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Microbiology and Molecular Biology Reviews, September 2006, p. 789-829, Vol. 70, No. 3
1092-2172/06/$08.00+0 doi:10.1128/MMBR.00040-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Nuclear ADP-Ribosylation Reactions in Mammalian Cells: Where Are We Today and Where Are We Going?
Paul O. Hassa,
Sandra S. Haenni,
Michael Elser, and
Michael O. Hottiger*
Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
Since poly-ADP ribose was discovered over 40 years ago, there has been significant progress in research into the biology of mono- and poly-ADP-ribosylation reactions. During the last decade, it became clear that ADP-ribosylation reactions play important roles in a wide range of physiological and pathophysiological processes, including inter- and intracellular signaling, transcriptional regulation, DNA repair pathways and maintenance of genomic stability, telomere dynamics, cell differentiation and proliferation, and necrosis and apoptosis. ADP-ribosylation reactions are phylogenetically ancient and can be classified into four major groups: mono-ADP-ribosylation, poly-ADP-ribosylation, ADP-ribose cyclization, and formation of O-acetyl-ADP-ribose. In the human genome, more than 30 different genes coding for enzymes associated with distinct ADP-ribosylation activities have been identified. This review highlights the recent advances in the rapidly growing field of nuclear mono-ADP-ribosylation and poly-ADP-ribosylation reactions and the distinct ADP-ribosylating enzyme families involved in these processes, including the proposed family of novel poly-ADP-ribose polymerase-like mono-ADP-ribose transferases and the potential mono-ADP-ribosylation activities of the sirtuin family of NAD+-dependent histone deacetylases. A special focus is placed on the known roles of distinct mono- and poly-ADP-ribosylation reactions in physiological processes, such as mitosis, cellular differentiation and proliferation, telomere dynamics, and aging, as well as "programmed necrosis" (i.e., high-mobility-group protein B1 release) and apoptosis (i.e., apoptosis-inducing factor shuttling). The proposed molecular mechanisms involved in these processes, such as signaling, chromatin modification (i.e., "histone code"), and remodeling of chromatin structure (i.e., DNA damage response, transcriptional regulation, and insulator function), are described. A potential cross talk between nuclear ADP-ribosylation processes and other NAD+-dependent pathways is discussed.
Over 40 years ago, P. Chambon and colleagues discovered that the addition of NAD to hen liver nuclear extracts stimulated the synthesis of poly-ADP-ribose (65, 433), paving the way for research into the biology of mono- and poly-ADP-ribose. A landmark meeting on ADP-ribosylation reactions, held in October 2005 in Newcastle, United Kingdom, commemorated this important scientific anniversary. For the first 30 to 35 years, research on ADP-ribosylation reactions was a relatively esoteric field. However, the development of new approaches, such as the generation of different knockout mice, has changed the situation in the past 5 years. Recent data show that ADP-ribosylation reactions play important roles in many physiological and pathophysiological processes, including inter- and intracellular signaling, transcription, DNA repair pathways, cell cycle regulation, and mitosis, as well as necrosis and apoptosis.
ADP-ribosylation reactions are phylogenetically ancient and can be divided into four major groups: mono-ADP-ribosylation, poly-ADP-ribosylation, ADP-ribose cyclization, and formation of O-acetyl-ADP-ribose. Mono-ADP-ribosylation of proteins and generation of free ADP-ribose or O-acetyl-ADP-ribose are the most conserved evolutionarily and are common to both prokaryotes and eukaryotes. ADP-ribosyl cyclase activities occur in unicellular and multicellular eukaryotes but not in bacteria and archaea (77, 140, 256, 257, 309; reviewed in references 88, 109, and 354). Poly-ADP-ribosylation reactions occur in multicellular eukaryotes and may also be present in unicellular eukaryotes (204, 309, 434; reviewed in reference 20). Poly-ADP-ribosylation or genes encoding poly-ADP-ribosylating enzymes have not been identified in bacteria. Despite the fact that no genes encoding poly-ADP-ribosylating enzymes have been identified in the archaeal genomes analyzed so far (309), recent studies provided evidence that poly-ADP-ribosylation-like reactions may exist in archaea (116, 118).
This review focuses mainly on the nuclear enzymatic mono-ADP-ribosylation and poly-ADP-ribosylation reactions occurring in mammalian cells and on the ADP-ribosylating enzyme families involved in these processes. Cytoplasmic and extracellular membrane-associated mono-ADP-ribosylation reactions, mediated by the ecto-mono-ADP-ribosyltransferase (e-MART) family and ADP-ribose cyclases, will not be discussed in detail. The reader is referred to recent excellent reviews on this topic (57, 100, 151, 305, 356). The first part of this review offers a brief overview of the molecular mechanisms of poly-ADP-ribosylation and discusses the ADP-ribosylating enzyme families, including novel ADP-ribosylating enzymes. Studies performed exclusively with poly-ADP-ribosylation/poly-ADP-ribose polymerase (PARP) inhibitors will only be partially addressed here, due to the off-target effects of poly-ADP-ribosylation inhibitors and nonspecific inhibition of both poly-ADP-ribosylation and certain mono-ADP-ribosylation reactions (179, 447). Since the term "covalent poly-ADP-ribosylation of proteins" is currently a subject of debate, we include a special section on "covalent poly-ADP-ribosylation of proteins." We discuss new technologies and strategies, as well as new models that might help to clarify whether poly-ADP-ribosylation is a covalent and reversible posttranslational modification of proteins. A special focus on the known and proposed physiological roles of distinct mono-ADP-ribosylation and poly-ADP-ribosylation reactions will be given in the last sections. Since it is not yet clear whether proteins are covalently poly-ADP-ribosylated or can just bind to free poly-ADP-ribose, we use the established term PARP instead of the term poly-ADP-ribosyltransferase recently suggested by Glowacki et al. and Otto et al. (140, 309).
The presence of poly-ADP-ribose was first suggested in 1963 by P. Chambon and coworkers, who reported that NAD+ stimulated the incorporation of labeled ATP into an acid-insoluble fraction of poly(A)-containing products in hen liver nuclear extracts (65). The enzyme responsible for the synthesis of poly-ADP-ribose was named PARP. The structure of poly-ADP-ribose was later solved by three independent laboratories (111, 288, 330, 383). Several years later, mono-ADP-ribosylation reactions were discovered during studies of bacterial toxins. These enzymes turned out to be mono-ADP-ribosyltransferases (MARTs) (136, 169). Subsequently, the existence of endogenous mono-ADP-ribosyltransferases was reported (reviewed in references 151, 304, 305, and 356). At first, mono-ADP-ribosylation and poly-ADP-ribosylation were postulated to serve as reversible posttranslational modifications of proteins, acting as regulatory mechanism for proteins. During the 1970s and 1980s, several laboratories partially purified several enzymes associated with mono-ADP-ribosylation and poly-ADP-ribosylation activities. In 1971, M. Miwa and T. Sugimura discovered poly-ADP-ribose glycohydrolase (PARG), which cleaves the ribose-ribose bonds in poly-ADP-ribose (278). Eight years later, the same group described the branched structure of poly-ADP-ribose in detail (277). However, it took an additional decade to isolate the genes encoding proteins responsible for these ADP-ribosylation reactions. In the late 1980s, the gene encoding a poly-ADP-ribose synthetase (initially named PARP, poly-ADP-ribose synthetase, or poly-ADP-ribosyltransferase and now named PARP-1) was isolated (9, 217, 414). At the same time, H. C. Lee and coworkers described an additional ADP-ribosylation reaction, the cyclization of ADP-ribose, which leads to cyclic-ADP-ribose. Cyclic-ADP-ribose is formed following NAD+ cleavage by NAD+ glycohydrolases/ADP-ribosylcyclases (223), and it serves as an important second messenger involved in the regulation of calcium signaling and homeostasis (reviewed in reference 354). In the early 1990s, the groups of J. Moss and F. Koch-Nolte identified several genes encoding e-MARTs and one gene encoding an ADP-ribosylarginine hydrolase (203, 302, 303, 385, 467).
