Identification of an inhibitor binding site of poly(ADP-ribose) glycohydrolase

Polymers of ADP-ribose involved in the maintenance of genomic integrity are converted to free ADP-ribose by the action of poly(ADP-ribose) glycohydrolase (PARG). As an approach to mapping functions of PARG onto the amino acid sequence of the protein, we report here experiments that identify an amino acid residue involved in the binding of potent PARG inhibitors. A photoreactive inhibitor, [alpha-(32)P]-8-azidoadenosine diphosphate (hydroxymethyl)pyrrolidinediol (8-N(3)-ADP-HPD), was used to photolabel a recombinant bovine PARG catalytic fragment (rPARG-CF). N-Terminal sequencing of tryptic and subtilitic peptides of photoderivatized rPARG-CF identified tyrosine 796 (Y796), a residue conserved in PARG across a wide range of organisms, as a site of photoderivatization. Site-directed mutants where this tyrosine residue was replaced with an alanine residue (Y796A) had a nearly 8-fold decrease in catalytic efficiency (k(cat)/K(M)), while replacement with a tryptophan residue (Y796W) had little effect on catalytic efficiency. Surface plasmon resonance spectroscopy using the PARG inhibitor 8-(aminohexyl)amino-ADP-HPD demonstrated that the binding constant of the inhibitor for Y796A was 21-fold lower (K(D) = 170 nM) than that of wild-type PARG (K(D) = 8.2 nM), while Y796W displayed a binding affinity similar to that of the wild-type enzyme. Our results indicate that Y796 is involved in inhibitor binding to PARG via a ring stacking interaction and identify a highly conserved region of the protein that putatively contains other residues involved in catalytic activity and/or substrate recognition.     

The occurence of a neutral protease and its inhibitor in rat peritoneal macrophages.

The lysate of the glycogen-induced macrophages in rat peritoneal exudate was fractionated by centrifugation and extraction into a water extract, 1 M KCl extract and residue fractions. Approximately 50% of the neutral protease activity toward casein in the lysate was recovered in the KCl extract fraction, which was practically devoid of acid protease, cathepsin D. The pH optimum of the neutral protease toward casein and urea-denatured hemoglobin was pH 8.5. The activity was inhibited strongly by DFP or chymostatin and only partially by HgCl2 or PCMB. Addition of a salt to the reaction medium caused enhancement of the activity with an optimum concentration of 0.25 M: KCl, KBr, KI, NaCl, NaBr, NaI, and MgCl2 were all almost equally effective. When the enzyme preparation was filtered through a column of Sephadex G-75 gel in the presence of 1 M KCl, a larger molecular weight fraction at the void volume was obtained in addition to a smaller molecular weight fraction showing a caseinolytic activity insensitive to KCl concentration. The former was found to have a specific inhibitory effect on the latter activity.     

Sustained depolarization and ADP-ribose activate a common ionic current in rat peritoneal macrophages

Phagocytosis is associated with large changes in the membrane potential of macrophages, but the functional significance of this is unknown. Whole cell recordings were made from rat peritoneal macrophages. Sustained (>30 s) depolarization of the cells progressively activated a conductance that remained high (several nanoSeimens) for several tens of seconds. This current: 1) was linearly dependent on potential between -100 and +50 mV; 2) reversed close to 0 mV in a physiological external solution; 3) could also be carried in part by N-methyl-D-glucamine (P(NMDG)/P(Na) 0.7), chloride (P(Cl)/P(Na) 0.5), or calcium (P(Ca)/P(Na) 1.3); and 4) was blocked by intracellular ATP (5 mM) or ADP (10 mM) and by extracellular lanthanum (half-maximal concentration 1 mM). A current with all the same properties was recorded in cells when the intracellular solution contained ADP-ribose (10-300 micro M) or beta-NAD (1 mM) (but not any other nucleotide analogs tested). The results suggest that prolonged depolarization leads to an increased intracellular level of ADP-ribose, which in turn activates this nonselective conductance(s).     

Identification of aminoethyl pyrrolo dihydroisoquinolinones as novel poly(ADP-ribose) polymerase-1 inhibitors

PARP inhibitors have been demonstrated to retard intracellular DNA repair and therefore sensitize tumor cells to cytotoxic agents or ionizing radiation. We report the identification of a novel class of PARP1 inhibitors, containing a pyrrolo moiety fused to a dihydroisoquinolinone, derived from virtual screening of the proprietary collection. SAR exploration around the nitrogen of the aminoethyl appendage chain of 1 led to compounds that displayed low nanomolar activity in a PARP1 enzymatic assay.     

