Peroxynitrite, a cytotoxic oxidant formed from nitric oxide (NO) and superoxide, induces DNA strand breakage, which activates the nuclear enzyme poly(ADPribose) synthase (PARS; EC 2.4.2.30). The cellular function of PARS was determined in fibroblast lines from PARS knockout animals (PARS ؊/؊ ) and corresponding wild-type animals (PARS ؉/؉ ), with the aid of the lipophilic PARS inhibitor 5-iodo-6-amino-1,2-benzopyrone (INH 2 BP). We investigated the role of PARS in peroxynitrite-induced fibroblast injury in vitro and also in the development of arthritis in vivo. Nitric oxide (NO), superoxide, and their cytotoxic reaction product peroxynitrite (ONOO Ϫ ) are terminal mediators of cellular injury in various forms of inflammation. In vitro studies employing conventional inhibitors of the nuclear enzyme poly(ADP-ribose) synthase (PARS; EC 2.4.2.30) suggested that the oxidative injury in response to oxy radicals and peroxynitrite is related to DNA single-strand breakage and consequent activation of PARS (1, 2). Massive ADPribosylation of nuclear proteins by PARS then results in cellular energy depletion and injury, reminiscent of necrosis (1-3). However, objections can be raised against the conclusions of these studies, because the commonly used relatively high concentrations of PARS inhibitors (e.g., nicotinamide and benzamide analogs), have additional effects as free radical scavengers, and have short cellular residence time (4-6).More recently a potent pharmacologically active inhibitor of PARS, the lipophilic 6-iodo-5-amino-1,2-benzopyrone (INH 2 BP), was developed (7, 8). Moreover, a genetically engineered mouse line that lacks PARS is now available: a fibroblast cell line from these animals can be used for in vitro investigations (9). These tools allow a direct testing of the role of PARS. The present work was designed to elucidate (i) whether inhibition of PARS by INH 2 BP protects against cellular oxidant injury triggered by peroxynitrite, a cytotoxic oxidant produced by the reaction of superoxide and NO (10-14); (ii) whether the PARS The results of the current study support the role of PARS activation in the peroxynitrite-mediated cellular oxidant injury and inflammation, and they demonstrate that either deletion of PARS or its selective inhibition by INH 2 BP protects against inflammatory cell injury.

Retroviral nucleocapsid and gag-precursor proteins from all known strains of retroviruses contain one or two copies of an invariant sequence, Cys-X2-Cys-X4-His-X4-Cys, that is populated with zinc in mature particles. Modification of cysteine or histidine residues results in defective packaging of genomic viral RNA and formation of non-infectious particles, making these structures potentially attractive targets for antiviral therapy. We recently reported that aromatic C-nitroso ligands of poly(ADP-ribose) polymerase preferentially destabilize one of the two (Cys-X2-Cys-X28-His-X2-Cys) zinc-fingers with concomitant loss of enzymatic activity, coincidental with selective cytocidal action of the C-nitroso substituted ligands on cancer cells. Based on the occurrence of (3Cys, 1His) zinc-binding sites in both retroviral nucleocapsid and gag proteins and in poly(ADP-ribose) polymerase, we reasoned that the C-nitroso compounds may also have antiretroviral effects. We show here that two such compounds, 3-nitrosobenzamide and 6-nitroso-1,2-benzopyrone, inhibit infection of human immunodeficiency virus HIV-1 in human lymphocytes and also eject zinc from isoalted HIV-1 nucleocapsid zinc fingers and from intact HIV-1 virions. Thus the design of zinc-ejecting agents that target retroviral zinc fingers represents a new approach to the chemotherapy of AIDS.

The enzymatic mechanism of poly(ADP-ribose) polymerase (PARP-1) has been analyzed in two in vitro systems: (a) in solution and (b) when the acceptor histones were attached to a solid surface. In system (a), it was established that the coenzymatic function of dsDNAs was sequence-independent. However, it is apparent from the calculated specificity constants that the AT homopolymer is by far the most effective coenzyme and randomly damaged DNA is the poorest. Rates of auto(poly-ADP-ribosylation) with dsDNAs as coenzymes were nearly linear for 20 min, in contrast to rates with dcDNA, which showed product [(ADPR)n] inhibition. An allosteric activation of auto(poly-ADP-ribosylation) by physiologic cellular components, Mg2+, Ca2+, and polyamines, was demonstrated, with spermine as the most powerful activator. On a molar basis, histones H(1) and H(3) were the most effective PARP-1 activators, and their action was abolished by acetylation of lysine end groups. It was shown in system (b) that oligo(ADP-ribosyl) transfer to histone H(1) is 1% of that of auto(poly-ADP-ribosylation) of PARP-1, and this trans(ADP-ribosylation) is selectively regulated by putrescine (activator). Physiologic cellular concentrations of ATP inhibit PARP-1 auto(poly-ADP-ribosylation) but less so the transfer of oligo(ADP-ribose) to histones, indicating that PARP-1 auto(ADP-ribosylation) activity is dormant in bioenergetically intact cells, allowing only trans(ADP-ribosylation) to take place. The inhibitory mechanism of ATP on PARP-1 consists of a noncompetitive interaction with the NAD site and competition with the coenzymic DNA binding site. A novel regulation of PARP-1 activity and its chromatin-related functions by cellular bioenergetics is proposed that occurs in functional cells not exposed to catastrophic DNA damage.

