PARP的生物学与病理学功能

PARP1是真核细胞内具有多聚腺苷酸二磷酸核糖基（PAR）催化活性的蛋白酶,目前发现18个具有该活性的蛋白．多聚腺苷酸二磷酸核糖基化反应是细胞内进行的翻译后修饰,该修饰作用于许多蛋白,涉及到染色体的稳定,DNA损伤修复,基因转录,细胞的增长,死亡和凋亡等方面．在生理病理方面与炎症,肿瘤,衰老等疾病相关联．本文针对以上方面进行了总结和讨论．     

New insights into the molecular and cellular functions of poly(ADP-ribose) and PARPs

Poly(ADP-ribose) polymerases (PARPs) are enzymes that transfer ADP-ribose groups to target proteins and thereby affect various nuclear and cytoplasmic processes. The activity of PARP family members, such as PARP1 and PARP2, is tied to cellular signalling pathways, and through poly(ADP-ribosyl)ation (PARylation) they ultimately promote changes in gene expression, RNA and protein abundance, and the location and activity of proteins that mediate signalling responses. PARPs act in a complex response network that is driven by the cellular, molecular and chemical biology of poly(ADP-ribose) (PAR). This PAR-dependent response network is crucial for a broad array of physiological and pathological responses and thus is a good target for chemical therapeutics for several diseases.     

Poly(ADP-ribose) polymerase plays a differential role in DNA damage-response and cell death pathways in Trypanosoma cruzi

Poly(ADP-ribosyl)ation is a post-translational modification of proteins. Poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are the enzymes responsible for poly(ADP-ribose) (PAR) polymer metabolism and are present in most higher eukaryotes. The best understood role of PARP is the maintenance of genomic integrity either via promotion of DNA repair at low levels of genotoxic stress or via promotion of cell death at higher levels of damage. The unicellular eukaryote Trypanosoma cruzi, as opposed to humans and other organisms, has only one PARP (TcPARP) and one PARG (TcPARG). In the present study we show that under different DNA-damaging agents (H(2)O(2) or UV-C radiation) TcPARP is activated and translocated from the cytosol to the nucleus, while TcPARG always shows a nuclear localisation. Parasites in the presence of PARP or PARG inhibitors, as well as parasites over-expressing either TcPARP or TcPARG, suggested that PAR metabolism could be involved in different phases of cell growth, even in the absence of DNA damage. We also believe that we provide the first reported evidence that different proteins could be poly(ADP-ribosyl)ated in response to different stimuli, leading to different cell death pathways.     

Poly(ADP-ribose): PARadigms and PARadoxes

Poly(ADP-ribosyl)ation (PARylation) is a posttranslational protein modification (PTM) catalyzed by members of the poly(ADP-ribose) polymerase (PARP) enzyme family. PARPs use NAD⁺ as substrate and upon cleaving off nicotinamide they transfer the ADP-ribosyl moiety covalently to suitable acceptor proteins and elongate the chain by adding further ADP-ribose units to create a branched polymer, termed poly(ADP-ribose) (PAR), which is rapidly degraded by poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3). In recent years several key discoveries changed the way we look at the biological roles and mode of operation of PARylation. These paradigm shifts include but are not limited to (1) a single PARP enzyme expanding to a PARP family; (2) DNA-break dependent activation extended to several other DNA dependent and independent PARP-activation mechanisms; (3) one molecular mechanism (covalent PARylation of target proteins) underlying the biological effect of PARPs is now complemented by several other mechanisms such as protein–protein interactions, PAR signaling, modulation of NAD⁺ pools and (4) one principal biological role in DNA damage sensing expanded to numerous, diverse biological functions identifying PARP-1 as a real moonlighting protein. Here we review the most important paradigm shifts in PARylation research and also highlight some of the many controversial issues (or paradoxes) of the field such as (1) the mostly synergistic and not antagonistic biological effects of PARP-1 and PARG; (2) mitochondrial PARylation and PAR decomposition, (3) the cross-talk between PARylation and signaling pathways (protein kinases, phosphatases, calcium) and the (4) divergent roles of PARP/PARylation in longevity and in age-related diseases.     