3'-Deoxy-NAD+ as a substrate for poly(ADP-ribose)polymerase and the reaction mechanism of poly(ADP-ribose) elongation

Adenyl-32P-Labeled 3'-deoxy-NAD+ was utilized as a substrate by pure DNA-dependent poly(ADP-ribose)polymerase (EC 2.4.2.30) from calf thymus in the automodification reaction with an apparent Km of 20 microM and a Vmax of 80 nmol/min/mg of protein. Analysis by lithium lauryl sulfate-polyacrylamide gel electrophoresis revealed a single 32P-labeled protein of 116-kDa which comigrated with automodified enzyme. Addition of increasing amounts of histone H1 up to a concentration of 15 micrograms/ml stimulated the synthesis of protein-bound polymers of 3'-deoxy-ADP-ribose. However, the average polymer size was equal to 2 in the presence and 4 in the absence of histone H1, respectively. The synthesis of protein-bound oligomers of 3'-deoxy-ADP-ribose was inhibited by the polymerase inhibitors benzamide, nicotinamide, thymidine, and NaCl. A pulse labeling of polymer synthesis with 40 microM [32P]3'-deoxy-NAD+ either in the presence or absence of 15 micrograms/ml of histone H1, followed by a chase with 1 mM [3H]NAD+, was used to determine the mechanism of poly(ADP-ribose) elongation. Following enzyme digestion of these polymers with phosphodiesterase, it was found that 52 and 24% of the total 32P radiolabel was associated with the 3'-deoxy-AMP termini of the polymers synthesized in the pulse reactions, in the presence or absence of histone H1, respectively. In contrast, less than 10% of the total radioactivity was associated with 3'-deoxy-AMP in the product of the chase reactions. These results are consistent with the conclusion that the initially attached residue of 3'-deoxy-ADP-ribose to either the polymerase or histone H1, is elongated by the "protein-distal" addition of ADP-ribose residues to the AMP terminus of the growing polymer chain.     

Identification and characterization of a mammalian 39-kDa poly(ADP-ribose) glycohydrolase

ADP-ribosylation is a post-translational modification resulting from transfer of the ADP-ribose moiety of NAD to protein. Mammalian cells contain mono-ADP-ribosyltransferases that catalyze the formation of ADP-ribose-(arginine) protein, which can be cleaved by a 39-kDa ADP-ribose-(arginine) protein hydrolase (ARH1), resulting in release of free ADP-ribose and regeneration of unmodified protein. Enzymes involved in poly(ADP-ribosylation) participate in several critical physiological processes, including DNA repair, cellular differentiation, and carcinogenesis. Multiple poly(ADP-ribose) polymerases have been identified in the human genome, but there is only one known poly(ADP-ribose) glycohydrolase (PARG), a 111-kDa protein that degrades the (ADP-ribose) polymer to ADP-ribose. We report here the identification of an ARH1-like protein, termed poly(ADP-ribose) hydrolase or ARH3, which exhibited PARG activity, generating ADP-ribose from poly-(ADP-ribose), but did not hydrolyze ADP-ribose-arginine, -cysteine, -diphthamide, or -asparagine bonds. The 39-kDa ARH3 shares amino acid sequence identity with both ARH1 and the catalytic domain of PARG. ARH3 activity, like that of ARH1, was enhanced by Mg(2+). Critical vicinal acidic amino acids in ARH3, identified by mutagenesis (Asp(77) and Asp(78)), are located in a region similar to that required for activity in ARH1 but different from the location of the critical vicinal glutamates in the PARG catalytic site. All findings are consistent with the conclusion that ARH3 has PARG activity but is structurally unrelated to PARG.     

Properties of poly(ADP-ribose) synthetase from rat pancreas and poly(ADP-ribosylation) of basic nuclear proteins.

