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)

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

The anti-apoptotic molecule Bcl-2 is located in the mitochondrial and endoplasmic reticulum membranes as well as the nuclear envelope. Although its location has not been as rigorously defined, the pro-apoptotic molecule Bax appears to be mainly a cytosolic protein which translocates to the mitochondria upon induction of apoptosis. Here we identify a protease activity in mitochondria-enriched membrane fractions from HL-60 cells capable of cleaving Bax which is absent from the cytosolic fraction. Bax protease activity is blocked in vitro by cysteine protease inhibitors including E-64 which distinguishes it from all known caspases and granzyme B, both of which are involved in apoptosis. Protease activity is also blocked by inhibitors against the calcium-activated neutral cysteine endopeptidase calpain. Partial purification of the Bax protease activity from HL-60 cell membrane fractions by column chromatography revealed that a calpain-like activity was the protease responsible for Bax cleavage. In addition, purified calpain enzymes cleaved Bax in a calcium-dependent manner. Pretreatment of HL-60 cells with the specific calpain inhibitor calpeptin effectively blocked both drug-induced Bax cleavage and calpain activation, but not PARP cleavage or cell death. These results suggest that calpains and caspases are activated during drug-induced apoptosis and that calpains, along with caspases, may be involved in modulating cell death by acting selectively on cellular substrates.
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PMID:Bax cleavage is mediated by calpain during drug-induced apoptosis. 976 17

Apoptosis is a mechanism of cell death that occurs in normal development and on the regulation of vertebrate tissues and organ cellularity. Neurons undergo p53-dependent and p53-independent apoptosis, depending upon the stimulus that triggers DNA fragmentation. Many neurons in the developing nervous system suffer apoptosis, with the cyclin D1 being an essential mediator of neuronal cell death. Other characteristics of apoptosis are: condensation of the nucleus, fragmentation of chromatin at nucleosome linkage sites, membrane blebbing, and the formation of apoptotic bodies. Among the possible molecular mechanisms are: (a) activation of proteases, as ICE (Il-1 beta converting enzyme); (b) calpain is activated in several cells, with PARP (Poly-ADP-ribose polymerase) and a small U1 Ribonucleoprotein, being substrates for ICE and its homologs such as ICH and others proteins. The p53 gene encodes a transcription factor that contributes to several different cellular activities, including apoptosis, the cellular response to radiation, and the activation of proteins such as GADD, Bcl-2 (represses to apoptosis) and Bax. P53 exerts a role as inductor of apoptosis by transactivating expression of the Bax gene. The p53 gene tumor suppressor limits cellular proliferation by including either the arrest of cell cycle in G1, or apoptosis, depending on the cellular context. The p21 is an inhibitor of cyclin-dependent kinase, which is transactivated by p53. During apoptosis, there is an activation of both, c-myc, and the transcription factor NF-kB, which is a important regulator of apoptosis. As an example of signalization of apoptosis we have selected to illustrate the problem related to the system Fas/APO in thymocytes.
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PMID:[Molecular bases of the programmed cell death process: implications of tumor suppressor protein p53 and other proteins in the control of cell cycle. Mechanisms of apoptotic action. Review]. 992 5

We have previously demonstrated that calpain is responsible for the cleavage of Bax, a proapoptotic protein, during drug-induced apoptosis of HL-60 cells (Wood, D. E., Thomas, A., Devi, L. A., Berman, Y., Beavis, R. C., Reed, J. C., and Newcomb, E. W. (1998) Oncogene 17, 1069-1078). Here we show the sequential activation of caspases and calpain during drug-induced apoptosis of HL-60 cells. Time course experiments using the topoisomerase I inhibitor 9-amino-20(S)-camptothecin revealed that cleavage of caspase-3 substrates poly(ADP-ribose) polymerase (PARP) and the retinoblastoma protein as well as DNA fragmentation occurred several hours before calpain activation and Bax cleavage. Pretreatment with the calpain inhibitor calpeptin blocked calpain activation and Bax cleavage but did not inhibit PARP cleavage, DNA fragmentation, or 9-amino-20(S)-camptothecin-induced morphological changes and cell death. Pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) inhibited PARP cleavage, DNA fragmentation, calpain activation, and Bax cleavage and increased cell survival by 40%. Interestingly, Z-VAD-fmk-treated cells died in a caspase- and calpain-independent manner that appeared morphologically distinct from apoptosis. Our results suggest that excessive or uncontrolled calpain activity may play a role downstream of and distinct from caspases in the degradation phase of apoptosis.
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PMID:Caspase-dependent activation of calpain during drug-induced apoptosis. 1007 37