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Suspensions of morphologically intact isolated rat-liver cells were used in conjunction with specific inhibitors to identify and quantitate the hepatic hydrogen-trans'ocating systems involved in the transfer ofreducing-equivalents from sorbitol or glycerol to 0,. Rates of hydrogen translocation were derived either from measurement of the major products of substrate metabolism or from rates of substrate utilization. It was found that a t saturating substrate concentrations, rates of sorbitol or glycerol 3-phosphate oxidation were closely similar (about 1.8 pmol x g wet weight-l x min-I). There was an inverse relationship between rates of sorbitol and glycerol uptake so that the rate of hydrogen flux to 0, from substrate mixtures was no greater than that from either substrate added separately. Rates of sorbitol and glycerol consumption were increased by pyruvate acting as a cytoplasmic hydrogen acceptor.It is concluded from these observations that a t high substrate levels and in the absence of a cytoplasmic hydrogen acceptor, hydrogen translocation is the rate-limiting process in the hepatic metabolism of sorbitol and glycerol and that the flux of reducing equivalents to 0, from these two substrates involves shared hydrogen-translocating systems. At low levels of substrate, more likely to be encountered in vivo, the rate of sorbitol or glycerol metabolism is dependent also on substrate concentration, but even under these circumstances it was found that the capacity of the hydrogen-translocating systems governs the over-all rate of metabolism whenever substrate mixtures were present.The nature of these systems was assessed by the use of specific inhibitors. About 1501, of the flux of reducing equivalents to 0, involved antimycin-insensitive pathways, presumably microsomal. A further 40°/, passed to 0, by a rotenone-insensitive path, most likely involving flavinlinked mitochondrial glycerolphosphate dehydrogenase. The remainder of the flux was rotenonesensitive, but less than half of this utilized malate-oxaloacetate or malate-aspartate shuttles.The pathway for this residual rotenone-sensitive fraction (about 20-30 ,Ilo of the total flux) remains to be clarified. These data suggest that in parenchymal cells from normal rat liver the glycerol 3-phosphate shuttle may be more important for the transfer of reducing equivalents from cytoplasm to mitochondria than has been previously recognized.Sorbitol uptake by the cells was inhibited up to 7001, by uncoupling agents. This inhibition could be overcome by addition of pyruvate as a cytoplasmic hydrogen acceptor or by artificial electron acceptors. This implies that uncoupling agents prevent the oxidation of cytoplasmic NADH by interfering with the operation of the normal hydrogen shuttles between cytoplasm and mitochondria and that these shuttles are energy-dependent.The rate-limiting and energy-dependent nature of the hydrogen translocating systems as revealed by these studies identify them as potential sites for metabolic regulation and as possible targets for hormonal...