For a long time it was thought that PARP-1 was the only enzyme with poly-ADP-ribosylation activity in mammalian cells. However, after nearly 15 years of intensive characterization of PARP-1, five different genes encoding "bona fide" PARP enzymes were identified (19, 178, 185, 196, 376), indicating that PARP-1 belongs to a family of PARPs. A similar situation is found in the case of MARTs. Although the mammalian e-MARTs (e-MART1 to -5/6) so far represent the only MART family for which enzymatic activities are well characterized (88, 89, 356), several reports predicted that distinct families of mono-ADP-ribosylating enzymes with no obvious sequence similarity to the well-known e-MARTs must exist in mammalian cells (88, 89, 356). Indeed, several members of the sirtuin family of NAD+-dependent histone deacetylases (SIRTs) were found to posses mono-ADP-ribosyltransferase activities and thus could represent a putative novel family of intracellular MARTs (128, 132, 234, 391). Moreover, very recent reports described 11 additional novel mammalian Parp-like genes (5, 20, 140) that may be good candidates to be members of a putative large family of intracellular PARP-like MARTs (5, 6, 241, 309, 452; reviewed in references 20 and 140). Although clear biochemical evidence for protein-mono-ADP-ribosylation by the SIRTs and PARP-like ADP-ribosyltransferases has yet to be established (309, 371), growing families of MARTs and PARPs exist and may be responsible for distinct mono-ADP-ribosylation and poly-ADP-ribosylation reactions in mammalian cells (20, 140, 161, 309).
A unique reaction catalyzed by distinct SIRT family members, in which the cleavage of NAD+ and the deacetylation of substrates are coupled to the formation of O-acetyl-ADP-ribose (O-AADP-ribose), was recently described (46, 390). O-AADP-ribose was shown to serve as small-molecule effector, involved in the modulation of heterochromatin formation (165, 231).
In eukaryotic cells, NAD+ has been shown to play a pivotal role as an essential coenzyme/transmitter molecule in bioenergetics (reviewed in references 243, 339, and 466). The synthesis of ATP and the balance of redox potential depend directly on NAD+ levels in cells. The chemistry of this molecule allows it to serve both as an electron acceptor (in its oxidized form, NAD+) and as an electron donor (in its reduced form, NADH) in reactions catalyzed by enzymes of the mitochondrial electron transport chain, leading to the generation of ATP during oxidative phosphorylation. In addition to its well-known roles in energy metabolism, NAD+ also has a distinct role as a precursor or immediate substrate for multiple ADP-ribosylation reactions. Such reactions are involved in cell regulation and metabolic processes and in the formation of various metabolites, including nicotinamide, free mono-ADP-ribose, mono-ADP-ribosylated proteins, cyclic-ADP-ribose, NAADP+, O-AADP-ribose, and poly-ADP-ribose (reviewed in references 32, 339, 354, and 466). Hydrolysis of the high-energy bond between the nicotinamide and ribose moieties of NAD+ produces a free energy of –34.3 kJ/mol (–8.2 kcal/mol) (457). This energy is used by distinct NAD+-metabolizing ADP-ribosylation enzymes to drive the transfer of the ADP-ribose moiety to proteins and the synthesis of ADP-ribose polymers. The multiple roles of NAD+ in bioenergetics and production of secondary messengers as well as in protein modifications and generation of free and protein-associated poly-ADP-ribose have important physiological consequences in the regulation of multiple cellular processes, as demonstrated by various studies performed on the molecular functions of NAD+-dependent enzyme families (reviewed in references 32, 243, 339, 354, and 466).
The involvement of NAD+ in these regulatory processes as a donor of ADP-ribose requires a constant resynthesis of NAD+ to avoid depletion of the intracellular NAD+ pool. In higher eukaryotes, the biosynthesis of NAD+ occurs through one de novo pathway and three distinct salvage pathways (243, 245, 339). NAD+ can be synthesized from four distinct precursors: L-tryptophan (thought to represent the de novo pathway) and nicotinic acid, nicotinamide (Nam), and nicotinamide riboside (thought to represent the three salvage pathways) (243, 245, 339). The Nam salvage pathway, leading from Nam to NAD+, goes through a single intermediate, nicotinamide mononucleotide (NMN). The nicotinic acid salvage pathway, known as the Preiss-Handler pathway, goes through two intermediates, nicotinic acid mononucleotide (NaMN) and nicotinic acid adenine dinucleotide. The nicotinamide riboside salvage pathway uses nicotinamide riboside as a precursor and is connected to the Nam salvage pathway through NMN (36). The de novo pathway leads from tryptophan to quinolinate and is connected to the Preiss-Handler pathway through NaMN. The presence of these multiple NAD+ biosynthetic routes most likely reflects differences in tissue distribution and/or intracellular compartmentalization of NAD+ metabolism (31, 243, 339, 465, 466). However, because nicotinamide is a product of NAD+ hydrolysis by numerous NAD+-glycohydrolases, including ADP-ribosylating enzymes, and no nicotinamidase-producing nicotinic acid exists in vertebrates, Nam is probably the major source for the biosynthesis of NAD+ in most mammalian cells (243, 339). A scheme for the NAD+ biosynthetic pathways and metabolism is shown in Fig. 1. The common enzymes of both the de novo and salvage pathways, the family of NMN adenylyltransferases (Na/NMNATs) which catalyze the production of NAD+ from NMN and ATP and represent the final step in the biosynthesis of NAD+, also play a crucial regulatory function for ADP-ribosylation processes in the cytoplasm and nucleus (reviewed in references 32, 243, 339, and 466). The predominant form of mammalian Na/NMNATs, Na/NMNAT-1, is localized in the nucleus, whereas Na/NMNAT-2 and Na/NMNAT-3 are cytoplasmic (460), being preferentially localized to Golgi complex and mitochondria, respectively (31). Their localization suggests that local production of NAD+ is important for the NAD+-dependent processes in those compartments (31, 243, 339). It is likely that local NAD+ production is strictly controlled under normal physiological conditions by the recruitment of biosynthetic enzymes to sites of NAD+-glycohydrolase activities, such as mono- and poly-ADP-ribosylation reactions (31, 198). The Na/NMNATs could sense the level of free mono-ADP-ribose or more likely free or bound poly-ADP-ribose and may be recruited in a poly-ADP-ribose-binding-dependent manner (31, 198). In this respect, PARP-4/vault-PARP and PARP-5/tankyrase-1 are the only members of the "bona fide" PARP family that have been localized to the cytoplasm. PARP-4/vault-PARP is present in cytoplasmic ribonucleoprotein particles (vaults) and cytoplasmic clusters (vault-PARP rods) as well as in the nuclear matrix (196, 235). PARP-5/tankyrase-1 was shown to be associated, at least in part, with the Golgi complex (75). Under normal physiological conditions, all other "bona fide" PARP family members seem to be localized exclusively to the nucleus (20).
