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)

Inhibitors of poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) are of clinical interest because they have potential for improving radiation therapy and chemotherapy of cancer. The refined binding structures of four such inhibitors are reported together with the refined structure of the unligated catalytic fragment of the enzyme. Following their design, all inhibitors bind at the position of the nicotinamide moiety of the substrate NAD+. The observed binding mode suggests inhibitor improvements that avoid other NAD(+)-binding enzymes. Because the binding pocket of NAD+ has been strongly conserved during evolution, the homology with ADP-ribosylating bacterial toxins could be used to extend the bound nicotinamide, which is marked by the inhibitors, to the full NAD+ molecule.
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PMID:Inhibitor and NAD+ binding to poly(ADP-ribose) polymerase as derived from crystal structures and homology modeling. 952 10

The binding site for the acceptor substrate poly(ADP-ribose) in the elongation reaction of the ADP-ribosyl transferase poly(ADP-ribose) polymerase (PARP) was detected by cocrystallizing the enzyme with an NAD+ analogue. The site was confirmed by mutagenesis studies. In conjunction with the binding site of the donor NAD+, the bound acceptor reveals the geometry of the elongation reaction. It shows in particular that the strictly conserved glutamate residue of all ADP-ribosylating enzymes (Glu988 of PARP) facilitates the reaction by polarizing both, donor and acceptor. Moreover, the binding properties of the acceptor site suggest a mechanism for the branching reaction, that also explains the dual specificity of this transferase for elongation and branching, which is unique among polymer-forming enzymes.
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PMID:The mechanism of the elongation and branching reaction of poly(ADP-ribose) polymerase as derived from crystal structures and mutagenesis. 957 Oct 33

NAD+ glycohydrolase (NADase) and non-enzymic ADP-ribosylation have been thought to be involved in the regulation of mitochondrial Ca2+ fluxes. In this study it was found that several conditions (5 mM nicotinamide, 5 mM 3-aminobenzamide, 2 mM EDTA, 1 mM ATP, 10 mM dithiothreitol) known to strongly inhibit the NADase decreased ADP-ribosylation in bovine liver mitochondrial membranes with [32P]NAD+ as substrate to only a limited extent, if at all. The reaction led to the specific modification of two proteins with apparent molecular masses of approx. 26 and 53 kDa. An excess of added free ADP-ribose diminished the incorporation of label from [32P]NAD+ only slightly. Dithiothreitol inactivated the NADase, whereas ADP-ribosylation was unaffected. At low concentrations (25 microM) ADP-ribosylation was efficient with NAD+, but not ADP-ribose, as substrate. Under these conditions mitochondrial ADP-ribosylation seems to occur as an enzymic reaction rather than a non-enzymic transfer of ADP-ribose previously liberated from NAD+ by NAD+ glycohydrolase. The chemical stability of the protein-ADP-ribose bonds in the mitochondrial membranes indicated that cysteine residues are the predominant acceptors. Moreover, yeast aldehyde dehydrogenase, known to be a substrate for thiol-associated ADP-ribosylation, was efficiently ADP-ribosylated by using the mitochondrial activity and NAD+ as substrate. The modification of a cysteine residue in the aldehyde dehydrogenase was verified by the observation that pretreatment of this acceptor protein with N-ethylmaleimide substantially decreased its modification. It is therefore concluded that bovine liver mitochondria contain a cysteine-specific ADP-ribosyltransferase.
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PMID:Enzymic, cysteine-specific ADP-ribosylation in bovine liver mitochondria. 957 67

The DNA-dependent protein kinase (DNA-PK) is a heterotrimeric enzyme that binds to double-stranded DNA and is required for the rejoining of double-stranded DNA breaks in mammalian cells. It has been proposed that DNA-PK functions in this DNA repair pathway by binding to the ends of broken DNA molecules and phosphorylating proteins that bind to the damaged DNA ends. Another enzyme that binds to DNA strand breaks and may also function in the cellular response to DNA damage is the poly(ADP-ribose) polymerase (PARP). Here, we show that PARP can be phosphorylated by purified DNA-PK, and the catalytic subunit of DNA-PK is ADP-ribosylated by PARP. The protein kinase activity of DNA-PK can be stimulated by PARP in the presence of NAD+ in a reaction that is blocked by the PARP inhibitor 1, 5-dihydroxyisoquinoline. The stimulation of DNA-PK by PARP-mediated protein ADP-ribosylation occurs independent of the Ku70/80 complex. Taken together, these results show that PARP can modify the activity of DNA-PK in vitro and suggest that these enzymes may function coordinately in vivo in response to DNA damage.
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PMID:Stimulation of the DNA-dependent protein kinase by poly(ADP-ribose) polymerase. 960 59

