Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P42574 (caspase-3)
 45,978 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The apoptotic cysteine protease, caspase-3, is expressed in cells as an inactive 32-kDa precursor from which 17 kDa (p17) and 12 kDa (p12) subunits of the mature caspase-3 are proteolytically generated during apoptosis. Two amino acid sequences, ESMD downward arrowS (amino acids 25-29) and IETD downward arrowS (amino acids 172-176), in the precursor have been defined as the cleavage sites for the production of the p17 and p12 subunits. Using a cell-free assay system, we demonstrate that the caspase-3 precursor appears to be cleaved first at the IETD downward arrowS site, producing the p12 subunit and a 20-kDa (p20) peptide. Subsequently, the p20 is cleaved at the ESMD downward arrowS site, generating the mature p17 subunit. The cleavage at the IETD downward arrowS site required a protease activity that was selectively inhibited by the peptide, Ac-IETD-CHO (acetyl-IETD-aldehyde), and other protease inhibitors, such as the cowpox viral serine protease inhibitor, CrmA, and N-alpha-tosyl-L-phenylalanine chloromethyl ketone. The protease that catalyzed the cleavage at the ESMD/S site was selectively inhibited by another peptide, Ac-ESMD-CHO (acetyl-ESMD-aldehyde). More interestingly, the caspase-3 inhibitor, Ac-DEVD-CHO, but not the caspase-1 inhibitor, Ac-YVAD-CHO, also selectively inhibited the protease activity that cleaves at the ESMD downward arrowS site. This indicated that the cleavage at the ESMD downward arrowS site was either autocatalytic or that it required a caspase-3-like activity. In summary, we demonstrate that production of the p17:p12 form of caspase-3 is a sequential two-step process and appears to require two distinct enzymatic activities.
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PMID:A sequential two-step mechanism for the production of the mature p17:p12 form of caspase-3 in vitro. 914 68

The human leukemia cell line, HL60 is very sensitive to various apoptotic stimuli and p53-null. The death-related cysteine proteases of the caspases family play a central role in the execution phase of apoptosis, and we recently reported the importance of serine protease activation in camptothecin-induced apoptotic endonuclease activation in HL60 cells. In the present study, we investigated the role of caspases (ICE/CED-3-related cysteine proteases) and serine proteases in cell death induced by the topoisomerase I inhibitor, camptothecin, in HL60 cells and in a cell-free system. We found that CPP32 is activated during camptothecin-induced apoptosis, and that N-benzyloxycarbony-Val-Ala-Asp (O-methyl) -fluoromethyketone (Z-VAD-fmk), a cell permeable caspase inhibitor blocks all features of apoptosis: morphological changes, cleavage of caspase 3 (CPP32/Yama/Apopain) and poly(ADP-ribose) polymerase, lamin B degradation and DNA fragmentation. However, Z-VAD-fmk and two other ICE/CED-3 inhibitors, YVAD-CHO and DEVD-CHO, were inactive in a cell-free system reconstituted from nuclei of untreated HL60 cells and cytosol from camptothecin-treated cells, suggesting that caspases are not required for endonuclease activation or lamin B cleavage in the cell-free system. By contrast, the serine protease inhibitors, 3,4-dichloroisocoumarin (DCI) and L-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone tosyl-L-phenylalanine chloromethyl ketone (TPCK), abolished the apoptosis-associated biochemical changes induced by camptothecin both in whole cells and in a cell-free system. DCI also inhibited CPP32 cleavage. Taken together, these results suggest that in HL60 cells, both CPP32 and serine proteases are activated in camptothecin-induced apoptosis.
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PMID:Camptothecin-induced apoptosis in p53-null human leukemia HL60 cells and their isolated nuclei: effects of the protease inhibitors Z-VAD-fmk and dichloroisocoumarin suggest an involvement of both caspases and serine proteases. 926 76

