by its activation of an endogenous protease component of a programmed cell death pathway.
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PMID:Proteolytic activation of the cell death protease Yama/CPP32 by granzyme B. 870 Aug 69

Cytotoxic T lymphocytes (CTLs) are able to kill target cells bearing foreign antigen through two distinct mechanisms: granule- and Fas-mediated cytotoxicity. The exact events involved in the induction of target cell apoptosis remain elusive, but research indicates a role for members of the interleukin-1beta converting enzyme (ICE)/Ced-3 family of cysteine proteases. The exact nature of the protease(s) involved is yet to be determined. Here we use activity assays and peptide inhibitors of ICE/Ced-3 proteases to study their role in Fas-mediated killing. We find that while certain inhibitors block DNA fragmentation and chromium release, others do not. Most notably, potent inhibitors of CPP32 and ICE could not inhibit DNA fragmentation during all cases of Fas-mediated cytotoxicity although an "ICE" inhibitor could suppress 51Cr release. Additionally, we find that CPP32 is not cleaved in all target cells during Fas killing. Although ICE activity (as measured by a fluorogenic substrate) is present in cell lysates from anti-Fas-treated cells, we found no pro-IL-1beta-cleaving activity in these lysates. Taken together, our results suggest that an alternate pathway to DNA fragmentation exists, which does not involve CPP32 activity, and that CPP32 and ICE activities are not essential to Fas-mediated killing.
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PMID:An interleukin-1beta converting enzyme-like protease is a key component of Fas-mediated apoptosis. 870 62

Cytotoxic T lymphocytes (CTLs) are able to recognize and destroy target cells bearing foreign antigen using one of two distinct mechanisms: granule- or Fas-mediated cytotoxicity. The exact mechanisms involved in the induction of apoptotic cell death remain elusive; however, it seems likely that a family of cysteine proteases related to interleukin-1beta converting enzyme are involved. One family member, CPP32, has been identified as an intracellular substrate for granzyme B, a CTL-specific serine protease responsible for the early induction of target cell DNA fragmentation. Here we use cytolytic cells from granzyme B-deficient mice to confirm that cleavage and activation of CPP32 represents a nonredundant role for granzyme B and that this activation plays a role in the induction of DNA fragmentation in target cells, a signature event for apoptotic cell death. A peptide inhibitor of CPP32-like proteases confirmed the function of these enzymes in fragmentation. 51Cr release was not suppressed under these conditions, suggesting that granzyme B cleavage of CPP32 is primarily involved in the induction of DNA fragmentation and not membrane damage during CTL-induced apoptosis.
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PMID:Cleavage of CPP32 by granzyme B represents a critical role for granzyme B in the induction of target cell DNA fragmentation. 870 64

Intracellular activation of sphingomyelinase, leading to ceramide generation, and ICE-like proteases have been implicated in TNF and Fas-induced apoptosis, but the links between these intracellular apoptotic mediators remain undefined. We show here that a specific peptide inhibitor of the ICE-like protease CPP32/Yama (DEVD-CHO) blocks anti-Fas-induced apoptosis in Jurkat and U937 cells, while having no effect on TNF-induced apoptosis in U937 cells. This peptide also prevents ceramide accumulation induced by Fas engagement. Jurkat and U937 cells, as well as their mtDNA-depleted derived lines (rho degree cells), were sensitive to ceramide toxicity, which was not prevented by ICE-like protease inhibitors. These results, taken together, suggest that ICE-like protease activation is a prerequisite for ceramide generation and subsequent apoptosis, at least in the case of Fas-induced cell death.
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PMID:CPP32 inhibition prevents Fas-induced ceramide generation and apoptosis in human cells. 870 67