Poly(ADP-ribose)polymerase (PARP) has been implicated in DNA repair mechanisms and the associated activity shown to markedly increase after DNA damage in carcinogen-treated cells. A defective DNA repair has been associated to the aetiology of human cancers. In order to assess the potential role of this enzyme in cellular response to DNA damage by gamma-radiation, we studied the activity of PARP in patients with familial adenomatous polyposis (FAP). We compared poly(ADP-ribose)polymerase activity by the rate of incorporation of radioactivity from [3H]adenine-NAD+ into acid-insoluble material in permeabilized leucocytes from FAP patients and healthy volunteers. Concomitantly, the intracellular levels of NAD+--the substrate for the PARP--and the reduced counterpart NADH were determined using an enzymatic cycling assay 30 min after [60Co] gamma-ray cells irradiation. Our results demonstrate that a marked stimulation of PARP activity is produced upon radiation of the cells from healthy subjects but not in the FAP leucocytes, which concomitantly show a marked decrease in total NAD-/NADH content. Our observations point to a role of PARP in the repair of the gamma-radiation-induced DNA lesions through a mechanism that is impaired in the cells from FAP patients genetically predisposed to colon cancer. The differences observed in PARP activation by gamma-radiation in patients and healthy individuals could reflect the importance of PARP activity dependent on treatment with gamma-rays. The absence of this response in FAP patients would seem to suggest a possible defect in the role of PARP in radiation-induced DNA repair in this cancer-prone disease.
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PMID:Absence of stimulation of poly(ADP-ribose) polymerase activity in patients predisposed to colon cancer. 963 38

During apoptosis, DNA undergoes fragmentation and caspase-3 cleaves poly(ADP-ribose) polymerase (PARP) into both a 24-kDa fragment containing the DNA binding domain and an 89-kDa fragment containing the catalytic and automodification domains. Atomic force microscopy revealed that recombinant full-length PARP bound to plasmid DNA fragments and linked them into chainlike structures. Automodification of PARP in the presence of NAD+ resulted in its dissociation from the DNA fragments, which, nevertheless, remained physically aligned. A recombinant 28-kDa fragment of PARP containing the DNA binding domain but lacking the automodification domain irreversibly bound to and linked DNA fragments in the absence or presence of NAD+. Identical results were obtained on incubation of internucleosomal DNA fragments from apoptotic cells with the products of cleavage of recombinant PARP by purified caspase-3. The 24-kDa product of PARP cleavage by caspase-3 may contribute to the irreversibility of apoptosis by blocking the access of DNA repair enzymes to DNA strand breaks.
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PMID:Irreversible binding of poly(ADP)ribose polymerase cleavage product to DNA ends revealed by atomic force microscopy: possible role in apoptosis. 972 47

1. Arginine-specific ADP-ribosyltransferase (ART) activity has been implicated in white cell chemotaxis. In this study, we examined the capacity of a panel of structurally unrelated inhibitors and pseudosubstrates of ART to inhibit chemotaxis of A7r5 rat vascular smooth muscle cells in response to PDGF-BB. 2. The IC50 values for nicotinamide (12 mM) and novobiocin (165 microM) were similar to those observed for inhibition of chemotaxis by human polymorphonuclear neutrophil leucocytes (PMN), whereas vitamins K3 (IC50=22 microM) and K1 (IC50=95 microM) were less potent than previously described in PMNs. The pseudo-substrates for the enzyme (DEA-BAG, agmatine and arginine-methylester) also inhibited A7r5 chemotaxis, and in addition inhibited cell adhesion at similar concentrations. Vitamin K3 was unique among the inhibitors of ART, in that it also inhibited cell adhesion. 3. A rat ART1 transcript was amplified by rtPCR from rat skeletal muscle, and was noted to share 94% homology with the mouse ART1 cDNA sequence. No such transcript could be detected in A7r5 cells by Northern blot analysis or rtPCR. 4. Evidence for ART activity on the surface of A7r5 cells was investigated using 32P-NAD+ as substrate, and labelled membrane proteins were observed with MWt values of 116, 100, 90 and 70 kDa. Exposure of the labelled proteins to phosphodiesterase yielded 32P-AMP, and hydrolysis with NaOH yielded 32P-NAD+. These results indicated that the labelled proteins were adducts with NAD+, and not the products of ART activity. The absence of ART catalytic activity in A7r5 cells was confirmed in protocols designed to show ADP-ribosylation of agmatine. 5. We conclude that the chemotactic activity of A7r5 cells is independent of ART activity, and the mechanism whereby the novel panel of inhibitors reduced cell migration remains undefined.
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PMID:Inhibition of chemotaxis in A7r5 rat smooth muscle cells by a novel panel of inhibitors. 977 55

