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.     

Are PARPs promiscuous?

Post-translational modifications exist in different varieties to regulate diverse characteristics of their substrates, ultimately leading to maintenance of cell health. The enzymes of the intracellular poly(ADP-ribose) polymerase (PARP) family can transfer either a single ADP-ribose to targets, in a reaction called mono(ADP-ribosyl)ation or MARylation, or multiple to form chains of poly(ADP-ribose) or PAR. Traditionally thought to be attached to arginine or glutamate, recent data have added serine, tyrosine, histidine and others to the list of potential ADP-ribose acceptor amino acids. PARylation by PARP1 has been relatively well studied, whereas less is known about the other family members such as PARP7 and PARP10. ADP-ribosylation on arginine and serine is reversed by ARH1 and ARH3 respectively, whereas macrodomain-containing MACROD1, MACROD2 and TARG1 reverse modification of acidic residues. For the other amino acids, no hydrolases have been identified to date. For many PARPs, it is not clear yet what their endogenous targets are. Better understanding of their biochemical reactions is required to be able to determine their biological functions in future studies. In this review, we discuss the current knowledge of PARP specificity in vitro and in cells, as well as provide an outlook for future research.     

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.     

Distribution of protein poly(ADP-ribosyl)ation systems across all domains of life

Poly(ADP-ribosyl)ation is a post-translational modification of proteins involved in regulation of many cellular pathways. Poly(ADP-ribose) (PAR) consists of chains of repeating ADP-ribose nucleotide units and is synthesized by the family of enzymes called poly(ADP-ribose) polymerases (PARPs). This modification can be removed by the hydrolytic action of poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3). Hydrolytic activity of macrodomain proteins (MacroD1, MacroD2 and TARG1) is responsible for the removal of terminal ADP-ribose unit and for complete reversion of protein ADP-ribosylation.Poly(ADP-ribosyl)ation is widely utilized in eukaryotes and PARPs are present in representatives from all six major eukaryotic supergroups, with only a small number of eukaryotic species that do not possess PARP genes. The last common ancestor of all eukaryotes possessed at least five types of PARP proteins that include both mono and poly(ADP-ribosyl) transferases. Distribution of PARGs strictly follows the distribution of PARP proteins in eukaryotic species. At least one of the macrodomain proteins that hydrolyse terminal ADP-ribose is also always present. Therefore, we can presume that the last common ancestor of all eukaryotes possessed a fully functional and reversible PAR metabolism and that PAR signalling provided the conditions essential for survival of the ancestral eukaryote in its ancient environment.PARP proteins are far less prevalent in bacteria and were probably gained through horizontal gene transfer. Only eleven bacterial species possess all proteins essential for a functional PAR metabolism, although it is not known whether PAR metabolism is truly functional in bacteria. Several dsDNA viruses also possess PARP homologues, while no PARP proteins have been identified in any archaeal genome.Our analysis of the distribution of enzymes involved in PAR metabolism provides insight into the evolution of these important signalling systems, as well as providing the basis for selection of the appropriate genetic model organisms to study the physiology of the specific human PARP proteins.     

Design and synthesis of potent inhibitors of the mono(ADP-ribosyl)transferase,PARP14

A series of (Z)-4-(3-carbamoylphenylamino)-4-oxobut-2-enyl amides were synthesized and tested for their ability to inhibit the mono-(ADP-ribosyl)transferase, PARP14 (a.k.a. BAL-2; ARTD-8). Two synthetic routes were established for this series and several compounds were identified as sub-micromolar inhibitors of PARP14, the most potent of which was compound 4t, IC₅₀ = 160 nM. Furthermore, profiling other members of this series identified compounds with >20-fold selectivity over PARP5a/TNKS1, and modest selectivity over PARP10, a closely related mono-(ADP-ribosyl)transferase.     

Inhibitors and activators of ADP-ribosylation reactions

ADP-ribosylation reaction, that is the transfer of the ADP-ribose moiety of NAD⁺ to acceptor protein, is catalyzed by two classes of ADP-ribosyltransferases,i.e., poly(ADP-ribose) synthetase and mono (ADP-ribosyl)transferases. These two types differ not only in the number of transferring ADP-ribose units but also in the acceptor amino acid(s) and protein. Their in hibitors, particularly those of poly(ADP-ribose) synthetase, have been successfully employed in studies on biological functions of the enzymes and other related fields of research. Recently, we found many potent and specific inhibitors of poly-(ADP-ribose) synthetase, and broadened their chemical as well as biochemical variety. More recently, we found several potent inhibitors of arginine-specific mono(ADP-ribosyl)transferases and activators of poly(ADP-ribose) synthetase.     

