Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.22.61 (caspase-8)
 6,833 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have identified and characterized CLARP, a caspase-like apoptosis-regulatory protein. Sequence analysis revealed that human CLARP contains two amino-terminal death effector domains fused to a carboxyl-terminal caspase-like domain. The structure and amino acid sequence of CLARP resemble those of caspase-8, caspase-10, and DCP2, a Drosophila melanogaster protein identified in this study. Unlike caspase-8, caspase-10, and DCP2, however, two important residues predicted to be involved in catalysis were lost in the caspase-like domain of CLARP. Analysis with fluorogenic substrates for caspase activity confirmed that CLARP is catalytically inactive. CLARP was found to interact with caspase-8 but not with FADD/MORT-1, an upstream death effector domain-containing protein of the Fas and tumor necrosis factor receptor 1 signaling pathway. Expression of CLARP induced apoptosis, which was blocked by the viral caspase inhibitor p35, dominant negative mutant caspase-8, and the synthetic caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone (zVAD-fmk). Moreover, CLARP augmented the killing ability of caspase-8 and FADD/MORT-1 in mammalian cells. The human clarp gene maps to 2q33. Thus, CLARP represents a regulator of the upstream caspase-8, which may play a role in apoptosis during tissue development and homeostasis.
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PMID:CLARP, a death effector domain-containing protein interacts with caspase-8 and regulates apoptosis. 938 Jul 1

The inhibitor of apoptosis (IAP) family of proteins are highly conserved through evolution. However, the mechanisms by which these proteins interfere with apoptotic cell death have been enigmatic. Recently, we showed that one of the human IAP family proteins, XIAP, can bind to and potently inhibit specific cell death proteases (caspases) that function in the distal portions of the proteolytic cascades involved in apoptosis. In this study, we investigated three of the other known members of the human IAP family, c-IAP-1, c-IAP-2 and NAIP. Similarly to XIAP, in vitro binding experiments indicated that c-IAP-1 and c-IAP-2 bound specifically to the terminal effector cell death proteases, caspases-3 and -7, but not to the proximal protease caspase-8, caspases-1 or -6. In contrast, NAIP failed to bind tightly to any of these proteases. Recombinant c-IAP-1 and c-IAP-2 also inhibited the activity of caspases-3 and -7 in vitro, with estimated Kis of <=0.1 microM, whereas NAIP did not. The BIR domain-containing region of c-IAP-1 and c-IAP-2 was sufficient for inhibition of these caspases, though proteins that retained the RING domain were somewhat more potent. Utilizing a cell-free system in which caspases were activated in cytosolic extracts by addition of cytochrome c, c-IAP-1 and c-IAP-2 inhibited both the generation of caspase activities and proteolytic processing of pro-caspase-3. Similar results were obtained in intact cells when c-IAP-1 and c-IAP-2 were overexpressed by gene transfection, and apoptosis was induced by the anticancer drug, etoposide. Cleavage of c-IAP-1 or c-IAP-2 was not observed when interacting with the caspases, implying a different mechanism from the baculovirus p35 protein, the broad spectrum suicide inactivator of caspases. Taken together, these findings suggest that c-IAP-1 and c-IAP-2 function similarly to XIAP by inhibiting the distal cell death proteases, caspases-3 and -7, whereas NAIP presumably inhibits apoptosis via other targets.
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PMID:The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases. 938 71

Many forms of apoptosis, including that caused by the death receptor CD95/Fas/APO-1, depend on the activation of caspases, which are proteases that cleave specific intracellular proteins to cause orderly cellular disintegration. The requirements for activating these crucial enzymatic mediators of death are not well understood. Using molecular chimeras with either CD8 or Tac, we find that oligomerization at the cell membrane powerfully induces caspase-8 autoactivation and apoptosis. Death induction was abrogated by the z-VAD-fmk, z-IETD-fmk, or p35 enzyme inhibitors or by a mutation in the active site cysteine but was surprisingly unaffected by death inhibitor Bcl-2. Amino acid substitutions that prevent the proteolytic separation of the caspase from its membrane-associated domain completely blocked apoptosis. Thus, oligomerization at the membrane is sufficient for caspase-8 autoactivation, but apoptosis could involve a death signal conveyed by the proteolytic release of the enzyme into the cytoplasm.
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PMID:Membrane oligomerization and cleavage activates the caspase-8 (FLICE/MACHalpha1) death signal. 946 83