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FIG. 1. Mammalian NAD+ metabolic pathways. The biosynthesis of NAD+ occurs through both de novo and salvage pathways (339). In mammalian cells, 90% of free tryptophan is metabolized through the kynurenine pathway, leading to the de novo synthesis of NAD+. The three different salvage pathways start either from nicotinamide (Nam), nicotinic acid (Na), or nicotinamide riboside (NR). In mammals, the origin of nicotinic acid is mainly nutritional. Nicotinamide, a product of NAD+ hydrolysis, is first converted into nicotinamide mononucleotide (NMN) and then into NAD+ by nicotinamide phosphoribosyl transferase (NamPRT) and nicotinamide mononucleotide adenylyl transferases (Na/NMNAT-1, -2, and -3), respectively. Nicotinamide riboside was recently shown to serve as a precursor for NAD+ synthesis, connected to the Nam salvage pathway through NMN (36). Nicotinamide riboside is converted to NMN by the ATP-consuming nicotinamide riboside kinases 1 and 2 (NRK-1 and -2) (36). Nicotinic acid can be converted through the Preiss-Handler salvage pathway into nicotinic acid mononucleotide (NaNM) and nicotinate adenine dinucleotide by the concerted actions of nicotinic acid phosphoribosyl transferase (NaPRT) and Na/NMNAT-1, -2, and -3, respectively. Nicotinate adenine dinucleotide is directly transformed into NAD+ by the glutamine-hydrolyzing NAD+ synthetase (NADS). Na/NMNATs are ATP-consuming enzymes, using either NaMN or NMN as a substrate. Whether both NamPRT and NaPRT are also ATP-consuming enzymes in vivo is not certain. Thus, when the Preiss-Handler salvage pathway is used, the cell invests three or four molecules of ATP from Na to NAD+, depending on whether NaPRT is also an ATP-consuming enzyme in vivo. In mammalian cells, under the conditions where NAD+ is used as a glycohydrolase substrate, the Nam salvage pathway is required, since there is no nicotinamidase to produce nicotinic acid. Depending on whether NamPRT uses one ATP molecule to convert Nam into NMN, the Nam salvage pathway consumes two or three ATP molecules from Nam to NAD+. The de novo pathway is connected to the Preiss-Handler salvage pathway through NaMN. NAD+ can be hydrolyzed by various enzymatic activities, such as PARPs, MARTs, SIRTs, and ADP-ribosyl cyclases, which release the Nam moiety from NAD+ to produce poly-ADP-ribose, mono-ADP-ribosyl-protein, acetyl-ADP-ribose (O-AADPR), or cyclic-ADP-ribose (cADPR) and nicotinate adenine dinucleotide phosphate (NAADP), respectively. These products are then further metabolized by different hydrolase activities, yielding ADP-ribose (ADPR), which, in turn, can be transformed into 5-phosphribosyl-1-pyrophosphate (PRPP) by the ATP-consuming ADP-ribose pyrophosphatase (ARPP)/ribose phosphate pyrophosphokinase (RPPK) pathway. PRPP is used by the Nam salvage pathway enzymes NamPRT and NaPRT.
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It has been suggested that the most important factor affecting the maintenance of the NAD+ pool is the level of poly-ADP-ribosylation in cells (32, 243, 466). The catabolism of NAD+ in mammalian cells occurs mainly via poly-ADP-ribosylation reactions. The concentration of NAD+ in undamaged, proliferating mammalian cells is approximately 400 to 500 µM, and its half-life is about 1 to 2 h (115, 329, 438). However, when cells were exposed to high doses of genotoxic agents, sustained activation of poly-ADP-ribosylation reactions, coinciding with an increase in the levels of poly-ADP-ribose polymers generated following DNA damage, was shown to rapidly decrease the half-life of NAD+ in a dose-dependent manner. In fact, intracellular NAD+ levels undergo a decrease to 10 to 20% of their normal levels within 5 to 15 min upon exposure of cells to very high doses of DNA-damaging agents (143, 369). NAD+ depletion also results in ATP depletion, as NAD+ is an essential coenzyme/transmitter for the generation of ATP. The resynthesis of NAD+ requires two to four molecules of ATP per molecule of NAD+, depending on which salvage pathway is used in the cell and whether NamPRTase or NaPRTase is the ATP-consuming enzyme in vivo (70) (Fig. 1). It should be noted, however, that several studies indicate that under moderate levels of DNA damage, intracellular NAD+ levels undergo a decrease of only 5 to 10%. For a more detailed description of the NAD+ metabolism and enzymology, the reader is referred to the recent excellent reviews on this topic (243-245, 339).
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MONO-ADP-RIBOSYLTRANSFER REACTIONS
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Mono-ADP-ribosylation of proteins is a phylogenetically ancient, reversible, and covalent posttranslational modification of proteins in which the ADP-ribose moiety of NAD+ is transferred to a specific amino acid of an acceptor protein with the simultaneous release of nicotinamide (reviewed in references 304, 305, and 356). The reaction can occur through both enzymatic and nonenzymatic mechanisms (reviewed in references 302 and 304). Enzymatic mono-ADP-ribosylation reactions, originally identified as the pathogenic mechanism of several bacterial toxins, including pertussis toxin, cholera toxin, and certain clostridial toxins, are catalyzed by MARTs. Such enzymes have been detected in many prokaryotic and eukaryotic species and in viruses (reviewed in references 88, 140, 304, and 305). The extent of posttranslational modification by mono-ADP-ribosylation depends on the activity of cellular mono-ADP-ribose-protein hydrolases (MARHs), which reverse the reaction by hydrolyzing the protein-ADP-ribose bond (reviewed in references 88, 202, 304, and 305). The simultaneous presence of mono-ADP-ribosyltransferase and mono-ADP-ribose-protein hydrolase activities in the same cell suggests that mono-ADP-ribosylation of proteins acts as a reversible regulatory mechanism (306, 374; reviewed in references 202 and 304). MARTs and MARHs are opposing arms of an ADP-ribosylation cycle (306, 374). In contrast to the case for the prokaryotic ADP-ribosylation cycle, the functional relationship between MARTs and MARHs in eukaryotes is poorly documented (304, 305). Thus, the detailed mechanisms of coupling of MARTs and MARHs in eukaryotic mono-ADP-ribosylation cycles need to be investigated further.
Mono-ADP-ribosylation occurs at a number of different amino acid residues, directed by the specificity of the individual MARTs. The best-studied mono-ADP-ribosylation reactions are catalyzed by bacterial toxins (reviewed in references 88, 304, and 356). At least six amino acid-specific subclasses of bacterial MARTs have been characterized or identified so far. The amino acid residues of crucial host cell protein acceptors modified by specific bacterial MARTs include arginine, asparagine, glutamate, aspartate, cysteine, and modified histidine (diphthamide) (reviewed in references 88, 89, 304, and 305). Mono-ADP-ribosylation of cellular proteins through nonenzymatic mechanisms usually involves the conjugation of ADP-ribose to lysine or cysteine residues (59, 60, 184, 214; reviewed in reference 174). Amino acid-mono-ADP-ribose-specific MARHs cleave the ribose-amino acid bond, leading to release of free mono-ADP-ribose and regeneration of the free reactive group of the corresponding amino acid residue. A detailed description of distinct protein mono-ADP-ribosylation products is shown in Fig. 2. In eukaryotic cells, endogenous protein-mono-ADP-ribosyltransferase activities that modify arginine, glutamate, cysteine, phosphoserine and potentially aspartate and asparagine residues of acceptor proteins have also been detected (308, 373; reviewed in references 88, 89, 304, 305, and 356). For instance, intracellular mono-ADP-ribosylation has been demonstrated for heterotrimeric GTP-binding proteins, small GTPases, endoplasmic reticulum-resident glucose regulatory protein 78, tubulin, actin, elongation factor 2, mitochondrial glutamate dehydrogenase (GDH), and histones. A detailed list of distinct intracellular protein substrates is given in Table 1. A number of mono-ADP-ribosyltransferases have also been shown to ribosylate small molecules such as free amino acids, DNA, or RNA (reviewed in references 88, 304, and 356). Pierisin-1 and its homolog pierisin-2, two unique ADP-ribosylation toxins from the cabbage butterflies (Pieris rapae and Pieris brassicae), were shown to catalyze mono-ADP-ribosylation of 2'-deoxyguanosine in DNA to form N2-(C1-ADP-ribosyl)-2'-deoxyguanosine (386, 387).
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FIG. 2. Mono-ADP-ribosylation cycle and the corresponding products of protein mono-ADP-ribosylation. (A) Mono-ADP-ribosyl transferases catalyze the transfer of the ADP-ribose moiety of NAD+ to an acceptor molecule (free amino acids, proteins, DNA, and RNA, etc.). The action of mono-ADP-ribosyl-amino acid hydrolases, which regenerate the corresponding free acceptor molecule, is consistent with the presence of a mono-ADP-ribosylation cycle. (B) The transferase-catalyzed reaction of protein mono-ADP-ribosylation results in a stereo-specific formation of  -N-(C-1-ADP-ribosyl)-L-arginine,  -N-(C-1-ADP-ribosyl)-L-asparagine,  -N-(C-1-ADP-ribosyl)-L-diphtamide,  -S-(C-1-ADP-ribosyl)-L-cysteine, -O-(C-1-ADP-ribosyl)-L-glutamate,  -O-(C-1-ADP-ribosyl)-L-aspartate, or O-(ADP-ribosyl)-L-phosphoserine.
