Poly(ADP-ribose)polymerase 1 stimulates the AP-site cleavage activity of tyrosyl-DNA phosphodiesterase 1

The influence of poly(ADP-ribose)polymerase 1 (PARP1) on the apurinic/apyrimidinic (AP)-site cleavage activity of tyrosyl–DNA phosphodiesterase 1 (TDP1) and interaction of PARP1 and TDP1 were studied. The efficiency of single or clustered AP-site hydrolysis catalysed by TDP1 was estimated. It was shown that the efficiency of AP-site cleavage increases in the presence of an additional AP-site in the opposite DNA strand depending on its position. PARP1 stimulates TDP1; the stimulation effect was abolished in the presence of NAD⁺. The interaction of these two proteins was characterized quantitatively by measuring the dissociation constant for the TDP1–PARP1 complex using fluorescently-labelled proteins. The distance between the N-termini of the proteins within the complex was estimated using FRET. The data obtained suggest that PARP1 and TDP1 bind in an antiparallel orientation; the N-terminus of the former protein interacts with the C-terminal domain of the latter. The functional significance of PARP1 and TDP1 interaction in the process of DNA repair was demonstrated for the first time.     

Poly(ADP-ribose) polymerase activity in intact or permeabilized leukocytes from mammalian species of different longevity

Poly(ADP-ribosyl)ation is a eukaryotic posttranslational protein modification catalyzed by poly(ADP-ribose) polymerase (PARP), a highly conserved nuclear enzyme which uses NAD as substrate. We have previously tested PARP activity in permeabilized mononuclear blood cells (MNC) from 13 mammalian species as a function of the species-specific life span. A direct and maximal stimulus of PARP activation was provided by including saturating amounts of a double-stranded ollgonucleotide in the PARP-reaction buffer. The data yielded a strong positive correlation between PARP activities and the species' maximal life spans (r=0.84; p0.001). Here, we investigated the formation of poly(ADP-ribose) inliving MNC from two mammalian species with widely differing longevity (rat and man) by immunofluorescence detection of poly(ADP-ribose). The fraction of positive cells was recorded, following -irradiation of intact MNC, as a semiquantitative estimation of poly(ADP-ribose) formation. Human samples displayed a significantly higher percentage of positivity than did those from rats, consistent with our previous results on permeabilized cells. While rat MNC had a higher NAD content than human MNC, the number of radiation-induced DNA strand breaks was not significantly different in the two species. Since poly(ADP-ribosyl)ation is apparently involved in DNA repair and the cellular recovery from DNA damage, we speculate that the higher poly(ADP-ribosyl)ation capacity of long-lived species might more efficiently help to slow down the accumulation of unrepaired DNA damage and of genetic alterations, as compared with short-lived species. (Mol Cell Biochem138: 85–90, 1994)     

Poly(ADP-ribose) polymerase in plants affects energy homeostasis, cell death and stress tolerance

Plants contain two genes that code for poly(ADP-ribose) polymerase (PARP): parp1 and parp2. Both PARPs are activated by DNA damage caused by, example reactive oxygen species. Upon activation polymers of ADP-ribose are synthesized on a range of nuclear enzymes using NAD(+) as substrate. Here, we show that in plants stresses such as drought, high light and heat activate PARP causing NAD(+) breakdown and ATP consumption. When the PARP activity is reduced by means of chemical inhibitors or by gene silencing, cell death is inhibited and plants become tolerant to a broad range of abiotic stresses like high light, drought and heat. Plant lines with low poly(ADP-ribosyl)ation activity maintain under stress conditions their energy homeostasis by reducing NAD(+) breakdown and consequently energy consumption. The higher energy-use efficiency avoids the need for a too intense mitochondrial respiration and consequently reduces the formation of reactive oxygen species. From these results it can be concluded that breeding or engineering for a high energy-use efficiency under stress conditions is a valuable, but until today nearly unexploited, approach to enhance overall stress tolerance of crops.     