The isolated nuclei of rat pancreas contain an enzyme system that will incorporate 3H-labeled NAD into an acid-insoluble product, which is shown to be poly(ADP-ribose). The enzyme has an optimum pH of 7.8 and the optimum temperature is between 20 and 30 degrees C. Optimum Mg2+ concentration is 8 mM and dithiothreitol also stimulates the enzyme at a concentration of 8 mM. Under standard conditions, the Km value for the reaction is 0.25 mM and an inhibition by the substrate is observed at high substrate concentrations. It has also been found that only one basic nuclear protein, that is, histone H1, is modified by the synthetase. An average chain length of 5.0 is found in the nuclei and of 4.5 on histone H1. Radioautographic studies show that poly(ADP-ribose) is closely associated with chromatin.     

Determination of ADP-ribose and poly(ADP-ribose) by a new radioimmunoassay

A specific and sensitive radioimmunoassay for ADP-ribose has been developed on the basis of the selective conversion of ADP-ribose to 5'-AMP by alkaline treatment. Antibodies highly specific against 5'-AMP allowed quantification of ADP-ribose converted to 5'-AMP in the range of 1-40 pmol, and in the presence of large quantities of nucleic acids or 3'-AMP. Poly(ADP-ribose) could also be determined when degraded to ADP-ribose by poly(ADP-ribose) glycohydrolase. Determination of the chain length of purified polymer was possible by a parallel determination of ADP-ribose residues after glycohydrolase treatment and of 5'-AMP from the non-reducing end obtained by phosphodiesterase catalyzed hydrolysis. The high specificities of the alkaline conversion of ADP-ribose to 5'-AMP and of the radioimmunoassay for 5'-AMP allowed quantification of protein-bound ADP-ribose residues in crude tissue extracts as verified by comparison with chromatographically purified samples.     

Identification of ring-fused pyrazolo pyridin-2-ones as novel poly(ADP-ribose)polymerase-1 inhibitors

A novel class of PARP-1 inhibitors was identified containing a non-aromatic heterocycle or carbocycle fused to a pyrazolo pyridin-2-one. Compounds displayed low nanomolar binding activity in the PARP-1 binding assay and submicromolar activity in a cell based chemosensitization assay.     

Primary structure of human poly(ADP-ribose) synthetase as deduced from cDNA sequence

Human poly(ADP-ribose) synthetase consists of three proteolytically separable domains, the first for binding of DNA, the second for automodification, and the third for binding of the substrate, NAD (Ushiro, H., Yokoyama, Y., and Shizuta, Y. (1987) J. Biol. Chem. 262, 2352-2357). We have isolated and sequenced cDNA clones for the enzyme using synthesized oligodeoxyribonucleotide probes based on the partial amino acid sequence of the protein. The open reading frame determined encodes a protein of 1,013 amino acid residues with a molecular weight of 113,203. The deduced amino acid sequence is consistent with the partial amino acid sequences of tryptic or alpha-chymotryptic peptides and the total amino acid composition of the purified enzyme. The native enzyme is relatively hydrophilic as judged from the hydrophilicity profile of the total amino acid sequence. The net charge of the NAD binding domain is neutral but the DNA binding domain and the automodification domain are considerably rich in lysine residue and quite basic. The DNA binding domain involves a homologous repeat in the sequence and exhibits a sequence homology with localized regions of transforming proteins such as c-fos and v-fos. Furthermore, this domain contains a unique sequence element which resembles the essential peptide sequences for nuclear location of SV40 and polyoma virus large T antigens. These facts suggest the possibility that the physiological function of poly(ADP-ribose) synthetase lies in its ability to bind to DNA and to control transformation of living eukaryotic cells like the cases of those oncogene products.     

Poly(etheno ADP-ribose) blocks poly(ADP-ribose) glycohydrolase activity

Poly(ADP-ribose) is a biopolymer synthesized by poly(ADP-ribose) polymerases. Recent findings suggest the possibility for modulation of cellular functions including cell death and mitosis by poly(ADP-ribose). Derivatization of poly(ADP-ribose) may be useful for investigating the effects of poly(ADP-ribose) on various cellular processes. We prepared poly(etheno ADP-ribose) (poly(epsilonADP-ribose)) by converting the adenine moiety of poly(ADP-ribose) to 1-N(6)-etheno adenine residues. Poly(epsilonADP-ribose) is shown to be highly resistant to digestion by poly(ADP-ribose) glycohydrolase (Parg). On the other hand, poly(epsilonADP-ribose) could be readily digested by phosphodiesterase. Furthermore, poly(epsilonADP-ribose) inhibited Parg activity to hydrolyse ribose-ribose bonds of poly(ADP-ribose). This study suggests the possibility that poly(epsilonADP-ribose) might be a useful tool for studying the poly(ADP-ribose) dynamics and function of Parg. This study also implies that modification of the adenine moiety of poly(ADP-ribose) abrogates the susceptibility to digestion by Parg.     

