Biochimica et Biophysica Acta 1804 (2010) 1617–1625

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Review

Function and metabolism of sirtuin metabolite O-acetyl-ADP-ribose

Lei Tong, John M. Denu ⁎Department of Biomolecular Chemistry, University of Wisconsin, School of Medicine and Public Health, Madison, WI 53706, USA

⁎ Corresponding author. Department of BiomolecuWisconsin, School of Medicine and Public Health, 130053706, USA. Tel.: +1 608 265 1859; fax: +1 608 262 5

E-mail address: jmdenu@wisc.edu (J.M. Denu).

1570-9639/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.bbapap.2010.02.007

a b s t r a c t

a r t i c l e i n f o

Article history:Received 10 September 2009Received in revised form 4 February 2010Accepted 7 February 2010Available online 20 February 2010

Keywords:SirtuinSir2NADDeacetylationDeacetylaseO-acetyl-ADP-ribose

Sirtuins catalyze the NAD+-dependent deacetylation of target proteins, which are regulated by thisreversible lysine modification. During deacetylation, the glycosidic bond of the nicotinamide ribose is cleavedto yield nicotinamide and the ribose accepts the acetyl group from substrate to produce O-acetyl-ADP-ribose(OAADPr), which exists as an ∼50:50 mixture of 2′ and 3′ isomers at neutral pH. Discovery of this metabolitehas fueled the idea that OAADPr may play an important role in the biology associated with sirtuins, acting asa signaling molecule and/or an important substrate for downstream enzymatic processes. Evidence forOAADPr-metabolizing enzymes indicates that at least three distinct activities exist that could modulate thecellular levels of this NAD+-derived metabolite. In Saccharomyces cerevisiae, NUDIX hydrolase Ysa1 cleavesOAADPr to AMP and 2- and 3-O-acetylribose-5-phosphate, lowering the cellular levels of OAADPr. A buildupof OAADPr and ADPr has been linked to a metabolic shift that lowers endogenous reactive oxygen speciesand diverts glucose towards preventing oxidative damage. In vitro, the mammalian enzyme ARH3 hydrolyzesOAADPr to acetate and ADPr. A third nuclear-localized activity appears to utilize OAADPr to transfer theacetyl-group to another small molecule, whose identity remains unknown. Recent studies suggest thatOAADPr may regulate gene silencing by facilitating the assembly and loading of the Sir2–4 silencing complexonto nucleosomes. In mammalian cells, the Trpm2 cation channel is gated by both OAADPr and ADP-ribose.Binding is mediated by the NUDIX homology (NudT9H) domain found within the intracellular portion of thechannel. OAADPr is capable of binding the Macro domain of splice variants from histone protein MacroH2A,which is highly enriched at heterochromatic regions. With recently developed tools, the pace of newdiscoveries of OAADPr-dependent processes should facilitate new molecular insight into the diversebiological processes modulated by sirtuins.

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1. Introduction

Sirtuins are a conserved family of protein/histone deacetylasesfound in organisms ranging from bacteria to humans [1–3]. Membersof this family share a similar enzymatic core domain of ∼250 aminoacids. The founding member Silencing Information Regulator 2 (Sir2)was originally discovered as a gene required for efficient mating inSaccharomyces cerevisiae [4]. Yeast Sir2p is involved in manyprocesses that include gene silencing, ribosomal DNA recombination,DNA repair and longevity [5]. In higher organisms, Sir2 orthologs arealso linked to lifespan regulation [6,7]. Expressing extra copies of Sir2homologues leads to apparent lifespan extension in both C. elegansand D. melanoganster [2]. Although it is not yet clear if Sir2 homologsmodulate lifespan in mammals, mammalian sirtuins are reported toinduce positive effects on physiological processes, such as DNA repair,cell survival, stress resistance, metabolic control and insulin sensitiv-ity [2]. Moreover, sirtuins may mediate the beneficial effects of caloric

restriction (CR) [7–9], which is the only non-genetic method thatextends lifespan in nearly all organisms where this regimen has beeninvestigated.

1.1. Discovery of OAADPr

Once yeast Sir2 was identified as an essential factor in genesilencing [4,10,11], it was noted that Sir2 shared sequence homologyto a bacterial enzyme, CobB that could partially complement aphosphoribosyltransferase defect in cobalamin biosynthesis [12–14].Initial investigations of Sir2 enzymatic function reported a proteinADP-ribosylation activity, which required NAD+ [15,16]. However, anumber of subsequent reports began to reveal a more robust activity,NAD+-dependent histone deacetylation [17–19]. In 2000, the sur-prising molecular role of NAD+ in protein deacetylation was revealed[20,21]. The efficient histone/protein deacetylase reaction is tightlycoupled to the formation of a novel acetyl-ADP ribose product O-acetyl-ADP ribose (OAADPr, also abbreviated as AAR and AADPR bysome authors). One molecule of NAD+ and acetyl-lysine are readilyconverted to one molecule of deacetylated lysine, nicotinamide, andOAADPr (Fig. 1). The details of the chemical reaction have beenreported [22,23], and will not be reviewed here. However, the idea

Fig. 1. OAADPr production by Sir2/sirtuins deacetylation reaction. Sir2 and sirtuins catalyze NAD+ dependent deacetylation of histone tails, or other non-histone acetylated proteins.The reaction transfers the acetyl group from acetylated lysine residues to the ADP-ribose moiety of NAD+, generating deacetylated histone tails, nicotinamide, and the novelmetabolite OAADPr.