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Mono-ADP-Ribosyltransferase Families
The mammalian e-MARTs (e-MART-1 to -5/6) represent the only mammalian mono-ADP-ribosyltransferase family of structurally related proteins characterized on the molecular level (reviewed in references 88, 202, 304, and 356). Human and mouse e-MARTs identified to date represent either glycosylphosphatidylinositol-anchored surface proteins or secretory proteins. e-MART-1, e-MART-2a, e-MART-2b, and e-MART-5 were found to transfer ADP-ribose to arginine residues in extracellular target proteins, on cell surfaces, or circulating in body fluids (reviewed in references 88, 140, and 356). In contrast, e-MART-3 and e-MART-4 did not display any arginine-specific enzymatic activity (reviewed in references 88, 140, and 356). Mono-ADP-ribosyltransferase activity on the surface of human monocytes correlated with the presence of e-MART-3 in unstimulated monocytes (146). Consistent with its expression in lymphatic tissues, e-MART-4 is expressed only in response to stimulation with lipopolysaccharide (146). Interestingly, cell surface mono-ADP-ribosylated proteins on human monocytes are covalently modified at cysteine residues, which strongly suggests that e-MART-3 and e-MART-4 may be cysteine-specific e-MARTs (146). Thus, it is unlikely that members of the known e-MART family are involved in mono-ADP-ribosylation of intracellular protein targets, such as heterotrimeric G proteins or histones, although one cannot exclude the possible existence of alternatively spliced or processed cytosolic isoforms (88, 109). Corda and Di Girolamo (88, 89) proposed that these novel intracellular MARTs most likely constitute different families of proteins with no obvious sequence similarity to the well-known e-MARTs (88, 89, 109). Although a preliminary study reported partial purification of an arginine-specific mono-ADP-ribosyltransferase from hen liver nuclei with activity towards histones H2A, H2B, H3, and H4 (389), no arginine-directed cytosolic or nuclear mono-ADP-ribosyltransferases from mammalian cells have been unambiguously identified and analyzed on a molecular level. Thus, most authors refer to these mono-ADP-ribose modifications as "poly-ADP-ribosylation," presuming that these mono-ADP-ribose modifications represent remnants of poly-ADP-ribose polymers catalyzed by poly-ADP-ribosylation enzymes (see "Covalent poly-ADP-ribosylation of nuclear substrates?" below). However, at least two distinct families of putative intracellular MARTs may exist in mammalian cells. The first family is represented by the well-known sirtuin family of NAD+-dependent histone deacetylases and ADP-ribosyltransferases (SIRT1 to -7) (128, 132, 234, 391). The second possible family consists of 11 novel PARP-like gene products, referred to here as Pl-MARTs (Pl-MART-1 to -11) (20, 140, 309).
The family of putative PARP-like mono-ADP-ribosyltransferases.
During the last 10 years, more than 16 novel Parp-like genes were cloned or described based on thorough searches of nonredundant databases (5, 6, 20, 130, 140, 241, 309, 452). Among all 17 human and 16 mouse Parp-like genes, only the 6 human and mouse "bona fide" poly-ADP-ribose polymerase gene products contain an evolutionarily conserved catalytic glutamate residue (309). The crystal structure of chicken PARP-1 and amino acid replacement analysis of human PARP-1 demonstrated that the conserved residue, E988, is essential for poly-ADP-ribose chain elongation (255, 338, 342). The absence of this crucial residue in PARP-1 was shown to restrict its enzymatic activity to mono-ADP-ribosylation. Surprisingly, several residues important for poly-ADP-ribose chain elongation, including E988 and residues thought to be required for the PARP branching activities (255, 342, 366), seem to be missing in 11 of the Parp-like genes identified (5, 6, 20, 241, 452). Otto and coworkers recently suggested that these 11 human and 10 mouse Parp-like gene family members may possess mono-ADP-ribosyltransferase rather than poly-ADP-ribosyltransferase activity (309). However, as those authors pointed out, one cannot exclude the possibility that some of these 11 novel Parp-like gene family members are not enzymatically active or may even have acquired novel functions (309). There is preliminary experimental evidence that at least one of the proposed Pl-MARTs, BAL-1, lacks any auto-ADP-ribosylation activity (5, 6). On the other hand, most of the Pl-MART enzymes investigated on a biochemical level (PARP-10, TiPARP, BAL-2, and BAL-3) possess auto-ADP-ribosylation activity (5, 6, 241, 452). Thus, these novel PARP-like gene products represent candidates for a possible large family of intracellular PARP-like mono-ADP-ribosyltransferases.
The members of the proposed Pl-MART family can be divided according to their domain structures and the sequences of their catalytic domains into at least four subgroups. Figure 3 shows a proposed classification of the family based on recent literature and database searches (309). Although all putative Pl-MARTs share an evolutionarily conserved "PARP signature" motif in their catalytic domains (20, 140), most family members are structurally distinct from the "classical" 114-kDa PARP-1 and other previously described "bona fide" PARPs. The Pl-MARTs contain a diversity of adaptor domains and additional motifs, including WWE domains, macroH2-like domains, and ubiquitin- or RNA-binding motifs, suggesting that they possess unique properties and are involved in many biological functions in which the previously described PARP family might not participate. Subgroup I contains Pl-MART-1 (also known as PARP-6 [20]), Pl-MART-2 and possibly its alternatively transcribed or spliced short isoform Pl-MART-2b (formerly PARP-8 [20]), and Pl-MART-3 (also known as PARP-16 [20]). The functions of Pl-MART-1 to -3 are not yet known. Pl-MART-4 (also known as PARP-10), a Myc-interacting protein that inhibits transformation (452), is the only member of subgroup II. Subgroup III includes four members that contain a C3H1-type zinc finger and/or a WWE domain: Pl-MART-5 (initially described as PARP-11 [20]), which has an unknown function; Pl-MART-6 (also known as TiPARP and PARP-7 [241]), whose homolog in Arabidopsis thaliana, CEO1/RCD1, is involved in hormonal signaling and stress-response pathways (7, 30); Pl-MART-7 (ZC3HDC1, formerly PARP-12), which has an unknown function (20); and Pl-MART-8, previously described as the C3H1-type zinc finger-containing antiviral protein-1 (130). The nuclear-localized family members Pl-MART-9, Pl-MART-10, and Pl-MART-11, initially described as risk-related proteins in diffuse large B-cell lymphomas that enhance cellular migration (B-aggressive lymphoma proteins 1 to 3 [BAL-1, BAL-2, and BAL-3, also known as PARP-9, PARP-14, and PARP-15] [5, 6, 20]), belong to the macroH2A and/or WWE domain-containing Pl-MART subgroup VI. Interestingly, Pl-MART-11 may exist only in the human genome (309). Since the biochemical evidence for mono-ADP-ribosylation by Pl-MARTs has yet to be established and because several different classifications have already been proposed for Parp-like genes (20, 309), a final, consistent classification of all PARP-like proteins needs to be made in agreement with the entire PARP/MART community once their different enzymatic activities are characterized.
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FIG. 3. Domain structures of the human Pl-MART family. A new classification and a schematic comparison of protein structures of the 11 members of the Pl-MART family, based on the literature and database searches, are shown. The most significant domains detected have been indicated. The WWE domain is named after three of its conserved residues (W/W/E) and is predicted to mediate specific protein-protein interactions in ubiquitin- and ADP-ribose conjugation systems (24). Although the exact roles of the conserved macroH2A/A1pp domains remain unknown, they have been proposed to have ADP-ribose 1"-phosphate (Appr-1"p)-processing activity and may regulate mono-ADP-ribosylation (219). ZF, C3H-type zinc finger domain; RRM, RNA recognition motif (252); UIM, ubiquitin interaction motif (22); MVP-ID, M-vault particle interaction domain; TPH, Ti-PARP homologous domain; GRD, glycine-rich domain.