Regulatory mechanisms of poly(ADP-ribose) polymerase

Here, we describe the latest developments on the mechanistic characterization of poly(ADP-ribose) polymerase (PARP) [EC 2.4.2.30], a DNA-dependent enzyme that catalyzes the synthesis of protein-bound ADP-ribose polymers in eucaryotic chromatin. A detailed kinetic analysis of the automodification reaction of PARP in the presence of nicked dsDNA indicates that protein-poly(ADP-ribosyl)ation probably occurs via a sequential mechanism since enzyme-bound ADP-ribose chains are not reaction intermediates. The multiple enzymatic activities catalyzed by PARP (initiation, elongation, branching and self-modification) are the subject of a very complex regulatory mechanism that may involve allosterism. For instance, while the NAD+ concentration determines the average ADP-ribose polymer size (polymerization reaction), the frequency of DNA strand breaks determines the total number of ADP-ribose chains synthesized (initiation reaction). A general discussion of some of the mechanisms that regulate these multiple catalytic activities of PARP is presented below.     

The 193-kD vault protein, VPARP, is a novel poly(ADP-ribose) polymerase.

Mammalian vaults are ribonucleoprotein (RNP) complexes, composed of a small ribonucleic acid and three proteins of 100, 193, and 240 kD in size. The 100-kD major vault protein (MVP) accounts for >70% of the particle mass. We have identified the 193-kD vault protein by its interaction with the MVP in a yeast two-hybrid screen and confirmed its identity by peptide sequence analysis. Analysis of the protein sequence revealed a region of approximately 350 amino acids that shares 28% identity with the catalytic domain of poly(ADP-ribose) polymerase (PARP). PARP is a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage. The catalytic domain of p193 was expressed and purified from bacterial extracts. Like PARP, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction; thus, the 193-kD protein is a new PARP. Purified vaults also contain the poly(ADP-ribosyl)ation activity, indicating that the assembled particle retains enzymatic activity. Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP. Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s). A portion of p193 is nuclear and localizes to the mitotic spindle.     

Amino acids stimulate glycyl-tRNA synthetase nuclear localization for mammalian target of rapamycin expression in bovine mammary epithelial cells

Amino acids are required for the activation of mammalian target of rapamycin (mTOR) to increase cell growth, protein and lipid synthesis, and inhibit autophagy. However, the mechanism through which amino acids activate the mTOR signaling is still largely unknown. In our previous study, we discovered that glycyl-tRNA synthetase (GlyRS) is a key mediator of amino-acid-induced mTOR expression and activation in bovine mammary epithelial cells (BMECs). Here we show that amino acids stimulate GlyRS nuclear localization for mTOR expression in BMECs. Met stimulates GlyRS nuclear localization, and the nuclear GlyRS is cleaved into a C-terminus-containing truncated form. We prove that GlyRS has a bipartite nuclear leading sequences, and GlyRS is phosphorylated at Thr544 and Ser704 in the cytoplasm under the stimulation of amino acids (Met, Leu, and Lys). The nuclear GlyRS physically binds to nuclear factor kappa B1, triggers its phosphorylation, thereby enhancing mRNA expression of its target genes including mTOR, S6K1, and 4EBP1. We further demonstrate that GlyRS is required for the inhibition of autophagy by Met. Thus our work elucidates that amino acids trigger GlyRS phosphorylation and nuclear localization to enhance the mRNA expression of mTOR.     

聚腺苷二磷酸核糖聚合酶抑制剂耐药性研究进展

聚腺苷二磷酸核糖聚合酶（PARP）抑制剂可选择性杀死同源重组功能缺陷的肿瘤细胞,而对正常细胞的危害较小,这是“合成致死”理论应用于临床的典型范例。尽管 PARP 抑制剂作为一种新型靶向药物,极具应用潜力,但其临床应用也面临诸多问题,其中耐药性的产生被认为是限制 PARP 抑制剂临床应用的重要原因之一。简介 PARP-1 的功能及 PARP-1 抑制剂研究进展,着重综述 PARP-1 抑制剂耐药的临床表现、可能的发生机制及逆转策略,为 PARP-1 抑制剂的临床合理应用提供参考。     

Altered poly(ADP-ribose) metabolism impairs cellular responses to genotoxic stress in a hypomorphic mutant of poly(ADP-ribose) glycohydrolase