The enzymatic transfer of ADP-ribose from NAD to histone H 1 (defined as trans-poly(ADP-ribosylation)) or to PARP I (defined as auto-poly(ADP-ribosylation)) was studied with respect to the nature of the DNA required as a coenzyme. Linear double-stranded DNA (dsDNA) containing the MCAT core motif was compared with DNA containing random nicks (discontinuous or dcDNA). The dsDNAs activated trans-poly(ADP-ribosylation) about 5 times more effectively than dcDNA as measured by V max . Activation of auto-poly(ADP-ribosylation) by dcDNA was 10 times greater than by dsDNA. The affinity of PARP I toward dcDNA or dsDNA in the auto-poly(ADP-ribosylation) was at least 100-fold lower than in trans-poly(ADP-ribosylation) (K a ‫؍‬ 1400 versus 3-15, respectively). Mg 2؉ inhibited trans-poly(ADP-ribosylation) and so did dcDNA at concentrations required to maximally activate auto-poly(ADP-ribosylation). Mg 2؉ activated auto-poly(ADP-ribosylation) of PARP I. These results for the first time demonstrate that physiologically occurring dsDNAs can serve as coenzymes for PARP I and catalyze preferentially trans-poly(ADPribosylation), thereby opening the possibility to study the physiologic function of PARP I.The activity of poly(ADP-ribose) polymerase (PARP I, 1 EC 2.4.2.30) was originally considered to represent a novel form of covalent protein modification analogous to phosphorylation, acetylation, etc. (1). Immunochemical analysis of normal adult rat tissues for poly(ADP-ribose) (pADPR) showed that 99% of covalently bound polymer was found in the non-histone fraction of nuclear proteins, with histone H 1 being the major acceptor among the remaining 1% (2). The actual quantities of protein-bound pADPR in normal tissues, however, are small (200 -250 ng/g, dry weight) as compared with the large abundance of PARP I protein itself (0.5 ϫ 10 6 copies/cell (cf. Ref. 4). This difference suggests that PARP I activity in vivo is highly regulated. The secondary structure of pADPR (3) predicts that binding of modified proteins to other proteins or to DNA will be altered by poly(ADP-ribosylation). Furthermore, unmodified PARP I avidly binds to a large number of nuclear proteins (4), an association that is completely blocked by added histone H 1 . In addition, an assortment of transcription factors was shown to bind to PARP I (cf. Ref. 5), and a transcriptional regulatory role of PARP I in muscle differentiation has been identified (6). The MCAT element derived from this work (cf. Ref. 6) served as a basic DNA sequence for the construction of dsDNAs studied in the present experiment. Recently the topology of the direct binding of PARP I to Topo 1 has been demonstrated in detail (7). The binding of PARP I to histone H 1 induces conformational changes in the latter coinciding with new phosphorylation sites for Cdc2-kinase (8).Although the enzymatic transfer of ADP-ribose from NAD to PARP I and to several nuclear proteins has been well established (1), it has remained unclear what factors determine protein auto-or heteromodification by poly(ADP-ribos...

1 Activation of poly(ADP-ribose) synthetase (PARS, also termed polyADP-ribose polymerase or PARP) has been proposed as a major mechanism contributing to b-cell destruction in type I diabetes. In the present study, we have investigated the role of PARS in mediating the induction of diabetes and b-cell death in the multiple-low-dose-streptozotocin (MLDS) model of type I diabetes. 2 Mice genetically de®cient in PARS were found to be less sensitive to MLDS than wild type mice, with a lower incidence of diabetes and reduced hyperglycemia. 3 A potent inhibitor of PARS, 5-iodo-6-amino-1,2-benzopyrone (INH 2 BP), was also found to protect mice from MLDS and prevent b-cell loss, in a dose-dependent manner. Paradoxically, in the PARS de®cient mice, the compound increased the onset of diabetes. 4 In vitro the cytokine combination; interleukin-1b, tumor necrosis factor-a and interferon-g inhibited glucose-stimulated insulin secretion from isolated rat islets of Langerhans and decreased RIN-5F cell viability. The PARS inhibitor, INH 2 BP, protected both the rat islets and the b-cell line, RIN-5F, from these cytokine-mediated e ects. These protective e ects were not mediated by inhibition of cytokine-induced nitric oxide formation. 5 Inhibition of PARS by INH 2 BP was unable to protect rat islet cells from cytokine-mediated apoptosis. 6 Cytokines, peroxynitrite and streptozotocin were all shown to induce PARS activation in RIN-5F cells, an e ect suppressed by INH 2 BP. 7 The present study provides evidence for in vivo PARS activation contributing to b-cell damage and death in the MLDS model of diabetes, and indicates a role for PARS activation in cytokinemediated depression of insulin secretion and cell viability in vitro. British Journal of Pharmacology (2001) 133, 909 ± 919

Cellular proteins extracted from normal and cancer cells bind polymerizing ADP-ribose transferase (pADPRT) on nitrocellulose membrane transblots. Histones at 1 mg/ml concentration completely prevent the binding of pADPRT to cellular proteins, indicating that the binding of histones to pADPRT sites competitively blocks the association of pADPRT to proteins other than histones. The direct binding of pADPRT to histones is shown by cross-linking with glutaraldehyde. The COOH-terminal basic histone H1 tail binds to the basic polypeptide domain of pADPRT. The basic domain present in the NH2-terminal part of core histones is the probable common structural feature of all core histones that accounts for their binding to pADPRT. Two polypeptide domains of pADPRT were identified, by way of CNBr fragments, to bind histones. These two domains are located within the 64-kDa fragment of pADPRT and are contiguous with the polypeptide domains that were shown to participate in self-association of pADPRT, ending at the 606th amino acid residue. The polypeptide domains of pADPRT which participate in DNA binding are thus shown to associate also with other proteins. Intact pADPRT binds to both the zinc-free or zinc-reconstituted basic polypeptide fragments of pADPRT. Histones activate auto-poly(ADP)-ribosylation of pADPRT by increasing the number of short oligomers on pADPRT. This reaction is also dependent in a biphasic manner on the concentration of pADPRT. Histones in solution are only marginally poly(ADP)-ribosylated but are good polymer acceptors when incorporated into artificial nucleosome structures.