Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.22.62 (caspase-9)
 7,507 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Understanding how oncogenic transformation sensitizes cells to apoptosis may provide a strategy to kill tumor cells selectively. We previously developed a cell-free system that recapitulates oncogene dependent apoptosis as reflected by activation of caspases, the core of the apoptotic machinery. Here, we show that this activation requires a previously identified apoptosis-promoting complex consisting of caspase-9, APAF-1, and cytochrome c. As predicted by the in vitro system, preventing caspase-9 activation blocked drug-induced apoptosis in cells sensitized by E1A, an adenoviral oncogene. Oncogenes, such as E1A, appear to facilitate caspase-9 activation by several mechanisms, including the control of cytochrome c release from the mitochondria.
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PMID:Oncogene-dependent apoptosis is mediated by caspase-9. 981 57

Apoptosis is a genetically programmed cell death that is required for morphogenesis during embryogenic development and for tissue homeostasis in adult organisms. In most cases, apoptosis involves cytochrome c release from mitochondria. In the cytosol, cytochrome c combines with APAF-1 in the presence of ATP to activate caspase-9 that, in turn, activates effectors caspases such as caspase-3. Bcl-2 and related proteins control cytochrome c release from the mitochondria whereas IAP (for Inhibitor of APoptosis) molecules modulate the activity of caspases. Plasma membrane receptors such as Fas (CD95, APO-1), characterized by a so-called "death domain" in their cytoplasmic domain, can activate the caspase cascade through adaptator molecules such as FADD (Fas-Associated protein with a Death Domain). Dysregulation of the apoptotic machinery plays a role in the pathogenesis of various diseases and molecules involved in cell death pathways are potential therapeutic targets in immunologic, neurologic, cancer, infectious and inflammatory diseases.
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PMID:[Apoptosis: molecular mechanisms]. 1010 3

We report here the reconstitution of the de novo procaspase-9 activation pathway using highly purified cytochrome c, recombinant APAF-1, and recombinant procaspase-9. APAF-1 binds and hydrolyzes ATP or dATP to ADP or dADP, respectively. The hydrolysis of ATP/dATP and the binding of cytochrome c promote APAF-1 oligomerization, forming a large multimeric APAF-1.cytochrome c complex. Such a complex can be isolated using gel filtration chromatography and is by itself sufficient to recruit and activate procaspase-9. The stoichiometric ratio of procaspase-9 to APAF-1 is approximately 1 to 1 in the complex. Once activated, caspase-9 disassociates from the complex and becomes available to cleave and activate downstream caspases such as caspase-3.
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PMID:An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. 1020 61

Apoptosis and platelet activation share common morphological and biochemical features. Because caspases are essential mediators of apoptosis, we examined whether platelets contain these proteinases and use them during platelet activation. Human platelets contained caspase-9, caspase-3, and the caspase activators APAF-1 and cytochrome c as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Upon treatment with cytochrome c and dATP, platelet cytoplasmic extracts recapitulated apoptotic events, including sequential activation of procaspase-9 and procaspase-3 and subsequent proteolysis of caspase substrates. Calcium ionophore-stimulated platelets also recapitulated apoptotic events, including cell shrinkage, plasma membrane microvesiculation, phosphatidyl serine externalization, and proteolysis of procaspase-9, procaspase-3, gelsolin, and protein kinase C-delta. Strikingly, however, these events occurred without caspase activation or release of mitochondrial cytochrome c, suggesting a role for a noncaspase proteinase. Supporting this, inhibition of the calcium-dependent proteinase, calpain, prevented caspase proteolysis, 'apoptotic' substrate cleavage, and platelet microvesiculation. In vitro, purified calpain cleaved recombinant procaspase-9 and procaspase-3 without activating either caspase, confirming the inhibitor studies. These data implicate calpain as a potential regulator of caspases and suggest that calpain, not caspases, promotes apoptosis-like events during platelet activation.
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PMID:Calpain functions in a caspase-independent manner to promote apoptosis-like events during platelet activation. 1047 93