ICE family proteases have been implicated as important effectors of the apoptotic pathway, perhaps acting hierarchically in a protease cascade. Using cleavage of endogenous protease substrates as probes, three distinct tiers of ICE-like activity were observed after Fas ligation in Jurkat cells. The earliest cleavage detected (30 min) was of fodrin, and produced a 150 kDa fragment. The second phase of cleavage (50 min) involved PARP, U1-70kDa and DNA-PKcs, all substrates of the CPP32-like proteases. Lamin B cleavage was observed during the third cleavage phase (90 min). Distinct inhibition profiles obtained using a panel of peptide-based inhibitors of ICE-like proteases clearly distinguished the three different cleavage phases. These studies provide evidence for a sequence of ICE-like proteolytic activity during apoptosis. The early fodrin cleavage, producing a 150 kDa fragment, identifies an ICE-like activity proximal to CPP32 in Fas-induced Jurkat cell apoptosis.
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PMID:Sequential activation of three distinct ICE-like activities in Fas-ligated Jurkat cells. 870 81

Although proteases related to the interleukin 1 beta-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cell-free model system for apoptosis (S/M extracts). First, we compared the substrate specificities of two recombinant human IRPs, CPP32 and Mch2 alpha. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Mch2 alpha also cleaved recombinant and nuclear lamin A at a conserved VEID decreases NG sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, CPP32 did not cleave lamin A. The cleavage of lamin A by Mch2 alpha and by S/M extracts was inhibited by millimolar concentrations of Zn2+, which had a minimal effect on cleavage of poly (ADP-ribose) polymerase by CPP32 and by S/M extracts. We also found that N-(acetyltyrosinylvalinyl-N epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone, which derivatizes the larger subunit of active ICE, can affinity label up to five active IRPs in S/M extracts. Together, these observations indicate that the processing of nuclear proteins in apoptosis involves multiple IRPs having distinct preferences for their apoptosis-associated substrates.
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PMID:Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis. 871 Aug 82

Emerging evidence suggests that an amplifiable protease cascade consisting of multiple aspartate specific cysteine proteases (ASCPs) is responsible for the apoptotic changes observed in mammalian cells undergoing programmed cell death. Here we describe the cloning of two novel ASCPs from human Jurkat T-lymphocytes. Like other ASCPs, the new proteases, named Mch4 and Mch5, are derived from single chain proenzymes. However, their putative active sites contain a QACQG pentapeptide instead of the QACRG present in ail known ASCPs. Also, their N termini contain FADD-like death effector domains, suggesting possible interaction with FADD. Expression of Mch4 in Escherichia coli produced an active protease that, like other ASCPs, was potently inhibited (Kj = 14 nM) by the tetrapeptide aldehyde DEVD-CHO. Interestingly, both Mch4 and the serine protease granzyme B cleave recombinant proCPP32 and proMch3 at a conserved IXXD-S sequence to produce the large and small subunits of the active proteases. Granzyme B also cleaves proMch4 at a homologous IXXD-A processing sequence to produce mature Mch4. These observations suggest that CPP32 and Mch3 are targets of mature Mch4 protease in apoptotic cells. The presence of the FADD-like domains in Mch4 and Mch5 suggests a role for these proteases in the Fas-apoptotic pathway. In addition, these proteases could participate in the granzyme B apoptotic pathways.
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PMID:In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains. 875 96