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that some sirtuins mediate protein ADP-ribosylation, and notdeacetylation, remains active. The potential ADP-ribosylating activityof sirtuins stems primarily from twomain observations: Some sirtuinsexhibit little or no detectable protein deacetylase activity on testedsubstrates, and low levels of ADPr transfer to protein are oftendetected under extremely long incubations with NAD+. Severalpublished studies have provided evidence that the reported ADP-ribosylation activity of sirtuins represents a slow side reaction thatmay not reflect an important physiological function of these enzymes[24–28].

From several mechanistic studies, the deacetylation productreleased by sirtuins appears to be the 2′-O-acetyl-ADP-ribose isomerof OAADPr [21,23,29]. At neutral pH, 2′-O-acetyl-ADP-ribose and 3′-O-acetyl-ADP-ribose were found to be the solution products, existing in∼1:1 ratio and generated through a non-enzymatic intramoleculartransesterification [23,29] (Fig. 1). The ability of sirtuins to produceOAADPr is well conserved ranging from bacterial CobB to mammaliansirtuins ([3,19,27,30]). It is important to note that the energy releasedby hydrolysis of NAD+ is 8.2 kcal/mol, in a comparable range for thehydrolysis of ATP to ADP [15,31]. It is striking that Sir2 enzymes (ClassIII protein deacetylases) retain this energetically expensive mecha-nism, compared to the Class I and II deacetylases, which directlyproduce acetate and do not require NAD+. There is accumulatingevidence that the elaborate mechanism of sirtuin catalysis rendersOAADPr which can elicit downstream responses that might synergizeor antagonize the biological functions of sirtuin genes. In the followingsections, we will discuss several studies published over the last fewyears focusing on the metabolism and cellular functions of OAADPr.

1.2. OAADPr metabolism

After the discovery of OAADPr, a quantitative microinjection assayof OAADPr into starfish oocytes caused a delay/block in oocytematuration [32], supporting the hypothesis that OAADPr can evokebiological activity. It is worth noting that microinjection of purifiedsirtuin enzymes also led to the identical effect. Although themolecular basis behind these observations is unknown, it wasreasonable to postulate that the metabolism of OAADPr in vivomight be tightly controlled. Several OAADPr-metabolizing enzymeshave been reported (Fig. 2). The best characterized are the NUDIXhydrolases (hydrolysis of a nucleoside diphosphate linked to anothermoiety x), including Ysa1 from yeast and NudT5 from mouse [33].Recombinant Ysa1 and mNudT5 cleave the pyrophosphate bond ofOAADPr, generating 2- and 3-O-acetylribose-5-phosphate and AMP.ARH3 from the human ADP-ribosyl hydrolase (ARH) family hydro-lyzes the acetyl group of OAADPr, generating ADPr, whichmight act as

a negative feedback inhibitor of ARH3 [34]. Additionally, twouncharacterized enzyme activities detected in both yeast andhuman cells have been reported, namely, a cytoplasmic esterasethat removes the acetyl group from OAADPr, and a nuclear acetyl-transferase that transfers the acetyl group from OAADPr to anunknown molecule [33]. Future identification and detailed analysisof those activities will likely be important for clarifying thephysiological roles of OAADPr.

1.2.1. OAADPr hydrolysis by NUDIX hydrolasesNUDIX hydrolases catalyze the hydrolysis of a nucleoside

diphosphate linked to another moiety x, thus the abbreviation“NUDIX” [35,36]. They are found in more than 250 organisms andcharacterized by the highly conserved array of amino acids GX5EX7-REUXEEXGU (U represents a bulky, hydrophobic amino acid and Xrepresents any amino acid). The nucleoside diphosphate linkage iscommon to the broad range of substrates utilized by the family,including NADH, dinucleoside polyphosphates, and nucleotide sugarssuch as ADPr. NUDIX hydrolases are further divided into sub-familiesbased on their substrate specificity. Comparison among the membersof the ADPr pyrophosphatase subfamily revealed a conserved proline,16 amino acid residues downstream of the NUDIX box, whichdetermines a catalytic preference for ADPr. Considering the structuralsimilarity between OAADPr and ADPr, several members from theADPr pyrophosphatase subfamily were analyzed for their activityagainst OAADPr [33]. Ysa1 from S. cerevisiae [37], NudT5 from mouse[38] and NudT9 from human [39] belong to this family and exhibitADPrase activity. Ysa1 and mouse NUDT5 (mNUDT5) are able tocatalyze hydrolysis of a variety of ADP sugar conjugates with apreference for ADPr. Human NUDT9 (hNUDT9) is highly specific forADPr and non-physiological IDPR. The shared mechanism amongADPr pyrophosphatases is a nucleophilic attack by water on thepyrophosphate linkage of the substrate, producing AMP and ribose-5-phosphate. Thus, OAADPr hydrolysis yields the correspondingproducts AMP and 2- and 3-O-acetylribose-5-phosphate ([40–42]).