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The SIRTs, a family of putative intracellular mono-ADP-ribosyltransferases.
The silent information regulator SIR2-like proteins, also named SIRTs, represent a family of NAD+-dependent deacetylases (SIRT1 to -7) (reviewed in references 37, 106, and 147). The SIRT family regulates a wide range of cellular processes, including development, metabolism, heterochromatin formation, chromosome segregation, DNA transcription, DNA repair, DNA recombination, cellular differentiation, apoptosis, and the determination of life span (reviewed in references 37, 106, and 147). The sirtuins are phylogenetically conserved in eukaryotes, prokaryotes, and archaea. The first discovered member of this protein family is the yeast SIR2 histone deacetylase of Saccharomyces cerevisiae, which is required for transcriptional silencing (reviewed in reference 37). Most of the mammalian SIRTs were found to have intrinsic histone deacetylation activity in vitro and in vivo. Of the seven mammalian SIR2-like proteins, SIRT1, SIRT2, SIRT3, and SIRT5 have been shown to have NAD+-dependent deacetylase activities in vitro (271; reviewed in references 37 and 106). However, SIRTs often have nonhistone substrates, and not all mammalian SIRT members are localized to the nucleus. Consequently, the SIRTs have a diversity of substrates that reflect the various biological processes in which the enzymes function. For example, human SIRT1 and its mouse homolog SIR2
were reported to deacetylate, in vivo, acetylated transcription factors such as p53 and DNA repair factors such as Ku70, while acetylated -tubulin was found to be an in vivo deacetylation target for hSIRT2 (82, 289, 422). For a detailed description of SIRTs and their functional roles, see the recent reviews on this topic (37, 106, 147).
Based on extensive analyses of the NAD+-dependent deacetylation reaction, an unusual mechanism has been proposed (371, 462, 463; reviewed in reference 106). SIRTs consume one NAD+ cosubstrate molecule per acetyl group, which is removed from a lysine side chain. SIRTs cleave the glycosidic bond between the Nam and ADP-ribose portions of NAD+. The ADP-ribose intermediate is necessary for deacetylation to take place (371). Subsequently, the acetyl group removed from the target substrate can be transferred to the ADP-ribose moiety to form 2'-O-AADP-ribose and 3'-O-AADP-ribose. Several reports suggested that the mammalian SIRT family members SIRT1, SIRT2, and SIRT6 might possess intrinsic mono-ADP-ribosyltransferase activity (128, 132, 234, 284, 391). SIRT1, SIRT2, and SIRT6 were shown to transfer mono-ADP-ribose to bovine serum albumin and histones in vitro (128, 284, 391). Furthermore, a point mutation in yeast SIR2 that abolished the observed histone ADP-ribosyltransferase activities in vitro resulted in a complete loss of silencing in vivo, suggesting that the potential histone-ADP-ribosyltransferase activity of yeast SIR2 could be required for silencing (391). SIRT6 does undergo auto-mono-ADP-ribosylation via an intramolecular reaction mechanism (234). All seven human SIRTs characterized so far share a conserved histidine, which may be important not only for their NAD+-dependent deacetylase activities but also for their mono-ADP-ribosyltransferase activities (128; reviewed in reference 347). Whether NAD+-dependent deacetylation of proteins, acetylation of ADP-ribose, or the potential mono-ADP-ribosylation of proteins can occur simultaneously or depends on the conditions and the type of SIRT protein involved remains to be investigated. The mono-ADP-ribosyltransferase activity of yeast SIR2 (as well as its NAD+-glycohydrolase activity) has been shown to require the presence of an acetyl-lysine-containing substrate in the reaction, suggesting that it is linked to deacetylation (391). In contrast, SIRT6 and SIRT7 did not possess in vitro protein deacetylase activity, indicating that the enzymatic mechanisms are different for each SIRT member (234, 271). SIRT6 catalyzed in vitro ADP-ribosylation of nonacetylated bovine serum albumin much more efficiently than SIRT1. In contrast, histone H1 was a better substrate for SIRT1 than for SIRT6 (284). However, the possible mono-ADP-ribosyltransferase activities of the different SIRT members have not been adequately addressed on a molecular level (106). Further investigation is needed to determine if the SIRTs do indeed have bona fide protein mono-ADP-ribosyltransferase activity in vitro. The in vivo relevance of the mono-ADP-ribosylation activity of SIRTs remains a subject of debate, although it has been speculated to be involved in DNA double-strand break and base excision repair (132, 160, 284). Indeed, a recent study provided evidence that certain SIRTs may function in vivo as potential mono-ADP-ribosyltransferases in DNA damage response pathways (132). TbSIR2RP1, a SIR2-related protein from the protozoan parasite Trypanosoma brucei, has been shown to catalyze mono-ADP-ribosylation of histones in vitro, particularly H2A and H2B (132). Treatment of trypanosomal nuclei with a DNA-alkylating agent resulted in a significant increase in the level of histone mono-ADP-ribosylation, specifically that of H2A and H2B, and a concomitant increase in chromatin sensitivity to micrococcal nuclease. Both of these responses correlated with the level of TbSIR2RP1 expression (132). A possible O-AADP-ribose and mono-ADP-ribosylation metabolism is schematically drawn in Fig. 4, based on the literature (46, 106, 327, 371).
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FIG. 4. Possible metabolism of acetyl-ADP-ribose (O-AADPR) and mono-ADP-ribosylation of proteins by SIRTs. SIRTs cleave the glycosidic bond between the Nam and ADP-ribose portions of NAD+. The ADP-ribose intermediate is necessary for the deacetylation reaction. Following hydrolysis of the glycosidic bond, nicotinamide is released, and ADP-ribose binds the acetyl-peptide, forming of an O-alkylamidate intermediate. The acetyl group removed from the target substrate is transferred to the ADP-ribose moiety to form 2'-acetyl-ADP-ribose (2'-O-AADPR) and then subsequently released together with the deacetylated protein from the enzyme-intermediate complex. 2'-O-AADPR spontaneously equilibrates with the regioisomer 3'-acetyl-ADP-ribose (3'-O-AADPR) through trans-esterification. In mammalian cells, acetyl-ADP-ribose can be deacetylated by esterases to the ATP precursor ADP-ribose or can function as an acetyl donor to acetylate unknown substrates by nuclear trans-acetylases. Transformation of acetyl-ADP-ribose into AMP and acetyl-ribose-5-phosphate by ADP-ribose hydrolases of the Nudix family is not clearly established. The possible deacetylation-dependent and -independent transfers of mono-ADP-ribose to proteins catalyzed by SIRTs are shown in the lower part of the figure.
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Nuclear substrates for covalent mono-ADP-ribosylation of proteins.