The implication of oxidative damage and/or intact mitochondrial function in physiological Fas-based cytotoxicity has been tested using the cytolytic hybridoma d11S and the CD8(+) CTL clone KB5.C20, previously stimulated to express Fas ligand (FasL) on their surface, as effectors and U937 or U937-rho0 cells (depleted of mitochondrial DNA) as targets. Immobilized anti-Fas mAb, which induced death of U937 cells, inhibited the growth of U937-rho0 cells but without inducing cell death. By contrast, FasL-expressing effectors readily killed both targets, with induction of DNA fragmentation, in 20 h assays. These results demonstrate the lack of involvement of mitochondrial-derived free radicals and/or intact mitochondrial function in physiological Fas-based cytotoxicity. Supplementation of Fas-sensitive cells (Jurkat, U937, L1210Fas) with a polyunsaturated fatty acid, which induces cell death through the generation of lipid free radicals, resulted in the potentiation of Fas-based cytotoxicity. This potentiating effect, but not Fas-based cytotoxicity itself, was eliminated by the physiological antioxidant vitamin E. On the other hand, the IL-1beta-converting enzyme (ICE)-like protease tetrapeptide inhibitor Ac-YVAD-cmk partially inhibited Fas-based cytotoxicity, while the specific inhibitor of CPP32/Yama Ac-DEVD-CHO was a much more effective inhibitor of Fas-induced apoptosis. It was concluded that Fas-induced cytotoxicity was clearly dependent on ICE-like protease activation, and especially on that of CPP32 in Fas-sensitive cells, including mitochondrial DNA-depleted ones.
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PMID:Role of oxidative damage and IL-1 beta-converting enzyme-like proteases in Fas-based cytotoxicity exerted by effector T cells. 875 63

Cytotoxic T cells (CTL) represent the major defense mechanism against the spread of virus infection. It is believed that the pore-forming protein, perforin, facilitates the entry of a series of serine proteases (particularly granzyme B) into the target cell which ultimately leads to DNA fragmentation and apoptosis. We demonstrate here that during CTL-mediated cytolysis the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an enzyme implicated in the repair of double strand breaks in DNA, is specifically cleaved by an interleukin (IL)-1 beta-converting enzyme (ICE)-like protease. A serine protease inhibitor, 3,4-dichloroisocoumarin (DCl), which is known to block granzyme B activity, inhibited CTL-induced apoptosis and prevented the degradation of DNA-PKcs in cells but failed to prevent the degradation of purified DNA-PKcs by CTL extracts. However, Tyr-Val-Ala-Asp-CH2Cl (YVAD-CMK) and other cysteine protease inhibitors prevented the degradation of purified DNA-PKcs by CTL extracts. Furthermore, incubation of DNA-PKcs with granzyme B did not produce the same cleavage pattern observed in cells undergoing apoptosis and when this substrate was incubated with either CTL extracts or the ICE-like protease, CPP32. Sequence analysis revealed that the cleavage site in DNA-PKcs during CTL killing was the same as that when this substrate was exposed to CPP32. This study demonstrates for the first time that the cleavage of DNA-PKcs in this intact cell system is exclusively due to an ICE-like protease.
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PMID:Interleukin-1 beta-converting enzyme-like protease cleaves DNA-dependent protein kinase in cytotoxic T cell killing. 876 Aug 15

Interleukin-1 beta converting enzyme (ICE) defines a new class of mammalian cysteine protease that shares strong homology with the Caenorhabditis elegans death gene ced-3. Both ICE and CED-3, when introduced into cultured cells, induce apoptosis, indicating that this type of cysteine protease may play an important role in the process of programmed cell death. Here, we report the cloning of a mouse and rat gene encoding a novel cysteine protease. The putative proteins encoded by these cDNAs contain the conserved sequence (QACRG) necessary for covalent linkage to the substrate as well as the three amino acids responsible for substrate binding and catalysis in ICE. Amino acid sequence analysis indicates that this rodent cysteine protease is the homolog of human CPP32 beta. Mouse CPP32 beta mRNA is highly expressed in spleen, and to a lesser degree in brain, lung, liver, and kidney. The mouse CPP32 beta genomic locus spans a region of approximately 20 kb, including seven exons and six introns. Mouse interspecific backcross mapping allowed localization of CPP32 beta to the central region of mouse chromosome 8, linked to Scvr, Lpl, Jund1 and Mlr.
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PMID:Molecular characterization of mouse and rat CPP32 beta gene encoding a cysteine protease resembling interleukin-1 beta converting enzyme and CED-3. 876 Dec 96


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