Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.2.30 (PARP)
13,611 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cysteine proteases of the CED-3 and ICE family have been recently proposed as the ultimate executioners in several mammalian cell death pathways. Among them, the cysteine protease CPP32 has been shown to participate in programmed cell death (PCD), or apoptosis, affecting lymphoid cells in vitro. In the thymus, negative selection is a mechanism through which developing thymocytes expressing a TcR with high affinity for self peptide-MHC complexes are eliminated by PCD. In order to investigate the role of CPP32 in thymic apoptosis, isolated thymocytes were submitted to cell surface CD3 crosslinking by immobilized anti-CD3 mAb or to dexamethasone treatment. Although apoptosis occurred in the absence or after crosslinking with anti-CD3 mAb, specific activation of CPP32, as assessed by the extent of proteolytic cleavage of the p32 zymogen, was only detected in thymocytes cultured in the presence of the immobilized antibody or dexamethasone. This activation was a very early event during apoptosis as it occurred before the exposure of phosphatidyl serine to the upper side of the cell membrane. This was observed both in anti-CD3- and dexamethasone-induced apoptosis. Moreover, using mice transgenic for pigeon cytochrome C (PCC)-specific TcR, we were able to show that, after injection of PCC, the activation of CPP32 and cleavage of its substrate occurred in thymocytes obtained from mice expressing a permissive MHC haplotype for PCC presentation (H-2k). Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD. While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F. Taken together, these results support the concept that CPP32 is among the earliest effectors of the pathway leading to negative selection of autoreactive thymocytes. Our results also suggest the involvement of a distinct CPP32-like cysteine protease in spontaneous apoptosis of thymocytes.
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PMID:Specific activation of the cysteine protease CPP32 during the negative selection of T cells in the thymus. 934 8

Caspase-3 is an ICE-like protease activated during apoptosis induced by different stimuli. Poly(ADP-ribose) polymerase (PARP), the first characterized substrate of caspase-3, shares a region of homology with the large subunit of Replication Factor C (RF-C), a five-subunit complex that is part of the processive eukaryotic DNA polymerase holoenzymes. Caspase-3 cleaves PARP at a DEVD-G motif present in the 140 kDa subunit of RF-C (RFC140) and evolutionarily conserved. We show that cleavage of RFC140 during Fas-mediated apoptosis in Jurkat cells and lymphocytes results in generation of multiple fragments. Cleavage is inhibited by the caspase-3-like protease inhibitor Ac-DEVD-CHO but not the caspase-1/ICE-type protease inhibitor Ac-YVAD-CHO. In addition, recombinant caspase-3 cleaves RFC140 in vitro at least at three different sites in the C-terminal half of the protein. Using amino-terminal microsequencing of radioactive fragments, we identified three sites: DEVD723G, DLVD922S and IETD1117A. We did not detect cleavage of small subunits of RF-C of 36, 37, 38 and 40 kDa by recombinant caspase-3 or by apoptotic Jurkat cell lysates. Cleavage of RFC140 during apoptosis inactivates its function in DNA replication and generates truncated forms that further inhibit DNA replication. These results identify RFC140 as a critical target for caspase-3-like proteases and suggest that caspases could mediate cell cycle arrest.
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PMID:The large subunit of replication factor C is a substrate for caspase-3 in vitro and is cleaved by a caspase-3-like protease during Fas-mediated apoptosis. 935 17

An in vitro system has been employed to study the apoptotic mechanisms in the AK-5 tumor which is a spontaneously regressing rat histiocytoma. Cytosolic extracts of tumor cells primed for apoptosis using dexamethasone and immune serum from tumor-regressing animals were able to induce apoptosis in intact nuclei and reproduce the classical morphological and biochemical features typical of apoptotic cells. The cleavage of lamin A and PARP to signature fragments by these extracts and the inhibition of the same using peptide inhibitors signify the pivotal role of ICE and ICE-related proteases in apoptosis. Lamin A cleavage was insensitive to YVAD but PARP cleavage was blocked by both YVAD and DEVD. Cell extracts derived from cells overexpressing the Bcl-2 gene and Nedd-2 antisense gene, respectively, failed to induce apoptosis in exogenously added nuclei, suggesting that Bcl-2 gene product is downregulating a key event in apoptotic cascade. The study also demonstrates the coherent action of different ICE-related proteases in apoptosis and their functional redundancy. This system may prove useful for analyzing complex molecular mechanisms underlying apoptosis in tumor cells.
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PMID:Caspase-mediated apoptosis in AK-5 tumor cells: a cell-free study using peptide inhibitors and antisense strategy. 936 20