Poly(ADP-Ribose) polymerase 1 as a key regulator of DNA repair

Poly(ADP-ribosyl)ation (PARylation) of proteins is one of the immediate cell responses to DNA damage and is catalyzed by poly(ADP-ribose) polymerases (PARPs). When bound to damaged DNA, some members of the PARP family are activated and use NAD⁺ as a source of ADP to catalyze synthesis of poly(ADP-ribose) (PAR) covalently attached to a target protein. PAR synthesis is considered as a mechanism that provides a local signal of DNA damage and modulates protein functions in response to genotoxic agents. PARP1 is the best-studied protein of the PARP family and is widely known аs a regulator of repair of damaged bases and single-strand nicks. Data are accumulating that PARP1 is additionally involved in double-strand break repair and nucleotide excision repair. The review summarizes the literature data on the role that PARP1 and PARylation play in DNA repair and particularly in base excision repair; original data obtained in our lab are considered in more detail.     

Poly(ADP-ribosyl)ation in mammalian ageing

Poly(ADP-ribose) polymerases (PARPs) catalyze the post-translational modification of proteins with poly(ADP-ribose). Two PARP isoforms, PARP-1 and PARP-2, display catalytic activity by contact with DNA-strand breaks and are involved in DNA base-excision repair and other repair pathways. A body of correlative data suggests a link between DNA damage-induced poly(ADP-ribosyl)ation and mammalian longevity. Recent research on PARPs and poly(ADP-ribose) yielded several candidate mechanisms through which poly(ADP-ribosyl)ation might act as a factor that limits the rate of ageing.     

Differential and Concordant Roles for Poly(ADP-Ribose) Polymerase 1 and Poly(ADP-Ribose) in Regulating WRN and RECQL5 Activities

Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) catalyzes the poly(ADP-ribosyl)ation (PARylation) of proteins, a posttranslational modification which forms the nucleic acid-like polymer PAR. PARP1 and PAR are integral players in the early DNA damage response, since PARylation orchestrates the recruitment of repair proteins to sites of damage. Human RecQ helicases are DNA unwinding proteins that are critical responders to DNA damage, but how their recruitment and activities are regulated by PARPs and PAR is poorly understood. Here we report that all human RecQ helicases interact with PAR noncovalently. Furthermore, we define the effects that PARP1, PARylated PARP1, and PAR have on RECQL5 and WRN, using both in vitro and in vivo assays. We show that PARylation is involved in the recruitment of RECQL5 and WRN to laser-induced DNA damage and that RECQL5 and WRN have differential responses to PARylated PARP1 and PAR. Furthermore, we show that the loss of RECQL5 or WRN resulted in increased sensitivity to PARP inhibition. In conclusion, our results demonstrate that PARP1 and PAR actively, and in some instances differentially, regulate the activities and cellular localization of RECQL5 and WRN, suggesting that PARylation acts as a fine-tuning mechanism to coordinate their functions in time and space during the genotoxic stress response.     

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.     

Quantitative proteomics profiling of the poly(ADP-ribose)-related response to genotoxic stress

Upon DNA damage induction, DNA-dependent poly(ADP-ribose) polymerases (PARPs) synthesize an anionic poly(ADP-ribose) (pADPr) scaffold to which several proteins bind with the subsequent formation of pADPr-associated multiprotein complexes. We have used a combination of affinity-purification methods and proteomics approaches to isolate these complexes and assess protein dynamics with respect to pADPr metabolism. As a first approach, we developed a substrate trapping strategy by which we demonstrate that a catalytically inactive Poly(ADP-ribose) glycohydrolase (PARG) mutant can act as a physiologically selective bait for the isolation of specific pADPr-binding proteins through its macrodomain-like domain. In addition to antibody-mediated affinity-purification methods, we used a pADPr macrodomain affinity resin to recover pADPr-binding proteins and their complexes. Second, we designed a time course experiment to explore the changes in the composition of pADPr-containing multiprotein complexes in response to alkylating DNA damage-mediated PARP activation. Spectral count clustering based on GeLC-MS/MS analysis was complemented with further analyses using high precision quantitative proteomics through isobaric tag for relative and absolute quantitation (iTRAQ)- and Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics. Here, we present a valuable resource in the interpretation of systems biology of the DNA damage response network in the context of poly(ADP-ribosyl)ation and provide a basis for subsequent investigations of pADPr-binding protein candidates.     

