UNIPROT:P42574 - FACTA Search

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Query: UNIPROT:P42574 (caspase-3)
45,978 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent genetic and biochemical studies have implicated cysteine-dependent aspartate-directed proteases (caspases) in the active phase of apoptosis. In the present study, three complementary techniques were utilized to follow caspase activation during the course of etoposide-induced apoptosis in HL-60 human leukemia cells. Immunoblotting revealed that levels of procaspase-2 did not change during etoposide-induced apoptosis, whereas levels of procaspase-3 diminished markedly 2-3 h after etoposide addition. At the same time, cytosolic peptidase activities that cleaved DEVD-aminotrifluoromethylcoumarin and VEID-aminomethylcoumarin increased 100- and 20-fold, respectively; but there was only a 1. 5-fold increase in YVAD-aminotrifluoromethylcoumarin cleavage activity. Affinity labeling with N-(Nalpha-benzyloxycarbonylglutamyl-Nepsilon-biotin yllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone indicated that multiple active caspase species sequentially appeared in the cytosol during the first 6 h after the addition of etoposide. Analysis on one- and two-dimensional gels revealed that two species comigrated with caspase-6 and three comigrated with active caspase-3 species, suggesting that several splice or modification variants of these enzymes are active during apoptosis. Polypeptides that comigrate with the cytosolic caspases were also labeled in nuclei of apoptotic HL-60 cells. These results not only indicate that etoposide-induced apoptosis in HL-60 cells is accompanied by the selective activation of multiple caspases in cytosol and nuclei, but also suggest that other caspase precursors such as procaspase-2 are present but not activated during apoptosis.
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PMID:Activation of multiple interleukin-1beta converting enzyme homologues in cytosol and nuclei of HL-60 cells during etoposide-induced apoptosis. 905 43

Cloning of interleukin-1 beta converting enzyme (ICE) and Caenorhabditis elegans death protein CED-3 revealed the structural and functional homology between these two proteases. It also suggested the involvement of ICE-like cysteine proteases in apoptosis. Several CED-3- and ICE-like cysteine proteases have been described, including Nedd2/Ich-1, CPP32 beta, Tx, ICErel3, and Mch2. We have previously described a mouse ortholog of cysteine protease CPP32 beta that shares strong homology with ICE and CED-3. Here, we describe the cloning of mouse and human Casp7, another member of this family of cysteine proteases. Mouse Casp7 encodes a putative 340-amino-acid polypeptide that contains all the known conserved residues required for protease function, including the QACRG sequence, aspartic acid residues for internal cleavage sites, and the residues required for substrate binding. Three RNA variants of human Casp7 were also cloned. Amino acid sequence analysis indicated that Casp7 shared high homology with CPP32 beta/Casp3 and Mch2/Casp6. Northern blot analysis demonstrated that a 2.6-kb Casp7 mRNA was expressed in various tissues except brain. Mouse interspecific backcross mapping allowed localization of Casp7 to the distal region of mouse chromosome 19, linked to Mxi1, Adra2a, and Aop1.
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PMID:Identification and mapping of Casp7, a cysteine protease resembling CPP32 beta, interleukin-1 beta converting enzyme, and CED-3. 907 Sep 23

The Fas/Fas ligand (FasL) pathway is widely involved in apoptotic cell death in lymphoid and nonlymphoid cells. It has recently been postulated that many chemotherapeutic agents also induce cell death by activating the Fas/FasL pathway. In the present study we compared apoptotic pathways induced by anti-Fas or chemotherapeutic agents in the Jurkat human T-cell leukemia line. Immunoblotting showed that treatment of wild-type Jurkat cells with anti-Fas or the topoisomerase II-directed agent etoposide resulted in proteolytic cleavage of precursors for the cysteine-dependent aspartate-directed proteases caspase-3 and caspase-7 and degradation of the caspase substrates poly(ADP-ribose) polymerase (PARP) and lamin B1. Likewise, affinity labeling with N-(N(alpha)-benzyloxycarbonylglutamyl-N(epsilon)-biotinyllysyl+ ++)aspartic acid [(2,6-dimethyl-benzoyl)oxy]methyl ketone [Z-EK(bio)D-amok] labeled the same five active caspase species after each treatment, suggesting that the same downstream apoptotic pathways have been activated by anti-Fas and etoposide. Treatment with ZB4, an antibody that inhibits Fas-mediated cell death, failed to block etoposide-induced apoptosis, raising the possibility that etoposide does not initiate apoptosis through Fas/FasL interactions. To further explore the relationship between Fas- and chemotherapy-induced apoptosis, Fas-resistant Jurkat cells were treated with various chemotherapeutic agents. Multiple independently derived Fas-resistant Jurkat lines underwent apoptosis that was indistinguishable from that of the Fas-sensitive parental cells after treatment with etoposide, doxorubicin, topotecan, cisplatin, methotrexate, staurosporine, or gamma-irradiation. These results indicate that antineoplastic treatments induce apoptosis through a Fas-independent pathway even though Fas- and chemotherapy-induced pathways converge on common downstream apoptotic effector molecules.
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PMID:Comparison of apoptosis in wild-type and Fas-resistant cells: chemotherapy-induced apoptosis is not dependent on Fas/Fas ligand interactions. 924 21

