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)

Granzyme B plays an essential role in cytotoxic T lymphocyte (CTL)-mediated cell killing. Recent studies suggest that granzyme B may exert its effect by cleaving and activating CPP32, a member of the interleukin-1 beta-converting enzyme/Ced-3 family of cysteine proteases. We have examined the processing and activation of CMH-1, a close homologue of CPP32, by granzyme B in vitro. We have found that granzyme B specifically cleaves CMH-1 at Asp198-Ser199 between the p20 and p12 and activates the cysteine protease. Cleavage between p20 and the prosequence of CMH-1 at Asp23-Ala24 is autocatalytic and is not required for CMH-1 activity in vitro. The cleavage and activation of CMH-1 by granzyme B in vitro sugge st that, in addition to CPP32, CMH-1 may also play a role in CTL-mediated cell killing.
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PMID:Processing and activation of CMH-1 by granzyme B. 863 95

Employing the degenerate primer-dependent polymerase chain reaction approach used recently to clone human Mch2, we have identified and cloned the insect Spodoptera frugiperda target of the baculovirus antiapoptotic protein p35. This protein named Sf caspase-1 belongs to the family of caspases and is highly related to human Mch3 and CPP32 in sequence and specific activity. The proenzyme of Sf caspase-1 is 299 amino acids in length and can undergo autocatalytic processing in Escherichia coli to an active enzyme heterocomplex. Autoprocessing occurs at Asp-28, Asp-184, and Asp-195 to generate the large p19/p18 and small p12 subunits. Sf caspase-1 is able to induce apoptosis in Sf9 cells and is capable of cleaving p35 to similar sized fragments as observed with extracts from p35 null mutant baculovirus-infected Sf9 cells. Sf caspase-1 activity is potently inhibited by p35, suggesting that it is an important target of this antiapoptotic protein. Finally, the Sf9 nuclear immunophilin FKBP46 was identified as a death-associated substrate for Sf caspase-1.
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PMID:Spodoptera frugiperda caspase-1, a novel insect death protease that cleaves the nuclear immunophilin FKBP46, is the target of the baculovirus antiapoptotic protein p35. 899 5

The cysteine protease CPP32 has been expressed in a soluble form in Escherichia coli and purified to >95% purity. The three-dimensional structure of human CPP32 in complex with the irreversible tetrapeptide inhibitor acetyl-Asp-Val-Ala-Asp fluoromethyl ketone was determined by x-ray crystallography at a resolution of 2.3 A. The asymmetric unit contains a (p17/p12)2 tetramer, in agreement with the tetrameric structure of the protein in solution as determined by dynamic light scattering and size exclusion chromatography. The overall topology of CPP32 is very similar to that of interleukin-1beta-converting enzyme (ICE); however, differences exist at the N terminus of the p17 subunit, where the first helix found in ICE is missing in CPP32. A deletion/insertion pattern is responsible for the striking differences observed in the loops around the active site. In addition, the P1 carbonyl of the ketone inhibitor is pointing into the oxyanion hole and forms a hydrogen bond with the peptidic nitrogen of Gly-122, resulting in a different state compared with the tetrahedral intermediate observed in the structure of ICE and CPP32 in complex with an aldehyde inhibitor. The topology of the interface formed by the two p17/p12 heterodimers of CPP32 is different from that of ICE. This results in different orientations of CPP32 heterodimers compared with ICE heterodimers, which could affect substrate recognition. This structural information will be invaluable for the design of small synthetic inhibitors of CPP32 as well as for the design of CPP32 mutants.
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PMID:Structure of recombinant human CPP32 in complex with the tetrapeptide acetyl-Asp-Val-Ala-Asp fluoromethyl ketone. 904 80

