Title: Identification of Macrodomain Proteins as Novel O-Acetyl-ADP-ribose Deacetylases
Abstract: Sirtuins are a family of protein lysine deacetylases, which regulate gene silencing, metabolism, life span, and chromatin structure. Sirtuins utilize NAD+ to deacetylate proteins, yielding O-acetyl-ADP-ribose (OAADPr) as a reaction product. The macrodomain is a ubiquitous protein module known to bind ADP-ribose derivatives, which diverged through evolution to support many different protein functions and pathways. The observation that some sirtuins and macrodomains are physically linked as fusion proteins or genetically coupled through the same operon, provided a clue that their functions might be connected. Indeed, here we demonstrate that the product of the sirtuin reaction OAADPr is a substrate for several related macrodomain proteins: human MacroD1, human MacroD2, Escherichia coli YmdB, and the sirtuin-linked MacroD-like protein from Staphylococcus aureus. In addition, we show that the cell extracts derived from MacroD-deficient Neurospora crassa strain exhibit a major reduction in the ability to hydrolyze OAADPr. Our data support a novel function of macrodomains as OAADPr deacetylases and potential in vivo regulators of cellular OAADPr produced by NAD+-dependent deacetylation. Sirtuins are a family of protein lysine deacetylases, which regulate gene silencing, metabolism, life span, and chromatin structure. Sirtuins utilize NAD+ to deacetylate proteins, yielding O-acetyl-ADP-ribose (OAADPr) as a reaction product. The macrodomain is a ubiquitous protein module known to bind ADP-ribose derivatives, which diverged through evolution to support many different protein functions and pathways. The observation that some sirtuins and macrodomains are physically linked as fusion proteins or genetically coupled through the same operon, provided a clue that their functions might be connected. Indeed, here we demonstrate that the product of the sirtuin reaction OAADPr is a substrate for several related macrodomain proteins: human MacroD1, human MacroD2, Escherichia coli YmdB, and the sirtuin-linked MacroD-like protein from Staphylococcus aureus. In addition, we show that the cell extracts derived from MacroD-deficient Neurospora crassa strain exhibit a major reduction in the ability to hydrolyze OAADPr. Our data support a novel function of macrodomains as OAADPr deacetylases and potential in vivo regulators of cellular OAADPr produced by NAD+-dependent deacetylation. IntroductionMacrodomains are evolutionarily conserved structural domains found in proteins with diverse cellular functions (1Till S. Ladurner A.G. Front. Biosci. 2009; 14: 3246-3258Crossref PubMed Google Scholar, 2Ladurner A.G. Mol. Cell. 2003; 12: 1-3Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Prior evidence suggested that macrodomains function as binding modules of NAD+ metabolites, including ADP-ribose/poly(ADP-ribose) (3Karras G.I. Kustatscher G. Buhecha H.R. Allen M.D. Pugieux C. Sait F. Bycroft M. Ladurner A.G. EMBO J. 2005; 24: 1911-1920Crossref PubMed Scopus (382) Google Scholar, 4Egloff M.P. Malet H. Putics A. Heinonen M. Dutartre H. Frangeul A. Gruez A. Campanacci V. Cambillau C. Ziebuhr J. Ahola T. Canard B. J. Virol. 2006; 80: 8493-8502Crossref PubMed Scopus (176) Google Scholar, 5Neuvonen M. Ahola T. J. Mol. Biol. 2009; 385: 212-225Crossref PubMed Scopus (149) Google Scholar, 6Ahel D. Horejsí Z. Wiechens N. Polo S.E. Garcia-Wilson E. Ahel I. Flynn H. Skehel M. West S.C. Jackson S.P. Owen-Hughes T. Boulton S.J. Science. 