Apoptosis is a major form of cell death, characterized initially by a series of stereotypic morphological changes. In the nematode Caenorhabditis elegans, the gene ced-3 encodes a protein required for developmental cell death. Since the recognition that CED-3 has sequence identity with the mammalian cysteine protease interleukin-1 beta-converting enzyme (ICE), a family of at least 10 related cysteine proteases has been identified. These proteins are characterized by almost absolute specificity for aspartic acid in the P1 position. All the caspases (ICE-like proteases) contain a conserved QACXG (where X is R, Q or G) pentapeptide active-site motif. Capases are synthesized as inactive proenzymes comprising an N-terminal peptide (prodomain) together with one large and one small subunit. The crystal structures of both caspase-1 and caspase-3 show that the active enzyme is a heterotetramer, containing two small and two large subunits. Activation of caspases during apoptosis results in the cleavage of critical cellular substrates, including poly(ADP-ribose) polymerase and lamins, so precipitating the dramatic morphological changes of apoptosis. Apoptosis induced by CD95 (Fas/APO-1) and tumour necrosis factor activates caspase-8 (MACH/FLICE/Mch5), which contains an N-terminus with FADD (Fas-associating protein with death domain)-like death effector domains, so providing a direct link between cell death receptors and the caspases. The importance of caspase prodomains in the regulation of apoptosis is further highlighted by the recognition of adapter molecules, such as RAIDD [receptor-interacting protein (RIP)-associated ICH-1/CED-3-homologous protein with a death domain]/CRADD (caspase and RIP adapter with death domain), which binds to the prodomain of caspase-2 and recruits it to the signalling complex. Cells undergoing apoptosis following triggering of death receptors execute the death programme by activating a hierarchy of caspases, with caspase-8 and possibly caspase-10 being at or near the apex of this apoptotic cascade.
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PMID:Caspases: the executioners of apoptosis. 933 44

Previous studies have shown that K562 chronic myelogenous leukemia cells are resistant to induction of apoptosis by a variety of agents, including the topoisomerase II (topo II) poison etoposide, when examined 4 to 24 hours after treatment with an initiating stimulus. In the present study, the responses of K562 cells and apoptosis-proficient HL-60 acute myelomonocytic leukemia cells to etoposide were compared, with particular emphasis on determining the long-term fate of the cells. When cells were treated with varying concentrations of etoposide for 1 hour and subsequently plated in soft agar, the two cell lines displayed similar sensitivities, with a 90% reduction in colony formation at 5 to 10 mu mol/L etoposide. After treatment with 17 mu mol/L etoposide for 1 hour, cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP), DNA fragmentation, and apoptotic morphological changes were evident in HL-60 cells in less than 6 hours. After the same treatment, K562 cells arrested in G2 phase of the cell cycle but otherwise appeared normal for 3 to 4 days before developing similar apoptotic changes. When the etoposide dose was increased to 68 mu mol/L, apoptotic changes were evident in HL-60 cells after 2 to 3 hours, whereas the same changes were observed in K562 cells after 24 to 48 hours. This delay in the development of apoptotic changes in K562 cells was accompanied by delayed release of cytochrome c to the cytosol and delayed appearance of peptidase activity that cleaved the fluorogenic substrates Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC) and Val-Glu-Ile-Asp-aminomethylcoumarin (VEID-AMC) as well as an altered spectrum of active caspases that were affinity labeled with N-(Nalpha-benzyloxycarbonylglutamyl-Nepsilon-biotin yllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone [z-EK(bio)D-aomk]. On the other hand, the activation of caspase-3 under cell-free conditions occurred with indistinguishable kinetics in cytosol prepared from the two cell lines. Collectively, these results suggest that a delay in the signaling cascade upstream of cytochrome c release and caspase activation leads to a long latent period before the active phase of apoptosis is initiated in etoposide-treated K562 cells. Once the active phase of apoptosis is initiated, the spectrum and subcellular distribution of active caspase species differ between HL-60 and K562 cells, but a similar proportion of cells are ultimately killed in both cell lines.
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PMID:Comparison of caspase activation and subcellular localization in HL-60 and K562 cells undergoing etoposide-induced apoptosis. 937 39