Genotoxic stress activates nuclear poly(ADP-ribose) (PAR) metabolism leading to PAR synthesis catalyzed by DNA damage activated poly(ADP-ribose) polymerases (PARPs) and rapid PAR turnover by action of nuclear poly(ADP-ribose) glycohydrolase (PARG). The involvement of PARP-1 and PARP-2 in responses to DNA damage has been well studied but the involvement of nuclear PARG is less well understood. To gain insights into the function of nuclear PARG in DNA damage responses, we have quantitatively studied PAR metabolism in cells derived from a hypomorphic mutant mouse model in which exons 2 and 3 of the PARG gene have been deleted (PARG-Delta2,3 cells), resulting in a nuclear PARG containing a catalytic domain but lacking the N-terminal region (A domain) of the protein. Following DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), we found that the activity of both PARG and PARPs in intact cells is increased in PARG-Delta2,3 cells. The increased PARG activity leads to decreased PARP-1 automodification with resulting increased PARP activity. The degree of PARG activation is greater than PARP, resulting in decreased PAR accumulation. Following MNNG treatment, PARG-Delta2,3 cells show reduced formation of XRCC1 foci, delayed H2AX phosphorylation, decreased DNA break intermediates during repair, and increased cell death. Our results show that a precise coordination of PARPs and PARG activities is important for normal cellular responses to DNA damage and that this coordination is defective in the absence of the PARG A domain.     

聚腺苷二磷酸核糖基聚合酶──在DNA损伤修复及程序性死亡中的作用

聚腺苷二磷酸核糖基聚合酶(poly (ADP-ribose) polyerase, PARP)是存在于多数真核细胞中的一个蛋白质翻译后修饰酶,它可催化组蛋白H₁等重要核蛋白及它自身的聚腺苷二磷酸核糖基化作用.细胞受到外界损伤因子作用时, DNA发生链断裂,PARP结合到DNA断裂口,其催化活性被激活,修饰受体蛋白,进而引发一系列级联反应.这种性质使PARP有可能作为细胞内的分子感受器和传感器,启动细胞内对损伤作出反应的信号传导机制,从而根据细胞受损程度决定细胞的命运：修复或是死亡．     

Growth-phase-dependent response to DNA damage in poly(ADP-ribose) polymerase deficient cell lines: basis for a new hypothesis describing the role of poly(ADP-ribose) polymerase in DNA replication and repair

We have studied the role of poly(ADP-ribose) polymerase in the repair of DNA damage induced by x-ray and N-methyl N-nitro-N-nitrosoguanidine (MNNG) by using V79 chinese hamster cells, and two derivative mutant cell lines, ADPRT54 and ADPRT351, that are deficient in poly(ADP-ribose) polymerase activity. Under exponentially growing conditions these mutant cell lines are hypersensitive to x-irradiation and MNNG compared to their parental V79 cells which could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in the repair of DNA damage. However, the level of DNA strand breaks induced by x-irradiation and MNNG and their rates of repair are similar in all the cell lines, thus suggesting that it may not be the difference in strand break formation or in its rate of repair that is contributing to the enhanced cell killing in exponentially growing poly(ADP-ribose) polymerase deficient cell lines. In contrast, under growth-arrested conditions, all three cell lines become similarly sensitive to both x-irradiation and MNNG, thus suggesting that poly(ADP-ribose) polymerase may not be involved in the repair of DNA damage in growth-arrested cells. These paradoxical results could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in DNA repair in a cell-cycle-dependent fashion, however, it is functionally active throughout the cell cycle. To resolve this dilemma and explain these results and those obtained by many others, we propose that the normal function of poly(ADP-ribose) polymerase is to prevent DNA recombination processes and facilitate DNA ligation.     

Pharmacological inhibition of poly(ADP-ribose) polymerase inhibits angiogenesis

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which plays an important role in regulating cell death and cellular responses to DNA repair. Pharmacological inhibitors of PARP are being considered as treatment for cancer both in monotherapy as well as in combination with chemotherapeutic agents and radiation, and were also reported to be protective against untoward effects exerted by certain anticancer drugs. Here we show that pharmacological inhibition of PARP with 3-aminobenzamide or PJ-34 dose-dependently reduces VEGF-induced proliferation, migration, and tube formation of human umbilical vein endothelial cells in vitro. These results suggest that treatment with PARP inhibitors may exert additional benefits in various cancers and retinopathies by decreasing angiogenesis.     