Steady-state kinetic analyses were performed with Ysa1, mNudT5and NudT9 using OAADPr and ADPr as substrates (Table 1) [33]. Ysa1hydrolyzesOAADPrwith a∼2 fold lower kcat value (37 s−1) and a∼3.5fold lower kcat/Km value (4.5×104 M−1 s−1) compared to ADPr.mNudT5 catalyzes the hydrolysis of both ADPr andOAADPr at a similarmaximal rate (0.8–0.9 s−1) and kcat/Kmvalue (1.96×104 vs. 1.73×104,respectively). This kinetic analysis demonstrates that Ysa1 andmNudT5 bind and hydrolyze OAADPr and ADPr with similar catalyticefficiency (Table 1). In contrast, NudT9 poorly catalyzes hydrolysis ofOAADPr compared with ADPr. The Km value for NudT9 catalysis ofOAADPr is ∼500-fold higher than that of ADPr, suggesting that the low

Fig. 2. OAADPr metabolism by the ADPr pyrophosphatase, unknown esterase and acetyl transferase. The pyrophosphatase cleaves the pyrophosphate bond of OAADPr, generatingacetyl-ribose-phosphate and AMP; the esterase removes the acetyl group of OAADPr; the unknown acetyl-transferase transfers the acetyl group from OAADPr to an unknownmolecule X.

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catalytic activity results from weak binding affinity [33] (Table 1).Consistent with poor binding affinity, OAADPr was unable to inhibitsignificantly ADPr hydrolysis by NudT9. These results suggest thatsteric clash from the acetyl group leads to decreased affinity.

Ysa1 is the major endogenous NUDIX hydrolase for both OAADPrand ADPr in yeast [43]. Using an HPLC based assay, cell extracts withendogenous Ysa1 cleaved OAADPr/ADPr to AMP, but cells lackingYsa1 (Δysa1) exhibited no significant NUDIX activity. Utilizing anOAADPr analog that is resistant to esterase, N-acetyl-ADPr analog wascleaved into AMP by cell extracts containing endogenous Ysa1,supporting the conclusion that OAADPr is directly hydrolyzed byYsa1 [43]. Given that Ysa1 is the major metabolizing enzyme forOAADPr/ADPr in yeast, cellular concentrations of these metabolitesunder different levels of Ysa1 were determined by LC/MS-MS. BothOAADPr and ADPr levels increase when Ysa1 was deleted [43,44]. Inthe Δysa1 strain, OAADPr concentration increased 49% and ADPrconcentration increased 50% compared to wild type cells. AMP, thehydrolysis product of Ysa1, decreased 22% in the deletion strain,demonstrating that the increase in OAADPr/ADPr concentration wasthe direct result of loss of Ysa1 activity. In comparison, whole cellconcentrations of NAD+, NADH, and ATP were not significantlyaffected [43].

1.2.2. ARH3The human ADP-ribosyl hydrolase (ARH) family contains three

members, ARH1–3, which share considerable sequence homology [45].Based on the available EST (http://www.ncbi.nlm.nih.gov/unigene)sequences, human ARH1 (UGID:142774 UniGene Hs.99884), ARH2(UGID:142438 UniGene Hs.98669) and ARH3 (UGID:133769 UniGeneHs.18021) are widely expressed in multiple tissues including brain,heart, liver, and uterus. ARH1 cleaves the ADP-ribose-arginine bond inthe α-ADP-ribosyl-(arginine) protein to generate ADPr and (arginine)protein [46,47]. Substrates of ARH2 have not been identified [45]. ARH3

Table 1Kinetic parameters for YSA1, mNudT5, and NUDT9 using ADPr or OAADPr as substrates (ad

Enzyme (substrate) Vmax (μmol min−1 mg−1) kc

YSA1 (OAADPr)a 83.9 ± 3.7 36YSA1 (ADPr)a 175.7 ± 25.1 76YSA1 (ADPr)b 182.3 ± 5.5 79mNudT5 (OAADPr)a 1.95 ± 0.46 0.mNudT5 (ADPr)a 2.35 ± 0.48 0.mNudT5 (ADPr)c 5.67 ± 0.47NUDT9 (OAADPr)a,d