Apoptosis induced by numerous cancer chemotherapeutic and other toxic agents has been shown to proceed through a cascade of proteases, now termed caspases, culminating in cleavage of a set of proteins. The ability of photodynamic treatment (PDT) with the phthalocyanine Pc 4 to activate cellular caspases has been assessed during the rapid apoptosis in murine lymphoma L5178Y-R cells. Cells were exposed to combinations of Pc 4 and activating red light that result in > or =90% cell death, as judged by a clonogenic assay. The rate of entry of cells into apoptosis was dose dependent. For 0.5 microM Pc 4 and either 2.1 or 3 kJ/m2, which kill 90 or 99.9% of the cells, oligonucleosomal fragmentation was visible on agarose gels as early as 60 or 30 min after PDT, respectively. To assess caspase activation, cells were harvested at various times after PDT, and cell proteins were subjected to electrophoresis and Western blot analysis, using an antibody to poly(ADP-ribose) polymerase (PARP). The cleavage of the normally Mr 116,000 PARP into fragments of Mr approximately 90,000 was observed at approximately the same time as the earliest DNA fragmentation. An antibody to the polymer, poly(ADP-ribose), did not recognize the Mr approximately 90,000 PARP cleavage products, in contrast to the parent enzyme. This analysis also revealed that levels of a poly(ADP-ribosylated) Mr 100,000 protein, tentatively identified as topoisomerase I, were maintained in cells after PARP was fully cleaved. Caspase-3 (and/or caspase-7) activity, as measured in cell lysates with the fluorogenic substrate DEVD-AMC, was elevated almost immediately after PDT. The cell-permeable, irreversible caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoro-methylketone, inhibited PDT-induced apoptosis and PARP cleavage, whereas the inactive peptide analogue, benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone, was without effect. The results indicate that PDT-induced apoptosis is mediated by activation of caspase-3 and/or other similar caspases.
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PMID:Protease activation and cleavage of poly(ADP-ribose) polymerase: an integral part of apoptosis in response to photodynamic treatment. 950 Apr 54

Calcium is involved in several steps of the apoptotic process. In nuclei, endonucleases are presumed to be the main targets of calcium; however, little is known about its role during the cytosolic phase of apoptosis. We used a cell-free system to address this question. Our results show that CaCl2 triggered nuclear apoptosis (i.e. typical morphological change and DNA fragmentation) at concentrations of 5 mM. This concentration was lowered 10-fold by the co-incubation with cytosolic extracts from nonapoptotic cells. Apoptotic changes induced by the incubation of nuclei with CaCl2 in the presence of these cytosols were strongly reduced in the presence of an inhibitor of caspase-3 and to a lesser extent by an inhibitor of caspase-1. We also show that calcium-induced apoptosis is affected by protease inhibitors such as N-tosyl-L-phenylalanine chloromethyl ketone, but not by calpain or several lysosomal protease inhibitors. The addition of CaCl2 to the cell-free system increased a caspase-3 activity in nonapoptotic cytosols as shown by specific antibodies and an enzymatic assay. No activation of a caspase-3-like activity by the addition of cytochrome c was observed in these extracts under similar conditions. The enhanced caspase-3 activity induced by calcium was inhibited by protease inhibitors affecting morphological nuclear apoptosis except for those responsible for the degradation of lamin A. These results suggest that CaCl2 could trigger, in normal cells, an apoptotic cascade through the activation of cytosolic caspase-3 activity.
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PMID:Induction of a caspase-3-like activity by calcium in normal cytosolic extracts triggers nuclear apoptosis in a cell-free system. 965 49

