Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Stably transfected Jurkat T cells were produced in which Bax expression is inducible by muristerone A. The cell death resulting from induction of the overexpression of Bax was prevented by inhibition of the mitochondrial permeability transition (MPT) with cyclosporin A (CyA) in combination with the phospholipase A2 inhibitor aristolochic acid (ArA). The caspase-3 inhibitor Z-Asp-Glu-Val aspartic acid fluoromethylketone (Z-DEVD-FMK) had no effect on the loss of viability. The MPT was measured as the CyA plus ArA-preventable loss of the mitochondrial membrane potential (DeltaPsim). The MPT was accompanied by the release of cytochrome c from the mitochondria, caspase-3 activation in the cytosol, cleavage of the nuclear enzyme poly(ADP-ribose)polymerase (PARP), and DNA fragmentation, all of which were inhibited by CyA plus ArA. Z-DEVD-FMK had no effect on the loss of DeltaPsim and the redistribution of cytochrome c but did prevent caspase-3 activation, PARP cleavage, and DNA fragmentation. It is concluded that Bax induces the MPT, a critical event in the loss of cell viability. In addition to the cell death, the MPT mediates other typical manifestations of apoptosis in this model, namely release of cytochrome c, caspase activation with PARP cleavage, and DNA fragmentation.
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PMID:The overexpression of Bax produces cell death upon induction of the mitochondrial permeability transition. 951 87

Interleukin-1beta-converting enzyme is a member of a family of human cysteine proteases with specificity for aspartic acid, which have been named caspases. Within this family of enzymes, transcript X (TX) and transcript Y (TY) (caspases 4 and 5, respectively) are very similar to ICE (caspase 1) and form the ICE subfamily. Given the high degree of conservation in the sequences of these proteases (more than 50% amino acid identity in the mature enzymes), it was of interest to examine whether they shared similar substrate specificities. The three enzymes, ICE, TX and TY, were therefore expressed in baculovirus-infected insect cells, as 30-kDa proteins lacking the propeptide. Automaturation into p20 and p10 subunits occurred within the cells. Active ICE, TX and TY were collected in the cell culture supernatants. In addition, their production induced the activation of an endogenous 32-kDa putative cysteine protease (CPP32) like caspase. T7-tagged ICE, TX and TY were purified by immunoaffinity and tested for their catalytic efficiency on YVAD-containing synthetic substrates and on the ICE natural substrate, pro-interleukin-1beta. TX cleaved the same synthetic substrates as ICE (Km of 90 microM and k(cat) of 0.4 s(-1) for Suc-YVAD-NH-Mec, where Suc represents succinyl and NH-Mec represents amino-4-methylcoumarin) and could cleave pro-interleukin-1beta into the same peptides as ICE but less efficiently. On the other hand, TY showed very little efficacy on the different ICE substrates (Km of 860 microM for Suc-YVAD-NH-Mec). These results show that the ICE/TX/TY subfamily has functional heterogeneity and that ICE remains the preferred enzyme for pro-interleukin-1beta cleavage.
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PMID:Enzymatic activity of two caspases related to interleukin-1beta-converting enzyme. 957 63

The requirement for caspases (ICE-like proteases) were investigated in mediating apoptosis of WEHI7.2 mouse lymphoma cells in response to two death inducers with different mechanisms of action, the glucocorticoid hormone dexamethasone (DX) and the calcium-ATPase inhibitor thapsigargin (TG). Apoptosis induction by these agents followed different kinetics, and was closely correlated with in vivo activation of caspase-3 (CPP32/Yama/Apopain) and cleavage of the caspase target protein poly(ADP-ribose) polymerase (PARP). Caspase activation and PARP cleavage were inhibited by Bcl-2 overexpression. Cell extracts from DX- and TG-treated cells cleaved the in vitro synthesized baculovirus p35 ICE-like protease target, producing 25 and 10 kDa fragments. p35 cleavage was inhibited by mutating the active site aspartic acid to alanine, and by a panel of protease inhibitors that inhibit caspase-3-like proteases, including iodoacetamide, N-ethylmaleimide, and Ac-DEVD-cho. Treatment of cells in vivo with two cell permeant peptide fluoromethylketone inhibitors of caspase activity, Z-VAD-fmk and Z-DEVD-fmk, inhibited DX- and TG-induced apoptotic nuclear changes and maintained plasma membrane integrity, whereas the cathepsin inhibitor, Z-FA-fmk, and two calpain inhibitors failed to inhibit apoptosis. An unexpected observation was that due to the delayed time course of DX-induced apoptosis, optimal preservation of plasma membrane integrity was achieved by adding caspase inhibitors beginning 8 h after DX addition. In summary, the findings indicate that two diverse apoptosis-inducing signals converge into a common Bcl-2-regulated pathway that leads to caspase activation and apoptosis.
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PMID:Apoptosis induction by the glucocorticoid hormone dexamethasone and the calcium-ATPase inhibitor thapsigargin involves Bc1-2 regulated caspase activation. 970 90