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.     

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.     

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.     

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.     

ADP-ribosylation of RNA and DNA: from in vitro characterization to in vivo function

The functionality of DNA, RNA and proteins is altered dynamically in response to physiological and pathological cues, partly achieved by their modification. While the modification of proteins with ADP-ribose has been well studied, nucleic acids were only recently identified as substrates for ADP-ribosylation by mammalian enzymes. RNA and DNA can be ADP-ribosylated by specific ADP-ribosyltransferases such as PARP1–3, PARP10 and tRNA 2′-phosphotransferase (TRPT1). Evidence suggests that these enzymes display different preferences towards different oligonucleotides. These reactions are reversed by ADP-ribosylhydrolases of the macrodomain and ARH families, such as MACROD1, TARG1, PARG, ARH1 and ARH3. Most findings derive from in vitro experiments using recombinant components, leaving the relevance of this modification in cells unclear. In this Survey and Summary, we provide an overview of the enzymes that ADP-ribosylate nucleic acids, the reversing hydrolases, and the substrates’ requirements. Drawing on data available for other organisms, such as pierisin1 from cabbage butterflies and the bacterial toxin–antitoxin system DarT–DarG, we discuss possible functions for nucleic acid ADP-ribosylation in mammals. Hypothesized roles for nucleic acid ADP-ribosylation include functions in DNA damage repair, in antiviral immunity or as non-conventional RNA cap. Lastly, we assess various methods potentially suitable for future studies of nucleic acid ADP-ribosylation.     

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.     

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.     

Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

Genotoxic stress activates PARP1, resulting in the post-translational modification of proteins with poly(ADP-ribose) (PAR). We genetically deleted PARP1 in one of the most widely used human cell systems, i.e. HeLa cells, via TALEN-mediated gene targeting. After comprehensive characterization of these cells during genotoxic stress, we analyzed structure–function relationships of PARP1 by reconstituting PARP1 KO cells with a series of PARP1 variants. Firstly, we verified that the PARP1\E988K mutant exhibits mono-ADP-ribosylation activity and we demonstrate that the PARP1\L713F mutant is constitutively active in cells. Secondly, both mutants exhibit distinct recruitment kinetics to sites of laser-induced DNA damage, which can potentially be attributed to non-covalent PARP1–PAR interaction via several PAR binding motifs. Thirdly, both mutants had distinct functional consequences in cellular patho-physiology, i.e. PARP1\L713F expression triggered apoptosis, whereas PARP1\E988K reconstitution caused a DNA-damage-induced G2 arrest. Importantly, both effects could be rescued by PARP inhibitor treatment, indicating distinct cellular consequences of constitutive PARylation and mono(ADP-ribosyl)ation. Finally, we demonstrate that the cancer-associated PARP1 SNP variant (V762A) as well as a newly identified inherited PARP1 mutation (F304L\V762A) present in a patient with pediatric colorectal carcinoma exhibit altered biochemical and cellular properties, thereby potentially supporting human carcinogenesis. Together, we establish a novel cellular model for PARylation research, by revealing strong structure–function relationships of natural and artificial PARP1 variants.     

Biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase

Here, we report the biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase (PARP) (EC 2.4.2. 30). PARP was effectively mono(ADP-ribosyl)ated both in solution and via an activity gel assay following SDS-PAGE with 20 microM or lower concentrations of [32P]-3'-dNAD+ as the ADP-ribosylation substrate. We observed the exclusive formation of [32P]-3'-dAMP and no polymeric ADP-ribose molecules following chemical release of enzyme-bound ADP-ribose units and high-resolution polyacrylamide gel electrophoresis. The reaction in solution (i) was time-dependent, (ii) was activated by nicked dsDNA, and (iii) increased with the square of the enzyme concentration. Stoichiometric analysis of the reaction indicated that up to four amino acid residues per mole of enzyme were covalently modified with single units of 3'-dADP-ribose. Peptide mapping of mono(3'-dADP-ribosyl)ated-PARP following limited proteolysis with either papain or alpha-chymotrypsin indicated that the amino acid acceptor sites for chain initiation with 3'-dNAD+ as a substrate are localized within an internal 22 kDa automodification domain. Neither the amino-terminal DNA-binding domain nor the carboxy-terminal catalytic fragment became ADP-ribosylated with [32P]-3'-dNAD+ as a substrate. Finally, the apparent rate constant of mono(ADP-ribosyl)ation in solution indicates that the initiation reaction catalyzed by PARP proceeds 232-fold more slowly than ADP-ribose polymerization.     

