Nuclear ADP-Ribosylation Reactions in Mammalian Cells: Where Are We Today and Where Are We Going?

Nuclear ADP-Ribosylation Reactions in Mammalian Cells: Where Are We Today and Where Are We Going?
Paul O. Hassa, Sandra S. Haenni,{dagger} Michael Elser,{dagger} 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).

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).

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).

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).

NAD+ stimulated the incorporation of ATP onto Poly(A) products in nuclear extracts…

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).

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.

NAD+ Metabolism

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).

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 (243245, 339).  

Mono Reactions 

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.

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).

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{alpha} were reported to deacetylate, in vivo, acetylated transcription factors such as p53 and DNA repair factors such as Ku70, while acetylated {alpha}-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). [Can NRF2/KEAP1 be ribosylated..what experimental evidence suggest that this may happen?]

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).

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.

Free poly-ADP-ribose and the “poly-ADP-ribose code.” Several reports suggested that many types of poly-ADP-ribose structures observed in vitro also exist in vivo. Although the functional relevance of this heterogeneity is not yet known, it could play a crucial role in determining specific functional outcomes. It was suggested that certain types of free poly-ADP-ribose are involved in stress-dependent signaling processes in vivo (13, 97, 168, 246).

 Theoretically, in silico structures of poly-ADP-ribose need to be experimentally confirmed by in-depth analyses of poly-ADP-ribose structures generated in vitro and in vivo. This would be the first step in deciphering the potential “poly-ADP-ribose code.” Direct-binding experiments with each type of poly-ADP-ribose will be needed to detect poly-ADP-ribose-binding proteins, as well as the poly-ADP-ribose structures recognized by them. Direct-binding experiments will also be required to understand the cross-reactivity between related types of poly-ADP-ribose structures. These studies can be performed only with sensitive high-throughput methods. New technologies are therefore needed for the detection of poly-ADP-ribose-protein interactions. Recently, T. Feizi and W. Chai described a microarray platform for deciphering the glyco code for oligosaccharide moieties of glycoproteins, glycolipids, proteoglycans, and polysaccharides (119). Their technology involves the generation of oligosaccharide microarrays from entire glycomes, which could then be probed for protein binding. The authors suggested that these microarrays could also be coupled to techniques that allow determination of the range of proteins in proteomes that interact with carbohydrates and identification of the oligosaccharide sequences recognized by these proteins (119). Such a microarray technology adapted for poly-ADP-ribose would allow the large-scale identification of poly-ADP-ribose-binding proteins and poly-ADP-ribose-binding motifs and would enable the molecular identification of specific poly-ADP-ribose-recognition systems in whole organisms. This would allow the elucidation of the putative “poly-ADP-ribose code” for specific poly-ADP-ribose structures. Knowledge of these poly-ADP-ribose-recognition systems, including the cross-reactivity between related poly-ADP-ribose structures, would provide the tools to develop therapeutic drugs which would act through manipulation of poly-ADP-ribose-protein interactions.


About this entry