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)

Constitutive expression of human nuclear NAD+: protein ADP-ribosyltransferase (polymerizing) [pADPRT; poly(ADP-ribose)polymerase; EC 2.4.2.30] as an active enzyme in Saccharomyces cerevisiae, under the control of the alcohol dehydrogenase promoter, was only possible with simultaneous inhibition of ADP-ribosylation by 3-methoxybenzamide. Induction of fully active pADPRT from the inducible galactose epimerase promoter resulted in inhibition of cell division and morphological changes reminiscent of cell cycle mutants. Expression of a pADPRT cDNA truncated at its 5' end had no influence on cell proliferation at all. Obviously the amino-terminal part of the DNA binding domain containing the first "zinc finger", which is essential for inducibility of pADPRT activity by DNA breaks, is also required for inhibition of cell growth on expression in yeast. Full-length as well as truncated pADPRT molecules were directed to the cell nucleus where the fully active enzyme produced large amounts of poly(ADP-ribose) by automodification. Since pADPRT turned out to be the only target for ADP-ribosylation in these cells, elevated levels of poly(ADP-ribose) were the most likely cause of inhibition of cell division, presumably resulting from interaction with chromosomal proteins.
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PMID:Inhibition of cell proliferation in Saccharomyces cerevisiae by expression of human NAD+ ADP-ribosyltransferase requires the DNA binding domain ("zinc fingers"). 155 29

We have overproduced the full-length human poly(ADP-ribose) polymerase (PARP) in Spodoptera frugiperda (Sf9) cells using a baculovirus expression vector system. Approx. 20 mg of purified protein from 5 x 10(8) Sf9 cells were obtained by a simple three-step purification procedure including 3-aminobenzamide affinity chromatography. The recombinant protein (rePARP), which migrates as a unique 116-kDa band on SDS-polyacrylamide gels, was identified as PARP by Western blotting using either polyclonal or monoclonal antibodies raised against the purified human and calf thymus enzymes. Furthermore, rePARP is a functional protein, as demonstrated by its ability to specifically bind Zn2+ and DNA, and to recognize single-strand breaks in DNA. The purified enzyme has the same affinity for NAD+ and turnover number as the human placental PARP. Thus, rePARP produced in insect cells is biologically active and suitable for functional analysis. The reproducibility of the overproduction and the simplicity of the purification protocol, as well as the yield of the produced protein, should greatly facilitate physicochemical and structural studies.
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PMID:Overproduction and large-scale purification of the human poly(ADP-ribose) polymerase using a baculovirus expression system. 160 10

The role of ADP ribosylation of proteins in the physiological regulation of sporulation in Streptomyces griseus was studied. We report here that both the activity of NAD+: arginine ADP-ribosyltransferase (ADPRT) and the pattern of ADP-ribosylated proteins showed characteristic changes during the life cycle in S. griseus 2682. Analysis off ADP-ribosylated proteins revealed that in a nonsporulating mutant of the parental wild-type (wt) strain (Bld7 mutant), both the activity of ADPRT and the pattern of ADP-ribosylated proteins were different from those of the parental strain. Addition of 3-aminobenzamide (3AB), the most potent inhibitor of ADPRT, inhibited sporulation of S. griseus 2682 and the A-factor (AF)-induced sporulation of S. griseus Bld7, but in both cases the inhibitory effect of 3AB was strictly age-dependent. Using [alpha-32P]GTP, we have demonstrated the presence of GTP-binding proteins in purified cell membranes of S. griseus 2682 and S. griseus Bld7. The same GTP-binding proteins were observed in Bld7 and the wt. AF stimulated the basal GTPase activity of cell membranes of S. griseus 2682 in a concentration-dependent manner, suggesting that GTP-binding proteins might be involved in the AF-induced sporulation process.
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PMID:The possible role of ADP ribosylation in physiological regulation of sporulation in Streptomyces griseus. 161 34

