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

Cytotoxic T lymphocytes (CTL) can induce apoptosis through a granzyme B-based killing mechanism. Here we show that in cells undergoing apoptosis by granzyme B, both p45 pro-interleukin 1 beta converting enzyme (ICE) and pro-CPP32 are processed. Using ICE deficient (ICE -/-) mice, embryonic fibroblasts exhibit high levels of resistance to apoptosis by granzyme B or granzyme 3, while B lymphoblasts are granzyme B-resistant, thus identifying an ICE-dependent apoptotic pathway that is activated by CTL granzymes. In contrast, an alternative ICE-independent pathway must also be activated as ICE -/- thymocytes remain susceptible to apoptosis by both granzymes. In ICE -/- B cells or HeLa cells transfected with mutant inactive ICE or Ich-1S that exhibit resistance to granzyme B, CPP32 is processed to p17 and poly(ADP-ribose) polymerase is cleaved indicating that this protease although activated was not associated with an apoptotic nuclear phenotype. Using the peptide inhibitor Ac-DEVD-CHO, apoptosis as well as p45 ICE hydrolysis are suppressed in HeLa cells, suggesting that a CPP32-like protease is upstream of ICE. In contrast, p34cdc2 kinase, which is required for granzyme B-induced apoptosis, remains inactive in ICE -/- B cells indicating it is downstream of ICE. We conclude that granzyme B activates an ICE-dependent cell death pathway in some cell types and requires a CPP32-like Ac-DEVD-CHO inhibitable protease acting upstream to initiate apoptosis.
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PMID:Activation of an interleukin 1 converting enzyme-dependent apoptosis pathway by granzyme B. 885 98

Recent evidence suggests that CPP32 is an essential component of an aspartate-specific cysteine protease (ASCP) cascade responsible for apoptosis execution in mammalian cells. Activation of CPP32 could lead to activation of other downstream ASCPs, resulting in late morphological changes such as lamin cleavage and DNA fragmentation, observed in cells undergoing apoptosis. Here we describe the identification and cloning of a novel human ASCP named Mch6 from Jurkat T lymphocytes. We demonstrate that the pro-enzymes of Mch6 and the lamin-cleaving enzyme Mch2alpha are substrates for mature CPP32. Site-directed mutagenesis revealed that CPP32 processes pro-Mch6 preferentially at Asp330 to generate two subunits of molecular masses 37 kDa (p37) and 10 kDa (p10). However, CPP32 processes pro-Mch2alpha at three aspartate processing sites (Asp23, Asp179, and Asp193) to produce the large (p18) and small (p11) subunits of the mature Mch2alpha enzyme. The CPP32-processed Mch2alpha is capable of cleaving the VEIDN lamin cleavage site, indicating that CPP32 can, in fact, activate pro-Mch2alpha. Granzyme B at a concentration that allows processing and activation of CPP32 failed to process pro-Mch2alpha. However, incubation of pro-Mch2alpha with granzyme B in the presence of a cellular extract containing pro-CPP32 resulted in activation of pro-CPP32 and subsequent processing of pro-Mch2alpha. Interestingly, granzyme B can also process pro-Mch6 but at a site N-terminal to that cleaved by CPP32. These data suggest that Mch2alpha and Mch6 are downstream proteases activated in CPP32- and granzyme B-mediated apoptosis. This is the first demonstration of a protease cascade involving granzyme B, CPP32, Mch2alpha, and Mch6 and evidence that the lamin-cleaving enzyme Mch2 is a target of mature CPP32.
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PMID:The Ced-3/interleukin 1beta converting enzyme-like homolog Mch6 and the lamin-cleaving enzyme Mch2alpha are substrates for the apoptotic mediator CPP32. 890 Feb 1

Lymphocyte granule-mediated apoptosis is postulated to entail the formation of membrane pores by perforin. Then soluble granzyme reaches the cytosol either through these pores or by reparative pinocytosis. We demonstrate here that Jurkat cells bind and internalize granzyme B via high affinity binding sites without toxic consequence. Apoptosis occurs, however, if sublytic perforin is added to targets washed free of soluble granzyme B. We suggest that granule-mediated apoptosis mimics viral strategies for cellular entry. Accordingly, co-internalization of granzyme B with adenovirus, a virus that escapes endosomes to reach the cytosol, also induced apoptosis. Poly(ADP-ribose) polymerase cleavage and processing of CPP32, ICE-LAP3, and Mch2 were detected at 30 min, while cytosolic acidification and DNA fragmentation occurred at 60 min. Annexin V binding and membrane permeabilization arose at 4 h. The concurrent activation of the Ced-3 proteases differed from the rate at which each cysteine protease is cleaved in vitro by granzyme B. Thus, granzyme B may not directly process these proteases in whole cells but rather may function by activating a more proximal enzyme. These results indicate that adenovirus-mediated delivery of granzyme B is suitable for elucidating biochemical events that accompany granule-mediated apoptosis.
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PMID:New paradigm for lymphocyte granule-mediated cytotoxicity. Target cells bind and internalize granzyme B, but an endosomolytic agent is necessary for cytosolic delivery and subsequent apoptosis. 891 May 61