2009; 325: 1240-1243Crossref PubMed Scopus (434) Google Scholar, 7Kraus W.L. Nat. Struct. Mol. Biol. 2009; 16: 904-907Crossref PubMed Scopus (31) Google Scholar) and O-acetyl-ADP-ribose (OAADPr) 5The abbreviations used are: OAADPr, O-acetyl-ADP-ribose; PEI, polyethyleneimine; aa, amino acids; PDB, Protein Data Bank. (8Kustatscher G. Hothorn M. Pugieux C. Scheffzek K. Ladurner A.G. Nat. Struct. Mol. Biol. 2005; 12: 624-625Crossref PubMed Scopus (244) Google Scholar, 9Comstock L.R. Denu J.M. Org. Biomol. Chem. 2007; 5: 3087-3091Crossref PubMed Scopus (21) Google Scholar). OAADPr is produced in reactions catalyzed by NAD+-dependent protein/histone deacetylases (10Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 14178-14182Crossref PubMed Scopus (491) Google Scholar, 11Jackson M.D. Denu J.M. J. Biol. Chem. 2002; 277: 18535-18544Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), which regulate gene silencing, metabolic enzymes, life span, and many other cellular processes (12Smith B.C. Hallows W.C. Denu J.M. Chem. Biol. 2008; 15: 1002-1013Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 13Oberdoerffer P. Michan S. McVay M. Mostoslavsky R. Vann J. Park S.K. Hartlerode A. Stegmuller J. Hafner A. Loerch P. Wright S.M. Mills K.D. Bonni A. Yankner B.A. Scully R. Prolla T.A. Alt F.W. Sinclair D.A. Cell. 2008; 135: 907-918Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar, 14Hallows W.C. Smith B.C. Lee S. Denu J.M. Cell. 2009; 137: 404-406Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). OAADPr has been implicated as a signaling molecule, modulating cellular processes affected by NAD+-dependent protein/histone deacetylation (15Hoff K.G. Wolberger C. Nat. Struct. Mol. Biol. 2005; 12: 560-561Crossref PubMed Scopus (17) Google Scholar, 16Tong L. Denu J.M. Biochim. Biophys. Acta. 2010; 1804: 1617-1625Crossref PubMed Scopus (88) Google Scholar, 17Tong L. Lee S. Denu J.M. J. Biol. Chem. 2009; 284: 11256-11266Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The binding of OAADPr and other NAD+ metabolites to macrodomains such as the histone variant macroH2A1.1 (1Till S. Ladurner A.G. Front. Biosci. 2009; 14: 3246-3258Crossref PubMed Google Scholar, 5Neuvonen M. Ahola T. J. Mol. Biol. 2009; 385: 212-225Crossref PubMed Scopus (149) Google Scholar, 8Kustatscher G. Hothorn M. Pugieux C. Scheffzek K. Ladurner A.G. Nat. Struct. Mol. Biol. 2005; 12: 624-625Crossref PubMed Scopus (244) Google Scholar, 9Comstock L.R. Denu J.M. Org. Biomol. Chem. 2007; 5: 3087-3091Crossref PubMed Scopus (21) Google Scholar) suggests a possible connection between metabolic regulation, gene activity, and chromatin structure. The mechanism by which cells regulate and utilize OAADPr is not well understood. In yeast, the NUDIX ADP-ribose pyrophosphatase Ysa1 modulates the cellular levels of both ADPr and OAADPr, converting each to AMP and the corresponding ribose-phosphate (17Tong L. Lee S. Denu J.M. J. Biol. Chem. 2009; 284: 11256-11266Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Cells lacking ysa1 exhibit an increased resistance to oxidative insults and produce lower levels of endogenous reactive oxygen species. In vitro, the poly (ADP-ribose) glycohydrolase ARH3 was capable of removing the acetyl group from OAADPr (18Ono T. Kasamatsu A. Oka S. Moss J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16687-16691Crossref PubMed Scopus (91) Google Scholar), though the activity was orders of magnitude slower than that observed for the NUDIX family (19Rafty L.A. Schmidt M.T. Perraud A.L. Scharenberg A.M. Denu J.M. J. Biol. Chem. 2002; 277: 47114-47122Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). In eukaryotic cell extracts at least two different cellular activities were shown to contribute to OAADPr deacetylation (19Rafty L.A. Schmidt M.T. Perraud A.L. Scharenberg A.M. Denu J.M. J. Biol. Chem. 2002; 277: 47114-47122Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), but the identities of these factors remain unknown.Here we report a direct functional connection between sirtuins and a family of macrodomain proteins. We demonstrate that proteins belonging to this distinct branch of macrodomains are OAADPr deacetylases that efficiently catalyze the hydrolysis