The molecular mechanisms for sensitivity and resistance of tumor cells towards chemotherapy are only partially understood. In chemosensitive leukemias and solid tumors, anticancer drugs have been shown to induce apoptosis. We previously identified activation of the CD95 (APO-1/Fas) receptor/CD95 ligand (CD95/CD95-L) system as a key mechanism for drug-induced apoptosis. Here, we show that therapeutic concentrations of doxorubicin, methotrexate and cytarabine also induce apoptosis via activation of the CD95 system in primary leukemia cells in vivo. CD95-resistant and doxorubicin-resistant leukemia and neuroblastoma cells display cross-resistance for induction of cell death. Down-regulation of CD95 expression was found in drug-resistant and CD95-resistant cell lines. Furthermore, up-regulation of CD95-L, previously shown to mediate drug-induced apoptosis in a variety of tumor cells, was completely blocked in doxorubicin-resistant cells. The prototype caspase (ICE/Ced-3 protease) substrate, poly(ADP-ribose)polymerase (PARP), was cleaved in sensitive, but not in resistant tumor cells following CD95 triggering or drug treatment. Since failure to activate CD95-L was not due to decreased drug uptake or increased drug efflux, non-multi-drug resistance (non-MDR) mechanisms are involved in this type of resistance. These findings suggested that an intact CD95 system plays a key role in determining sensitivity or resistance towards anticancer therapy.
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PMID:Deficient activation of the CD95 (APO-1/Fas) system in drug-resistant cells. 936 15

Children with congenital homozygous deficiency of purine nucleoside phosphorylase (PNP) have abnormalities in purine metabolism that result in T-cell selective immune deficiency. The mechanism of action for cell death has been attributed to intracellular accumulation of dGTP, a potent inhibitor of ribonucleotide reductase and subsequently DNA synthesis, in thymocytes and T-cells but not B-cells. However, the mode of cell death has not been determined to be either necrosis or apoptosis. To examine the involvement of apoptosis in T-cells following PNP inhibition, MOLT-4 cells, a human T cell leukemia cell line, were co-treated with the PNP inhibitor, CI-1000 (2-amino 3,5-dihydro-7-(3-thienylmethyl)-4H-pyrrolo[3,2-d]-pyrimidin-4-one HCl), and 2'-deoxyguanosine (dGuo) which resulted in a concentration-dependent loss of cell viability (trypan blue) and inhibition of tritiated thymidine ([3H]-TdR) uptake. Staining of cells with the DNA dye Hoechst 33,258 showed nuclear morphology characteristic of apoptosis. Western blots (24 h lysates) were probed with antibodies against several proteins implicated in apoptosis. Anti-PARP revealed the presence of an 85 kD PARP breakdown product while, anti-alpha-spectrin revealed the accumulation a 120 kD breakdown product, both suggestive of CPP32 cleavage (caspase-3; an ICE-like cysteine protease). Western blots also detected the loss of the intact 32 kD caspase-3 isoform, a biochemical event associated with caspase-3 activation. Corresponding fluorometric activity assays detected a marked increase in caspase-3-like activity using the substrate Ac-DEVD-MCA. Lastly, a pan caspase inhibitor (Z-D-DCB) and 2'-deoxycytidine (dCyd), which is known to prevent dGTP accumulation following PNP inhibition, were able to prevent cell death and all indicators of caspase-3-like activity in MOLT-4 cells co-treated with dGuo and CI-1000. In summary, we provided several lines of evidence for the role of apoptosis and the contribution of caspase-3-like proteases in T-cell death following PNP inhibition.
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PMID:A purine nucleoside phosphorylase (PNP) inhibitor induces apoptosis via caspase-3-like protease activity in MOLT-4 T cells. 940 42