Autocatalytic activation of initiator caspases is the link between pro-apoptotic signals and the destruction machinery of apoptosis. Activation of caspase-9, which mediates oncogene and drug-induced apoptosis, requires binding to the protein APAF-1. We found that the proteolytic activity of caspase-9 in a complex with APAF-1 is several orders of magnitude higher than that of the free enzyme. Thus, this complex functions as a holoenzyme in which caspase-9 is the catalytic subunit and APAF-1 its allosteric regulator. We argue that caspase-9 is activated by allosteric regulation and suggest that this mechanism is common for other initiator caspases.
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PMID:Caspase-9 and APAF-1 form an active holoenzyme. 1061 66

Fold recognition algorithm FFAS (Rychlewski et al., Protein Sci, 2000;9:232-241) was used to match the nucleotide-binding adaptor shared by APAF-1, certain R gene products and CED-4 (NB-ARC domain) to the structure of the D2 domain of N-ethylemaleimide-Sensitive Fusion Protein and the delta; subunit of clamp loader of DNA polymerase III. The predicted structure consists of the p-loop ATP-binding domain, followed by two alpha-helical domains that regulate the oligomerization process. This prediction suggests a detailed molecular mechanism for the "induced proximity" hypothesis (Salvesen and Dixit, Proc Natl Acad Sci USA 1999;96:10964-10967) for CED3/caspase-9 activation by CED4/APAF-1 complex. According to this model, the ATP binding acts as a trigger in CED-4 oligomerization and the helical domain immediately following the ATP-binding domain provides additional mechanisms for regulation of the oligomerization process. This model explains most of known experimental data about CED-4-mediated caspase activation and, at the same time, suggest experiments that could test this hypothesis.
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PMID:ATP-activated oligomerization as a mechanism for apoptosis regulation: fold and mechanism prediction for CED-4. 1073 40

Cytogenetic and molecular analyses have shown that the chromosome band 12q22 is recurrently deleted in male germ cell tumors (GCTs), indicating the presence of a candidate tumor suppressor gene (TSG) in this region. To identify the TSG, we mapped the APAF1 gene, a proapoptotic mammalian homologue of ced-4, to chromosomal band 12q22, that suggested that this might be the candidate deleted gene in GCTs. We further localized the gene between the polymorphic markers D12S1671 and D12S1082 at 12q22 to determine the role of APAF1 in the pathogenesis of GCT, and we characterized its normal genomic structure and analyzed its alterations in GCTs. The APAF1 gene comprises 27 exons, with the coding region spanning 26. The region containing APAF1 was found to be deleted in GCT by fluorescence in situ hybridization analysis, but without evidence of coding sequence alterations. RT-PCR and Western blot analysis showed APAF1 gene expression at detectable levels in all GCT cell lines analyzed. An aberrant-sized APAF1 protein was seen in one cell line. This and 2 other cell lines carrying APAF1 deletions also exhibited defects in dATP-mediated caspase-3 activation. Caspase-3 activity was effectively restored by addition of recombinant caspase-9 and APAF1 proteins, and to a lesser extent by caspase-9 alone, but not by APAF1 alone. These data do not support a TSG role for APAF1, but defects in other components of the apoptotic pathway that may be related to 12q22 deletion cannot be ruled out. Genes Chromosomes Cancer 28:258-268, 2000.
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PMID:Genetic analysis of the APAF1 gene in male germ cell tumors. 1086 31

Apaf1/CED4 family members play central roles in apoptosis regulation as activators of caspase family cell death proteases. These proteins contain a nucleotide-binding (NB) self-oligomerization domain and a caspase recruitment domain (CARD). A novel human protein was identified, NAC, that contains an NB domain and CARD. The CARD of NAC interacts selectively with the CARD domain of Apaf1, a caspase-activating protein that couples mitochondria-released cytochrome c (cyt-c) to activation of cytosolic caspases. Cyt-c-mediated activation of caspases in cytosolic extracts and in cells is enhanced by overexpressing NAC and inhibited by reducing NAC using antisense/DNAzymes. Furthermore, association of NAC with Apaf1 is cyt c-inducible, resulting in a mega-complex (>1 MDa) containing both NAC and Apaf1 and correlating with enhanced recruitment and proteolytic processing of pro-caspase-9. NAC also collaborates with Apaf1 in inducing caspase activation and apoptosis in intact cells, whereas fragments of NAC representing only the CARD or NB domain suppress Apaf1-dependent apoptosis induction. NAC expression in vivo is associated with terminal differentiation of short lived cells in epithelia and some other tissues. The ability of NAC to enhance Apaf1-apoptosome function reveals a novel paradigm for apoptosis regulation.
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PMID:A novel enhancer of the Apaf1 apoptosome involved in cytochrome c-dependent caspase activation and apoptosis. 1111 15

