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)

We have used two different approaches to study the consequences of NAD/poly(ADP-ribose) deficiency on p53 expression and its activity in V79-derived cell lines. In the first approach, we have used two cell lines that are deficient in poly(ADP-ribose) (pADPR) synthesis because of deficiency in the enzyme poly(ADP-ribose) polymerase (PARP). In a second approach, we have used a cell line that is deficient in NAD/pADPR metabolism due to unavailability of NAD, the substrate for PARP. These NAD/PARP-deficient cell lines exhibit a significant reduction in both baseline p53 expression and its activity compared to their parental V79 cells. Furthermore, etoposide, a topoisomerase II inhibitor that was shown to cause an increase in p53 expression and subsequent apoptosis in V79 cells, failed to produce any significant increase in p53 expression or apoptotic DNA fragmentation in NAD/PARP-deficient cell lines. Thus, our studies suggest that NAD/pADPR synthesis may be involved in the regulation of p53 and its dependent pathways.
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PMID:Involvement of NAD-poly(ADP-ribose) metabolism in p53 regulation and its consequences. 764 Nov 78

The evolution of ADP-ribosyltransferase (NAD+) pseudogene 1 (ADPRTP1) was studied among higher primates. When the human pseudogene was used to probe genomic DNA from chimpanzee, gorilla, macaque, howler monkey and lemur, a fragment from gorilla produced the most intense hybridization signal. The resultant hybridization pattern indicated a modified pseudogene structure in these primates relative to the human and gorilla loci. Sequence comparison of this new DNA locus (ADPRTP1 and surrounding retroposons) showed a nucleotide (nt) identity of 98.13% (over 5.8 kb) between the genomic regions of human and gorilla. A unique duplicated region of 30 base pairs (bp) was found in gorilla ADPRTP1, separate from the duplicated region (193 bp) responsible for the restriction-fragment length polymorphism (RFLP) previously reported in humans, and which appeared to represent a marker for a predisposition to cancer. An endogenous pol (gene encoding polymerase) related element that flanked the human pseudogene was used as a probe to identify a fragment from this retroviral family in New World monkeys. Altogether, analysis of these retroposons will provide an opportunity for future studies on the molecular phylogenetic relationship of higher primates.
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PMID:Conservation of sequences between human and gorilla lineages: ADP-ribosyltransferase (NAD+) pseudogene 1 and neighboring retroposons. 772 Oct 98

Activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) is an early response of cells exposed to DNA-damaging compounds such as nitric oxide (NO) or reactive oxygen intermediates (ROI). Excessive poly-(ADP-ribose) formation by PARP has been assumed to deplete cellular NAD+ pools and to induce the death of several cell types, including the loss of insulin-producing islet cells in type I diabetes. In the present study we used cells from mice with a disrupted and thus inactivated PARP gene to provide direct evidence for a causal relationship between PARP activation, NAD+ depletion, and cell death. We found that mutant islet cells do not show NAD+ depletion after exposure to DNA-damaging radicals and are more resistant to the toxicity of both NO and ROI. These findings directly prove that PARP activation is responsible for most of the loss of NAD+ following such treatment. The ADP-ribosylation inhibitor 3-aminobenzamide partially protected islet cells with intact PARP gene but not mutant cells from lysis following either NO or ROI treatment. Hence the protective action of 3-aminobenzamide must be due to inhibition of PARP and does not result from its other pharmacological properties such as oxygen radical scavenging. Finally, the use of mutant cells an alternative pathway of cell death was discovered which does not require PARP activation and NAD+ depletion. In conclusion, the data prove the causal relationship of PARP activation and subsequent islet cell death and demonstrate the existence of an alternative pathway of cell death independent of PARP activation and NAD+ depletion.
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PMID:Inactivation of the poly(ADP-ribose) polymerase gene affects oxygen radical and nitric oxide toxicity in islet cells. 774 49

A full-length Arabidopsis thaliana cDNA (app) encoding a protein with high similarity (about 60%) to the catalytic domain of vertebrate poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) has been cloned. The N-terminal extension of the Arabidopsis protein shows similarities with domains of different nuclear and DNA binding proteins in agreement with nuclear localization and putative function of a plant PARP. APP is encoded by a single gene mapped at the top of chromosome 4 of the Arabidopsis genome and mRNA is abundant in cell suspension culture compared to its accumulation in whole plant.
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PMID:Characterization of an Arabidopsis thaliana cDNA homologue to animal poly(ADP-ribose) polymerase. 775 May 52

Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalyzed by poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30), with NAD+ serving as the substrate. PARP is strongly activated upon recognition of DNA strand breaks by its DNA-binding domain. Experiments with low-molecular-weight inhibitors of PARP have led to the view that PARP activity plays a role in DNA repair and possibly also in DNA replication, cell proliferation, and differentiation. Accumulating evidence for nonspecific inhibitor effects prompted us to develop a molecular genetic system to inhibit PARP in living cells, i.e., to overexpress selectively the DNA-binding domain of PARP as a dominant negative mutant. Here we report on a cell culture system which allows inducible, high-level expression of the DNA-binding domain. Induction of this domain leads to about 90% reduction of poly(ADP-ribose) accumulation after gamma-irradiation and sensitizes cells to the cytotoxic effect of gamma-irradiation and of N-methyl-N'-nitro-N-nitrosoguanidine. In contrast, induction does not affect normal cellular proliferation or the replication of a transfected polyomavirus replicon. Thus, trans-dominant inhibition of the poly(ADP-ribose) accumulation occurring after gamma-irradiation or N-methyl-N'-nitro-N-nitrosoguanidine is specifically associated with a disturbance of the cellular recovery from the inflicted damage.
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PMID:trans-dominant inhibition of poly(ADP-ribosyl)ation sensitizes cells against gamma-irradiation and N-methyl-N'-nitro-N-nitrosoguanidine but does not limit DNA replication of a polyomavirus replicon. 776 Aug 11

Poly(ADP-ribose) polymerase [PARP; NAD+ ADP-ribosyltransferase; NAD+:poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] is a zinc-dependent eukaryotic DNA-binding protein that specifically recognizes DNA strand breaks produced by various genotoxic agents. To study the biological function of this enzyme, we have established stable HeLa cell lines that constitutively produce the 46-kDa DNA-binding domain of human PARP (PARP-DBD), leading to the trans-dominant inhibition of resident PARP activity. As a control, a cell line was constructed, producing a point-mutated version of the DBD, which has no affinity for DNA in vitro. Expression of the PARP-DBD had only a slight effect on undamaged cells but had drastic consequences for cells treated with genotoxic agents. Exposure of cell lines expressing the wild-type (wt) or the mutated PARP-DBD, with low doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in an increase in their doubling time, a G2 + M accumulation, and a marked reduction in cell survival. However, UVC irradiation had no preferential effect on the cell growth or viability of cell lines expressing the PARP-DBD. These PARP-DBD-expressing cells treated with MNNG presented the characteristic nucleosomal DNA ladder, one of the hallmarks of cell death by apoptosis. Moreover, these cells exhibited chromosomal instability as demonstrated by higher frequencies of both spontaneous and MNNG-induced sister chromatid exchanges. Surprisingly, the line producing the mutated DBD had the same behavior as those producing the wt DBD, indicating that the mechanism of action of the dominant-negative mutant involves more than its DNA-binding function. Altogether, these results strongly suggest that PARP is an element of the G2 checkpoint in mammalian cells.
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PMID:A dominant-negative mutant of human poly(ADP-ribose) polymerase affects cell recovery, apoptosis, and sister chromatid exchange following DNA damage. 776 96

The effect of inhibition of poly(ADP-ribose) polymerase (PARP) on the growth arrest and cell killing induced by N-methyl-N-nitrosourea (MNU) was studied in L929 fibroblasts. Depletion of NAD and ATP preceded the cell killing by a 1-h exposure to 10 or 15 mM MNU. 3-Aminobenzamide (ABA), an inhibitor of PARP, spared the depletion of NAD and ATP and prevented the cell killing. With 5 mM MNU, a depletion of NAD was promptly reversed, and there was no loss of ATP and no cell death. Aphidicolin, a DNA polymerase inhibitor, prevented the restoration of NAD, with resulting depletion of ATP and death of the cells, effects that were prevented by ABA. Azide together with 2-deoxyglucose depleted ATP, followed by a loss of NAD and cell death, changes that occurred in the absence of DNA single strand breaks (DNA SSB). ABA prevented the depletion of NAD, but not that of ATP, nor the cell killing. MNU (2.5 mM) inhibited cell growth without effect on the viability of the cells. ABA potentiated the cell growth inhibition. Thus, inhibition of PARP potentiates cell growth inhibition by limiting DNA repair mechanisms. Alternatively, inhibition of the DNA repair response to more extensive DNA damage prevents cell killing. The ATP depletion caused by poly(ADP-ribosyl)ation, rather than DNA SSB and the loss of NAD, is the more critical event in the cell killing.
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PMID:Growth inhibition and cell killing by N-methyl-N-nitrosourea: metabolic alterations that accompany poly(ADP-ribosyl)ation. 778 36