In eukaryotes, mono-ADP-ribosylation of arginine residues occurs on extracellular, cytoplasmic, and nuclear target proteins, whereas mono-ADP-ribosylation of cysteines occurs on extracellular, cytoplasmic, and mitochondrial target proteins. Mono-ADP-ribosylation of asparagine residues may be restricted to the cytoplasm, while mono-ADP-ribosylation of glutamate residues and potentially of the phosphate group of phosphoserine may be restricted to the nucleus (Tables 1 and 2). During the last two decades, several studies indicated that histones are covalently modified by mono-ADP-ribose in response to genotoxic stress, while other reports proposed that the extent of mono-ADP-ribosylation of histones varied depending on the cell cycle stage, proliferation activity, and degree of terminal differentiation (1, 142, 211, 212, 373, 379, 395, 435). For instance, when cells were exposed to damage by · OH radicals or methylating/alkylating agents, total covalent mono-ADP-ribosylation of histones increased 3 to 15 times, whereas the levels of histone H1-linked mono-ADP-ribosyl groups were even elevated by more than 30-fold (211, 212). Initial reports suggested that histone H1 (H1.1/H1.2/H1.3/H1.4/H1.5) was covalently mono-ADP-ribosylated on glutamate residues E2, E15, and E114/E115/E117 and arginine residue R33 (H1.3) and histone H2B on E2 (55, 292, 293, 335, 416). These modified sites were identified by "in vivo" ADP-ribosylation of histones, using radiolabeled NAD+ and subsequent high-pressure liquid chromatography-coupled Edman sequencing analysis of the radiolabeled single peptides (55, 292, 293, 416). Other studies indicated that mono-ADP-ribosylation also occurs on glutamic acid residues of H2A; on arginine residues of H2A, H2B, H3, and H4; and on the phosphate group of phosphoserine in histones H1, H2A, H2B, H3, and H4 (141, 308, 373, 389). Several reports indicated that the nonhistone, high-mobility group (HMG) proteins HMGA1a, HMGA1b, HMGA2, HMGB1, HMGB2, HMGN1, and HMGN2 might also serve as targets for mono-ADP-ribosylation in intact cultured cells (394, 396, 398). However, the specific amino acid residues in HMG proteins that were thought to be mono-ADP-ribosylated were not identified. To date, endogenous mono-ADP-ribosylation of HMGB1 and HMGB2 has been observed following DNA damage in intact cells (394, 396, 398). The same group also reported that treatment of cells with glucocorticoids quickly decreased endogenous mono-ADP-ribosylation on HMGN1 and HMGN2 proteins, while mono-ADP-ribosylation on HMGB1, HMGB2, and histone H1 was less susceptible to hydrolysis during glucocorticoid treatment (394). Mono-ADP-ribosylation of chromosomal proteins may influence the regulation of human myeloid cell maturation (239). A detailed list of distinct nuclear protein substrates for mono-ADP-ribosylation is given in Table 2.
Substrate specificities of the SIRT and putative Pl-MART families.
The enzymatic activities and bond specificities of SIRTs and Pl-MARTs have not yet been experimentally determined. Thus, it is quite difficult to predict any substrate specificities for these enzymes. Based on the assumption that the SIRTs have arginine-specific and cysteine-specific MART activities, while the Pl-MARTs have glutamate- and aspartate-specific MART activities, one may propose that mono-ADP-ribosylation on arginine residue R33 of histone H1.3 and at arginine residues of histones H2A, H2B, H3, and H4 is mediated by nuclear members of the SIRT family, such as SIRT1, SIRT6, or SIRT7, whereas mono-ADP-ribosylation on glutamate residues E2, E15, and E114/115/117 of H1 (H1.1/H1.2/H1.3/H1.4/H1.5) and on E2 of histone H2B is mediated by nuclear members of the Pl-MART family. The mitochondrial SIRT3, SIRT4, or SIRT5 (271) may be responsible for the cysteine-specific mono-ADP-ribosylation of GDH on cysteine 119 (Table 1) (78). SIRT2, a predominantly cytoplasmic protein associated with microtubules and acting as a bona fide tubulin deacetylase (289), could be responsible for the arginine-specific mono-ADP-ribosylation of both alpha and beta chains of tubulin. Several studies provided evidence that both deacetylation and arginine-specific mono-ADP-ribosylation of tubulins are involved in depolymerization of the microtubules (260, 289, 351, 403). The heterotrimeric GTP-binding proteins and small GTPases may represent substrates for the predominantly cytoplasmic members of the Pl-MART family (Table 1).
The diversity of substrate and amino acid specificity is reflected by the diverse biological activities of specific SIRTs and Pl-MARTs. Additionally, substrate specificity could also be regulated by cofactors, such as small GTPases and ADP-ribosylation factors (ARFs). ARFs are 20-kDa guanine nucleotide-binding proteins that play a critical role in many vesicular trafficking events and were initially identified as stimulators of bacterial toxin-catalyzed ADP-ribosylation of GTP-binding proteins (45, 179, 280, 281, 421). ARFs were shown to activate, allosterically, bacterial toxin mono-ADP-ribosyltransferases (45, 307).
Mono-ADP-Ribose-Protein Hydrolases
Several reports demonstrated the existence of distinct intracellular mono-ADP-ribose-protein hydrolase activities (reviewed in references 202, 304, 305, and 306). The best-characterized intracellular mono-ADP-ribose-protein hydrolase activity is represented by mono-ADP-ribose-arginine-hydrolase-1 (MARH-1), which specifically hydrolyzes ADP-ribose-arginine bonds, leading to the release of free mono-ADP-ribose and regeneration of the guanidino group of arginine (385; reviewed in reference 283). In mammalian cells, most ADP-ribose-arginine hydrolase activities are cytosolic, although some are located on the cell surface. Besides the well-characterized MARH-1, additional proteins exhibiting hydrolase activity towards mono-ADP-ribose linked to glutamates or cysteines were identified and partially purified. A mono-ADP-ribosyl protein-lyase, catalyzing the cleavage of the bond between mono-ADP-ribose and glutamate residues in histone H2B or H1, was purified from rat liver and characterized (296, 300). The purified enzyme exhibited a single protein band at the position of 83 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This putative enzyme was hypothesized to act as a mono-ADP-ribose-glutamate-lyase, whose cleavage product is 5'-ADP-3"-deoxypent-2"-enofuranose, a dehydrated form of ADP-ribose (296). In addition, hydrolysis of linkages between mono-ADP-ribose and cysteine residues in the alpha subunits of heterotrimeric GTP-binding proteins was found in the cytosol of human erythrocytes (392). This putative mono-ADP-ribose-cysteine-hydrolase was tentatively named ADP-ribose-protein hydrolase C. Moreover, mitochondrial GDH was recently identified as a specific target for enzymatic cysteine-specific mono-ADP-ribosylation (78, 163). This covalent mono-ADP-ribosylation was removed by an ADP-ribose-cysteine hydrolase activity present in mitochondria (163).
Until recently, the only cloned and characterized mammalian gene encoding a soluble, intracellular cytosolic mono-ADP-ribose-protein hydrolase was the Marh1 gene, whose product hydrolyzes mono-ADP-ribose-arginine bonds (385). Two additional ADP-ribosyl-hydrolase-like genes, Arh2 and Arh3, have recently been identified by screening the human genome sequence and cDNA databases for homologues of the Marh1 gene (140). A recent report demonstrated that ARH-2 and ARH-3 did not hydrolyze ADP-ribose-arginine, -cysteine, -diphthamide, or -asparagine bonds (297). ARH-3 may possess intrinsic poly-ADP-ribose-ribose-glycohydrolase activity, generating free ADP-ribose from PARP-1-bound poly-ADP-ribose (see "PARGs" below) (297). Thus, ARH-2 may be a candidate for a glutamate- or aspartate-specific mono-ADP-ribose-protein hydrolase. The presence of distinct intracellular mono-ADP-ribose-protein hydrolase activities which are not connected to the identified Arh-like genes in the human genome suggests that mono-ADP-ribose-protein hydrolases might encompass different families.
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POLY-ADP-RIBOSYLATION REACTIONS
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Poly-ADP-Ribosylation Cycle
Poly-ADP-ribose is a homopolymer of ADP-ribose units linked by glycosidic bonds and synthesized by members of the PARP family (20, 93, 161). Like mono-ADP-ribose synthesis, poly-ADP-ribose synthesis requires NAD+ as a precursor and immediate substrate of the reaction. The constitutive levels of poly-ADP-ribose are usually very low in unstimulated cells (122, 164, 212; reviewed in reference 93). However, in response to mitogenic stimuli or genotoxic stress (i.e., in the presence of DNA strand breaks), the PARP activity and the levels of poly-ADP-ribose may increase 10- to 500-fold, while cellular NAD+ levels are correspondingly reduced. Both constitutive and activated levels of poly-ADP-ribose are functions of the concentration of NAD+ in cells (15, 164, 436, 437). However, most free or protein-associated poly-ADP-ribose polymers synthesized upon genotoxic stress are rapidly degraded in vivo, with a half-life of >40 s to 6 min, which accounts for their transient nature in living cells (15, 173, 175, 435). There may also be a biphasic decay: 85% of poly-ADP-ribose polymers synthesized due to genotoxic stress have a half-life of less than 40 s, while the residual fraction is catabolized with a half-life of approximately 6 min (15, 435). This rapid turnover contrasts with the slow catabolism of the constitutive fraction of poly-ADP-ribose, with a much longer half-life of 7.7 h (15). It is likely that the degradation starts immediately upon initiation of poly-ADP-ribose synthesis, suggesting that poly-ADP-ribose-metabolizing enzymes are tightly regulated.