Brefeldin A (BFA) has recently been shown to induce apoptosis in human tumor cells in a p53-independent fashion. In this study, BFA-induced apoptosis was analyzed in the human Jurkat T-cell line. Apoptosis occurred 8 h after treatment with BFA and at concentrations as low as 10 ng/ml and increased with the duration of BFA exposure. Forskolin, an inhibitor of BFA-induced deaggregation of the Golgi-microtubular complex in some cell lines, failed to reverse BFA-mediated apoptosis. Further study of the mechanism of BFA-induced apoptosis was conducted by using a series of peptide protease inhibitors. Complete inhibition of cell death was achieved with benzyloxycarbonyl-Val-Ala-Asp-fluromethylketone, a peptide inhibitor of the caspase protease family, and Z-Asp-Glu-Val-Asp-FMK, a specific inhibitor of caspase-3. Both Acetyl-Tyr-Val-Ala-Asp-chloromethylketone and Acetyl-Tyr-Val-Ala-Asp-aldehyde, selective caspase-1 (interleukin-1beta converting enzyme) inhibitors, exerted only partial protection of cells from apoptosis at higher concentrations. Z-Phe-Ala-FMK, a cysteine protease inhibitor lacking aspartate at the P1 position, did not have any impact on BFA-induced apoptosis. Furthermore, Jurkat cells transfected with the proto-oncoprotein Bcl-2, which is able to block various apoptotic conditions, showed remarkable resistance to the apoptotic effect of BFA. Thus, the data indicate that BFA-induced apoptosis requires caspase(s) activation, primarily the activation of caspase-3, and is inhibited by overexpression of Bcl-2.
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PMID:Brefeldin A-mediated apoptosis requires the activation of caspases and is inhibited by Bcl-2. 982 1

Most of malignant lymphomas have been shown to be relatively radiosensitive clinically, but some, especially recurrent ones, are frequently highly radioresistant. To clarify the origin of the difference, we examined ionizing radiation (IR)-induced apoptosis in three closely related human lymphoma cell lines (DL-40, DL-95, and DL-110) that differ in p53 status. DL-95 and DL-110 cells have a wild-type p53, whereas DL-40 cells carry a T to G transition in exon 5 of the p53 gene, resulting in a change of Cys to Phe at codon 176. Irradiation of DL-40 cells (mutant-type p53) with 5 Gy gamma-rays resulted in delayed apoptosis with membrane changes (annexin-V expression) 13 h after IR, and caspase-3 activation 23 h after IR, whereas apoptotic response was not identified in DL-95 cells until 48 h after IR in spite of their normal p53 status. Concerning DL-110 cells, delayed and reduced apoptotic response was revealed both microscopically and by DNA fragmentation assay. These results suggested that IR-induced apoptosis in DL-40 cells is mediated by a mechanism involving the caspase-3 cascade of the p53-independent pathway, and that some unknown mechanism inhibited IR-induced apoptosis in existence of wild-type p53, especially for DL-95 cells.
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PMID:Ionizing radiation-induced apoptosis in human lymphoma cell lines differing in p53 status. 1063 91

We have shown that reoxygenation of hypoxic rat kidney proximaltubule cells leads to apoptosis. This is mediated by translocation ofBax from the cytosol to mitochondria, accompanied by release ofmitochondrial cytochrome c (cyt.c). The present studyhas examined the proteolytic mechanisms responsible for apoptosisduring hypoxia-reoxygenation. Caspases were activated duringhypoxia, as shown by cleavage of fluorogenic peptide substrates. By5 h caspase-3-like activity to cleave carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin was increased approx. 30-fold. Thiswas accompanied by specific processing of pro-caspase-3, -8 and -9 intoactive forms. Caspase activation during hypoxia was blocked bycarbobenzoxy-Val-Ala-Asp-fluoromethyl ketone and overexpression of Bcl-2. Of particular interest, caspase activation was also suppressed bythe chymotryptic inhibitors N-tosyl-L-phenylalaninechloromethyl ketone (TPCK) and Ala-Pro-Phe chloromethyl ketone (APF),and the general serine protease inhibitor 4-(2-aminoethyl)benzenesulphonyl fluoride. Inhibition of caspase activationby these compounds resulted in arrest of apoptosis. On the other hand,the serine protease inhibitors did not prevent release of mitochondrialcyt.c during hypoxia, suggesting that these compounds blockeda critical step in post-mitochondrial caspase activation. Furtherstudies using an in vitro reconstitution model showedthat cyt. c/dATP stimulated caspase-9 processing and downstreamcaspase activation were significantly suppressed in the presence ofTPCK and APF. Based on these results, we speculate that serineproteases may be involved in post-mitochondrial apoptotic events thatlead to activation of the initiator, caspase-9.
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PMID:Serine protease inhibitors suppress cytochrome c-mediatedcaspase-9 activation and apoptosis during hypoxia-reoxygenation. 1076 69