The influence of poly (ADP-ribose) polymerase (PARP) and poly ADP-ribosylation on DNA synthesis supported by human replicative DNA polymerase (DNA pol) alpha, delta, and epsilon has been examined using the replication system containing poly(dA)4500-oligo(dT)12-18 as the template primer. PARP alone inhibited the pol activities in a dose-dependent manner even in the presence of the accessory factors for DNA pol delta, proliferating cell nuclear antigen (PCNA) and activator 1 (Al; RF-C). Both DNA pol alpha and epsilon activities were decreased approximately 10-fold under the poly ADP-ribosylating condition. In contrast, DNA synthesis by DNA pol delta holoenzyme was not affected by poly ADP-ribosylation like prokaryotic DNA pol's. The analysis of poly(dT) formed by DNA pol alpha and epsilon indicated that poly ADP-ribosylation mainly reduced the frequency of replication. These observations suggest a possibility that PARP acts as a negative regulator for the initiation of DNA replication upon cellular DNA damage.
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PMID:Poly (ADP-ribose) polymerase inhibits DNA replication by human replicative DNA polymerase alpha, delta and epsilon in vitro. 780 50

The structural gene for the 49-kDa form of exoenzyme S (exoS) isolated from Pseudomonas aeruginosa 388 was expressed in both Escherichia coli and P. aeruginosa PA103. Expression of exoS in E. coli under the transcriptional regulation of the T7 promoter yielded a soluble cytosolic protein with an apparent molecular mass of 49 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Expression of exoS in P. aeruginosa PA103 under the transcriptional regulation of the 0.9 kbp of Pseudomonas chromosomal DNA flanking the 5' end of exoS yielded a nitrilotriacetic acid-inducible extracellular protein with an apparent molecular mass of 49 kDa. Recombinant ExoS (rExoS) reacted with the anti-49-kDa form of exoenzyme S immunoglobulin G, existed as an aggregate as determined by gel filtration chromatography, and ADP-ribosylated soybean trypsin inhibitor at a specific activity that was similar (within twofold) to that of native exoenzyme S. Allelic exchange of exoS with a tetracycline gene cartridge yielded a strain of P. aeruginosa 388 that did not express detectable amounts of either ExoS in an immunoblot analysis using the anti-49-kDa form of exoenzyme S immunoglobulin G or ADP-ribosyltransferase activity under standard enzyme assay conditions. Expression of catalytically active rExoS in E. coli demonstrated that exoS was necessary and sufficient for the factor-activating exoenzyme S-dependent ADP-ribosyltransferase activity of exoenzyme S. Expression of nitrilotriacetic acid-inducible rExoS in P. aeruginosa PA103 demonstrated that the 0.9 kbp of Pseudomonas chromosomal DNA flanking the 5' end of exoS encoded a functional exoenzyme S promoter. Expression analysis and allelic exchange experiments suggest that the 49- and 53-kDa forms of exoenzyme S are encoded by separate genes.
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PMID:Expression of recombinant exoenzyme S of Pseudomonas aeruginosa. 780 44

Low-dose gamma-irradiation of mouse embryonic fibroblast C3D2F1 3T3-a cells caused G1 arrest along with G2 arrest and inhibition of replicative DNA synthesis. When the cells were cultured in the presence of inhibitors of poly(ADP-ribose) polymerase [EC 2.4.2.30], such as 3-aminobenzamide, benzamide and luminol, G1 arrest of C3D2F1 3T3-a cells was suppressed and enhancement of G2 arrest was observed. In contrast, 3-aminobenzoic acid, a non-inhibitory analog of 3-aminobenzamide, did not suppress G1 arrest following gamma-irradiation. These results suggest that the poly(ADP-ribosyl)ation reaction is critical for the pathway of G1 arrest and is also involved in the pathway of G2 arrest.
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PMID:Suppression of G1 arrest and enhancement of G2 arrest by inhibitors of poly(ADP-ribose) polymerase: possible involvement of poly(ADP-ribosyl)ation in cell cycle arrest following gamma-irradiation. 782 93


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