The structure of poly-ADP-ribose is well characterized. The ADP-ribose units in the polymer are linked by glycosidic ribose-ribose 1'-2' bonds. The chain length of polymers is heterogeneous and can reach 200 to 400 units in vitro and in vivo (16, 181, 182, 186, 276). Long polymers are branched in an irregular manner. Branching occurs in vitro and in vivo with a frequency of approximately one branch per linear section of 20 to 50 units of ADP-ribose (16, 181, 182, 186, 276). The chemical structure of the branching site of poly-ADP-ribose was determined by nuclear magnetic resonance and mass spectroscopy (MS) and found to be the same as in the linear regions of the polymer (276). The branching site of poly-ADP-ribose is O-D-ribofuranosyl-(1'-2')-O-D-ribofuranosyl-(1'-2')-adenosine-5',5',5-triphosphate, commonly known as Ado-(P)-Rib-(P)-Rib-P (276). The global structures of the branched poly-ADP-ribose can be very complex. T. Minaga and E. Kun postulated that the structures of certain types of long poly-ADP-ribose chains can have helicoidal secondary structures and thus may have some similarity to the structures of RNA and DNA (272, 273). Interestingly, many antibodies against poly-ADP-ribose can recognize RNA and DNA and vice versa (188, 362, 363). The functional significance of the structural heterogeneity of poly-ADP-ribose is not known, but it could play a crucial role in determining specific functional outcomes in vivo (i.e., stress-dependent signaling in regard to survival or cell death).
At least four distinct enzymatic activities were postulated to be required for the synthesis of free or PARP-associated linear and branched poly-ADP-ribose (17, 93, 264): (i) initiation or covalent auto-mono-ADP-ribosylation of the corresponding poly-ADP-ribose polymerase; (ii) elongation of the polymer, whereby the covalently bound mono-ADP-ribose serves as a starting unit; (iii) branching of the polymer; and (iv) release of the enzyme-bound branched poly-ADP-ribose, either through poly-ADP-ribose-ribose-glycohydrolase activities (intrinsic or by PARG [see below]) or through a putative intrinsic poly-ADP-ribosyl-protein-hydrolase activity. Based on experimental evidence in vitro, it was suggested that the classical PARP enzyme, PARP-1, possesses (i) auto-mono-ADP-ribosylation, (ii) elongation, and (iii) branching activities. Whether poly-ADP-ribose polymerases also possess intrinsic poly-ADP-ribose-ribose-glycohydrolase or poly-ADP-ribosyl-protein-hydrolase activities that release the polymers remains to be investigated.
To date, two different enzymes or enzymatic activities are known or hypothesized to degrade free (non-protein-bound) or protein-bound linear or branched poly-ADP-ribose (reviewed in references 93 and 340). The major enzymatic activity is the well-characterized poly-ADP-ribose glycohydrolysis carried out by PARGs (53). Poly-ADP-ribose phosphodiesterase/ADP-ribose pyrophosphatase was suggested to possess pyrophosphatase activity and to cleave the pyrophosphate linkages to release 5'-AMP from chain termini, phosphoribosyl-AMP from internal residues, and diphosphoribosyl-AMP from branching points (49). Of these two enzymatic activities, only the Parg genes and their gene products have been identified and characterized to date (18, 230, 297). The major mammalian poly-ADP-ribose-ribose-glycohydrolase, PARG, has both endoglycosidase and exoglycosidase activities (18, 52, 53), which are responsible for the hydrolysis of glycosidic ribose-ribose bonds internal to and at the ends of ADP-ribose polymers, respectively. The endoglycosidase activity releases free poly-ADP-ribose from PARPs and is of particular physiological importance because it may provide a mechanism for the generation of various types of free poly-ADP-ribose. These products may be important signaling molecules involved in distinct cellular processes, such as cell death or cell growth. In addition, branched and short polymers are degraded more slowly by PARGs than long and linear poly-ADP-ribose polymers (18, 52, 53). This mode of action of PARGs may explain the very short half-life of poly-ADP-ribose synthesized in the presence of DNA damage in vivo (<40 s) compared with the far longer half-life (
7.7 h) of constitutively synthesized poly-ADP-ribose in unstimulated cells (15, 435, 437). Thus, the biphasic degradation of poly-ADP-ribose in vivo clearly indicates that two major types of polymers (linear
branched) with different structures and distinct half-lives exist in vivo (16). The complexity and concentration of each distinct type and structure of poly-ADP-ribose may vary not only depending on the cellular context and stimuli, but also depending on specific branching activities of different PARPs in vivo. An overall view of poly-ADP-ribosylation reactions and metabolism is shown in Fig. 5.
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FIG. 5. Poly-ADP-ribose metabolism. Steps 1 to 3 and steps 4 to 7 of the poly-ADP-ribose cycle represent the anabolic and catabolic reactions, respectively, in the metabolism of poly-ADP-ribose. The synthesis of poly-ADP-ribose requires three distinct PARP activities: step 1, initiation or mono-ADP-ribosylation of a specific glutamic (?) acid residue(s) in the corresponding PARP enzyme (acceptor); step 2, elongation of the polymer; and step 3, branching of the polymer. The degradation requires at least four (alternative) PARG and (P/M)ARH activities: step 4, exoglycosidase and endoglycosidase (PARG) activities, respectively, that hydrolyze the glycosidic linkages between the ADP-ribose units; step 5, potential poly-ADP-ribosyl-protein hydrolase activities; and step 6, MARH, or step 7, mono-ADP-ribosyl-protein lyase activities. Chemical structures in this figure were drawn with MarvinSketch, version 4.0.4 (ChemAxon, Budapest, Hungary).
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In addition to the well-established model of synthesis of free or PARP-associated poly-ADP-ribose, several groups proposed that poly-ADP-ribosylation may also serve as a covalent posttranslational modification of proteins (reviewed in references 93, 160, and 161). It was suggested that different poly-ADP-ribose polymerases covalently attach poly-ADP-ribose to the side chain carboxyl groups of glutamic or aspartic acid residues of putative acceptor proteins (reviewed in reference 14). Similar to the synthesis of free poly-ADP-ribose, ADP-ribose units may be added successively to acceptor proteins to form branched protein-bound polymers (reviewed in reference 14). More than 30 years ago, several groups proposed that this putative covalent posttranslational modification is very transient but extensive in vivo, with the poly-ADP-ribose chains reaching more than 200 units on protein acceptors (reviewed in reference 14). The observed mono-ADP-ribose groups covalently bound to proteins in vivo were suggested to be remnants of poly-ADP-ribose polymers, and the major regulatory step of poly-ADP-ribosylation of proteins was proposed to be catalyzed in vivo by ADP-ribose-protein hydrolases (435, 436). More recently, it was postulated that PARG itself has the predicted ADP-ribose-protein hydrolase activity responsible for the hydrolysis of the most proximal unit of ADP-ribose on the protein acceptor (108). Thus, PARG was thought to modulate the level and complexity of poly-ADP-ribose on the different acceptor proteins, thereby preventing hypermodification of nuclear proteins with very long poly-ADP-ribose chains (108). However, no convincing in vitro data have been published, and to date, no ADP-ribose-protein hydrolase mutants of PARGs exist.
