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)

The prevention of apoptosis by Zn2+ has generally been attributed to its inhibition of an endonuclease acting in the late phase of apoptosis. In this study we investigated the effect of Zn2+ on an earlier event in the apoptotic process, the proteolysis of the "death substrate" poly(ADP-ribose) polymerase (PARP). Pretreatment of intact Molt4 leukemia cells with micromolar concentrations of Zn2+ caused an inhibition of PARP proteolysis induced by the chemotherapeutic agent etoposide. Using a cell-free system consisting of purified bovine PARP as a substrate and an apoptotic extract or recombinant caspase-3 as the PARP protease, Zn2+ inhibited PARP proteolysis in the low micromolar range. To rule out an effect of Zn2+ on PARP, a protein with two zinc finger domains, we used recombinant caspase-3 and a chromogenic tetrapeptide substrate containing the caspase-3 cleavage site. In this system, Zn2+ inhibited caspase-3 with an IC50 of 0.1 microM. These results identify caspase-3 as a novel target of Zn2+ inhibition in apoptosis and suggest a regulatory role for Zn2+ in modulating the upstream apoptotic machinery.
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PMID:Zinc is a potent inhibitor of the apoptotic protease, caspase-3. A novel target for zinc in the inhibition of apoptosis. 922 15

The observation that the nematode cell death effector gene product Ced-3 is homologous to human interleukin-1beta-converting enzyme (caspase-1) has led to the discovery of at least nine other human caspases, many of which are implicated as mediators of apoptosis. Significant interest has been given to aspects of the cell biology and substrate specificity of this family of proteases; however, quantitative descriptions of their biochemical characteristics have lagged behind. We describe the influence of a number of environmental parameters, including pH, ionic strength, detergent, and specific ion concentrations, on the activity and stability of four caspases involved in death receptor-mediated apoptosis. Based on these observations, we recommend the following buffer as optimal for investigation of their characteristics in vitro: 20 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 100 mM NaCl, 10 mM dithiothreitol, 1 mM EDTA, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid (CHAPS), 10% sucrose, pH 7.2. Caspase activity is not affected by concentrations of Ca2+ below 100 mM, but is abolished by Zn2+ in the submicromolar range, a common characteristic of cysteine proteases. Optimal pH values vary from 6.8 for caspase-8 to 7.4 for caspase-3, and activity of all is relatively stable between 0 and 150 mM NaCl. Consequently, changes in the physiologic pH and ionic strength would not significantly alter the activity of the enzymes, inasmuch as all four caspases are optimally active within the range of these parameters found in the cytosol of living and dying human cells.
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PMID:Biochemical characteristics of caspases-3, -6, -7, and -8. 932 97

Granzyme B (GzmB) is a neutral serine protease found in cytotoxic lymphocytes; this enzyme is critically involved in delivering the rapid apoptotic signal to susceptible target cells. GzmB has been difficult to study and has not yet been produced in non-mammalian systems because of the complex processing events that are thought to be required for its activation. In this report, we have successfully produced fully active, soluble recombinant GzmB (rGzmB) in a yeast-based system by fusing GzmB cDNA in frame with yeast alpha-factor cDNA, using the yeast KEX2 signal peptidase to release the processed enzyme into the supernatant of yeast cultures. We expressed the proenzyme form of GzmB as well and determined that pro-GzmB is efficiently converted to its active form by the cysteine proteinase dipeptidyl peptidase I. The fully processed enzyme was able to hydrolyze the synthetic substrate N-t-butyloxycarbonyl-L-alanyl-L-alanyl-L-aspartyl (Boc-Ala-Ala-Asp) thiobenzyl ester with a kcat of 17 s-1 and catalytic efficiency kcat/Km of 181,237 M-1 S-1; the recombinant enzyme is therefore at least twice as active as purified native GzmB. In addition, the recombinant enzyme hydrolyzes Boc-Ala-Ala-Met thiobenzyl ester with a kcat of 3.2 S-1 and a catalytic efficiency kcat/Km of 65,306 M-1 S-1. Purified rGzmB can also cleave the putative substrate caspase-3 into its signature p20/p10 forms. Unlike caspases, rGzmB is not sensitive to inhibition by several peptide-based inhibitors, including Ac-DEVD-CHO, Ac-YVAD-CMK, and ZIETD-FMK, as well as Zn2+ (a known inhibitor of caspase-3). Structural studies of rGzmB may allow us to better understand the substrate specificity of this enzyme and to design better inhibitors.
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PMID:Production of fully active recombinant murine granzyme B in yeast. 943 Jul 5

Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear enzyme which is responsible for synthesis of poly(ADP-ribose) in response to DNA damage caused by numerous agents and during DNA base excision repair. After DNA damage, the enzyme binds to nicks in DNA through its N-terminal zinc fingers and catalyzes the formation of poly(ADP-ribose) on various nuclear acceptors including itself. When DNA damage is extensive, cells induce their own demise by activating the proteases that induce apoptosis (caspases) which cleave PARP and other death substrates. Here we report the development of a new approach to investigate the sensitivity of mono(ADP-ribosyl)ated and DNA-bound PARP to cleavage during apoptosis. The development of a stoichiometric labeling procedure of the enzyme has allowed us to evaluate the catalytic properties of caspase 3 toward mono(ADP-ribosyl)ated PARP at various enzyme:substrate molar ratios. We show that low levels of automodification (< or = 3 U of ADP-ribose per chain) do not inhibit the proteolysis of the substrate. In addition, we demonstrate that binding of unmodified PARP to DNA influences the kinetics of its cleavage by caspase 3.
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PMID:Proteolysis of poly(ADP-ribose) polymerase by caspase 3: kinetics of cleavage of mono(ADP-ribosyl)ated and DNA-bound substrates. 965 May 95

Geranylgeraniol (GGO) at 50 microM induces apoptosis in HL-60 cells. We examined the effects of Zn2+ ions on this process. Treatment of HL-60 cells with Zn2+ ions inhibited subsequent GGO-induced fragmentation of DNA. In a cell-free system that consisted of a specific substrate for caspase-3 and a lysate of HL-60 cells that had been treated with 50 microM GGO, Zn2+ ions at concentrations above 0.1 mM inhibited the activity of caspase-3. The effect of Zn2+ ions on the processing of caspase-3 during GGO-induced apoptosis was investigated by Western blotting, which revealed that an inactive 32-kDa precursor of caspase-3 was cleaved, in response to GGO, to yield an activated 17-kDa enzyme. Treatment of HL-60 cells with Zn2+ ions inhibited the cleavage of the precursor by a protease that was induced by treatment with GGO, and inhibition of this processing was well correlated with the inhibition by Zn2+ ions of caspase-3 activity in the cell-free system. In cell-extracted cytosols, Zn2+ ions inhibited the cleavage of the 32-kDa precursor by caspase-9 (Aapf-3) that was activated by addition of cytochrome c and dATP. These results indicate that inhibition of GGO-induced apoptosis in HL-60 cells by Zn2+ ions might be due to inhibition by Zn2+ ions of the processing of a precursor to caspase-3.
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PMID:Zinc ions prevent processing of caspase-3 during apoptosis induced by geranylgeraniol in HL-60 cells. 968 18

During apoptosis, changes to the nucleus of the dying cell include DNA degradation and structural collapse. These changes are accomplished by caspase-mediated cleavage of DNA-fragmenting factor DFF45, an inhibitor of the effector molecule DFF40. DFF45 and, more efficiently, a mutant lacking one caspase-cleavage site (DFF45m) inhibited nuclear changes in a cell-free system when apoptosis was initiated by adding caspase-3 to cell extracts. In primary tissues from several mammalian species, human caspase-3 activated and human DFF45m blocked nuclear apoptosis demonstrating evolutionary conservation of this step. However, DFF45m did not significantly inhibit DNA-fragmenting activity in extracts from staurosporine-treated cells from the human cell line Jurkat. In extracts from normal Jurkat cells, DFF45m blocked caspase-triggered DNA cleavage efficiently only if added within a short time of the addition of the caspase. At later time points, this inhibition by DFF45m was strongly reduced in efficiency while Zn2+ still completely blocked DNA fragmentation. These results demonstrate the evolutionary conservation of a linear pathway in apoptosis and suggest the existence of more complex events as final effector machinery.
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PMID:Extent and limitation of the control of nuclear apoptosis by DNA-fragmenting factor. 992 Jul 77

Apoptosis is mediated by members of the caspase family of proteases which can be activated by release of mitochondrial cytochrome c. Additional members of the caspase family are activated at the cell surface in response to direct stimulus from the external environment such as by activation of the Fas receptor. It has been suggested that these upstream caspases directly activate the downstream caspases which would obviate a role for cytochrome c in apoptosis induced by the Fas receptor. We demonstrate that cytochrome c is released from mitochondria of Jurkat cells in response to both staurosporine and an agonistic anti-Fas antibody and that only the latter is inhibited by the caspase inhibitor z-VAD-FMK. This suggests that an upstream caspase such as caspase-8 is required for the Fas-mediated release of mitochondrial cytochrome c. The protein phosphatase inhibitor calyculin A prevented cytochrome c release and apoptosis induced by both agents, suggesting that release of cytochrome c is required in both models. Zinc, once thought of as an endonuclease inhibitor, has previously been shown to prevent the activation of caspase-3. We show that zinc prevents the activation of downstream caspases and apoptosis induced by both insults, yet does not prevent release of mitochondrial cytochrome c. The ability of calyculin A and zinc to prevent DNA digestion implies that the mitochondrial pathway is important for induction of apoptosis by both agents. These results do not support an alternative pathway in which caspase-8 directly activates caspase-3. These results also demonstrate that a critical protein phosphatase regulates the release of cytochrome c and apoptosis induced by both insults.
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PMID:The temporal relationship between protein phosphatase, mitochondrial cytochrome c release, and caspase activation in apoptosis. 1006 78