Inhibitors of poly(ADP-ribose) polymerase (PARP), including benzamide, protect against 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP)-induced dopamine neurotoxicity in vivo [Cosi et al., Brain Res. 729 (1996) 264-269]. In vitro, the activation of PARP by free radical damaged DNA has been shown to be correlated with rapid decreases in the cellular levels of its substrate nicotinamide adenine dinucleotide (NAD+), and ATP. Here, we investigated in vivo whether MPTP acutely caused region- and time-dependent changes in brain levels of NAD+, ATP, ADP and AMP in C57BL/6N mice killed by head-focused microwave irradiation, and whether such effects were modified by treatments with neuroprotective doses of benzamide. At 1 h after MPTP injections (4x20 mg/kg i.p.), NAD+ was reduced by 11-13% in the striatum and ventral midbrain, but not in the frontal cortex. The ATP/ADP ratio was reduced by 10% and 32% in the striatum and cortex, respectively, but was unchanged in the midbrain. All of these regional changes were prevented by co-treatment with benzamide (2x160 mg/kg i.p.), which by itself did not alter regional levels of NAD+, ATP, ADP or AMP in control mice. In a time-course study, a single dose of MPTP (30 mg/kg i.p.) resulted in maximal and transient increases in striatal levels of MPP+ and 3-methoxytyramine (+540%) at 0.5-2 h, followed by maximal and coincidental decreases in NAD+ (-10%), ATP (-11%) and dopamine content (-39%) at 3 h. Benzamide (1x640 mg/kg i. p., 30 min before MPTP) partially reduced MPP+ levels by 30% with little or no effect on MPTP or MPDP+ levels, did not affect or even slightly potentiated the increase in 3-methoxytyramine, and completely prevented the losses in striatal NAD+, ATP and dopamine content, without by itself causing any changes in these latter parameters in control mice. These results (1) confirm that MPTP reduces striatal ATP levels [Chan et al., J. Neurochem. 57 (1991) 348-351.]; (2) show that MPTP causes a regionally-dependent (striatal and midbrain) loss of NAD+; (3) indicate that the PARP inhibitor benzamide can prevent these losses without interfering with MPTP-induced striatal dopamine release; and (4) provide further evidence to suggest an involvement of PARP in MPTP-induced neurotoxicity in vivo.
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PMID:Decreases in mouse brain NAD+ and ATP induced by 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine (MPTP): prevention by the poly(ADP-ribose) polymerase inhibitor, benzamide. 979 36

Poly(ADP-ribose) polymerase (PARP) (EC 2.4.2.30), the only enzyme known to synthesize ADP-ribose polymers from NAD+, is activated in response to DNA strand breaks and functions in the maintenance of genomic integrity. Mice homozygous for a disrupted gene encoding PARP are viable but have severe sensitivity to gamma-radiation and alkylating agents. We demonstrate here that both 3T3 and primary embryo cells derived from PARP-/- mice synthesized ADP-ribose polymers following treatment with the DNA-damaging agent, N-methyl-N'-nitro-N-nitrosoguanidine, despite the fact that no PARP protein was detected in these cells. ADP-ribose polymers isolated from PARP-/- cells were indistinguishable from that of PARP+/+ cells by several criteria. First, they bound to a boronate resin selective for ADP-ribose polymers. Second, treatment of polymers with snake venom phosphodiesterase and alkaline phosphatase yielded ribosyladenosine, a nucleoside diagnostic for the unique ribosyl-ribosyl linkages of ADP-ribose polymers. Third, they were digested by treatment with recombinant poly(ADP-ribose) glycohydrolase, an enzyme highly specific for ADP-ribose polymers. Collectively, these data demonstrate that ADP-ribose polymers are formed in PARP-/- cells in a DNA damage-dependent manner. Because the PARP gene has been disrupted, these results suggest the presence of a previously unreported activity capable of synthesizing ADP-ribose polymers in PARP-/- cells.
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PMID:Poly(ADP-ribose) polymerase null mouse cells synthesize ADP-ribose polymers. 980 57

We have examined the domain-specific interactions between p53 and poly(ADP-ribose)polymerase (PARP) (E.C. 2.4.2.30) in apoptotic HeLa cells. Apoptosis was induced by exposing cells to 50 microM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for increasing lengths of time and was confirmed by: (a) oligonucleosomal fragmentation of chromatin; (b) increase in p53 levels; and (c) degradation of PARP into the characteristic M(r) 85,000 (COOH-terminal catalytic domain) and M(r) 29,000 (DNA-binding domain) peptide fragments. We also immunodetected p53 in immunoprecipitates obtained with a PARP-specific antibody. However, intact PARP coimmunoprecipitated with a p53-specific antibody during the initial 30 min of MNNG treatment. After 60 min, only the COOH-terminal fragment coimmunoprecipitated with p53, indicating that PARP noncovalently binds p53 via its M(r) 85,000 catalytic domain. Therefore, we next examined p53 as a covalent target for poly(ADP-ribosyl)ation. Although p53 was not endogenously poly (ADP-ribosyl)ated in situ, incubation of cell extracts with full-length PARP from calf thymus and [32P]beta NAD+ resulted in its time-dependent poly(ADP-ribosyl)ation. In summary, our results are consistent with the conclusion that PARP and p53 are activated with nonoverlapping kinetics during apoptosis.
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PMID:Functional interactions of p53 with poly(ADP-ribose) polymerase (PARP) during apoptosis following DNA damage: covalent poly(ADP-ribosyl)ation of p53 by exogenous PARP and noncovalent binding of p53 to the M(r) 85,000 proteolytic fragment. 982 14


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