The PARP Family
For a long time, the best-investigated PARP protein, PARP-1, has been thought to be the only enzyme with poly-ADP-ribosylation activity in mammalian cells. However, this view has been challenged recently by the development of mice deficient in the Parp1 gene (431, 432) and the identification of novel poly-ADP-ribosylating enzymes. Primary cells derived from Parp1–/– mice can still synthesize poly-ADP-ribose following treatment with DNA-damaging agents (360). Five new genes encoding "bona fide" PARP enzymes have been identified (19, 178, 185, 196, 376), indicating that PARP-1 belongs to a family of poly-ADP-ribose polymerases.
The six "bona fide" PARP family members can be divided into at least three subgroups according to their domain structures, the sequences of their catalytic domains, and their enzymatic activities. Figure 6 shows a classification and schematic comparison of protein structures of the PARP family based on the literature and on database searches. Subgroup I includes PARP-1; PARP-1b, which seems to be a product of an alternative transcription initiation site within the Parp1 gene (previously described as short PARP-1 [343]); PARP-2; and PARP-3 (19, 178). Experimental data suggest that both PARP-1 and PARP-2 play a major role in distinct stress response pathways (reviewed in references 20 and 161). Subgroup II contains a single member, PARP-4 (vault-PARP), which is the largest of the family (192.6 kDa) and was identified as a component of the vault complex. The vault complex is a cytoplasmic ribonucleoprotein complex of unknown function associated with an untranslated vault RNA and two other highly conserved proteins, major vault protein and telomerase-associated protein-1 (196). Tankyrase 1, tankyrase 2a, and perhaps its alternatively spliced or transcribed isoform tankyrase 2b, which are referred to in this review as PARP-5 and PARP-6a/b, respectively, belong to subgroup III (20, 185, 376). Both PARP-5 and PARP-6a were identified as components of the telomeric complex (185, 376).
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FIG. 6. Domain structures of the human PARP family. A classification and a schematic comparison of protein structures of the six members of the bona fide PARP family, based on literature and database searches, are shown. The most significant domains detected have been indicated. The PRD domain is called the PARP regulatory domain and may be involved in regulation of the PARP-branching activity. The WGR domain is named after the most conserved central motif (W/G/R) of the domain. This motif is found in a variety of poly(A) polymerases and other proteins of unknown function. The BRCT domain is named after the breast cancer suppressor protein-1 (BRCA1) carboxy-terminal domain and is found within many DNA damage repair and cell cycle checkpoint proteins (446). The unique diversity of this domain superfamily allows BRCT modules to interact by forming homo- or hetero-BRCT multimers and phosphorylation-dependent BRCT-non-BRCT interactions (139, 446). The sterile alpha motif (SAM) is a widespread domain in signaling and nuclear proteins and mediates homo- or heterodimerization in many cases (reviewed in reference 20). The ankyrin repeat domains (ARD) mediate protein-protein interactions in very diverse families of proteins (279). The number of ankyrin repeats in a protein can range from 2 to over 20 (279). The vault protein inter-alpha-trypsin (VIT) and von Willebrand type A (vWA) domains are conserved domains found in all inter-alpha-trypsin inhibitor (ITI) family members (261). Although the exact roles of these domains remain unknown, they are presumed to be involved in mediating protein-protein interactions (261). ZF-I and ZF-II, PARP-1-type zinc finger domains (they can act as DNA nick sensors and general DNA-binding domains [161]); SAP, SAF/Acinus/PIAS-DNA-binding domain; LZM, putative leucine zipper-like motif; MVP-ID, major-vault particle interaction domain; NLS, nuclear localization signal; CLS, centriole-localization signal; HPS, His-Pro-Ser region.
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All "bona fide" PARP enzymes (PARP-1, PARP-1b, PARP-2, PARP-3, PARP-4, PARP-5, and PARP-6a) have automodification activity and most likely covalent auto-ADP-ribosylation activity (19, 26, 185, 196, 343, 376). PARP-1 showed the strongest automodification activity in vitro. Based on this unique property, PARP-1 was identified as the main acceptor of radioactively labeled ADP-ribose in isolated nuclei and permeabilized cells (294). It was proposed that the covalently or noncovalently automodified forms of the enzyme do not serve as an intermediate in the synthesis of poly-ADP-ribose but play some biological role(s) as structural elements (294). The automodified domains were mapped for PARP-1 and PARP-2. Automodification takes place in the DNA-binding domains of PARP-1 and PARP-2 and in the so-called automodification domain of PARP-1 (107, 353). Earlier studies suggested that the auto-ADP-ribosylation activity targets the 25 to 30 glutamic acid residues in the automodification domain of PARP-1 (166). Moreover PARP-1 and PARP-2 were proposed to be able to trans-ADP-ribosylate each other at multiple sites, although it is not clear whether the modification is covalent or noncovalent (353; our unpublished observations). PARP-1 was shown to form different heteromers with PARP-2 and PARP-3 (26, 353). Another interesting aspect is the role of the PARPs in synthesis and branching of distinct types of poly-ADP-ribose. PARP-1 was suggested to synthesize complex, branched poly-ADP-ribose (reviewed in references 20 and 93). Further investigation is needed to see if the other PARPs of subgroup I, PARP-2 and PARP-3, have the same ability to catalyze all the reactions necessary to produce branched polymers or whether they can synthesize only linear polymers. Future studies will certainly clarify whether the PARP family could be subdivided into three classes of enzymatic activities: branched-polymer-synthesizing PARPs (i.e., PARP-1, PARP-2, and PARP-3), linear-polymer-synthesizing PARPs (PARP-4?), and linear-oligomer-synthesizing PARPs (PARP-5 and PARP-6) (334). The type of branched polymers might be characteristic for each branched-polymer-synthesizing PARP enzyme.
The enzymatic activities of PARP-1 and PARP-2 were initially proposed to be exclusively dependent on the presence of single-strand breaks and double-strand breaks in DNA (19, 145, 171). Recent studies have demonstrated that PARP-1 may be activated not only by DNA breaks induced by peroxynitrite, gamma radiation, and alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine or methylnitrosourea but also by other stimuli, such as D-myo-inositol-1,4,5-triphosphate, which does not directly involve DNA damage (167, 198, 215). These studies indicate that PARP-1 might be involved in new DNA damage-independent, poly-ADP-ribosylation-dependent signaling pathways. The PARP activities of the very closely related PARP-1b (short PARP-1) and PARP-3 are not stimulated by DNA strand breaks (26, 343). PARP-1 was thought to be the major anabolic activity responsible for poly-ADP-ribosylation in living cells. Activation of poly-ADP-ribose polymerases was proposed to be one of the earliest responses of mammalian cells to genotoxic stress (reviewed in reference 93). Poly-ADP-ribose formation following DNA damage in primary mouse embryos and mouse embryo fibroblasts from Parp1 knockout mice was observed at 2 to 50% of wild-type values, depending on the tissue and cell type (149), and was drastically reduced only in Parp1–/– brain, pancreas, liver, small intestine, colon, and testis. Moderate levels of residual poly-ADP-ribose formation were seen in Parp1–/– stomach, bladder, thymus, heart, lung, kidney, and spleen (149). Only limited data are available regarding the physiological roles and poly-ADP-ribosylation activity of the novel PARP family members. Generally, the contribution of each PARP member to the total cellular poly-ADP-ribosylation activity depends on tissue, cell type, and stimuli, and the new PARPs are likely involved in specific nuclear and cytoplasmic functions requiring limited levels of poly-ADP-ribosylation.
PARGs
The "classical" poly-ADP-ribose glycohydrolase, PARG, is known to represent the major PARG activity catalyzing the hydrolysis of poly-ADP-ribose polymers to free ADP-ribose in the cell (reviewed in references 44 and 269). While at least six genes encode different "bona fide" PARPs synthesizing ADP-ribose polymers, only one single gene coding for the "classical" PARG activity has been detected in mammalian cells until recently (268, 297). The mammalian Parg gene encodes at least 
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