The MDM2 oncogene product is a regulator of the p53 tumor suppressor. MDM2 is cleaved by Caspase 3 (CPP32) during apoptosis after aspartic acid-361, generating a 60 kd fragment. Here we report that human tumor cell lines often express high levels of a 60 kd MDM2 isoform (p60) in the absence of apoptosis. We demonstrate that p60 is a product of caspase cleavage of full length MDM2 after residue 361. The protease that cleaves MDM2 in non-apoptotic cells appears to be distinct from the apoptosis-specific Caspase 3, since Caspase 3 substrate poly(ADP-ribose) polymerase (PARP) is not cleaved in cells producing p60. The p60 form of MDM2 is a significant fraction of the p53-bound MDM2 protein in certain tumor cells, suggesting that it functions in the regulation of p53. p60 is also detected in breast tumors overexpressing MDM2. These observations suggest that MDM2 is regulated by caspase processing in non-apoptotic cells, and may account for the MDM2 proteins of similar mobility seen in tumors and other cell lines.
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PMID:A 60 kd MDM2 isoform is produced by caspase cleavage in non-apoptotic tumor cells. 984 Sep 26

Retinal ganglion cells die by apoptosis following axotomy. The molecular mechanisms of the retinal ganglion cell death are not well understood. In the present study using RT-PCR and in situ hybridization techniques we demonstrated that levels of mRNA for Bcl-2 and Bcl-x decreased after axotomy. Bax levels remained high until 4 days after axotomy, decreased by day 7 and remained low up to day 10. CPP32 levels increased at day 7 and remained high after optic nerve cut. We studied whether inhibitors of CPP32/caspase would save the axotomy induced ganglion cell death. DEVD-CHO (Ac-Asp-Glu-Val-aspartic acid aldehyde) and DEVD-FMK (Z-Asp-Glu-Val-Asp-FMK), caspase inhibitors, when administered intraocularly at the time of optic nerve cut, at days 3 and 7 protect about 30-35% the ganglion cells from death. We further demonstrated that the number of reactive microglia decrease in the retina when the inhibitors were given as compared with retina where no inhibitors were given. The present data offers new avenues for studying the complex interactions between the retinal ganglion cell death and the activation of resident microglia/macrophages.
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PMID:Caspase inhibitors block the retinal ganglion cell death following optic nerve transection. 1010 Dec 30

Apoptotic cell suicide initiated by ligation of CD95 (Fas/APO-1) occurs through recruitment, oligomerization and autocatalytic activation of the cysteine protease, caspase-8 (MACH, FLICE, Mch5). An endogenous mammalian regulator of this process, named Usurpin, has been identified (aliases for Usurpin include CASH, Casper, CLARP, FLAME-1, FLIP, I-FLICE and MRIT). This protein is ubiquitously expressed and exists as at least three isoforms arising by alternative mRNA splicing. The Usurpin gene is comprised of 13 exons and is clustered within approximately 200 Kb with the caspase-8 and -10 genes on human chromosome 2q33-34. The Usurpin polypeptide has features in common with pro-caspase-8 and -10, including tandem 'death effector domains' on the N-terminus of a large subunit/small subunit caspase-like domain, but it lacks key residues that are necessary for caspase proteolytic activity, including the His and Cys which form the catalytic substrates diad, and residues that stabilize the P1 aspartic acid in substrates. Retro-mutation of these residues to functional caspase counterparts failed to restore proteolytic activity, indicating that other determinants also ensure the absence of catalytic potential. Usurpin heterodimerized with pro-caspase-8 in vitro and precluded pro-caspase-8 recruitment by the FADD/MORT1 adapter protein. Cell death induced by CD95 (Fas/APO-1) ligation was attenuated in cells transfected with Usurpin. In vivo, a Usurpin deficit was found in cardiac infarcts where TUNEL-positive myocytes and active caspase-3 expression were prominent following ischemia/reperfusion injury. In contrast, abundant Usurpin expression (and a caspase-3 deficit) occurred in surrounding unaffected cardiac tissue, suggesting reciprocal regulation of these pro- and anti-apoptotic molecules in vivo. Usurpin thus appears to be an endogenous modulator of apoptosis sensitivity in mammalian cells, including the susceptibility of cardiac myocytes to apoptotic death following ischemia/ reperfusion injury.
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PMID:Cell death attenuation by 'Usurpin', a mammalian DED-caspase homologue that precludes caspase-8 recruitment and activation by the CD-95 (Fas, APO-1) receptor complex. 1020 Apr 73