NUDT9 (ADPr)a 11.0 ± 0.3 7NUDT9 (ADPr)e 11.8 ± 0.3

aThese data from Ref. [33].bThese data from Ref. [37].cThese data from Ref. [38].dDue to the inability to saturate, the kcat/Km for NUDT9 with OAADPr was determined from tKm value was determined by fixing the Vmax at the same rate or 2-fold slower than that obtaand YSA1, see above) and fitting the data to the Michaelis–Menten equation.eThese data from Ref. [76].

binds ADP-ribose but is incapable of hydrolyzing ADP-ribosylarginine[45]. Instead, ARH3 catalyzes the hydrolysis of poly(ADP-ribose). Ono etal. [34] examined the activity of ARH3 against OAADPr. The hydrolyzedproduct was consistent with ADPr as analyzed by HPLC, high resolutiongel electrophoresis and MALDI-TOF. ARH3-dependent OAADPr hydro-lysis required Mg2+, similar to the ARH1 catalyzed cleavage of ADP-ribosylarginine andARH3-catalyzedpoly(ADP-ribose) hydrolysis. ARH1is also capable of hydrolyzingOAADPr, albeit at a 250 fold slower rate. Incontrast, ARH2 did not display any activity for OAADPr under the sameconditions. ADPr competitively inhibited ARH3 catalyzed OAADPrreactions with a Ki of 2 µM, while NAD+, AMP and ADP showed littleor no inhibition. As shownpreviously, the vicinal carboxylic amino acidsAsp60/Asp61 in ARH1 and Glu755/Glu756 in PARG are critical for theircatalytic activity. To investigate the importance of similar groups inARH3, Asp77/Asp78, Glu261/Glu262 and Glu238/Glu239 were mutat-ed. Only the Asp77/Asp78 mutation completely abolished catalyticactivity, though their exact roles in OAADPr hydrolysis have not beenelucidated [34,45]. Whether ARH3 metabolizes OAADPr in vivo awaitsfurther clarification. Given these observations, it is possible that ARH3 isthe previously observed OAADPr esterase.

1.3. OAADPr function

1.3.1. OAADPr in gene silencingThe three regions of the S. cerevisiae genome that are silenced

include the two silent mating type loci, the telomeres, and the rRNA-encoding DNA (the rDNA). Silencing at telomere and mating type lociis mediated by a multi-protein nucleosomal binding complex calledthe Silent Information Regulator (SIR) complex, which contains theSir2, Sir3, and Sir4 proteins [48]. No obvious homologues to Sir3p andSir4 have been detected in multi-cellular eukaryotes [49]. Sir2p-dependent deacetylation of histone tails is essential for spreading theSir2–4 complex along silent chromatin (Fig. 3). Considerable evidence

apted from Ref. [33]).

at (s−1) Km (mM) kcat/Km (M−1 s−1)

.5 ± 1.6 0.081 ± 0.019 4.51 × 104

.4 ± 10.9 0.048 ± 0.011 1.59 × 105

.2 ± 2.4 0.040 ± 0.009 1.98 × 106

78 ± 0.18 0.045 ± 0.009 1.73 × 104

94 ± 0.19 0.048 ± 0.016 1.96 × 104

0.038 ± 0.0057.9–16.4 4.48 ± 0.24 × 102

.7 ± 0.2 0.033 ± 0.006 2.33 × 105

0.100 ± 0.010

he slope of the line fitted by linear-least squares regression. An estimated range for theined with ADPr as substrate (a reasonable assumption based on the results with NudT5

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indicates that Sir3 and Sir4 only interact with deacetylated histonetails, and that this interaction is required for the establishment andthe maintenance of silenced regions [50,51]. Therefore, in the analysisof Sir2 function in silencing, major attention has been drawn to thedeacetylase activity. However, loss of Sir2 activity cannot be rescuedby simultaneous deletion of a major histone acetyl transferase, Sas2[52,53], suggesting that other aspects of Sir2 activity, such as NAD+

hydrolysis and OAADPr synthesis, might function in gene silencing.

1.3.2. OAADPr facilitates SIR complex assembly and loading of SIRcomplex onto nucleosomes in vitro

The initial model of silent chromatin assembly proposed that theSir2/Sir4 complex is recruited to the silencer region where Sir2deacetylates adjacent histone tails. Additional Sir2–4 complexes arethen recruited via Sir4/Sir3 interactions, producing subsequentrounds of Sir2 deacetylation and further Sir2–4 recruitment [11,53–56]. Liou et al. measured the binding affinities of Sir–Sir interactionsusing full length proteins purified from yeast cells [57]. The Sir2/Sir4complex bound Sir3 with an apparent disassociation constant (Kd) of∼3×10−11 M, while the Kd for Sir2/Sir3 interaction was ∼6×10−8 Mand for the Sir3/Sir3 was ∼2×10−9 M. Using a number of analyticaland biochemical methods, multiple forms of Sir3 oligomers rangingfrom dimers to decamers were observed ([57,58] and Tong and Denu,unpublished observations). Using TAP-Sir4/HA-Sir2 immobilized onIgG-Sepharose beads, Sir3 bound to the resin and eluted with Sir4/Sir2, suggesting the possibility of forming of a stable SIR complexamong the three components in solution [57,58].