The apoptotic cysteine protease, caspase-3, is expressed in cells as an inactive 32-kDa precursor from which 17 kDa (p17) and 12 kDa (p12) subunits of the mature caspase-3 are proteolytically generated during apoptosis. Two amino acid sequences, ESMD downward arrowS (amino acids 25-29) and IETD downward arrowS (amino acids 172-176), in the precursor have been defined as the cleavage sites for the production of the p17 and p12 subunits. Using a cell-free assay system, we demonstrate that the caspase-3 precursor appears to be cleaved first at the IETD downward arrowS site, producing the p12 subunit and a 20-kDa (p20) peptide. Subsequently, the p20 is cleaved at the ESMD downward arrowS site, generating the mature p17 subunit. The cleavage at the IETD downward arrowS site required a protease activity that was selectively inhibited by the peptide, Ac-IETD-CHO (acetyl-IETD-aldehyde), and other protease inhibitors, such as the cowpox viral serine protease inhibitor, CrmA, and N-alpha-tosyl-L-phenylalanine chloromethyl ketone. The protease that catalyzed the cleavage at the ESMD/S site was selectively inhibited by another peptide, Ac-ESMD-CHO (acetyl-ESMD-aldehyde). More interestingly, the caspase-3 inhibitor, Ac-DEVD-CHO, but not the caspase-1 inhibitor, Ac-YVAD-CHO, also selectively inhibited the protease activity that cleaves at the ESMD downward arrowS site. This indicated that the cleavage at the ESMD downward arrowS site was either autocatalytic or that it required a caspase-3-like activity. In summary, we demonstrate that production of the p17:p12 form of caspase-3 is a sequential two-step process and appears to require two distinct enzymatic activities.
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PMID:A sequential two-step mechanism for the production of the mature p17:p12 form of caspase-3 in vitro. 914 68

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 have investigated the ability of Sf-caspase-1 and two mammalian caspases, caspase-1 and caspase-3, to induce apoptosis in Spodoptera frugiperda Sf-21 insect cells. While the transient expression of the pro-Sf-caspase-1 did not induce apoptosis, expression of the pro-domain deleted form, p31, or coexpression of the two subunits of mature Sf-caspase-1, p19 and p12, induced apoptosis in Sf-21 cells. The behavior of Sf-caspase-1 resembled that of the closely related mammalian caspase, caspase-3, and contrasted with that of the mammalian caspase-1, the pro-form of which was active in inducing apoptosis in Sf-21 cells. The baculovirus caspase inhibitor P35 blocked apoptosis induced by active forms of all three caspases. In contrast, members of the baculovirus inhibitor of apoptosis (IAP) family failed to block active caspase-induced apoptosis. However, during viral infection, expression of OpIAP or CpIAP blocked the activation of pro-Sf-caspase-1 and the associated induction of apoptosis. Thus, the mechanism by which baculovirus IAPs inhibit apoptosis is distinct from the mechanism by which P35 blocks apoptosis and involves inhibition of the activation of pro-caspases like Sf-caspase-1.
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PMID:Baculovirus inhibitors of apoptosis (IAPs) block activation of Sf-caspase-1. 939 Oct 73

Nitric oxide has been shown to inhibit apoptosis of human umbilical venous endothelial cells (HUVEC). Therefore we investigated the effect of different NO donors, PAPA NONOate (NOC-15; NO.) and nitrosodium tetrafluoroborate (NOBF4, NO+), and the reaction product of NO and O2-, peroxynitrite (ONOO- ), on TNF-alpha- or serum depletion-induced apoptosis of HUVEC. TNF-alpha-induced DNA fragmentation, determined by ELISA, was inhibited by NOC-15, NOBF4, and ONOO- in a concentration-dependent manner (maximal effects with 10 microM NO. and ONOO- and 100 microM NO+). The inhibition of apoptosis correlated with a protective effect on cell viability. The caspases, a cysteine protease family, play an important role in apoptotic processes. To determine whether the different NO donors and ONOO- regulate this enzyme, caspase-3-like activity was measured in homogenates of TNF-alpha-treated HUVEC. The TNF-alpha-induced enzyme activity was abrogated by NO., NO+, and ONOO-. Furthermore, caspase-3 activity was determined in vitro by reconstitution of the separately cloned, bacterially expressed, and purified active p17 and p12 subunits. The reconstituted caspase-3 exhibited enzyme activity, which was suppressed by the different NO donors and ONOO- with an IC50 of 50 microM for NOC-15, 1 mM for NOBF4, and 50 microM for ONOO-. The inhibition of caspase-3 activity correlated with a S-nitrosylation of the reactive cysteine residue and was reversed by further addition of dithiothreitol. This study suggests that the cellular regulatory processes of NO to protect cells from apoptosis may be independent of the redox state and that low concentrations of NO and ONOO- inhibit the cellular suicide program in HUVEC via S-nitrosylation of members of the caspase family.
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PMID:Effects of redox-related congeners of NO on apoptosis and caspase-3 activity. 944