of OAADPr to produce ADP-ribose and free acetate. This group comprises eukaryotic MacroD proteins (orthologues of human MacroD1 and MacroD2), bacterial YmdB proteins and sirtuin-linked macrodomain proteins from certain pathogenic bacteria and fungi. Sirtuin-linked macrodomains constitute a macrodomain subfamily that are either fusions with sirtuin proteins or are genetically coupled through the same operon.DISCUSSIONOAADPr is a sirtuin reaction product generated from the NAD+-dependent protein deacetylation reactions and has been implicated as a signaling molecule (15Hoff K.G. Wolberger C. Nat. Struct. Mol. Biol. 2005; 12: 560-561Crossref PubMed Scopus (17) Google Scholar, 16Tong L. Denu J.M. Biochim. Biophys. Acta. 2010; 1804: 1617-1625Crossref PubMed Scopus (88) Google Scholar). Consequently, intracellular levels of OAADPr might be tightly regulated. However, the detailed mechanisms by which cells regulate the levels of OAADPr are poorly understood. The observation that some macrodomains and sirtuins are physically linked provides an important clue to their possible functional connections. In this study we demonstrate that the product of the sirtuin reaction is a substrate for the macrodomain. A subfamily of macrodomain proteins, which we have named MacroD-like proteins, is capable of efficient deacetylation of OAADPr to yield free acetate and ADPr. Our mutagenic analysis and structural studies suggest that they evolved a catalytic mechanism that is distinct from other macrodomain protein subfamilies.Previously, only the 39 kDa human poly (ADP-ribose) glycohydrolase ARH3 had been reported to catalyze the deacetylation reaction of OAADPr in vitro (18Ono T. Kasamatsu A. Oka S. Moss J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16687-16691Crossref PubMed Scopus (91) Google Scholar). Unlike the deacetylation reaction catalyzed by MacroD-like enzymes, ARH3 requires the presence of magnesium ion and catalyzes OAADPr hydrolysis more than an order of magnitude slower than MacroD-like enzymes (18Ono T. Kasamatsu A. Oka S. Moss J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16687-16691Crossref PubMed Scopus (91) Google Scholar). Moreover, the action of ARH3 appears to be less specific, as this protein can also hydrolyze poly(ADP-ribose) (18Ono T. Kasamatsu A. Oka S. Moss J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16687-16691Crossref PubMed Scopus (91) Google Scholar). In contrast, MacroD-like enzymes are incapable of hydrolyzing poly(ADP-ribose) (supplemental Fig. S2). ARH3 is present only in vertebrates and cannot be responsible for hydrolysis of OAADPr in fungi (such as N. crassa), bacteria and archaea, which all encode sirtuins. On the other hand, MacroD-like proteins are highly conserved through all kingdoms of life. Within eukaryotic cells, sirtuins (and therefore OAADPr) are found in all cellular compartments. In this respect, it is interesting to note that highly related human MacroD1 and MacroD2 enzymes have quite distinct intracellular localization: MacroD1 is present in mitochondria and nucleus, whereas MacroD2 is mostly cytoplasmic (5Neuvonen M. Ahola T. J. Mol. Biol. 2009; 385: 212-225Crossref PubMed Scopus (149) Google Scholar). Therefore, together MacroD1 and MacroD2 co-exist in all cellular compartments where OAADPr is generated in human cells.Cellular OAADPr levels can be also regulated by NUDIX hydrolyzes. NUDIX hydrolase Ysa1 from S. cerevisiae was shown to hydrolyze ADPr/OAADPr to produce AMP and ribose phosphate and acetyl-ribose phosphate, respectively (19Rafty L.A. Schmidt M.T. Perraud A.L. Scharenberg A.M. Denu J.M. J. Biol. Chem. 2002; 277: 47114-47122Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). In yeast, Ysa1 modulated the levels of AMP and ADPr/OAADPr (17Tong L. Lee S. Denu J.M. J. Biol. Chem. 2009; 284: 11256-11266Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The