Design and synthesis of phenolic hydrazide hydrazones as potent poly(ADP-ribose) glycohydrolase (PARG) inhibitors

Poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are enzymes responsible for catalyzing the formation and degradation of poly(ADP-ribose) (PAR) polymers, respectively. Activation of PARP has been shown to be involved in cell death induced by genotoxic stimuli. On the other hand, genetic disruption of PARG also leads to increased level of cell death by accumulation of PAR. Unlike PARP, where significant medicinal effort has been expended to identify potent inhibitors, PARG has been insufficiently investigated as a molecular therapeutic target. In this study, we report the design, synthesis, and biological evaluation of phenolic hydrazide hydrazones as potent PARG inhibitors. Compounds 3d, 3e, 5d, 5e, 8a, 8b and 8c showed their ability to inhibit the catalytic activity of PARG in vitro with IC₅₀ values of 1.0, 2.1, 3.1, 3.2, 3.1, 2.8 and 1.6 μM, respectively.     

Diadenosine 5′, 5′′′-P1,P4-tetraphosphate (Ap4A) is synthesized in response to DNA damage and inhibits the initiation of DNA replication

The level of intracellular diadenosine 5′, 5′′′-P¹,P⁴-tetraphosphate (Ap₄A) increases several fold in mammalian cells treated with non-cytotoxic doses of interstrand DNA-crosslinking agents such as mitomycin C. It is also increased in cells lacking DNA repair proteins including XRCC1, PARP1, APTX and FANCG, while >50-fold increases (up to around 25 μM) are achieved in repair mutants exposed to mitomycin C. Part of this induced Ap₄A is converted into novel derivatives, identified as mono- and di-ADP-ribosylated Ap₄A. Gene knockout experiments suggest that DNA ligase III is primarily responsible for the synthesis of damage-induced Ap₄A and that PARP1 and PARP2 can both catalyze its ADP-ribosylation. Degradative proteins such as aprataxin may also contribute to the increase. Using a cell-free replication system, Ap₄A was found to cause a marked inhibition of the initiation of DNA replicons, while elongation was unaffected. Maximum inhibition of 70–80% was achieved with 20 μM Ap₄A. Ap₃A, Ap₅A, Gp₄G and ADP-ribosylated Ap₄A were without effect. It is proposed that Ap₄A acts as an important inducible ligand in the DNA damage response to prevent the replication of damaged DNA.     

DNA polymerase β-dependent cell survival independent of XRCC1 expression

Base excision repair (BER) is a primary mechanism for repair of base lesions in DNA such as those formed by exposure to the DNA methylating agent methyl methanesulfonate (MMS). Both DNA polymerase β (pol β)- and XRCC1-deficient mouse fibroblasts are hypersensitive to MMS. This is linked to a repair deficiency as measured by accumulation of strand breaks and poly(ADP-ribose) (PAR). The interaction between pol β and XRCC1 is important for recruitment of pol β to sites of DNA damage. Endogenous DNA damage can substitute for MMS-induced damage such that BER deficiency as a result of either pol β- or XRCC1-deletion is associated with sensitivity to PARP inhibitors. Pol β shRNA was used to knock down pol β in Xrcc1^+/+ and Xrcc1^−/− mouse fibroblasts. We determined whether pol β-mediated cellular resistance to MMS and PARP inhibitors resulted entirely from coordination with XRCC1 within the same BER sub-pathway. We find evidence for pol β-dependent cell survival independent of XRCC1 expression for both types of agents. The results suggest a role for pol β-dependent, XRCC1-independent repair. PAR immunofluorescence data are consistent with the hypothesis of a decrease in repair in both pol β knock down cell variants.