Poly(ADP-ribose) polymerase inhibition prevents both apoptotic-like delayed neuronal death and necrosis after H(2)O(2) injury

Toxic reactive oxygen species (ROS) such as hydrogen peroxide, nitric oxide, superoxide, and the hydroxyl radical are generated in a variety of neuropathological conditions and cause significant DNA damage. We determined the effects of 3-aminobenzamide (AB), an inhibitor of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), on cell death in differentiated PC12 cells, a model of sympathetic neurons, after H(2) O(2) injury. Exposure to 0.5 mm H(2) O(2) resulted in a significant decrease in intracellular NAD(H), NADP(H), and ATP levels. This injury resulted in the death of 90% of the cells with significant necrosis early (2 h) after injury and increased apoptosis (12-24 h after injury), as measured by PS exposure and the presence of cytoplasmic oligonucleosomal fragments. Treatment with 2.5 mm AB restored pyridine nucleotide and ATP levels and ameliorated cell death (65% versus 90%) by decreasing the extent of both necrosis and apoptosis. Interestingly, we observed that H(2) O(2) -induced injury caused a delayed cell death exhibiting features of apoptosis but in which caspase-3 like activity was absent. Moreover, pretreatment with AB restored caspase-3-like activity. Our results suggest that apoptosis and necrosis are both triggered by PARP overactivation, and that maintenance of cellular energy levels after injury by inhibiting PARP shifts cell death from necrosis to apoptosis.     

Naringin in Ganshuang Granule suppresses activation of hepatic stellate cells for anti‐fibrosis effect by inhibition of mammalian target of rapamycin

A previous study has demonstrated that Ganshuang granule (GSG) plays an anti‐fibrotic role partially by deactivation of hepatic stellate cells (HSCs). In HSCs activation, mammalian target of rapamycin (mTOR)‐autophagy plays an important role. We attempted to investigate the role of mTOR‐autophagy in anti‐fibrotic effect of GSG. The cirrhotic mouse model was prepared to demonstrate the anti‐fibrosis effect of GSG. High performance liquid chromatography (HPLC) analyses were used to identify the active component of GSG. The primary mouse HSCs were isolated and naringin was added into activated HSCs to observe its anti‐fibrotic effect. 3‐methyladenine (3‐MA) and Insulin‐like growth factor‐1 (IGF‐1) was added, respectively, into fully activated HSCs to explore the role of autophagy and mTOR. GSG played an anti‐fibrotic role through deactivation of HSCs in cirrhotic mouse model. The concentration of naringin was highest in GSG by HPLC analyses and naringin markedly suppressed HSCs activation in vitro, which suggested that naringin was the main active component of GSG. The deactivation of HSCs caused by naringin was not because of the autophagic activation but mTOR inhibition, which was supported by the following evidence: first, naringin induced autophagic activation, but when autophagy was blocked by 3‐MA, deactivation of HSCs was not attenuated or reversed. Second, naringin inhibited mTOR pathway, meanwhile when mTOR was activated by IGF‐1, deactivation of HSCs was reversed. In conclusion, we have demonstrated naringin in GSG suppressed activation of HSCs for anti‐fibrosis effect by inhibition of mTOR, indicating a potential therapeutic application for liver cirrhosis.     

Poly(ADP-ribose) turnover in quail myoblast cells: Relation between the polymer level and its catabolism by glycohydrolase

The concerted action of poly(ADP-ribose) polymerase (PARP) which synthesizes the poly(ADP-ribose) (pADPr) in response to DNA strand breaks and the catabolic enzyme poly(ADP-ribose) glycohydrolase (PARG) determine the level of polymer and the rate of its turnover. In the present study, we have shown that the quail myoblast cells have high levels of basal polymer as compared to the murine C3H10T1/2 fibroblasts. We have conducted this study to investigate how such differences influence polymer synthesis and its catabolism in the cells in response to DNA damage by alkylating agent. In quail myoblast cells, the presence of high MNNG concentration such as 200 \sgmaelig;M for 30 min induced a marginal decrease of 15% in the NAD content. For C3H10T1/2 cell line, 64 \sgmaelig;M MNNG provoked a depletion of NAD content by approximately 50%. The induction of the polymer synthesis in response to MNNG treatment was 6-fold higher in C3H10T1/2 cells than in quail myoblast cells notwithstanding the fact that 3-fold higher MNNG concentration was used for quail cells. The polymer synthesis thus induced in quail myoblast cells had a 4-5 fold longer half life than those induced in C3H10T1/2 cells. To account for the slow turnover of the polymer in the quail myoblast cells, we compared the activities of the polymer catabolizing enzyme (PARG) in the two cell types. The quail myoblast cells had about 25% less activity of PARG than the murine cells. This difference in activity is not sufficient to explain the large difference of the rate of catabolism between the two cell types implicating other cellular mechanisms in the regulation of pADPr turnover.