Treatment of U-937 promonocytic cells with the DNA topoisomerase II inhibitor etoposide rapidly caused death by apoptosis, as determined by changes in chromatin structure, production of DNA breaks, nucleosome-sized DNA degradation, decrease in mitochondrial membrane potential and phosphatidyl serine translocation in the plasma membrane, and at the same time induced intracellular acidification. Both the execution of the apoptotic process and the intracellular acidification were reduced by the addition of forskolin plus theophylline or other cAMP increasing agents. These agents also attenuated the induction of apoptosis by camptothecin, heat-shock, cadmium chloride and X-radiation. Although etoposide slightly increased the production of reactive oxygen intermediates, this increase was not prevented by forskolin plus theophylline, and the addition of antioxidant agents failed to inhibit apoptosis. Etoposide caused a great increase in NF-(kappa)B binding activity, which was not prevented by forskolin plus theophylline, while AP-1 binding was little affected by the topoisomerase inhibitor. The treatments did not significantly alter the levels of Bcl-2 and Bax. By contrast, the expression of c-myc, which was very high in untreated U-937 cells and only partially inhibited by etoposide, was rapidly and almost totally abolished by the cAMP increasing agents. Finally, it was observed that etoposide caused a transient dephosphorylation of retinoblastoma (Rb), which was associated with cleavage of poly(ADP-ribose) polymerase (PARP). Both Rb dephosphorylation and PARP cleavage were inhibited by forskolin plus theophylline. The inhibition of Rb (type I) phosphatase and ICE/CED-3-like protease activities, and the abrogation of c-myc expression, are mechanisms which could explain the anti-apoptotic action of cAMP increasing agents in myeloid cells.
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PMID:cAMP increasing agents attenuate the generation of apoptosis by etoposide in promonocytic leukemia cells. 945 37

c-Myc is a transcriptional activator implicated in the control of cell proliferation, differentiation and transformation, but is also involved in the regulation of programmed cell death, apoptosis. Despite intensive research, the molecular mechanisms by which c-Myc triggers and executes cell death remain still elusive. Here, we made use of Rat 1A MycER cells expressing a conditionally active c-Myc protein and tested first the hypothesis that ornithine decarboxylase (ODC), which is a transcriptional target of c-Myc, were a mediator of c-Myc-induced apoptosis. However, our results show that the activity of ODC is not required for the c-Myc-mediated apoptosis to occur in these cells. We also found that the expression of p53, p21waf1/cip1, Bcl-2, Bax, Bcl-xL, Bad and cyclins D1, E, A and B did not show any significant changes following c-Myc induction. But, our studies revealed that the c-Myc induced apoptosis is associated with a specific cleavage of poly(ADPribose) polymerase (PARP), suggesting that a cysteine protease of the ICE/CED-3 family is involved. Moreover, we found that the cysteine protease CPP32/Caspase-3, which is known to cleave PARP, is processed from its inactive form to an active protease composed of 17 and 12 kDa subunits; whilst Ich-1/Caspase-2 belonging to another subset of this protease family was not processed/ activated following c-Myc activation. The activation of CPP32 and apoptotic cell death were inhibited by addition of Z-VAD-fmk, a universal inhibitor of ICE-like proteases. Further, a selective inhibitor of CPP32-like proteases (Z-DEVD-fmk) partly inhibited apoptosis. These results provide evidence that the ICE/CED3-family proteases, CPP32 and likely others, play a critical role in the execution of a nuclear proto-oncogene, c-Myc-induced apoptosis.
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PMID:Involvement of CPP32/Caspase-3 in c-Myc-induced apoptosis. 946 64

