Toward specific functions of poly(ADP-ribose) polymerase-2

Poly(ADP-ribose) polymerase-2 (PARP-2) belongs to a family of enzymes that catalyze poly(ADP-ribosyl)ation of proteins. PARP-1 and PARP-2 are so far the only PARP enzymes whose catalytic activity has been shown to be induced by DNA-strand breaks, providing strong support for key shared functions in the cellular response to DNA damage. Accordingly, clinical trials for cancer, using PARP inhibitors that target the conserved catalytic domain of PARP proteins, are now ongoing. However, recent data suggest unique functions for PARP-2 in specific processes, such as genome surveillance, spermatogenesis, adipogenesis and T cell development. Understanding these physiological roles might provide invaluable clues to the rational development and exploitation of specific PARP-2 inhibitor drugs in a clinical setting and the design of new therapeutic approaches in different pathophysiological conditions.     

PARP inhibitors for cancer therapy

Poly(ADP-ribose) polymerase 1 (PARP-1) is a zinc-finger DNA-binding enzyme that is activated by binding to DNA breaks. Poly(ADP-ribosyl)ation of nuclear proteins by PARP-1 converts DNA damage into intracellular signals that activate either DNA repair by the base-excision pathway or cell death. A family of 18 PARPs has been identified, but only the most abundant, PARP-1 and PARP-2, which are both nuclear enzymes, are activated by DNA damage. PARP inhibitors of ever-increasing potency have been developed in the 40 years since the discovery of PARP-1, both as tools for the investigation of PARP-1 function and as potential modulators of DNA-repair-mediated resistance to cytotoxic therapy. Owing to the high level of homology between the catalytic domains of PARP-1 and PARP-2, the inhibitors probably affect both enzymes. Convincing biochemical evidence, which has been corroborated by genetic manipulation of PARP-1 activity, shows that PARP inhibition is associated with increased sensitivity to DNA-alkylating agents, topoisomerase I poisons and ionising radiation. Novel PARP inhibitors of sufficient potency and suitable pharmacokinetic properties to allow evaluation in animal models have been shown to enhance the antitumour activity of temozolomide (a DNA-methylating agent), topoisomerase poisons and ionising radiation; indeed, the combination with temozolomide resulted in complete tumour regression in two independent studies. The combination of a PARP inhibitor and temozolomide is currently undergoing clinical evaluation for the first time.     

The Role of PARP-1 and PARP-2 Enzymes in Metabolic Regulation and Disease

While originally described as DNA damage repair agents, recent data suggest a role for poly(ADP-ribose) polymerase (PARP) enzymes in metabolic regulation by influencing mitochondrial function and oxidative metabolism. Here we review how PARP activity has a major metabolic impact and the role of PARP-1 and PARP-2 in diverse metabolic complications.     

Effect of simulated microgravity conditions on poly(ADP-ribose) polymerase activity in primary cultures of adult rat hepatocytes

The possible involvement of poly(ADP-ribose) polymerase [PARP; E.C. 2.4.2.30] in the adaptive response to low-g conditions was studied in cultured adult rat hepatocytes exposed to simulated microgravity produced by the random positioning machine (RPM-3D-clinostat). Four different poly(ADP-ribose) polymerases (PARPs) have been identified recently. The best-studied member of this family is PARP-1, a highly conserved, multimodular 113 kDa protein. In multicellular organisms PARPs catalyze poly(ADP-ribose) synthesis from NAD+ to a number of structural and catalytic proteins. Moreover, PARP-1 can control its protein and DNA interactions by catalyzing its automodification with poly(ADP-ribose) molecules that can include up to 200 ADP-ribose residues and several branching points; by these polymers, PARP-1 may nocovalently interact with other proteins and alter their functions. PARP-1 binds to DNA and is activated by free ends interacting with several other DNA damage checkpoint proteins. Thus, PARPs may target specific signal network proteins via poly(ADP-ribose) and regulate their domain functions. Poly(ADP-ribosyl)ation plays a central role in genome stability and is involved in DNA replication and repair, gene expression, cell differentiation and transformation. We have shown that a loss of PARP-1 activity is a critical event in the early molecular steps of the hepatocarcinogenesis process. Moreover, a prompt increase in this enzymatic activity is linked not only to the presence of DNA free ends but is linked also to the start of DNA synthesis. More recently, we have reported that PARP-1 is involved in hormone-mediated gene expression in vitro and in vivo during rat liver regeneration.     