PARP2 Is the Predominant Poly(ADP-Ribose) Polymerase in Arabidopsis DNA Damage and Immune Responses

Poly (ADP-ribose) polymerases (PARPs) catalyze the transfer of multiple poly(ADP-ribose) units onto target proteins. Poly(ADP-ribosyl)ation plays a crucial role in a variety of cellular processes including, most prominently, auto-activation of PARP at sites of DNA breaks to activate DNA repair processes. In humans, PARP1 (the founding and most characterized member of the PARP family) accounts for more than 90% of overall cellular PARP activity in response to DNA damage. We have found that, in contrast with animals, in Arabidopsis thaliana PARP2 (At4g02390), rather than PARP1 (At2g31320), makes the greatest contribution to PARP activity and organismal viability in response to genotoxic stresses caused by bleomycin, mitomycin C or gamma-radiation. Plant PARP2 proteins carry SAP DNA binding motifs rather than the zinc finger domains common in plant and animal PARP1 proteins. PARP2 also makes stronger contributions than PARP1 to plant immune responses including restriction of pathogenic Pseudomonas syringae pv. tomato growth and reduction of infection-associated DNA double-strand break abundance. For poly(ADP-ribose) glycohydrolase (PARG) enzymes, we find that Arabidopsis PARG1 and not PARG2 is the major contributor to poly(ADP-ribose) removal from acceptor proteins. The activity or abundance of PARP2 is influenced by PARP1 and PARG1. PARP2 and PARP1 physically interact with each other, and with PARG1 and PARG2, suggesting relatively direct regulatory interactions among these mediators of the balance of poly(ADP-ribosyl)ation. As with plant PARP2, plant PARG proteins are also structurally distinct from their animal counterparts. Hence core aspects of plant poly(ADP-ribosyl)ation are mediated by substantially different enzymes than in animals, suggesting the likelihood of substantial differences in regulation.     

The expanding field of poly(ADP-ribosyl)ation reactions. 'Protein Modifications: Beyond the Usual Suspects' Review Series

Poly(ADP-ribosyl)ation is a post-translational modification of proteins that is mediated by poly(ADP-ribose) polymerases (PARPs). Although the existence and nature of the nucleic acid-like molecule poly(ADP-ribose) (PAR) has been known for 40 years, understanding its biological functions--originally thought to be only the regulation of chromatin superstructure when DNA is broken--is still the subject of intense research. Here, we review the mechanisms controlling the biosynthesis of this complex macromolecule and some of its main biological functions, with an emphasis on the most recent advances and hypotheses that have developed in this rapidly growing field.     

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.     

Poly(ADP-ribose) signaling in cell death

Poly(ADP-ribosyl)ation (PARylation) is a reversible protein modification carried out by the concerted actions of poly(ADP-ribose) polymerase (PARP) enzymes and poly(ADP-ribose) (PAR) decomposing enzymes such as PAR glycohydrolase (PARG) and ADP-ribosyl hydrolase 3 (ARH3). Reversible PARylation is a pleiotropic regulator of various cellular functions but uncontrolled PARP activation may also lead to cell death. The cellular demise pathway mediated by PARylation in oxidatively stressed cells has been described almost thirty years ago. However, the underlying molecular mechanisms have only begun to emerge relatively recently. PARylation has been implicated in necroptosis, autophagic cell death but its role in extrinsic and intrinsic apoptosis appears to be less predominant and depends largely on the cellular model used. Currently, three major pathways have been made responsible for PARP-mediated necroptotic cell death: (1) compromised cellular energetics mainly due to depletion of NAD, the substrate of PARPs; (2) PAR mediated translocation of apoptosis inducing factor (AIF) from mitochondria to nucleus (parthanatos) and (3) a mostly elusive crosstalk between PARylation and cell death/survival kinases and phosphatases. Here we review how these PARP-mediated necroptotic pathways are intertwined, how PARylation may contribute to extrinsic and intrinsic apoptosis and discuss recent developments on the role of PARylation in autophagy and autophagic cell death.     

Poly(ADP-ribose) Polymerase 1 Modulates Interaction of the Nucleotide Excision Repair Factor XPC-RAD23B with DNA via Poly(ADP-ribosyl)ation

Poly(ADP-ribosyl)ation is a reversible post-translational modification that plays an essential role in many cellular processes, including regulation of DNA repair. Cellular DNA damage response by the synthesis of poly(ADP-ribose) (PAR) is mediated mainly by poly(ADP-ribose) polymerase 1 (PARP1). The XPC-RAD23B complex is one of the key factors of nucleotide excision repair participating in the primary DNA damage recognition. By using several biochemical approaches, we have analyzed the influence of PARP1 and PAR synthesis on the interaction of XPC-RAD23B with damaged DNA. Free PAR binds to XPC-RAD23B with an affinity that depends on the length of the poly(ADP-ribose) strand and competes with DNA for protein binding. Using ³²P-labeled NAD⁺ and immunoblotting, we also demonstrate that both subunits of the XPC-RAD23B are poly(ADP-ribosyl)ated by PARP1. The efficiency of XPC-RAD23B PARylation depends on DNA structure and increases after UV irradiation of DNA. Therefore, our study clearly shows that XPC-RAD23B is a target of poly(ADP-ribosyl)ation catalyzed by PARP1, which can be regarded as a universal regulator of DNA repair processes.     