Intracellular cysteine proteases are important mediators of apoptosis. Indeed, some nuclear proteins and enzymes are cleaved during apoptosis, in particular poly(ADP-ribose) polymerase (PARP), which is activated by DNA strand interruptions and is involved in DNA repair. PARP is cleaved into two fragments of 29 and 85 kDa (apparent molecular mass) in human promyelomonocytic leukemia cells, HL-60, treated with etoposide to induce apoptosis. These cells possess protease activities, caspases, that share many features with the ICE/CED-3 family. The cleavage occurs between Asp-214 and Gly-215, a site that is conserved in human, bovine, and chicken PARP. This cleavage has been shown to be an early marker of apoptosis. To monitor apoptosis, to understand the role of PARP cleavage by caspases, and to study the role of the two fragments in DNA repair, members of our laboratory have developed two polyclonal antipeptide antibodies directed against the two human PARP sequences: [196-214] for LP96-22 and [215-228] for LP96-24. Moreover, these antibodies will be useful to map the necrotic cleavage of PARP, which generates fragments different from those obtained during apoptosis, and thus to discriminate between apoptotic and necrotic cell death.
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PMID:Characterization of antibodies specific for the caspase cleavage site on poly(ADP-ribose) polymerase: specific detection of apoptotic fragments and mapping of the necrotic fragments of poly(ADP-ribose) polymerase. 949 68

The cowpox virus (CPV) CrmA and the equivalent rabbitpox virus (RPV) SPI-2 proteins have anti-inflammatory and antiapoptosis activity by virtue of their ability to inhibit caspases, including the interleukin-1beta-converting enzyme (ICE; caspase-1). Infection of LLC-PK1 pig kidney cells with a CPV CrmA mutant, but not with wild-type (wt) CPV, results in the induction of many of the morphological features of apoptosis (C. A. Ray and D. J. Pickup, Virology 217:384-391, 1996). In our study, LLC-PK1 cells infected with CPV delta crmA, but not those infected with wt CPV, showed induction of poly(ADP-ribose) polymerase (PARP)- and lamin A-cleaving activities and processing of the CPP32 (caspase-3) precursor to a mature 18-kDa form. Surprisingly, infection of LLC-PK1 cells with either wt RPV (despite the presence of the SPI-2 protein) or RPV delta SPI-2 resulted in cleavage activity against PARP and lamin A and the appearance of the mature subunit of CPP32/caspase-3. The biotinylated specific peptide inhibitor Ac-Tyr-Val-Lys(biotinyl)-Asp-2,6-dimethylbenzoyloxymethylketone [AcYV(bio)KD-aomk] labeled active caspase subunits of 18, 19, and 21 kDa in extracts from LLC-PK1 cells infected with CPV delta crmA, wt RPV, or RPV delta SPI-2 but not wt CPV. Mixed infection of LLC-PK1 cells with wt RPV and wt CPV gave no PARP-cleaving activity, and all PARP cleavage mediated by SPI-2 and CrmA mutants of RPV and CPV, respectively, could be eliminated by coinfection with wt CPV. These results suggest that the RPV SPI-2 and CPV CrmA proteins are not functionally equivalent and that CrmA, but not SPI-2 protein, can completely prevent apoptosis in LLC-PK1 cells under these conditions.
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PMID:Activation of caspases in pig kidney cells infected with wild-type and CrmA/SPI-2 mutants of cowpox and rabbitpox viruses. 955 31

We have presently determined the effect of inhibition of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) on the occurrence of apoptosis in insulin-producing cells. The ADP-ribosylation activities of intact cells were decreased by incubation of RINm5F cells for 16 h with the PARP inhibitors nicotinamide (NA) (20-50 mM) or 3-aminobenzamide (3-ABA) (10 mM). Exposure to 20-50 mM NA or 10 mM 3-ABA both resulted in massive apoptosis in RINm5F cells. A 24 h exposure to 50 mM nicotinamide induced apoptosis in fetal but not adult rat islet cells. In addition, exposure of RINm5F cells to 50 mM NA for 12-24 h induced the appearance of the 85 kDa proteolytic PARP fragment, indicating activation of the ICE-like protease caspase-3. Incubation with 20-50 mM NA did not induce any consistent effects upon transcription factor NF-kappaB activity, demonstrating that this pathway is not involved in induction of apoptosis by NA. It is concluded that in insulin-producing cells with a high mitotic rate, inhibition of ADP-ribosylation--and consequently of auto-modification and release of PARP bound to DNA strand breaks--leads to activation of programmed cell death.
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PMID:Nicotinamide-induced apoptosis in insulin producing cells is associated with cleavage of poly(ADP-ribose) polymerase. 970 78


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