Current models for Fas (CD95)-mediated apoptosis suggest that FLICE/caspase-8 is recruited and activated, which results in cell death. However, the role of additional molecules in Fas signaling and FLICE activation is not clear. A chimeric Fas/FLICE (F/F) receptor, containing the extracellular/transmembrane portion of Fas and the caspase region of FLICE, mediated anti-Fas apoptosis. FLICE protease subunits were generated from the F/F precursor. Killing induced by Fas, but not F/F, was blocked by a dominant negative FADD. Apoptosis triggered through Fas and F/F was inhibited by coexpression of CrmA and p35, but not Bcl-xL. F/F bypassed Fas resistance in COS-7 cells and blocking by the death effector domain (DED)-containing viral protein MC159. These results show that: 1) F/F induces cell death, indicating that FLICE activation is sufficient for apoptosis and does not require additional Fas- or FADD-binding proteins; and 2) F/F bypasses proximal defects in Fas signaling that prevent FLICE recruitment or activation.
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PMID:Apoptosis induced by a chimeric Fas/FLICE receptor: lack of requirement for Fas- or FADD-binding proteins. 949 39

Several caspases are mediators of apoptotic cell death. We describe a novel murine member of this growing protein family. Based on homology and especially on the substrate specificity, this new procaspase is identified as the murine counterpart of human procaspase-8. The protein exhibits a rather low similarity (76%) and identity (70%) to human procaspase-8. Procaspase-8 mRNA is expressed in all adult mouse tissues examined, the highest levels being reached in kidney, liver and lung. Procaspase-8 mRNA expression is highest in seven-day old embryos, but also during later stages of development the expression was fairly high. Both human and murine procaspase-8 are very weak substrates for granzyme B as compared to procaspase-3. Murine procaspases-1, 2, 3, 6, 7, 8, 11/4 and 12 are processed by recombinant murine caspase-8, suggesting a key role in the procaspase activation cascade. In addition, murine caspase-8 induced cell death that was inhibited both by cytokine response modifier A and p35. In vitro experiments demonstrated that p35 inhibits caspase-8 directly.
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PMID:Molecular cloning and identification of murine caspase-8. 983 23

Caspases (aspartate-specific cysteine proteases) play a critical role in the execution of the mammalian apoptotic program. To address the regulation of human caspase activation, we used the yeast Saccharomyces cerevisiae, which is devoid of endogenous caspases. The apical procaspases, -8beta and -10, were efficiently processed and activated in yeast. Although protease activity, per se, was insufficient to drive cell death, caspase-10 activity had little effect on cell viability, whereas expression of caspase-8beta was cytotoxic. This lethal phenotype was abrogated by co-expression of the pan-caspase inhibitor, baculovirus p35, and by mutation of the active site cysteine of procaspase-8beta. In contrast, autoactivation of the executioner caspase-3 and -6 zymogens was not detected. Procaspase-3 activation required co-expression of procaspase-8 or -10. Surprisingly, activation of procaspase-6 required proteolytic activities other than caspase-8, -10, or -3. Caspase-8beta or -10 activity was insufficient to catalyze the maturation of procaspase-6. Moreover, a constitutively active caspase-3, although cytotoxic in its own right, was unable to induce the processing of wild-type procaspase-6 and vice versa. These results distinguish sequential modes of activation for different caspases in vivo and establish a yeast model system to examine the regulation of caspase cascades. Moreover, the distinct terminal phenotypes induced by various caspases attest to differences in the cellular targets of these apoptotic proteases, which may be defined using this system.
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PMID:Cascades of mammalian caspase activation in the yeast Saccharomyces cerevisiae. 991 59

Tumor necrosis (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines that promotes apoptosis. TRAIL induces apoptosis in a wide variety of tumor cells but not in normal cells. Oncogene Bcl-2 can protect cells from apoptosis induced by various stress stimuli. However, it is not clear whether Bcl-2 can regulate TRAIL-induced apoptosis. The objective of this study was to investigate whether Bcl-2 can regulate apoptosis induced by TRAIL. TRAIL initiates the activation of caspases, the loss of mitochondrial transmembrane potential (Delta psi(m)), and the redistribution of mitochondrial cytochrome c. TRAIL has no effect on Delta psi(m) and apoptosis in Jurkat cells deficient in either FADD or caspase-8, suggesting both FADD and caspase-8 are required for TRAIL signaling. Overexpression of Bcl-2 delays, but does not inhibit, TRAIL-induced Delta psi(m), cytochrome c release from mitochondria and apoptosis, whereas etoposide-induced apoptosis is blocked by Bcl-2. XIAP, cowpox virus CrmA and baculovirus p35 inhibits TRAIL-induced apoptosis. These data suggest that TRAIL can be used to kill Bcl-2 positive cells that can not be killed by other class of chemotherapeutic drugs.
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PMID:Failure of Bcl-2 to block mitochondrial dysfunction during TRAIL-induced apoptosis. Tumor necrosis-related apoptosis-inducing ligand. 1111 58