Utilizing an in vitro complex assembly approach, other factorsaffecting the SIR complex were examined [57,58]. With differentcombinations of acetyl-H4-peptide, H4-peptide and NAD+ present,only the addition of both NAD+ and acetyl-H4-peptide lead to anapparent increase of Sir3 bound to the Sir2/Sir4 complex, suggestingthe effect was either due to NAD+ hydrolysis or the production ofOAADPr. The role of OAADPr in the observed structural rearrangementwas shown by adding purified OAADPr. In solution, addition ofOAADPr together with unacetylated H4 peptide promoted changes inthe stoichiometry and in the appearance of SIR complex, visualized byelectron microscopy [57,58].

Martino et al. investigated the effect of OAADPr on silent chromatinformation at the nucleosomal level [59]. Preincubation of the Sir2/Sir3/Sir4 complexwithOAADPr resulted in amarked increase in SIR complexaffinity for the chromatin template. In comparison, preincubation withADPr, ATP, γATP-S or NAD+ had no effect.

Sir3 contains an AAA+ ATPase motif within the C-terminaldomain. AAA+ ATPase motif-containing proteins are associatedwith the assembly, operation, and disassembly of protein complexes[60]. Althoughmissing putative residues for catalysis, the motif in Sir3appears to retain conserved residues for ATP binding. Considering the

Fig. 3. Model for the role of OAADPr in facilitating Sir complex assembly and loading ontocomplex onto nucleosomes, probably through binding to Sir2/Sir3. OAADPr might bind Sir

structural similarity between OAADPr and ATP, this domain couldmediate OAADPr binding with Sir3. Consistent with this hypothesis,preincubating Sir3 with OAADPr led to increased affinity of Sir3 forchromatin [59], indicating that the SIR complex loading efficiencycould be mediated by the Sir3/OAADPr interaction (Fig. 3). With keycatalytic residues missing, no OAADPr hydrolytic activity wasdetected with the AAA+ ATPase domain of Sir3 [60,61].

The AAA+ ATPase domain of Sir3 is a potential binding site forOAADPr. Interaction between recombinant purified Sir3 AAA+domain and OAADPr was detected by photo-crosslinking using[32P]-8N3-OAADPr, though binding evidence from isothermal titrationcalorimetry was inconclusive (Tong and Denu, unpublished observa-tions). The role of this domain in OAADPr binding and regulatedchromatin assembly awaits further evaluation. However, interactionof Sir2 with OAADPrwas readily detected by both crosslinking, ITC (Kd

of ∼10 µM) and pull down assays ([43] and Tong and Denu,unpublished observations). Collectively, it is quite possible that eitherSir2 or Sir3, or both are involved in OAADPr binding and SIR complexassembly and spreading along the chromatin (Fig. 3).

While the studies referenced above provided evidence for animportant role of OAADPr in silent chromatin assembly in vitro, Chouet al. came to a different conclusion based on an engineered protein-chimera employed to examine the role of OAADPr in vivo [62]. Using aSir3 chimera bearing Hos3, an unrelated NAD+-independent histonedeacetylase, silencing was restored in a Δsir2 strain. Even in strainsthat lack all five sirtuin homologs, the Sir3–Hos3 chimera was able torepress gene expression at the HM loci. The study concluded thatOAADPr is not required for silencing in those systems and deacetyla-tion of histone tails is the essential function of Sir2 in HM locisilencing. Nevertheless, these apparently conflicting reports can berationalized. Considering that OAADPr is not essential for Sir3 tointeract with Sir2/Sir4 as determined by surface plasmon resonance(SPR) and in vitro complex assembly [57], silent chromatin can beestablished in the absence of OAADPr. However, the presence ofOAADPr might enhance the efficiency of Sir3 binding to Sir2/Sir4 andchromatin [57,59], and thus enhance the construction of silentchromatin. Consistent with this hypothesis, Sir3–Hos3 only partiallyrescued silencing in the strain lacking all five sirtuin homologs,presumably in the absence of any OAADPr [60,62].

1.3.3. OAADPr interaction with macroH2AIn higher eukaryotes, heterochromatin is functionally akin to silent

chromatin in yeast. Binding partners like macroH2A1 (mH2A1) alsoplay important roles in chromatin structure and transcriptionalactivity. MH2A1 is a histone variant that contains a ∼20 kDa macrodomain and is highly enriched in heterochromatin regions such as thefemale inactive X chromosome, suggesting functions in generepression [63,64]. There are currently ±300 proteins in the SMART

the chromatin. OAADPr interaction with the Sir2–4 complex facilitates loading of the3 alone and promote Sir3 loading onto nucleosomes.