C3 ADP-ribosyltransferase is an exoenzyme produced by certain strains of Clostridium botulinum types C and D, which specifically ADP-ribosylates rho proteins in eukaryotic cells. Using the photoaffinity probe [alpha-32P]nicotinamide-2-azidoadenine dinucleotide, we have identified the adenine ring binding domain of the NAD+ binding site. The specificity of labeling was demonstrated by saturation effects and protection by the natural compound at physiologically relevant concentrations. Saturation of labeling was observed at 50 microM. Protection experiments indicated an 80% protection of labeling by 100 microM NAD+ when protein was photolyzed in the presence of 10 microM probe. Trypsin or Staphylococcus aureus V8 protease digestion of the photolabeled protein, along with boronate affinity chromatography and immobilized metal affinity chromatography, was used to specifically isolate the peptide region photolabeled with the probe. The peptide corresponded to Phe9-Gly19 near the N terminus.
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PMID:NAD+ binding site of Clostridium botulinum C3 ADP-ribosyltransferase. Identification of peptide in the adenine ring binding domain using 2-azido NAD. 163 27

This study describes a combined immunochemical and genetic approach defining a site on Pseudomonas aeruginosa exotoxin A (ETA) which is critical to the ADP-ribosyltransferase (ADPRT) activity of the toxin. The sequential epitope of a monoclonal antibody (TO-1) which binds to domain III (residues 405-613), containing the ADPRT activity of ETA, has been defined using a series of synthetic peptides. This epitope spans residues 422-432 which composes the major alpha-helical segment of domain III and includes His426 which has previously been shown to be essential for ADPRT activity (Wozniak, D.J., Hsu, L.-Y., and Galloway, D. R. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8880-8884). The critical His426 residue which projects into a major cleft becomes exposed when the ETA protein is in an ADPRT-active configuration. Since the TC-1 mAb does not block the binding of NAD+, it is possible that the alpha-helix site containing the TC-1 epitope and the His426 residue is associated with the interaction between ETA and its elongation factor 2 substrate.
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PMID:Immunochemical analysis of Pseudomonas aeruginosa exotoxin A. Analysis of the His426 determinant. 170 36

Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of [14C]NAD+ and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the alpha and beta chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight mirotubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated [14C]ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD+ resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.
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PMID:Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization. 173 82

An arginine-specific ADP-ribosyltransferase, named ADP-ribosyltransferase A, was partially purified from human platelets using polyarginine as an ADP-ribose acceptor. When human platelet membranes were incubated with the transferase A in the presence of NAD+, Gs, a stimulatory guanine nucleotide-binding protein of the adenylate cyclase was specifically mono-ADP-ribosylated. ADP-ribose transfer to Gs by this enzyme was suppressed when membranes were pre-ADP-ribosylated by cholera toxin. Incubation of membranes with the transferase A resulted in activation of the adenylate cyclase system. This stimulatory effect of the transferase A on the adenylate cyclase system was inhibited by the presence of polyarginine. These results indicate a role of ADP-ribosyltransferase A in regulation of the adenylate cyclase system via endogenous mono-ADP-ribosylation of Gs.
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PMID:Mono-ADP-ribosylation of Gs by an eukaryotic arginine-specific ADP-ribosyltransferase stimulates the adenylate cyclase system. 190 36