Ionizing radiation activates not only signalling pathways in the nucleus as a result of DNA damage, but also signalling pathways initiated at the level of the plasma membrane. Proteins involved in DNA damage recognition include poly(ADP ribose) polymerase (PARP), DNA-dependent protein kinase, p53 and ataxia- telangiectasia mutated (ATM). Many of these proteins are inactivated by caspases during the execution phase of apoptosis. Signalling pathways outside the nucleus involve tyrosine kinases such as stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), protein kinase C, ceramide and reactive oxygen species. Recent evidence shows that tumour cells resistant to ionizing radiation-induced apoptosis have defective ceramide signalling. How these signalling pathways converge to activate the caspases is presently unknown, although in some cell types a role for calpain has been suggested.
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PMID:Molecular mechanisms of ionizing radiation-induced apoptosis. 1036 Dec 59

We demonstrate here that both procaspase-3 (32 kDa) and PARP are calpain substrates. In calcium-channel opener maitotoxin-treated cells, a 30 kDa caspase-3 fragment is produced in a time and concentration-dependent manner. Formation of this fragment is prevented by calpain inhibitors but not by the pancaspase inhibitor, carbobenzoxy-Asp-CH(2)OC(O)-2,6-dichlorobenzene (Z-D-DCB) nor the selective proteasome inhibitor lactacystin. In maitotoxin-treated cells, PARP (113 kDa) is also cleaved into a 40 kDa immunoreactive fragment, in a calpain-inhibitor-sensitive manner. Both procaspase-3 and PARP are also cleaved in vitro by purified micro-calpain to a 30 kDa fragment and a 40 kDa fragment, respectively. Finally, we show that staurosporine-mediated caspase-3 activation is interrupted by maitotoxin pretreatment.
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PMID:Procaspase-3 and poly(ADP)ribose polymerase (PARP) are calpain substrates. 1048 59

A short period of ischemia and reperfusion, called ischemic preconditioning, protects various tissues against subsequent sustained ischemic insults. We previously showed that apoptosis of hepatocytes and sinusoidal endothelial cells is a critical mechanism of injury in the ischemic liver. Because caspases, calpains, and Bcl-2 have a pivotal role in the regulation of apoptosis, we hypothesized that ischemic preconditioning protects by inhibition of apoptosis through down-regulation of caspase and calpain activities and up-regulation of Bcl-2. A preconditioning period of 10 minutes of ischemia followed by 15 minutes of reperfusion maximally protected livers subjected to prolonged ischemia. After reperfusion, serum aspartate transaminase (AST) levels were reduced up to 3-fold in preconditioned animals. All animals subjected to 75 minutes of ischemia died, whereas all those who received ischemic preconditioning survived. Apoptosis of hepatocytes and sinusoidal endothelial cells, assessed by in situ TUNEL assay and DNA fragmentation by gel electrophoresis, was dramatically reduced with preconditioning. Caspase activity, measured by poly (adenosine diphosphate ribose) polymerase (PARP) proteolysis and a specific caspase-3 fluorometric assay, was inhibited by ischemic preconditioning. The antiapoptotic mechanism did not involve calpain-like activity or Bcl-2 expression because levels were similar in control and preconditioned livers. In conclusion, ischemic preconditioning confers dramatic protection against prolonged ischemia via inhibition of apoptosis through down-regulation of caspase 3 activity, independent of calpain-like activity or Bcl-2 expression.
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PMID:Ischemic preconditioning protects the mouse liver by inhibition of apoptosis through a caspase-dependent pathway. 1053 44