Poly(ADP-ribose) Contributes to an Association between Poly(ADP-ribose) Polymerase-1 and Xeroderma Pigmentosum Complementation Group A in Nucleotide Excision Repair

Exposure to ultraviolet radiation (UVR) promotes the formation of UVR-induced, DNA helix distorting photolesions such as (6-4) pyrimidine-pyrimidone photoproducts and cyclobutane pyrimidine dimers. Effective repair of such lesions by the nucleotide excision repair (NER) pathway is required to prevent DNA mutations and chromosome aberrations. Poly(ADP-ribose) polymerase-1 (PARP-1) is a zinc finger protein with well documented involvement in base excision repair. PARP-1 is activated in response to DNA damage and catalyzes the formation of poly(ADP-ribose) subunits that assist in the assembly of DNA repair proteins at sites of damage. In this study, we present evidence for PARP-1 contributions to NER, extending the knowledge of PARP-1 function in DNA repair beyond the established role in base excision repair. Silencing the PARP-1 protein or inhibiting PARP activity leads to retention of UVR-induced photolesions. PARP activation following UVR exposure promotes association between PARP-1 and XPA, a central protein in NER. Administration of PARP inhibitors confirms that poly(ADP-ribose) facilitates PARP-1 association with XPA in whole cell extracts, in isolated chromatin complexes, and in vitro. Furthermore, inhibition of PARP activity decreases UVR-stimulated XPA chromatin association, illustrating that these relationships occur in a meaningful context for NER. These results provide a mechanistic link for PARP activity in the repair of UVR-induced photoproducts.     

Identification of candidate substrates for poly(ADP-ribose) polymerase-2 (PARP2) in the absence of DNA damage using high-density protein microarrays

Poly(ADP-ribose) polymerase-2 (PARP2) belongs to the ADP-ribosyltransferase family of enzymes that catalyze the addition of ADP-ribose units to acceptor proteins, thus affecting many diverse cellular processes. In particular, PARP2 shares with PARP1 and, as recently highlighted, PARP3 the sole property of being catalytically activated by DNA-strand breaks, implying key downstream functions in the cellular response to DNA damage for both enzymes. However, evidence from several studies suggests unique functions for PARP2 in additional processes, possibly mediated through its basal, DNA-damage unstimulated ADP-ribosylating activity. Here, we describe the development and application of a protein microarray-based approach tailored to identify proteins that are ADP-ribosylated by PARP2 in the absence of DNA damage mimetics and might thus represent useful entry points to the exploration of novel PARP2 functions. Several candidate substrates for PARP2 were identified and global hit enrichment analysis showed a clear enrichment in translation initiation and RNA helicase molecular functions. In addition, the top scoring candidates FK506-binding protein 3 and SH3 and cysteine-rich domain-containing protein 1 were selected and confirmed in a complementary assay format as substrates for unstimulated PARP2.     

Function of poly(ADP-ribose) polymerase in response to DNA damage: Gene-disruption study in mice

To elucidate the biological functions of poly(ADP-ribose) polymerase (PARP, [EC 2.4.2.30]) in DNA damage responses, genetic and biochemical approaches were undertaken. By disrupting exon 1 of the mouse PARP gene by a homologous recombination, PARP-deficient mouse embryonic stem (ES) cell lines and mice could be produced without demonstrating lethality. PARP-/- ES cells showed complete loss of PARP activity and increased sensitivity to -irradiation and an alkylating agents, indicating a physiological role for PARP in the response to DNA damage. p53, a key molecule in cellular DNA damage response, was found to stimulate PARP activity and became poly(ADP-ribosyl)ated in the presence of damaged DNA. However, PARP-/- ES cells showed p21 and Mdm-2 mRNA induction following -irradiation, indicating that PARP activity is not indispensable for p21 and Mdm-2 mRNA induction in the established p53-cascade. On the other hand, in a reconstituted reaction system, purified PARP from human placenta suppressed the pRB-phosphorylation activity in the presence of NAD and damaged DNA. Human PARP expressed in E. coli showed a similar effect on pRB-phosphorylation activity of cdk2. These findings suggest a direct involvement of PARP in the regulation of cdk activity for cell-cycle arrest.     