Previous studies of the S1 subunit of pertussis toxin, an NAD(+)-dependent ADP-ribosyltransferase, suggested that a small amino-terminal region of amino acid sequence similarity to the active fragments of both cholera toxin and Escherichia coli heat-labile enterotoxin represents a region containing critical active-site residues that might be involved in the binding of the substrate NAD+. Other studies of two other bacterial toxins possessing ADP-ribosyltransferase activity, diphtheria toxin and Pseudomonas exotoxin A, have revealed the presence of essential glutamic acid residues vicinal to the active site. To help determine the relevance of these observations to activities of the enterotoxins, the A-subunit gene of the E. coli heat-labile enterotoxin was subjected to site-specific mutagenesis in the region encoding the amino-terminal region of similarity to the S1 subunit of pertussis toxin delineated by residues 6 through 17 and at two glutamic acid residues, 110 and 112, that are conserved in the active domains of all of the heat-labile enterotoxin variants and in cholera toxin. Mutant proteins in which arginine 7 was either deleted or replaced with lysine exhibited undetectable levels of ADP-ribosyltransferase activity. However, limited trypsinolysis of the arginine 7 mutants yielded fragmentation kinetics that were different from that yielded by the wild-type recombinant subunit or the authentic A subunit. In contrast, mutant proteins in which glutamic acid residues at either position 110 or 112 were replaced with aspartic acid responded like the wild-type subunit upon limited trypsinolysis, while exhibiting severely depressed, but detectable, ADP-ribosyltransferase activity. The latter results may indicate that either glutamic acid 110 or glutamic acid 112 of the A subunit of heat-labile enterotoxin is analogous to those active-site glutamic acids identified in several other ADP-ribosylating toxins.
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PMID:Effect of site-directed mutagenic alterations on ADP-ribosyltransferase activity of the A subunit of Escherichia coli heat-labile enterotoxin. 190 25

The A subunit of cholera toxin contains the ADP-ribosyltransferase activity in its major constituent polypeptide A1 (Mr 23,000) which is responsible for the elevation of cAMP typically observed with most mammalian cell types after exposure to the toxin. The primary structure of the A subunit, recently established by sequence analyses, is presented and used as the basis for the secondary structure prediction according to the method of Chou and Fasman. The results indicated the presence of 27% alpha-helix, 25% beta-structure, 12% beta-turn, and 36% random coil. The majority of the beta-structure consisted of six strands located in the NH2-terminal portion of the molecule (residues 33-106) covering one-half of the region corresponding to the A1 polypeptide portion. The beta-sheet domain led immediately into the active site region characterized by the alternating structures of beta-pleated sheet and alpha-helix (residues 95-140) similar to that reported for other NAD+ binding proteins. The presence of this structural feature in the region was confirmed by the use of another predictive method (J. Garnier et al., J. Mol. Biol. 1978, 120, 97-120). In addition, two regions (residues 14-18 and 200-214), previously identified to contain binding sites for the B subunit as evidenced by chemical modification and monoclonal antibody studies, were found to be in alpha-helix configuration.
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PMID:Cholera toxin A subunit: functional sites correlated with regions of secondary structure. 240 74

Treatment of platelets with a prostacyclin analogue, iloprost, decreased the cholera-toxin-induced ADP-ribosylation of membrane-bound Gs alpha (alpha-subunit of G-protein that stimulates adenylate cyclase; 42 kDa protein) and a cytosolic substrate (44 kDa protein) [Molina y Vedia, Reep & Lapetina (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5899-5902]. This decrease is apparently not correlated with a significant change in the quantity of membrane Gs alpha, as detected by two Gs alpha-specific antisera. This finding contrasts with the suggestion in a previous report [Edwards, MacDermot & Wilkins (1987) Br. J. Pharmacol. 90, 501-510], indicating that iloprost caused a loss of Gs alpha from the membrane. Our evidence points to a modification in the ability of the 42 kDa protein to be ADP-ribosylated by cholera toxin. This modification of Gs alpha might be related to its ADP-ribosylation by endogenous ADP-ribosyltransferase activity. Here we present evidence showing that Gs alpha was ADP-ribosylated in platelets that had been electropermeabilized and incubated with [alpha-32P]NAD+. This endogenous ADP-ribosylation of Gs alpha is inhibited by nicotinamide and stimulated by iloprost.
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PMID:The effect of iloprost on the ADP-ribosylation of Gs alpha (the alpha-subunit of Gs). 247 20


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