Apoptosis induced by a variety of agents results in the proteolytic cleavage of a number of cellular substrates by enzymes related to interleukin 1beta-converting enzyme (ICE). A small number of substrates for these enzymes have been identified to date, including enzymes involved in DNA repair processes: poly(ADP-ribose) polymerase and DNA-dependent protein kinase. We describe here for the first time the specific cleavage of the heteronuclear ribonucleoproteins (hnRNPs) C1 and C2 in apoptotic cells induced to undergo apoptosis by a variety of stimuli, including ionizing radiation, etoposide, and ceramide. No cleavage was observed in cells that are resistant to apoptosis induced by ionizing radiation. Protease inhibitor data implicate the involvement of an ICE-like protease in the cleavage of hnRNP C. Using recombinant ICE-like proteases and purified hnRNP C proteins in vitro, we show that the C proteins are cleaved by Mch3alpha and CPP32 and, to a lesser extent, by Mch2alpha, but not by ICE, Nedd2, Tx, or the cytotoxic T-cell protease granzyme B. The results described here demonstrate that the hnRNP C proteins, abundant nuclear proteins thought to be involved in RNA splicing, belong to a critical set of protein substrates that are cleaved by ICE-like proteases during apoptosis.
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PMID:Heteronuclear ribonucleoproteins C1 and C2, components of the spliceosome, are specific targets of interleukin 1beta-converting enzyme-like proteases in apoptosis. 891 May 95

The aspartase granzyme B is one of the major components of the granules involved in cell killing by cytotoxic T lymphocytes. Granzyme B has been shown to activate the apoptotic death pathway in the target cell, and this involves activation of members of the caspase (CASP) protein family. Therefore, activational cleavage of mouse (m) CASP proforms by granzyme B was examined in vitro. CASP can be subdivided in the CASP-1 (interleukin-1 beta-converting enzyme; ICE) subfamily, the CASP-2 (Ich1) subfamily, and the CASP-3 (CPP32) subfamily. Our results reveal that the proforms of the CASP-3 subfamily members mCASP-3 and mCASP-7 are hydrolyzed by granzyme B, while proforms of CASP-2 and CASP-1 subfamily members are not directly cleaved. Only one CASP-3 subfamily member, pro-mCASP-6, was not proteolytically cleaved by granzyme B. These results indicate that two members of the CASP-3 subfamily, but no others, become activated by granzyme B.
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PMID:Cleavage of caspase family members by granzyme B: a comparative study in vitro. 917 24

In the granule exocytosis pathway of cell-mediated cytotoxicity, rapid apoptotic nuclear damage in target cells has been unequivocally linked to granzyme B activity. Direct cleavage and activation of caspase-3 and related proteases by granzyme B have been identified as a central event in apoptosis induction by cytotoxic granules. The Bcl-2 oncoprotein has been recently shown to act at the level or upstream of caspase-3 family activation to inhibit apoptosis induced by various stimuli including Fas ligation, an alternative cell-mediated lytic pathway. In this study, we have investigated whether activation of this caspase family by granzyme B, during human NK and lymphokine-activated killer cell granule-mediated apoptosis, could be influenced by Bcl-2 expression. Bcl-2-overexpressing clones were generated from parental K562 and U937 cell lines (K6 and U4 clones, respectively). Bcl-2 expression abrogated early 125I-DNA release and DNA fragmentation, these defects being compensated for by extended incubation times. Cleavage of poly(ADP-ribose) polymerase, a specific caspase-3 family substrate, was detected in parental K562 cells exposed to lymphokine-activated killer effectors but not in K6 targets, indicating that caspase-3 and related proteases function was inhibited by Bcl-2. Functional inhibition of caspase-3 family with benzyloxycarbonyl-Asp-Glu-Val-Asp(OMe) fluoromethylketone led to similar consequences on apoptotic nuclear events as for Bcl-2 expression. Thus, Bcl-2 antagonizes granzyme B-mediated apoptosis by a mechanism that interferes with caspase-3 activity. Finally, Bcl-2 expression or the Asp-Glu-Val-Asp peptide was much less efficient in preventing phosphatidylserine externalization, suggesting that despite impaired nuclear apoptosis, immediate recognition and elimination of Bcl-2-expressing cells by tissue phagocytes should remain partly unaffected.
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PMID:Bcl-2 expression in target cells leads to functional inhibition of caspase-3 protease family in human NK and lymphokine-activated killer cell granule-mediated apoptosis. 920 Apr 47

CPP32 (also called Yama and apopain) is a member of a growing family of cysteine proteases which includes the interleukin-1 beta-converting enzyme (ICE) and the product of the Caenorhabditis elegans cell death gene ced-3. CPP32 has been consistently implicated as a key protease of the ICE/CED-3 family that is activated in response to a variety of death stimuli. Active CPP32 consists of P17 and p12 subunits derived from a 32 kDa pro-enzyme. This activation can be mediated by some ICE-like proteases and the cytotoxic T-cell (CTL) protease granzyme B. Once activated, CPP32 can process some of the other ICE family members and cleaves several cellular proteins in apoptotic cells. Inhibitors of CPP32 and other ICE-like proteases are potent inhibitors of apoptosis and promise to be important therapeutic molecules for the treatment of diseases, such as neurodegenerative and autoimmune disorders, that arise from excessive ell death.
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PMID:The apoptotic cysteine protease CPP32. 920 18