The MDM2 oncoprotein encodes a 90 kDa nuclear phosphoprotein capable of abrogating the growth suppressive functions of p53 and pRb tumor suppressor proteins by direct interaction. Alternative splicing of MDM2 protein coding sequences has been documented during tumor progression in human ovarian and bladder carcinomas. The aim of this study was to determine whether alternative splicing of MDM2 occurs during breast tumorigenesis in mice and humans and whether protein coding sequences were affected. Specimens representing normal and malignant breast tissues from the murine D2 mammary tumor model system and human breast carcinomas were examined. Three distinct mdm2 mRNA transcripts of 3.3, 1.6 and 1.5 kb were detected in normal and malignant murine mammary tissues by Northern blot analysis using a full-length mdm2 cDNA probe. Additional Northern blot analysis using a probe derived from exon 12 of murine mdm2 demonstrated that the 1.5 and 1.6 kb transcripts lack sequences encoding the C-terminus of the protein. No evidence of internal deletions of protein coding sequences of mdm2 was detected in any of the normal mammary tissues or D2 murine mammary tumors examined by reverse transcription PCR (RT-PCR). Three distinct MDM2 transcripts of 6.7, 4.7 and 1.9 kb were detected in malignant human breast tissue by Northern blot analysis using a cDNA probe specific for the complete open reading frame of human MDM2. However, a cDNA probe specific for the last exon of human MDM2 hybridized only to the 6.7 and 4.7 kb transcripts, demonstrating that the 1.9 kb transcript lacked protein coding sequences contained in exon 12. Similarly, no internal deletions were detected in a panel of malignant human breast tissues using RT-PCR and analogous primers within human MDM2. Therefore, breast tumors differ from other solid tumors reported previously in that no internal deletions of MDM2 protein coding sequences were observed. However, the data document the presence of multiple MDM2 mRNA transcripts in both normal and malignant breast tissues. A subset of MDM2 transcripts were shown to lack the last exon which contains sequences coding for the RING and zinc fingers and domains which are targets for caspase-3 mediated proteolytic degradation and are required to target p53 for proteosomal degradation.
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PMID:Expression of MDM2 during mammary tumorigenesis. 1018 33

While investigating endonucleases potentially involved in apoptosis, an antisera was raised to bovine deoxyribonuclease II, but it recognized a smaller protein of 26 kDa protein in a variety of cell lines. The 26 kDa protein underwent proteolytic cleavage to 22 kDa concomitantly with DNA digestion in cells induced to undergo apoptosis. Sequencing of the 26 kDa protein identified it as the Rho GDP-dissociation inhibitor D4-GDI. Zinc, okadaic acid, calyculin A, cantharidin, and the caspase inhibitor z-VAD-fmk, all prevented the cleavage of D4-GDI, DNA digestion, and apoptosis. The 26 kDa protein resided in the cytoplasm of undamaged cells, whereas following cleavage, the 22 kDa form translocated to the nucleus. Human D4-GDI, and D4-GDI mutated at the caspase 1 or caspase 3 sites, were expressed in Chinese hamster ovary cells which show no detectable endogenous D4-GDI. Mutation at the caspase 3 site prevented D4-GDI cleavage but did not inhibit apoptosis induced by staurosporine. The cleavage of D4-GDI could lead to activation of Jun N-terminal kinase which has been implicated as an upstream regulator of apoptosis in some systems. However, the results show that the cleavage of D4-GDI and translocation to the nucleus do not impact on the demise of the cell.
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PMID:Cleavage and nuclear translocation of the caspase 3 substrate Rho GDP-dissociation inhibitor, D4-GDI, during apoptosis. 1038 42

The antitumor effect of immuno- and chemotherapeutic agents is executed through stimulation of apoptotic programs in susceptible cells. Apoptosis induced in tumor cells requires activation of members of the caspase family of proteases. Deficient expression or activation of caspases may account in part for the failure of many current anticancer therapies. However, recent studies suggest that cell death can proceed in the absence of caspases. We investigated the susceptibility of human renal cell carcinoma (RCC) lines to two distinct modes of cell death, apoptosis and necrosis. RCC lines displayed almost complete resistance to apoptosis in response to the intracellular zinc chelator, N,N,N'N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), which instead induced dramatic accumulation of nonapoptotic necrotic cells. Conversely, TPEN was a potent inducer of apoptosis in caspase-competent normal kidney cells (NK-72) and Jurkat T lymphocytes. Resistance to apoptosis in RCC lines correlated with almost complete loss of caspase-3 expression and variable down-regulation of caspase-7, caspase-8, and caspase-10. These data may explain the resistance of RCC to drugs inducing apoptosis and have important consequences for further attempts to manipulate tumor cell death.
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PMID:Dead or dying: necrosis versus apoptosis in caspase-deficient human renal cell carcinoma. 1038 43

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