Apoptosis is a highly regulated process that is crucial for normal development and homeostasis of multicellular organisms. The p35 protein from baculoviruses effectively prevents apoptosis by its broad-spectrum caspase inhibition. Here we report the crystal structure of p35 in complex with human caspase-8 at 3.0 A resolution, and biochemical and mutagenesis studies based on the structural information. The structure reveals that the caspase is inhibited in the active site through a covalent thioester linkage to p35, which we confirmed by gel electrophoresis, hydroxylamine treatment and mass spectrometry experiments. The p35 protein undergoes dramatic conformational changes on cleavage by the caspase. The repositioning of the amino terminus of p35 into the active site of the caspase eliminates solvent accessibility of the catalytic dyad. This may be crucial for preventing hydrolysis of the thioester intermediate, which is supported by the abrogation of inhibitory activity through mutations at the N terminus of p35. The p35 protein also makes conserved contacts with the caspase outside the active-site region, providing the molecular basis for the broad-spectrum inhibitory activity of this protein. We demonstrate a new molecular mechanism of caspase inhibition, as well as protease inhibition in general.
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PMID:Covalent inhibition revealed by the crystal structure of the caspase-8/p35 complex. 1126 Jul 20

Cerebellar granule neurons (CGN) cultured in the presence of serum and depolarizing potassium concentrations undergo apoptosis when switched to serum-free medium containing physiological potassium concentrations. Here we show that processing of the key protease, caspase-3, depends on the activation of caspase-9, but not of caspase-8. Selective peptide inhibitors of caspase-9 block processing of caspase-3 and caspase-8 and inhibit apoptosis, whereas a selective inhibitor of caspase-8 blocks neither processing of caspase-3 nor cell death. The data obtained with peptide inhibitors were confirmed by adenovirally mediated ectopic expression of the cytokine response modifier A (crmA), the baculovirus protein p35, and the X chromosome-linked inhibitor of apoptosis (XIAP). Further, caspase-8-activating death receptors do not mediate apoptosis in CGN and potassium withdrawal-induced apoptosis evolves unaltered in gld or lpr mice, which harbor mutations in the CD95/CD95 ligand system. Thus, neuronal apoptosis triggered by potassium deprivation is death receptor-independent but involves the mitochondrial pathway of caspase activation.
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PMID:Cascade of caspase activation in potassium-deprived cerebellar granule neurons: targets for treatment with peptide and protein inhibitors of apoptosis. 1131 7

Tumor necrosis (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines that promotes apoptosis. TRAIL induces apoptosis via death receptors (DR4 and DR5) in a wide variety of tumor cells but not in normal cells. The objectives of this study are to investigate the intracellular mechanisms by which TRAIL induces apoptosis. The death receptor Fas, upon ligand binding, trimerizes and recruits the adaptor protein FADD through the cytoplasmic death domain of Fas. FADD then binds and activates procaspase-8. It is unclear whether FADD is required for TRAIL-induced apoptosis. Here we show that the signaling complex of DR4/DR5 is assembled in response to TRAIL binding. FADD and caspase-8, but not caspase-10, are recruited to the receptor, and cells deficient in either FADD or caspase-8 blocked TRAIL-induced apoptosis. In addition, TRAIL initiates the activation of caspases, the loss of mitochondrial transmembrane potential (Deltapsi(m)), the cleavage of BID, and the redistribution of mitochondrial cytochrome c. Treatment of Jurkat cells with cyclosporin A delayed TRAIL-induced Deltapsi(m), caspase-3 activation and apoptosis. Similarly, Overexpression of Bcl-2 or Bcl-X(L) delayed, but did not inhibit, TRAIL-induced Deltapsi(m) and apoptosis. In contrast, XIAP, cowpox virus CrmA and baculovirus p35 inhibited TRAIL-induced apoptosis. These data suggest that death receptors (DR4 and DR5) and Fas receptors induced apoptosis through identical signaling pathway, and TRAIL-induced apoptosis via both mitochondrial-dependent and -independent pathways.
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PMID:Intracellular mechanisms of TRAIL: apoptosis through mitochondrial-dependent and -independent pathways. 1136 Jan 96


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