Activation of caspase-3 requires proteolytic processing of the inactive zymogen into p18 and p12 subunits. We generated a rabbit polyclonal antiserum, CM1, which recognizes the p18 subunit of cleaved caspase-3 but not the zymogen. CM1 demonstrated an apparent specificity for activated caspase-3 by specifically immunolabelling only apoptotic but not necrotic cortical neurons in vitro. In the embryonic mouse nervous system, CM1 immunoreactivity was detected in neurons undergoing programmed cell death and was markedly increased in Bcl-xL-deficient embryos and decreased in Bax-deficient embryos. CM1 immunoreactivity was absent in the nervous system of caspase-3-deficient mouse embryos and in neurons cultured from caspase-3-deficient mice. Along with neuronal somata, extensive neuritic staining was seen in apoptotic neurons. These studies indicate that caspase-3 is activated during apoptosis in the developing nervous system in vivo and that CM1 is a useful reagent for its in situ detection.
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PMID:In situ immunodetection of activated caspase-3 in apoptotic neurons in the developing nervous system. 989 7

Synthetic peptides containing the arginine-glycine-aspartate (RGD) motif have been used extensively as inhibitors of integrin-ligand interactions in studies of cell adhesion, migration, growth and differentiation, because the RGD motif is an integrin-recognition motif found in many ligands. Here we report that RGD-containing peptides are able to directly induce apoptosis without any requirement for integrin-mediated cell clustering or signals. We show that RGD-containing peptides enter cells and directly induce autoprocessing and enzymatic activity of procaspase-3, a pro-apoptotic protein. Using the breast carcinoma cell line MCF-7, which has a functional deletion of the caspase-3 gene, we confirm that caspase-3 is required for RGD-mediated cell death. In addition to an RGD motif, pro-caspase-3 also contains a potential RGD-binding motif, aspartate-aspartate-methionine (DDM), near the site of processing to produce the p12 and p17 subunits. On the basis of the ability of RGD-DDX interactions to trigger integrin activation, we suggest that RGD peptides induce apoptosis by triggering conformational changes that promote pro-caspase-3 autoprocessing and activation. These findings provide an alternative molecular explanation for the potent proapoptotic properties of RGD peptides in models of angiogenesis, inflammation and cancer metastasis.
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PMID:RGD peptides induce apoptosis by direct caspase-3 activation. 1002 64

Caspases are a family of cysteine proteases activated during apoptosis. Modification of caspases by nitric oxide and its relevance during apoptosis is currently a controversial subject. In this study we analyzed the S-nitrosated form of caspase-3 at a molecular level. By using electrospray ionization-mass spectrometry, we detected poly-S-nitrosation of caspase-3 with an average of about 2 molecules of NO bound per enzyme. Although NO treatment completely inhibited enzyme activity, S-nitrosation was not restricted to the active site cysteine. Rather, we detected multiple relative mass increases of 30 +/- 1 Da in both the p12 and p17 subunits of caspase-3, corresponding to single to triple S-nitrosation. The stability of these S-nitrosations differed in physiologically relevant concentrations of 5 mM glutathione. Whereas all S-nitroso bonds in the p12 subunit were cleaved with release of NO and partial formation of protein-mixed disulfides with glutathione, a single S-nitrosation in the p17 subunit remained stable. Since this S-nitrosation was not observed in a mutant form of caspase-3 lacking the active site cysteine, we conclude that NO nitrosates the active site cysteine of caspase-3 and that this modification is notably inert to fast trans-nitrosation with glutathione. Furthermore, we provide evidence that treatment of caspase-3 with NO can lead to mixed disulfide formation with glutathione, demonstrating the oxidative character of NO.
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PMID:Mass spectrometric analysis of nitric oxide-modified caspase-3. 1040 38


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