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database [65] that contain a macro domain with diverse functions. Achromatin-remodeling enzyme, Amplified in Liver Cancer 1 (ALC1,also known as CHD1L) interacts with poly(ADP-ribose) (PAR) andcatalyzes PARP1-stimulated nucleosome sliding. PAR binding wasdetected with the macro domain-containing ALC1 proteins but notwith a macro-domain mutant. As a consequence, recruitment of ALC1to DNA damage sites of the macro domain mutant was severelyimpaired [66]. B-aggressive lymphoma 1 (BAL1) encodes a nuclearprotein with N-terminal macro domains and a putative C-terminalpoly(ADP-ribose) polymerase (PARP) active site. BAL1 proteinrepresses transcriptionwhen tethered to a promoter. A BAL1 proximalN-terminal construct which lacks the macro domains lost transcrip-tional repression activity. In contrast, the macro domain-containingBAL1 N terminus and the full-length BAL1 protein were equallyeffective in repressing transcription [67]. Although other macrodomain containing proteins can bind poly(ADP-ribose), mH2A1does not [68]. Interestingly, the gene H2AFY, which expressesmH2A1, generates two isoforms, mH2A1.1 and mH2A1.2. Whenscreening for mH2A1 binding mononucleotides, Kustatscher et al.found that the Sir2 metabolite OAADPr binds mH2A1.1 in vitro with adissociation constant of 2.7 µM and stoichiometry of 1:1 [69]. ADPrbinds with similar affinity but with lower binding enthalpy. ADP,AMP, NAD+ and cyclic ADPr were also tested for binding withmH2A1.1, but only ADP showing moderate binding while the othermolecules revealed no detectable binding. Thus mH2A 1.1 appears tobind OAADPr and ADPr specifically. However, with the other isoformmH2A1.2, no binding was detected.

The X-ray crystal structure mH2A 1.1 with OAADPr modeled inFig. 4 revealed that Phe348 and Asp203 interact with the adenine ringthrough hydrophobic stacking and hydrogen bonding, respectively,whereas Gly224 and Gly314were predicted to contact the phosphatesof OAADPr [69]. F348A and D203R mutations reduced the bindingaffinity up to 30-fold, while mutations of residues Gly224 and Gly314to Glu abolished binding. The crystal structure in combination withmutagenesis predicts that the binding pocket would accommodatethe extended confirmation of OAADPr. The major differences betweenmH2A 1.1 and 1.2 are three residues (EIS) inserted into the adeninebinding portion of the mH2A1.1-OAADPr binding site, which flippedthe adenine stacking phenylalanine and replaced the phosphateinteracting residues Gly223 and Gly224 in mH2A1.1 with largerresidues (Lys226 and Asp227) in mH2A1.2. Although these changesare small, they abolished the interaction between mH2A1 andOAADPr (Fig. 4).

Prior to the observation of differential binding to OAADPr, moststudies considered macroH2A1.1 and 1.2 to have identical functions.In addition to this difference, the tissue distribution and temporalexpression differ noticeably [64]. As mH2A1 represents ∼3% of totalcellular H2A [63], it may compose a considerable amount ofchromatin, and presents a novel way for the NAD+ metaboliteOAADPr to affect chromatin properties, especially at the heterochro-matic regions with enrichedmacroH2A1.1. SIRT1 has reported roles inheterochromatin establishment [70]. The production of OAADPr bySIRT1 and the potential synergy with chromatin bound mH2A mayfacilitate the establishment and/or maintenance of heterochromatin.

1.3.4. OAADPr modulation of the TRPM2 ion channelTransient receptor potential malastatin-related channel 2 (TRPM2)

is a non-selective cation channel, enriched in brain, gastrointestinaltissues and immune cells [71]. Although its physiology functions are notfirmly established, TRPM2 is stimulated by oxidative and nitrative stressthat lead to calcium influx into the cell [72,73]. Though the exactmechanisms are disputed, ADPr binding to NudT9H appears to berequired for the well-known oxidative stress activation of the channel,as depletion of ADPr by ADPr hydrolase overexpression rescues cellsfrom TRPM2 over activation [74] In response to oxidative and nitrativestress, ADPr may be generated in large quantities when PARP and PARG

become hyperactivated in response to DNA damage [74,75]. TRPM2activation is induced by ADPr binding to its C-terminal cytoplasmicdomain [39,76]. This domain, termed NudT9H, shares significanthomology to the mitochondrial NUDIX hydrolase NudT9. Fromelectrophysiological experiments, ADPr activates the channel at lowµM levels, a process that requires the NudT9H domain [76,77]. TheNudT9H domain displays no enzymatic activity, suggesting that ADPrbinding to this domain is a key step for stress induced TRPM2 channelgating [74,78].