Interaction between PARP-1 and ATR in mouse fibroblasts is blocked by PARP inhibition

Inhibition of PARP activity results in extreme sensitization to MMS-induced cell killing in cultured mouse fibroblasts. In these MMS-treated cells, PARP inhibition is accompanied by an accumulation of S-phase cells that requires signaling by the checkpoint kinase ATR [J.K. Horton, D.F. Stefanick, J.M. Naron, P.S. Kedar, S.H. Wilson, Poly(ADP-ribose) polymerase activity prevents signaling pathways for cell cycle arrest following DNA methylating agent exposure, J. Biol. Chem. 280 (2005) 15773-15785]. Here, we examined mouse fibroblast extracts for formation of a complex that may reflect association between the damage responsive proteins PARP-1 and ATR. Co-immunoprecipitation of PARP-1 and ATR was observed in extracts prepared from MMS-treated cells, but not under conditions of PARP inhibition. Further, our experiments demonstrated PAR-adduction of ATR in extracts from control and MMS-treated cells. An interaction between purified ATR and PARP-1 was similarly demonstrated, suggesting that the observed co-immunoprecipitation of ATR and PARP-1 from cell extracts may be due to a direct interaction between the two enzymes. In addition, purified recombinant ATR is a substrate for poly(ADP-ribosyl)ation by PARP-1, and poly(ADP-ribose) adduction of PARP-1 and ATR resulted in an increase in PARP-1 and ATR co-immunoprecipitation.     

Interaction of PARP-2 with DNA structures mimicking DNA repair intermediates and consequences on activity of base excision repair proteins

Poly(ADP-ribosyl)ation is a posttranslational protein modification significant for genomic stability and cell survival in response to DNA damage. Poly(ADP-ribosyl)ation is catalyzed by poly(ADP-ribose)polymerases (PARPs). Among the 17 members of the PARP family, PARP-1 and PARP-2 are described as enzymes whose catalytic activity is stimulated by some types of DNA damages.     

Sirtuins与PARP-1在细胞凋亡过程中的相互作用

哺乳动物Sirtuins家族目前共发现7个成员：SIRT1～SIRT7,它们均为NAD+依 赖性且从细菌到人类都保守的一类酶.人们已经对这7种去乙酰化酶进行了亚细胞定位 .目前,对其研究主要集中在对细胞发育相关的重要转录因子如p53、FOXO家族及相关 蛋白的去乙酰化修饰.Sirtuins对许多生理过程有着重要的调节作用,尤其是当发现 它们对寿命延长的调控作用后,Sirtuins引起了人们极大的关注,且都发表在世界顶 级刊物上.聚ADP核糖聚合酶(poly ADP-ribose polymerase, PARP)是一类存在于大多 数真核细胞中的蛋白质翻译后修饰酶,尤其是聚ADP核糖聚合酶1(PARP-1)在细胞内 DNA损伤修复等过程中起着重要作用,该酶同样以NAD+作为催化反应的底物.有研究发 现,Sirtuins家族成员与PARP-1在细胞内某些重要生理过程中存在着相互作用.本文评 述了Sirtuins家族成员、PARP-1的生物学特点,并就其参与哺乳动物细胞凋亡的调控 机制和相关信号通路进行了详细的论述,以期对Sirtuins家族成员、PARP-1生物学功 能及其相互作用的研究提供理论指导.     

Role of PARP and protein poly-ADP-ribosylation process in regulation of cell functions

This review focuses on the biological role of enzymes involved in posttranslational modification of proteins by their poly-ADP-ribosylation, a NAD-consuming process with an emerging key role in providing fundamental cell functions. To this end, detailed analysis of structural organization in relation to basic functions of the poly(ADP-ribose) polymerase-1 (PARP-1), the founding member of the PARP family, and other poly(ADP-ribose) polymerase isoforms is presented here. These include the current views on the role of PARP family enzymes and processes of poly-ADP-ribosylation of proteins in chromatin structure remodeling, DNA damage repair, regulation of gene expression, and integration of cellular signaling pathways. Considerable attention is paid to the involvement of PARP in cellular functions, particularly in cell division, intracellular transport of macromolcules, proteasomal protein degradation, immune response and caspase-independent necrotic pathways defined as necroptosis (programmed necrosis). In the light of the remarkable successes that have been reported for treating inflammatory disorders and cancer with different classes of PARPs inhibitors, we discuss the prospects of targeting PARPs with therapeutic purposes.     