Molecular mechanism of poly(ADP-ribosyl)ation by PARP1 and identification of lysine residues as ADP-ribose acceptor sites

Poly(ADP-ribose) polymerase 1 (PARP1) synthesizes poly(ADP-ribose) (PAR) using nicotinamide adenine dinucleotide (NAD) as a substrate. Despite intensive research on the cellular functions of PARP1, the molecular mechanism of PAR formation has not been comprehensively understood. In this study, we elucidate the molecular mechanisms of poly(ADP-ribosyl)ation and identify PAR acceptor sites. Generation of different chimera proteins revealed that the amino-terminal domains of PARP1, PARP2 and PARP3 cooperate tightly with their corresponding catalytic domains. The DNA-dependent interaction between the amino-terminal DNA-binding domain and the catalytic domain of PARP1 increased V_max and decreased the K_m for NAD. Furthermore, we show that glutamic acid residues in the auto-modification domain of PARP1 are not required for PAR formation. Instead, we identify individual lysine residues as acceptor sites for ADP-ribosylation. Together, our findings provide novel mechanistic insights into PAR synthesis with significant relevance for the different biological functions of PARP family members.     

Poly(ADP-ribose) polymerases: homology, structural domains and functions. Novel therapeutical applications

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes, which show differences in structure, cellular location and functions. However, all these enzymes possess poly(ADP-ribosyl)ation activity. Overactivation of PARP enzymes has been implicated in the pathogenesis of several diseases, including stroke, myocardial infarction, diabetes, shock, neurodegenerative disorder and allergy. The best studied of these enzymes (PARP-1) is involved in the cellular response to DNA damage so that in the event of irreparable DNA damage overactivation of PARP-1 leads to necrotic cell death. Inhibitors of PARP-1 activity in combination with DNA-binding antitumor drugs may constitute a suitable strategy in cancer chemotherapy. In addition, PARP inhibitors may be also useful to restore cellular functions in several pathophysiological states and diseases. This review gives an update of the state-of-the-art concerning PARP enzymes and their exploitation as pharmacological targets in several illnesses.     

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.     

The emerging role of poly(ADP-ribose) polymerase-1 in longevity

In the present paper, the involvement of the family of poly(ADP-ribose) polymerases (PARPs), and especially of PARP-1, in mammalian longevity is reviewed. PARPs catalyse poly(ADP-ribosyl)ation, a covalent post-translational protein modification in eukaryotic cells. PARP-1 and PARP-2 are activated by DNA strand breaks, play a role in DNA base-excision repair (BER) and are survival factors for cells exposed to low doses of ionising radiation or alkylating agents. PARP-1 is the main catalyst of poly(ADP-ribosyl)ation in living cells under conditions of DNA breakage, accounting for about 90% of cellular poly(ADP-ribose). DNA-damage-induced poly(ADP-ribosyl)ation also functions as a negative regulator of DNA damage-induced genomic instability. Cellular poly(ADP-ribosyl)ation capacity in permeabilised mononuclear blood cells (MNC) is positively correlated with life span of mammalian species. Furthermore PARP-1 physically interacts with WRN, the protein deficient in Werner syndrome, a human progeroid disorder, and PARP-1 and WRN functionally cooperate in preventing carcinogenesis in vivo. Some of the other members of the PARP family have also been revealed as important regulators of cellular functions relating to ageing/longevity. In particular, tankyrase-1, tankyrase-2, PARP-2 as well as PARP-1 have been found in association with telomeric DNA and are able to poly(ADP-ribosyl)ate the telomere-binding proteins TRF-1 and TRF-2, thus blocking their DNA-binding activity and controlling telomere extension by telomerase.     

Poly(ADP-ribosyl)ation in regulation of chromatin structure and the DNA damage response

Poly(ADP-ribose) (PAR) is a post-translational modification of proteins and is synthesised by PAR polymerases (PARPs), which have long been associated with the coordination of the cellular response to DNA damage, amongst other processes. Binding of some PARPs such as PARP1 to broken DNA induces a substantial wave of PARylation, which results in significant re-structuring of the chromatin microenvironment through modification of chromatin-associated proteins and recruitment of chromatin-modifying proteins. Similarly, other DNA damage response proteins are recruited to the damaged sites via PAR-specific binding modules, and in this way, PAR mediates not only local chromatin architecture but also DNA repair. Here, we discuss the expanding role of PAR in the DNA damage response, with particular focus on chromatin regulation.