OAADPr produced by sirtuins induces TRPM2 channel gating [78].Nicotinamide, a commonly employed inhibitor of sirtuins [79–81],abolished cell death caused by puromycin-induced TRPM2 overactivation, consistent with inhibition of OAADPr production andabolished TRPM2 activation. Two mammalian sirtuins, SIRT2 andSIRT3, were examined as possible sources of OAADPR generation thatled to TRPM2-dependent cell death induced by the puromycin. RNAiknockdown of SIRT2 and SIRT3 in TRPM2 expressing cells suppressedcell death induced by puromycin treatment. At 20 µM puromycin, lossof SIRT2 or SIRT3 increased cell survival 2–3 fold. With 50 µM ofpuromycin, 30% of SIRT2 or SIRT3 knocked down cells survived thetreatment, while survival of control cells was negligible. Consistentwith the hypothesis that OAADPr production is critical for TRMP2gating, OAADPr induced currents in whole cell patch-clamp experi-ments in TRPM2 expressing cells, but not when TRPM2 was absent.The kinetics and dose dependence of OAADPr activation were similarto that of ADPr. Hence, OAADPr-dependent gating of the TRPM2channel is as effective as ADPr and may in fact function as aphysiological regulator of TRPM2. OAADPr gating of TRPM2 was alsoobserved in a genetically unmodified cell line CRI-G1, which has ahigh level of endogenous TRPM2 expression. Consistent with a role forsirtuins, nicotinamide protected CRI-G1 cells from puromycin inducedcell death [78].

OAADPr and ADPr bind directly to the NudT9H domain of TRPM2.Using recombinant NudT9H domain, a Kd for ADPr of 130 µM wasmeasured by ITC, whereas no NAD+ binding was detected [78]. A UVcrosslinking approach was utilized to examine OAADPr binding toNudT9-H, yielding a Kd of ∼100 µM. These findings provide directevidence that OAADPr/ADPr bind to the NudT9H domain of theTRPM2 channel, and are likely an important component of the gatingmechanism (Fig. 5).

Although OAADPr/ADPr modulate TRPM2 gating in a similarmanner, they could accumulate under different circumstances. Forinstance, ADPr may be generated in large quantities in response toH2O2 stress, when PARP and PARG are activated [74,75], whileOAADPr could accumulate during upregulation of sirtuin activity inresponse to metabolic adaptation. It is also plausible that OAADPr israpidly converted to ADPr [33], resulting in a buildup of ADPr, whichthen mediates TRPM2 gating in vivo (Fig. 5).

1.3.5. OAADPr/ADPr modulate cellular REDOX (reduction–oxidationstatus)

The biological significance of altered OAADPr and ADPr levels inwild type versus Δysa1 yeast strains [44] was examined recently [43].When cells were treated with H2O2, Δysa1 cells displayed higherresistance to exogenous oxidative stress compared to wild type cells.Similarly, Δysa1 cells were more resistant to endogenous reactiveoxygen species (ROS) resulting from treatment with CuSO4, acompound known to increase cellular ROS [82,83]. Collectively,increased concentration of OAADPr/ADPr appears to endow cellswith a higher capacity to resist ROS stress. In addition, basal levels ofROS under normal conditions were 40% lower in the Δysa1 cellscompared to wild type cells, providing a strong correlation betweenlowered ROS with increased levels of OAADPr/ADPr [43]. Themechanism(s) underlying enhanced ROS resistance at higherOAADPr/ADPr levels was explored and two explanations were offered(Fig. 6): (i) OAADPr/ADPr inhibit pathway(s) that generate ROS; (ii)

Fig. 4. Left: model of mH2A 1.1 in complex with ADPR. Model generated by overlaying mH2A 1.1 structure (PDB ID: 2FXK) onto a macro domain-ADPR complex (PDB ID: 2BFQ). Twoimportant glycine residues involved in pyrophosphate binding are colored magenta. F348 and D203 side chains are shown in stick representation. Right: the side chain of the Ile inthe EIS insertion clashes with the adenine ring. mH2A 1.1 colored cyan, mH2A 1.2 colored yellow.

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OAADPr/ADPr promote pathway(s) that suppress ROS accumulation.The mitochondrial membrane potential ΔΨm in wild type cells wassignificantly higher than in Δysa1 cells, suggesting that increasedOAADPr/ADPr down regulates efficient coupling of respiration, andtherefore could suppress electron leakage and ROS generation, whichwas substantially higher in wild type versus Δysa1 cells [43]. Thedescribed biological functions of free ADPr/OAADPr and NUDIXhydrolases are likely conserved among yeast and mammals. Mam-malian genomes encode two close homologs of yeast Ysa1, NudT5 andNudT9 [40], both of which display robust catalytic activity towardADPr in vitro. Consistent with an analogous function in mammaliancells, Formentini et al. recently reported the importance of NudT5 andNudT9 in the mitochondrial energy dysfunction caused by PARP-1hyperactivation [91].