We report that the serine protease granzyme B (GrB), which is crucial for granule-mediated cell killing, initiates apoptosis in target cells by first maturing caspase-10. In addition, GrB has a limited capacity to mature other caspases and to cause cell death independently of the caspases. Compared with other members, GrB in vitro most efficiently processes caspase-7 and -10. In a human cell model, full maturation of caspase-7 does not occur unless caspase-10 is present. Furthermore, GrB matured caspase-3 with less efficiency than caspase-7 or caspase-10. With the caspases fully inactivated by peptidic inhibitors, GrB induced in Jurkat cells growth arrest and, over a delayed time period, cell death. Thus, the primary mechanism by which GrB initiates cell death is activation of the caspases through caspase-10. However, under circumstances where caspase-10 is absent or dysfunctional, GrB can act through secondary mechanisms including activation of other caspases and direct cell killing by cleavage of noncaspase substrates. The redundant functions of GrB ensure the effectiveness of granule-mediated cell killing, even in target cells that lack the expression or function (e.g., by mutation or a viral serpin) of one or more of the caspases, providing the host with overlapping safeguards against aberrantly replicating, nonself or virally infected cells.
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PMID:Granule-mediated killing: pathways for granzyme B-initiated apoptosis. 933 72

Interleukin-16, a proinflammatory cytokine produced in CD8(+) lymphocytes, is synthesized as a precursor protein (pro-IL-16). It is postulated that the C-terminal region of pro-IL-16 is cleaved, releasing bioactive IL-16. To characterize IL-16 cleavage, we transfected COS cells with a cDNA encoding a approximately 50-kDa form of pro-IL-16. Transfected COS cells released a approximately 20-kDa IL-16 cleavage product shown to consist of the 121 C-terminal residues of pro-IL-16 by immunoblotting and amino acid sequencing. Cleaved IL-16, but not pro-IL-16, exhibited lymphocyte chemoattractant activity. A C-terminal approximately 20-kDa IL-16 polypeptide was also released when pro-IL-16 was treated with concanavalin A-stimulated CD8(+) lymphocyte lysate. Cleavage occurred after an Asp, suggesting involvement of a caspase (interleukin-1beta-converting enzyme/CED-3) family protease. Using recombinant caspases and granzyme B, we determined that pro-IL-16 cleavage is mediated only by caspase-3. Relevance to pro-IL-16 processing in primary lymphocytes was supported by identifying the p20 subunit of activated caspase-3 in stimulated CD8(+) lymphocytes and by inhibition of CD8(+) lymphocyte lysate-mediated cleavage with Ac-DEVD-CHO. Pro-IL-16 is a substrate for caspase-3, and cleavage by this enzyme releases biologically active IL-16 from its inactive precursor.
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PMID:Processing and activation of pro-interleukin-16 by caspase-3. 942 80

Granzyme B (GzmB) is a neutral serine protease found in cytotoxic lymphocytes; this enzyme is critically involved in delivering the rapid apoptotic signal to susceptible target cells. GzmB has been difficult to study and has not yet been produced in non-mammalian systems because of the complex processing events that are thought to be required for its activation. In this report, we have successfully produced fully active, soluble recombinant GzmB (rGzmB) in a yeast-based system by fusing GzmB cDNA in frame with yeast alpha-factor cDNA, using the yeast KEX2 signal peptidase to release the processed enzyme into the supernatant of yeast cultures. We expressed the proenzyme form of GzmB as well and determined that pro-GzmB is efficiently converted to its active form by the cysteine proteinase dipeptidyl peptidase I. The fully processed enzyme was able to hydrolyze the synthetic substrate N-t-butyloxycarbonyl-L-alanyl-L-alanyl-L-aspartyl (Boc-Ala-Ala-Asp) thiobenzyl ester with a kcat of 17 s-1 and catalytic efficiency kcat/Km of 181,237 M-1 S-1; the recombinant enzyme is therefore at least twice as active as purified native GzmB. In addition, the recombinant enzyme hydrolyzes Boc-Ala-Ala-Met thiobenzyl ester with a kcat of 3.2 S-1 and a catalytic efficiency kcat/Km of 65,306 M-1 S-1. Purified rGzmB can also cleave the putative substrate caspase-3 into its signature p20/p10 forms. Unlike caspases, rGzmB is not sensitive to inhibition by several peptide-based inhibitors, including Ac-DEVD-CHO, Ac-YVAD-CMK, and ZIETD-FMK, as well as Zn2+ (a known inhibitor of caspase-3). Structural studies of rGzmB may allow us to better understand the substrate specificity of this enzyme and to design better inhibitors.
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PMID:Production of fully active recombinant murine granzyme B in yeast. 943 Jul 5


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