Glioblastoma multiforme (GBM) is the most frequent malignant brain tumor in adults and is invariably fatal. We have investigated the effect of cyclo-(Arg-Gly-Asp-D-Phe-Val) (cRGDfV) peptide on survival of human malignant glioma cells in vitro and in vivo. Immunofluorescent analyses revealed the presence of alpha(v)beta3 integrin on U-87MG and U-373MG cells, but minimal expression on U-251MG cells. Treatment of U-87MG and U-373MG cells in vitro with cRGDfV (20 microg/ml), but not the linear peptide, resulted in the appearance of rounded and loosely attached cells with subsequent cell death. By comparison, neither this cyclic peptide nor its linear homolog had any significant effect on growth and morphology of U-251MG cells. The death of cRGDfV-treated (20 microg/ml) glioma cells was blocked by pretreatment (10 microM) of cells with DEVD-FMK and LEHD-FMK, inhibitors of caspase-3 and caspase-9, respectively. Moreover, when glioma cells grown as spheroids were treated with cRGDfV (50 microg/ml), spheroid formation was markedly reduced. Further, treatment of intracranial U-87MG tumors in scid mice with cyclic peptide significantly (p < 0.001) prolonged their survival. These results indicated (i) that cRGDfV induced apoptosis of human glioma cells by binding alpha(v)beta3 integrin expressed on their cell surfaces and (ii) that cRGDfV may be an effective and non-toxic direct anti-tumor therapy for alpha(v)beta3-expressing GBMs.
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PMID:Human malignant glioma therapy using anti-alpha(v)beta3 integrin agents. 1089 66

Huntington disease is a devastating neurodegenerative disease caused by the expansion of a polymorphic glutamine tract in huntingtin. The huntingtin interacting protein (HIP-1) was identified by its altered interaction with mutant huntingtin. However, the function of HIP-1 was not known. In this study, we identify HIP-1 as a proapoptotic protein. Overexpression of HIP-1 resulted in rapid caspase 3-dependent cell death. Bioinformatics analyses identified a novel domain in HIP-1 with homology to death effector domains (DEDs) present in proteins involved in apoptosis. Expression of the HIP-1 DED alone resulted in cell death indistinguishable from HIP-1, indicating that the DED is responsible for HIP-1 toxicity. Furthermore, substitution of a conserved hydrophobic phenylalanine residue within the HIP-1 DED at position 398 eliminated HIP-1 toxicity entirely. HIP-1 activity was found to be independent of the DED-containing caspase 8 but was significantly inhibited by the antiapoptotic protein Bcl-x(L), implicating the intrinsic pathway of apoptosis in HIP-1-induced cell death. Co-expression of a normal huntingtin fragment capable of binding HIP-1 significantly reduced cell death. Our data identify HIP-1 as a novel proapoptotic mediator and suggest that HIP-1 may be a molecular accomplice in the pathogenesis of Huntington disease.
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PMID:Huntingtin interacting protein 1 induces apoptosis via a novel caspase-dependent death effector domain. 1100 1

trans-4-Aminomethylcyclohexanecarbonyl-Tyr(O-Pic)-octylamide (YO-2) inhibited plasmin (PL) selectively, while trans-4-aminomethylcyclohexanecarbonyl-Phe-4-carboxymethylanili de (YO-1) inhibited plasma kallikrein (PK). YO-2 induced apoptosis of M1 (melanoma) cell line and HT29 colon carcinoma cells during 24 h through activation of caspase-3, while YO-1 did not affect either cell line even during 48 h.
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PMID:Development of plasmin and plasma kallikrein selective inhibitors and their effect on M1 (melanoma) and HT29 cell lines. 1101 33


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