Inhibitors of poly (ADP-ribose) polymerase suppress lipopolysaccharide-induced nitrite formation in macrophages.

Stimulating bone marrow derived macrophages with LPS results in the induction of NO-synthase as measured by NO2- formation. Inhibitors of poly(ADP-ribose)polymerase, namely nicotinamide, 3-aminobenzamide and 3-methoxybenzamide, prevented NO2- formation in a dose dependent manner. Inhibition was most effective if the inhibitors were added at the same time as LPS. When added 10 h after exposure to LPS, a time at which expression of the enzyme had reached its maximum, no inhibition was observed. The inhibitors also blocked early events in activation such as protein and RNA-synthesis as well as DNA-synthesis. Thus prevention of NO2- formation may be related to inhibition of these events. Activation of macrophages by LPS was not accompanied by an increase but rather by a small decrease in ADP-ribosyltransferase activity. Whether this decrease plays a physiological role in activation needs further exploration.     

Importance of poly(ADP-ribose) glycohydrolase in the control of poly(ADP-ribose) metabolism.

Poly(ADP-ribosyl)ation is a posttranslational modification that alters the functions of the acceptor proteins and is catalyzed by the poly(ADP-ribose) polymerase (PARP) family of enzymes. Following DNA damage, activated poly(ADP-ribose) polymerase-1 (PARP-1) catalyzes the elongation and branching of poly(ADP-ribose) (pADPr) covalently attached to nuclear target proteins. Although the biological role of poly(ADP-ribosyl)ation has not yet been defined, it has been implicated in many important cellular processes such as DNA repair and replication, modulation of chromatin structure, and apoptosis. The transient nature and modulation of poly(ADP-ribosyl)ation depend on the activity of a unique cytoplasmic enzyme called poly(ADP-ribose) glycohydrolase which hydrolyzes pADPr bound to acceptor proteins in free ADP-ribose residues. While the PARP homologues have been recently reviewed, there are relatively scarce data about PARG in the literature. Here we summarize the latest advances in the PARG field, addressing the question of its putative nucleo-cytoplasmic shuttling that could enable the tight regulation of pADPr metabolism. This would contribute to the elucidation of the biological significance of poly(ADP-ribosyl)ation.     

Proteome-wide identification of poly(ADP-ribose) binding proteins and poly(ADP-ribose)-associated protein complexes

Poly(ADP-ribose) (pADPr) is a polymer assembled from the enzymatic polymerization of the ADP-ribosyl moiety of NAD by poly(ADP-ribose) polymerases (PARPs). The dynamic turnover of pADPr within the cell is essential for a number of cellular processes including progression through the cell cycle, DNA repair and the maintenance of genomic integrity, and apoptosis. In spite of the considerable advances in the knowledge of the physiological conditions modulated by poly(ADP-ribosyl)ation reactions, and notwithstanding the fact that pADPr can play a role of mediator in a wide spectrum of biological processes, few pADPr binding proteins have been identified so far. In this study, refined in silico prediction of pADPr binding proteins and large-scale mass spectrometry-based proteome analysis of pADPr binding proteins were used to establish a comprehensive repertoire of pADPr-associated proteins. Visualization and modeling of these pADPr-associated proteins in networks not only reflect the widespread involvement of poly(ADP-ribosyl)ation in several pathways but also identify protein targets that could shed new light on the regulatory functions of pADPr in normal physiological conditions as well as after exposure to genotoxic stimuli.     