Poly(ADP-ribose) polymerases: managing genome stability

The importance of poly(ADP-ribose) metabolism in the maintenance of genomic integrity following genotoxic stress has long been firmly established. Poly(ADP-ribose) polymerase-1 (PARP-1) and its catabolic counterpart, poly(ADP-ribose) glycohydrolase (PARG) play major roles in the modulation of cell responses to genotoxic stress. Recent discoveries of a number of other enzymes with poly(ADP-ribose) polymerase activity have established poly(ADP-ribosyl)ation as a general biological mechanism in higher eukaryotic cells that not only promotes cellular recovery from genotoxic stress and eliminates severely damaged cells from the organism, but also ensures accurate transmission of genetic information during cell division. Additionally, emerging data suggest the involvement of poly(ADP-ribosyl)ation in the regulation of intracellular trafficking, memory formation and other cellular functions. In this brief review on PARP and PARG enzymes, emphasis is placed on PARP-1, the best understood member of the PARP family and on the relationship of poly(ADP-ribosyl)ation to cancer and other diseases of aging.     

Poly(ADP-ribose) polymerase: a guardian angel protecting the genome and suppressing tumorigenesis

Poly(ADP-ribosyl)ation is an immediate cellular response to DNA damage generated either exogenously or endogenously. This post-translational modification is catalyzed by poly(ADP-ribose) polymerase (PARP, PARP-1, EC 2.4.2.30). It is proposed that this protein plays a multifunctional role in many cellular processes, including DNA repair, recombination, cell proliferation and death, as well as genomic stability. Chemical inhibitors of the enzyme, dominant negative or null mutations of PARP-1 cause a high degree of genomic instability in cells. Inhibition of PARP activity by chemical inhibitors renders mice or rats susceptible to carcinogenic agents in various tumor models, indicating a role for PARP-1 in suppressing tumorigenesis. Despite the above observations, PARP-1 knockout mice are generally not prone to the development of tumors. An enhanced tumor development was observed, however, when the PARP-1 null mutation was introduced into severely compromised immune-deficient mice (a mutation in DNA-dependent protein kinase) or mice lacking other DNA repair or chromosomal guardian molecules, such as p53 or Ku80. These studies indicate that PARP-1 functions as a cofactor to suppress tumorigenesis via its role in stabilization of the genome, and/or by interacting with other DNA strand break-sensing molecules. Studies using PARP-1 mutants and chemical inhibitors have started to shed light on the role of this protein and of the specific protein post-translational modification in the control of genomic stability and hence its involvement in cancer.     

ABCA1, ABCG1 and SR-BI: hormonal regulation in primary rat hepatocytes and human cell lines

ATM and PARP-1 are two of the most important players in the cell's response to DNA damage. PARP-1 and ATM recognize and bound to both single and double strand DNA breaks in response to different triggers. Here we report that ATM and PARP-1 form a molecular complex in vivo in undamaged cells and this association increases after γ-irradiation. ATM is also modified by PARP-1 during DNA damage. We have also evaluated the impact of PARP-1 absence or inhibition on ATM-kinase activity and have found that while PARP-1 deficient cells display a defective ATM-kinase activity and reduced γ-H2AX foci formation in response to γ-irradiation, PARP inhibition on itself is able to activate ATM-kinase. PARP inhibition induced γ H2AX foci accumulation, in an ATM-dependent manner. Inhibition of PARP also induces DNA double strand breaks which were dependent on the presence of ATM. As consequence ATM deficient cells display an increased sensitivity to PARP inhibition. In summary our results show that while PARP-1 is needed in the response of ATM to gamma irradiation, the inhibition of PARP induces DNA double strand breaks (which are resolved in and ATM-dependent pathway) and activates ATM kinase.     