yeast Ysa1 enzyme hydrolyzes ADPr and OAADPr at similar efficiency in a Mg2+-dependent reaction. However, the human mitochondrial ortholog NudT9, prefers ADPr over OAADPr as substrate by 500-fold. To date, a NUDIX hydrolase specific for only OAADPr remains to be identified in higher eukaryotes.Enzymatic activities that consume OAADPr have been reported in mammalian cells (19Rafty L.A. Schmidt M.T. Perraud A.L. Scharenberg A.M. Denu J.M. J. Biol. Chem. 2002; 277: 47114-47122Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Although the identities of the responsible enzymes were not determined, cytoplasmic fractions of HeLa cell protein extracts contain an OAADPr hydrolase that produced acetate and ADPr. Nuclear fractionation enriched an enzymatic activity that removed the acetyl group from OAADPr to form ADPr, but the acetyl moiety was transferred to an unknown acceptor molecule (19Rafty L.A. Schmidt M.T. Perraud A.L. Scharenberg A.M. Denu J.M. J. Biol. Chem. 2002; 277: 47114-47122Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Both activities appeared to be independent of Mg2+, as reactions were performed in the presence of EDTA. Whether the enzymatic activity in HeLa cell extracts can be attributed to MacroD1/2 enzymes remain to be investigated. This possibility is supported by the fact that we were able to detect only Mg-independent hydrolysis in extracts of the lower eukaryote N. crassa and a significant fraction of this activity is due to a MacroD homologue in this organism (Fig. 5). Thus, it is plausible to suggest that MacroD-like proteins function as OAADPr-consuming enzymes in vivo. This role is further supported by genomic evidence, as MacroD-like proteins are found linked to sirtuin in many groups of pathogenic bacteria and fungi (S. aureus, Streptococci, Mycoplasma, C. albicans, and several Aspergillus species).The MacroD-like protein group is represented by numerous proteins from bacteria and eukaryotes, and we propose that dysregulation of MacroD-like protein function would disrupt OAADPr homeostasis and sirtuin pathways. The available literature suggests that the dysregulation of both human MacroD1 and MacroD2 levels in cells might lead to human disease. MacroD1 overexpression was linked to breast cancer progression, whereas deficiency in MacroD2 function has been linked to a developmental disorder called Kabuki syndrome (38Tada M. Kanai F. Tanaka Y. Sanada M. Nannya Y. Tateishi K. Ohta M. Asaoka Y. Seto M. Imazeki F. Yoshida H. Ogawa S. Yokosuka O. Omata M. Cancer Sci. 2010; 101: 1261-1269Crossref PubMed Scopus (23) Google Scholar, 39Maas N.M. Van de Putte T. Melotte C. Francis A. Schrander-Stumpel C.T. Sanlaville D. Genevieve D. Lyonnet S. Dimitrov B. Devriendt K. Fryns J.P. Vermeesch J.R. J. Med. Genet. 2007; 44: 562-569Crossref PubMed Scopus (54) Google Scholar, 40Han W.D. Mu Y.M. Lu X.C. Xu Z.M. Li X.J. Yu L. Song H.J. Li M. Lu J.M. Zhao Y.L. Pan C.Y. Endocr. Relat. Cancer. 2003; 10: 217-224Crossref PubMed Scopus (46) Google Scholar). Clearly, more investigation is needed to clarify the proposed links between macrodomain dysregulation and these diseases.In summary, this work identifies OAADPr deacetylation activities in MacroD-like proteins from bacteria and eukaryotes. Our data demonstrate that MacroD-like proteins regulate a variety of biochemical pathways by acting as potential in vivo regulators of cellular OAADPr produced by NAD+-dependent deacetylation. IntroductionMacrodomains are evolutionarily conserved structural domains found in proteins with diverse cellular functions (1Till S. Ladurner A.G. Front. Biosci. 