We investigated the ability of caspases (cysteine proteases with aspartic acid specificity) to induce cytochrome c release from mitochondria. When Jurkat cells were induced to undergo apoptosis by Fas receptor ligation, cytochrome c was released from mitochondria, an event that was prevented by the caspase inhibitor, zVAD-fmk (zVal-Ala-Asp-CH2F). Purified caspase-8 triggered rapid cytochrome c release from isolated mitochondria in vitro. The effect was indirect, as the presence of cytosol was required, suggesting that caspase-8 cleaves and activates a cytosolic substrate, which in turn is able to induce cytochrome c release from mitochondria. The cytochrome c releasing activity was not blocked by caspase inhibition, but was antagonized by Bcl-2 or Bcl-xL. Caspase-8 and caspase-3 cleaved Bid, a proapoptotic Bcl-2 family member, which gains cytochrome c releasing activity in response to caspase cleavage. However, caspase-6 and caspase-7 did not cleave Bid, although they initiated cytochrome c release from mitochondria in the presence of cytosol. Thus, effector caspases may cleave and activate another cytosolic substrate (other than Bid), which then promotes cytochrome c release from mitochondria. Mitochondria significantly amplified the caspase-8 initiated DEVD-specific cleavage activity. Our data suggest that cytochrome c release, initiated by the action of caspases on a cytosolic substrates, may act to amplify a caspase cascade during apoptosis.
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PMID:Caspases induce cytochrome c release from mitochondria by activating cytosolic factors. 1036 79

The diphtheria toxin A chain (DTA) is a potent cytocidal agent that inactivates elongation factor 2. This activity of DTA inhibits protein synthesis and rapidly leads to cell death through apoptosis. In this paper, we have developed a simple in vitro assay for DTA activity in which in vitro-translated DTA is used to inhibit the translation of proteins in wheat germ extracts. Inhibition of translation by DTA is dependent on cofactor NAD+, and the analysis of an attenuated DTA mutant indicates that this in vitro assay accurately reflects the in vivo activity of DTA. We have also identified aspartic acid at residue 8 (Asp-8) of DTA as a site of cleavage by the cell-death protease caspase-3. Cleavage of DTA by caspase-3 inactivates its ability to inhibit translation in wheat germ extracts. Conservative mutations at Asp-8 render DTA resistant to cleavage by caspase-3, but only slightly affect the ability of DTA to inhibit translation in vitro. Moreover, caspase-3-resistant DTA mutants are toxic in cells in tissue culture. The in vitro assay that we describe here will be useful for the rapid analysis of DTA activity and the development of DTA mutants with altered biological properties that may be of therapeutic value. Lastly, these studies serve as a prototype for the creation of caspase-resistant effector molecules.
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PMID:In vitro-translated diphtheria toxin A chain inhibits translation in wheat germ extracts: analysis of biologically active, caspase-3-resistant diphtheria toxin mutants. 1057 23

Treatment of U937 cells with various apoptosis-inducing agents, such as TNFalpha and beta-D-arabinofuranosylcytosine (ara-C) alone or in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), bryostatin 1 or cycloheximide, causes proteolytic cleavage of protein kinase Cmu (PKCmu) between the regulatory and catalytic domain, generating a 62 kDa catalytic fragment of the kinase. The formation of this fragment is effectively suppressed by the caspase-3 inhibitor Z-DEVD-FMK. In accordance with these in vivo data, treatment of recombinant PKCmu with caspase-3 in vitro results also in the generation of a 62 kDa fragment (p62). Treatment of several aspartic acid to alanine mutants of PKCmu with caspase-3 resulted in an unexpected finding. PKCmu is not cleaved at one of the typical cleavage sites containing the motif DXXD but at the atypical site CQND378/S379. The respective fragment (amino acids 379-912) was expressed in bacteria as a GST fusion protein (GST-p62) and partially purified. In contrast to the intact kinase, the fragment does not respond to the activating cofactors TPA and phosphatidylserine and is thus unable to phosphorylate substrates effectively.
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PMID:Proteolytic cleavage of protein kinase Cmu upon induction of apoptosis in U937 cells. 1062 42

Apoptosis, or programmed cell death, plays a central role in the development and homeostasis of an organism. The breakdown of cellular proteins in apoptosis is mediated by caspases, which comprise a highly conserved family of cysteine proteases with specificity for aspartic acid residues at the P1 positions of their substrates. Multiple lines of evidence show that caspase-9 is critical for an apoptosis pathway mediated via the mitochondria. In this study, the three-dimensional structure of the catalytic domain of caspase-9 and its interaction with the inhibitor acetyl-Asp-Val-Ala-Asp fluoromethyl ketone (Ac-DVAD-fmk) have been predicted by a segment matching modeling procedure. As expected, the predicted caspase-9 structure shows both a high similarity in the overall folding topology and remarkable differences in the surface loop regions as compared to other caspase family members such as caspase-1, -3 and -8, for which crystal structures have been determined. This kind of comparative analysis reflects the convergence-divergence duality among the caspases. Moreover, some subtle differences have been observed between caspase-9 and caspase-3 in the subsite contacts with the covalently linked inhibitor Ac-DVAD-fmk. Based on the X-ray structural analysis of caspase-8, a main chain carbonyl oxygen appears to be involved in a catalytic triad with the active site Cys and His residues. The corresponding carbonyl oxygen in caspase-9, together with other expected features of the catalytic apparatus, appears in our model. The predicted structure of caspase-9 can serve as a reference for subsite analysis relative to rational design of highly selective caspase inhibitors for therapeutic application.
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PMID:Prediction of the tertiary structure of a caspase-9/inhibitor complex. 1074 77


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