Activation of protein kinase C (PKC) by TPA in human U937 myeloid leukemia cells is associated with induction of adherence, differentiation, and G0/G1 cell cycle arrest. In this study, we demonstrate that in addition to these differentiating cells about 25% of U937 cells accumulated in the subG1 phase after TPA treatment. This effect proved to be phorbol ester-specific, since other compounds such as retinoic acid or vitamin D3 failed to induce apoptosis in conjunction with differentiation. Only a specific inhibitor of PKC, GF109203X, but not the broad-spectrum kinase inhibitor staurosporine or a tyrosine kinase inhibitor genistein could reverse the induction of apoptosis. Bryostatin-1, another specific PKC activator with distinct biochemical activity failed to induce apoptosis. Moreover, bryostatin-1 completely abolished the induction of apoptosis in U937 cells even if added 8 hours after TPA treatment. Apart from apoptosis induced by various chemotherapeutic drugs, TPA-related cell death is not mediated by an autocrine Fas-FasL loop and could not be prevented by a blocking antibody to the Fas receptor. However, a 75% reduction in the number of apoptotic cells after TPA stimulation was achieved by preincubation with a blocking antibody to the TNFalpha receptor. Tetrapeptide cleavage assays revealed a four-fold increase in the DEVD-cleavage activity in U937 cells compared to a three-fold increase in TUR cells. Immunoblotting demonstrated that TUR cells did not activate significant levels of caspase-3 or -7, whereas in U937 cells a 20-kDa cleavage product corresponding to activated caspase-3 was detectable after 3 d TPA exposure. Moreover, immunoblots revealed a strongly reduced expression of the adaptor molecule APAF-1, which is required for cytochrome c-dependent activation of caspase-9 and subsequently caspase-3. APAF-1 proved to be inducible after PKC activation with phorbol ester in U937, but not in TUR cells. Thus, APAF-1 expression may, at least in part, be regulated by PKC activity and reduced APAF-1 levels are associated with resistance to various inducers of apoptosis. Furthermore, TPA exposure of U937 cells is associated with increased levels of the pro-apoptotic proteins Bak and Bcl-xs, whereas simultaneously a decline in the Bcl-2 expression was noticable.
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PMID:Protein kinase C activation modulates pro- and anti-apoptotic signaling pathways. 1113 46

Several apoptosis-related genes have been reported to be involved in chemotherapy-induced apoptosis in cancers. An assessment of the relationship between expression of those genes and the degree of chemotherapy-induced apoptosis may be useful in improving the efficacy of cancer therapy. We transduced Apaf-1 (apoptotic protease-activating factor-1) and caspase-9 into U-373MG glioma cells using adenovirus (Adv) vectors in the presence of etoposide and evaluated the degree of apoptosis. The degree of apoptosis in etoposide-treated U-373MG cells infected with Adv for Apaf-1 (Adv-APAF1) was higher (27%) than that in cells infected with control Adv (14%), that in cells infected with Adv for caspase-9 (Adv-Casp9) was higher (34%) than that in cells infected with Adv-APAF1, and that in cells infected with both Adv-APAF1 and Adv-Casp9 was the highest (41%). Treatment with etoposide increased expression of p53 and decreased expression of Bcl-X(L) in U-373MG cells which harbored mutant p53. These results indicate that the expression of Apaf-1 and caspase-9 may be important determinants in predicting the sensitivity of cancers to chemotherapy. Adv-mediated co-transduction of Apaf-1 and caspase-9 should render cancer cells highly sensitive to chemotherapy.
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PMID:Co-transduction of Apaf-1 and caspase-9 augments etoposide-induced apoptosis in U-373MG glioma cells. 1134 70


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