PARG is recruited to DNA damage sites through poly(ADP-ribose)- and PCNA-dependent mechanisms

Post-translational poly(ADP-ribosyl)ation has diverse essential functions in the cellular response to DNA damage as it contributes to avid DNA damage detection and assembly of the cellular repair machinery but extensive modification eventually also induces cell death. While there are 17 human poly(ADP-ribose) polymerase (PARP) genes, there is only one poly(ADP-ribose) glycohydrolase (PARG) gene encoding several PARG isoforms located in different subcellular compartments. To investigate the recruitment of PARG isoforms to DNA repair sites we locally introduced DNA damage by laser microirradiation. All PARG isoforms were recruited to DNA damage sites except for a mitochondrial localized PARG fragment. Using PARP knock out cells and PARP inhibitors, we showed that PARG recruitment was only partially dependent on PARP-1 and PAR synthesis, indicating a second, PAR-independent recruitment mechanism. We found that PARG interacts with PCNA, mapped a PCNA binding site and showed that binding to PCNA contributes to PARG recruitment to DNA damage sites. This dual recruitment mode of the only nuclear PARG via the versatile loading platform PCNA and by a PAR dependent mechanism likely contributes to the dynamic regulation of this posttranslational modification and ensures the tight control of the switch between efficient DNA repair and cell death.     

Defective control of mitotic and post-mitotic checkpoints in poly(ADP-ribose) polymerase-1(-/-) fibroblasts after mitotic spindle disruption

Poly(ADP-ribose) polymerase-1 (PARP), a DNA damage-responsive nuclear enzyme present in higher eukaryotes, is well-known for its roles in protecting the genome after DNA damage. However, even without exogenous DNA damage, PARP may play a role in stabilizing the genome because cells or mice deficient in PARP exhibit various signs of genomic instability, such as tetraploidy, aneuploidy, chromosomal abnormalities and susceptibility to spontaneous carcinogenesis. Normally, cell cycle checkpoints ensure elimination of cells with genomic abnormalities. Therefore, we examined efficiency of mitotic and post-mitotic checkpoints in PARP-/- and PARP+/+ mouse embryonic fibroblasts treated with mitotic spindle disrupting agent colcemid. PARP+/+ cells, like most mammalian cells, eventually escaped from spindle disruption-induced mitotic checkpoint arrest by 60 h. In contrast, PARP-/- cells rapidly escaped from mitotic arrest within 24 h by downregulation of cyclin B1/CDK-1 kinase activity. After escaping from mitotic arrest; both the PARP genotypes arrive in G1 tetraploid state, where they face post-mitotic checkpoints which either induce apoptosis or prevent DNA endoreduplication. While all the G1 tetraploid PARP+/+ cells were eliminated by apoptosis, the majority of the G1 tetraploid PARP-/- cells became polyploid by resisting apoptosis and carrying out DNA endoreduplication. Introduction of PARP in PARP-/- fibroblasts partially increased the stringency of mitotic checkpoint arrest and fully restored susceptibility to G1 tetraploidy checkpoint-induced apoptosis; and thus prevented formation of polyploid cells. Our results suggest that PARP may serve as a guardian angel of the genome even without exogenous DNA damage through its role in mitotic and post-mitotic G1 tetraploidy checkpoints.     

Poly(ADP-ribose) polymerase-1 modulates DNA repair capacity and prevents formation of DNA double strand breaks

Although poly(ADP-ribose) polymerase-1 (PARP-1) has no enzymatic activity involved in DNA damage processing by the base excision repair (BER) pathway, PARP-1 deficient cells are genetically unstable and sensitive to DNA-damaging agents. To explain this paradox, we investigated the impact of PARP-1 on BER in mammalian cells. We reduced cellular PARP-1 protein levels using siRNA, then introduced DNA damage by hydrogen peroxide treatment and examined the repair response. We find that PARP-1 is not involved in recruitment of the major BER proteins to sites of DNA damage. However, we find that PARP-1 protects excessive DNA single strand breaks (SSBs) from converting into DNA double strand breaks (DSBs) thus preserving them for subsequent repair by BER enzymes. This suggests that PARP-1 plays an important role in BER by extending the ability of BER enzymes to process DNA single strand breaks arising directly after mutagen stress or during processing of DNA lesions following extensive DNA damage.