Type I and II keratins help maintain the structural integrity of epithelial cells. Since apoptosis involves progressive cell breakdown, we examined its effect on human keratin polypeptides 8, 18, and 19 (K8, K18, K19) that are expressed in simple-type epithelia as noncovalent type I (K18, K19) and type II (K8) heteropolymers. Apoptosis induces rapid hyperphosphorylation of most known K8/18 phosphorylation sites and delayed formation of K18 and K19 stable fragments. In contrast, K8 is resistant to proteolysis and remains associated with the K18 fragments. Transfection of phosphorylation/glycosylation-mutant K8 and K18 does not alter fragment formation. The protein domains of the keratin fragments were determined using epitope-defined antibodies, and microsequencing indicated that K18 cleavage occurs at a conserved caspase-specific aspartic acid. The fragments are found preferentially within the detergent-insoluble pool and can be generated, in a phosphorylation-independent manner, by incubating keratins with caspase-3 or with detergent lysates of apoptotic cells but not with lysates of nonapoptotic cells. Our results indicate that type I keratins are targets of apoptosis-activated caspases, which is likely a general feature of keratins in most if not all epithelial cells undergoing apoptosis. Keratin hyperphosphorylation occurs early but does not render the keratins better substrates of the downstream caspases.
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PMID:Apoptosis generates stable fragments of human type I keratins. 940 8

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

Cytototoxic T lymphocyte-induced apoptosis can occur either through the directed exocytosis of granzyme B and perforin or via ligation of Fas. Both pathways involve the activation of a family of cysteine proteinases, the caspases, that cleave substrates at aspartic acid and are themselves activated by cleavage at internal aspartate residues. Fas recruits caspase 8, which initiates the death program through the subsequent activation of caspase 3. Granzyme B can process both caspase 8 and 3 in vitro, suggesting that both Fas and granzyme B access the apoptotic program in the same way. Here we demonstrate that although the two mechanisms are similar, the events that lead to activation of caspase 3 can be distinguished in vivo on the basis of their sensitivities to both pharmacological and virus-encoded caspase inhibitors. In cytotoxic T lymphocytes-mediated death the initial cleavage event on caspase 3 is insensitive to benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (zVAD-fmk) inhibition in both mouse and human systems. During Fas-mediated death, however, activation of caspase 3 is completely inhibited to zVAD-fmk. In addition, the viral serpin SPI-2, a homologue of cytokine response modifier A (crmA), is an effective inhibitor of the Fas but not the granzyme pathway. Our results demonstrate that whereas Fas-mediated activation of caspase 3 requires an upstream caspase activity that is zVAD-fmk-sensitive, the initial cleavage of caspase 3 during granule-mediated cell death is insensitive to zVAD-fmk, suggesting that caspase 3 is cleaved directly by granzyme B in vivo.
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PMID:Cytotoxic T lymphocyte-assisted suicide. Caspase 3 activation is primarily the result of the direct action of granzyme B. 969 85

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

We report here that the Rad51 recombinase is cleaved in mammalian cells during the induction of apoptosis by ionizing radiation (IR) exposure. The results demonstrate that IR induces Rad51 cleavage by a caspase-dependent mechanism. Further support for involvement of caspases is provided by the finding that IR-induced proteolysis of Rad51 is inhibited by Ac-DEVD-CHO. In vitro studies show that Rad51 is cleaved by caspase 3 at a DVLD/N site. Stable expression of a Rad51 mutant in which the aspartic acid residues were mutated to alanines (AVLA/N) confirmed that the DVLD/N site is responsible for the cleavage of Rad51 in IR-induced apoptosis. The functional significance of Rad51 proteolysis is supported by the finding that, unlike intact Rad51, the N- and C-terminal cleavage products fail to exhibit recombinase activity. In cells, overexpression of the Rad51(D-A) mutant had no effect on activation of caspase 3 but did abrogate in part the apoptotic response to IR exposure. We conclude that proteolytic inactivation of Rad51 by a caspase-mediated mechanism contributes to the cell death response induced by DNA damage.
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PMID:Role for caspase-mediated cleavage of Rad51 in induction of apoptosis by DNA damage. 1008 66

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