The observation that OAADPr/ADPr inhibits electron transportchain (ETC) [43] supports the hypothesis that increased OAADPr/ADPr inhibits pathway(s) that generates ROS (Fig. 6). Additional datasuggests that OAADPr/ADPr might stimulate pathways that suppressROS damage and accumulation. To eliminate ROS damage from acutestress of H2O2 and Cu2+, yeast cells reroute glucose metabolism from

Fig. 5. OAADPr and ADPr modulate calcium influx through TRPM2 channel. In mammaliancalcium influx into the cells. ADPr, another metabolite of NAD+, is believed to gate TRPM2

glycolysis to the NADPH generating pentose phosphate pathway [84–87]. NADPH is utilized to reduce oxidized glutathione and thioredoxin,which are cofactors required by ROS scavenging enzymes [88,89].Therefore, rerouting of the metabolic flux in yeast minimizes thedamage caused by oxidative stress inducing reagents. With structuralsimilarity to cofactors (ATP, NAD+, etc.) utilized by the glycolyticenzymes, OAADPr/ADPr might modulate the activities of keyglycolytic enzymes through interacting with the co-enzyme orsubstrate binding sites. Using an affinity-binding procedure, majorproteins interacting with OAADPr/ADPr were identified as phospho-glycerate kinase (PGK), alcohol dehydrogenase (ADH), and glyceral-dehyde-3-phosphate dehydrogenase (GAPDH), which are glycolyticor glycolysis-related enzymes. Steady-state inhibition of GAPDH byADPr revealed competitive inhibition, with a Ki value of 60 µM.OAADPr inhibits the enzyme with a similar IC50 value [43]. Consistentwith the idea that increased levelsOAADPr/ADPr induced rerouting ofglucose to the pentose phosphate pathway, NADPH levels weresubstantially higher in the Δysa1 cells (Fig. 6). Although mitochon-drial proteins were not identified as the major proteins in the OAADPraffinity-binding experiments, it remains to be determined whether

cells, OAADPr produced by sirtuins binds the NudT9H domain of TRPM2 and inducesin a similar mechanism as OAADPr.

Fig. 6. OAADPr metabolism and proposed functions in S. cerevisiae. OAADPr is producedby sirtuins and can be metabolized by Ysa1, an unknown esterase, and unknown acetyltransferase. Cells lacking Ysa1 display accumulation of ADPr/OAADPr and acorresponding decrease in AMP. Increased ADPr/OAADPr levels might protect cellsagainst ROS via inhibition of the ETC and generation of higher NADPH levels fromincreased flux through the pentose phosphate pathway.

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the citric acid cycle or othermetabolic pathways are regulated directlyby ADPr/OAADPr.

1.4. Synthesis of OAADPr analogs

To date, evidence supports direct binding ofOAADPr tomacro H2A,TRPM2, NUDIX hydrolases, ARH3 and possibly Sir2 and Sir3 of the SIRcomplex. However, the complete picture of OAADPr cellular functionand full identification of interacting proteins are likely incomplete.Current analysis of OAADPr function is greatly hindered by itsinstability and conversion by at least three types of enzymaticreactions. ARH3 cleaves the acetyl group on the ribose sugar andNUDIX hydrolases cleave the pyrophosphate linkage. To overcomethese limitations in OAADPr research, Comstock and Denu [90]developed a synthetic method to produce authentic OAADPr andnon-hydrolyzable analogs by substitution of the O-acetyl moiety withan N-acetyl group. N-acetyl analogs at both 2′- and 3′-positions of theribose sugar were synthesized, which will facilitate the differentiationof the 2′- and 3′-OAADPr isomers by their protein targets. UsingmacroH2A 1.1, Comstock et al. demonstrated the ability of bothanalogs to mimic properties of authentic OAADPr. More recently,installment of P-C-P to replace the P-O-P pyrophosphatemoiety yieldsOAADPr analogs that are resistant to cleavage by NUDIX hydrolases(Comstock, Tong, Denu, manuscript in preparation). These syntheticand non-hydrolyzable OAADPr analogs should prove valuable inprobing the biological functions of this unique metabolite, derivedfrom the NAD+-consuming activity of Sirtuin protein deacetylases.

2. Conclusions and perspectives

Over the last two decades, increasing attention has been paid tonewly recognized enzymes that consume NAD+. The sirtuin proteindeacetylases represent a novel class that employs NAD+ as a co-substrate to produce nicotinamide, deacetylated product and OAADPr.Although protein deacetylation of targets proteins, like histones,transcription factors and metabolic enzymes, have received themajority of attention among researchers, it will be crucial to account

for all the potential functions of the product OAADPr. Given the recentwork suggesting OAADPr might regulate gene silencing, ion channelgating, and REDOX regulation, this molecule could play a central rolein sirtuin biology, perhaps in synergy with specific protein deacetyla-tion. Because of the structural similarity between OAADPr and ADPr, itis reasonable to suggest that many of OAADPr's functions andinteracting partners might be shared with ADPr. On the other hand,mitochondrial Nudt9 is an important example of an enzyme thatdisplays a 500-fold discrimination between ADPr and OAADPr,favoring ADPr. The rapid conversion of OAADPr into ADPr by severalreported enzymes adds another level of complexity to resolving theseissues. The non-hydrolyzable OAADPr analogs are thus potentiallyuseful for differentiating the effect of OAADPr and ADPr in futurestudies.

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