beta-Lapachone (beta-lap) effectively killed MCF-7 and T47D cell lines via apoptosis in a cell-cycle-independent manner. However, the mechanism by which this compound activated downstream proteolytic execution processes were studied. At low concentrations, beta-lap activated the caspase-mediated pathway, similar to the topoisomerase I poison, topotecan; apoptotic reactions caused by both agents at these doses were inhibited by zVAD-fmk. However at higher doses of beta-lap, a novel non-caspase-mediated "atypical" cleavage of PARP (i.e., an approximately 60-kDa cleavage fragment) was observed. Atypical PARP cleavage directly correlated with apoptosis in MCF-7 cells and was inhibited by the global cysteine protease inhibitors iodoacetamide and N-ethylmaleimide. This cleavage was insensitive to inhibitors of caspases, granzyme B, cathepsins B and L, trypsin, and chymotrypsin-like proteases. The protease responsible appears to be calcium-dependent and the concomitant cleavage of PARP and p53 was consistent with a beta-lap-mediated activation of calpain. beta-Lap exposure also stimulated the cleavage of lamin B, a putative caspase 6 substrate. Reexpression of procaspase-3 into caspase-3-null MCF-7 cells did not affect this atypical PARP proteolytic pathway. These findings demonstrate that beta-lap kills cells through the cell-cycle-independent activation of a noncaspase proteolytic pathway.
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PMID:Activation of a cysteine protease in MCF-7 and T47D breast cancer cells during beta-lapachone-mediated apoptosis. 1069 31

Inhibitors of proteases are currently emerging as a potential anti-cancer modality. Nonselective protease inhibitors are cytotoxic to leukemia and cancer cell lines and we found that this cytotoxicity is correlated with their potency as inhibitors of the proteasome but not as inhibitors of calpain and cathepsin. Highly selective inhibitors of the proteasome were more cytotoxic and fast-acting than less selective inhibitors (PS341>>ALLN>>ALLM). Induction of wt p53 correlated with inhibition of the proteasome and antiproliferative effect in MCF7, a breast cancer cell line, which was resistant to apoptosis caused by proteasome inhibitors. In contrast, inhibitors of the proteasome induced apoptosis in four leukemia cell lines lacking wt p53. The order of sensitivity of leukemia cells was: Jurkat>HL60> or =U937>>K562. The highly selective proteasome inhibitor PS-341 induced cell death with an IC50 as low as 5 nM in apoptosis-prone leukemia cells. Cell death was preceded by p21WAF1/CIP1 accumulation, an alternative marker of proteasome inhibition, and by cleavage of PARP and Rb proteins and nuclear fragmentation. Inhibition of caspases abrogated PARP cleavage and nuclear fragmentation and delayed, but did not completely prevent cell death caused by PS-341. Reintroduction of wt p53 into p53-null PC3 prostate carcinoma cells did not increase their sensitivity to proteasome inhibitors. Likewise, comparison of parental and p21-deficient cells demonstrated that p21WAF1/CIP1 was dispensable for proteasome inhibitor-induced cytotoxicity. We conclude that accumulation of wt p53 and induction of apoptosis are independent markers of proteasome inhibition.
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PMID:Protease inhibitor-induced apoptosis: accumulation of wt p53, p21WAF1/CIP1, and induction of apoptosis are independent markers of proteasome inhibition. 1091 53

The purposes of this experiment were (1) to determine if apoptosis was accelerated during formation of selenite cataract, and (2) to determine the role of calpains and caspases in lens apoptosis. Evidence for apoptosis in selenite-injected rats included: approximately 7-8% of epithelial cells in germinative zone were positive, disappearance of the nuclear membrane, condensation of the chromatin, and breakdown of PARP. Activation of calpains was indicated by characteristic limited proteolysis of crystallins, breakdown of alpha-spectrin to 150/145 kDa fragments, hydrolysis of vimentin, and autolytic breakdown of m-calpain. Selenite cataract did not have an appreciable effect on the mRNA levels for caspase-3, calpains, and calpastatin. This indicated the increased enzyme activity of m-calpain and caspase-3 in selenite cataract occurred at the enzyme level rather than by upregulation of mRNAs. Increased calpain and caspase activity may be linked to the selenite-induced apoptosis. Such data are important because they indicate that apoptosis may be a fairly early event in selenite cataract.
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PMID:Evidence for apoptosis in the selenite rat model of cataract. 1096 62


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