Cytoplasmic poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase in AEV-transformed chicken erythroblasts

Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase activities were both investigated in chicken erythroblasts transformed by Avian Erythroblastosis Virus. Respectively 21% and 58% of these activities were found to be present in the post-mitochondrial supernatant (PMS). Fractionation of the PMS on sucrose gradients and poly(A⁺) mRNA detection by hybridization to [³H] poly(U) show that cytoplasmic poly(ADP-ribose) polymerase is exclusively localized in free mRNP. The glycohydrolase activity sedimented mostly in the 6 S region but 1/3 of the activity was in the free mRNP zone. Seven poly(ADP-ribose) protein acceptors were identified in the PMS in the Mr 21000–120000 range. The Mr 120000 protein corresponds to automodified poly(ADP-ribose) polymerase. A Mr 21000 protein acceptor is abundant in PMS and a Mr 34000 is exclusively associated with ribosomes and ribosomal subunits. The existence of both poly(ADP-ribose) polymerase and glycohydrolase activities in free mRNP argues in favour of a role of poly(ADP-ribosylation) in mRNP metabolism. A possible involvement of this post translational modification in the mechanisms of repression-derepression of mRNA is discussed.Abbreviations ADP-ribose adenosine (5) diphospho(5)--D ribose - poly(ADP-ribose) polymer of ADP-ribose - mRNP messenger ribonucleoprotein particles - PMSF phenylmethylsulfonyl fluoride - LDS lithium dodecyl sulfate - TCA trichloroacetic acid     

Detection of poly(ADP-ribose) polymerase and its reaction product poly(ADP-ribose) by immunocytochemistry

Summary Poly(ADP-ribose) polymerase catalyses the formation of ADP-ribose polymers covalently attached to various nuclear proteins, using NAD⁺ as substrate. The activity of this enzyme is strongly stimulated upon binding to DNA single or double strand breaks. Poly(ADP-ribosyl)ation is an immediate cellular response to DNA damage and is thought to be involved in DNA repair, genetic recombination, apoptosis and other processes during which DNA strand breaks are formed. In recent years we and others have established cell culture systems with altered poly(ADP-ribose) polymerase activity. Here we describe immunocytochemistry protocols based on the use of antibodies against the DNA-binding domain of human poly(ADP-ribose) polymerase and against its reaction product poly(ADP-ribose). These protocols allow for the convenient mass screening of cell transfectants with overexpression of poly(ADP-ribose) polymerase or of a dominant-negative mutant for this enzyme, i.e. the DNA-binding domain. In addition, the immunocytochemical detection of poly(ADP-ribose) allows screening for cells with altered enzyme activity.     

Intracellular distribution of poly(ADP-ribose) synthetase in rat spermatogenic cells

The highest activity of poly(ADP-ribose) synthetase was found in the testis among several rat tissues tested. Subcellular fractionation of the testis demonstrated that the synthetase was localized primarily in the nucleus and partially in the microsomal-ribosomal fraction. This result was confirmed by immunocytochemical staining with the enzyme-specific antibody. The synthetase was localized in the nuclei of interstitial cells, Sertoli cells, spermatogonia, and spermatocytes. In addition, round spermatids showed a granular staining in the cytoplasm, which was comparable in intensity with that in the nucleus. The cytoplasmic synthetase had a molecular weight of 115,000 and synthesized oligomers of ADP-ribose on itself (automodification). The synthetase activity in the isolated cytoplasmic fraction was stimulated about threefold by the addition of DNA and depressed by treatment with DNase I, suggesting the presence of endogenous activator DNA. A candidate DNA for such an activator was isolated from the microsomal-ribosomal fraction, and identified tentatively as mitochondrial DNA on the basis of its size and restriction fragment patterns.     

Presence of poly (ADP-ribose) polymerase and poly (ADP-ribose) glycohydrolase in the dinoflagellate Crypthecodinium cohnii

Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase have been detected in chromatin extracts from the dinoflagellate Crypthecodinium cohnii. Poly(ADP-ribose) glycohydrolase was detected by the liberation of ADP-ribose from poly(ADP-ribose). Poly(ADP-ribose) polymerase was proved by (a) demonstration of phosphoribosyl-AMP in the phosphodiesterase digest of the reaction product, (b) demonstration of ADP-ribose oligomers by fractionation of the reaction product on DEAE-Sephadex. The (ADP-ribose)-protein transfer is dependent on DNA; it is inhibited by nicotinamide, thymidine, theophylline and benzamide. The protein-(ADP-ribose bond is susceptible to 0.1 M NaOH (70%) and 0.4 M NH2OH (33%). Dinoflagellates, nucleated protists, are unique in that their chromatin lacks histones and shows a conformation like bacterial chromatin [Loeblich, A. R., III (1976) J. Protozool. 23, 13--28]; poly(ADP-ribose) polymerase, however, has been found only in eucaryotes. Thus our results suggest that histones were not relevant to the establishment of poly(ADP-ribose) during evolution.