2009; 14: 3246-3258Crossref PubMed Google Scholar, 2Ladurner A.G. Mol. Cell. 2003; 12: 1-3Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Prior evidence suggested that macrodomains function as binding modules of NAD+ metabolites, including ADP-ribose/poly(ADP-ribose) (3Karras G.I. Kustatscher G. Buhecha H.R. Allen M.D. Pugieux C. Sait F. Bycroft M. Ladurner A.G. EMBO J. 2005; 24: 1911-1920Crossref PubMed Scopus (382) Google Scholar, 4Egloff M.P. Malet H. Putics A. Heinonen M. Dutartre H. Frangeul A. Gruez A. Campanacci V. Cambillau C. Ziebuhr J. Ahola T. Canard B. J. Virol. 2006; 80: 8493-8502Crossref PubMed Scopus (176) Google Scholar, 5Neuvonen M. Ahola T. J. Mol. Biol. 2009; 385: 212-225Crossref PubMed Scopus (149) Google Scholar, 6Ahel D. Horejsí Z. Wiechens N. Polo S.E. Garcia-Wilson E. Ahel I. Flynn H. Skehel M. West S.C. Jackson S.P. Owen-Hughes T. Boulton S.J. Science. 2009; 325: 1240-1243Crossref PubMed Scopus (434) Google Scholar, 7Kraus W.L. Nat. Struct. Mol. Biol. 2009; 16: 904-907Crossref PubMed Scopus (31) Google Scholar) and O-acetyl-ADP-ribose (OAADPr) 5The abbreviations used are: OAADPr, O-acetyl-ADP-ribose; PEI, polyethyleneimine; aa, amino acids; PDB, Protein Data Bank. (8Kustatscher G. Hothorn M. Pugieux C. Scheffzek K. Ladurner A.G. Nat. Struct. Mol. Biol. 2005; 12: 624-625Crossref PubMed Scopus (244) Google Scholar, 9Comstock L.R. Denu J.M. Org. Biomol. Chem. 2007; 5: 3087-3091Crossref PubMed Scopus (21) Google Scholar). OAADPr is produced in reactions catalyzed by NAD+-dependent protein/histone deacetylases (10Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 14178-14182Crossref PubMed Scopus (491) Google Scholar, 11Jackson M.D. Denu J.M. J. Biol. Chem. 2002; 277: 18535-18544Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), which regulate gene silencing, metabolic enzymes, life span, and many other cellular processes (12Smith B.C. Hallows W.C. Denu J.M. Chem. Biol. 2008; 15: 1002-1013Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 13Oberdoerffer P. Michan S. McVay M. Mostoslavsky R. Vann J. Park S.K. Hartlerode A. Stegmuller J. Hafner A. Loerch P. Wright S.M. Mills K.D. Bonni A. Yankner B.A. Scully R. Prolla T.A. Alt F.W. Sinclair D.A. Cell. 2008; 135: 907-918Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar, 14Hallows W.C. Smith B.C. Lee S. Denu J.M. Cell. 2009; 137: 404-406Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). OAADPr has been implicated as a signaling molecule, modulating cellular processes affected by NAD+-dependent protein/histone deacetylation (15Hoff K.G. Wolberger C. Nat. Struct. Mol. Biol. 2005; 12: 560-561Crossref PubMed Scopus (17) Google Scholar, 16Tong L. Denu J.M. Biochim. Biophys. Acta. 2010; 1804: 1617-1625Crossref PubMed Scopus (88) Google Scholar, 17Tong L. Lee S. Denu J.M. J. Biol. Chem. 2009; 284: 11256-11266Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The binding of OAADPr and other NAD+ metabolites to macrodomains such as the histone variant macroH2A1.1 (1Till S. Ladurner A.G. Front. Biosci. 2009; 14: 3246-3258Crossref PubMed Google Scholar, 5Neuvonen M. Ahola T. J. Mol. Biol. 2009; 385: 212-225Crossref PubMed Scopus (149) Google Scholar, 8Kustatscher G. Hothorn M. Pugieux C. Scheffzek K. Ladurner A.G. Nat. Struct. Mol. Biol. 2005; 12: 624-625Crossref PubMed Scopus (244) Google Scholar, 9Comstock L.R. Denu J.M. Org. Biomol. Chem. 2007; 5: 3087-3091Crossref PubMed Scopus (21) Google Scholar) suggests a possible connection between metabolic regulation, gene activity, and chromatin structure. The mechanism by which cells regulate and utilize OAADPr is not well understood. In yeast, the NUDIX ADP-ribose pyrophosphatase Ysa1 modulates the cellular levels of both ADPr and OAADPr, converting each to AMP and the corresponding ribose-phosphate (17Tong L. Lee S. Denu J.M. J. Biol. Chem. 2009; 284: 11256-11266Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Cells lacking ysa1 exhibit an increased resistance to oxidative insults and produce lower levels of endogenous reactive oxygen species. In vitro, the poly (ADP-ribose) glycohydrolase ARH3 was capable of removing the acetyl group from OAADPr (18Ono T. Kasamatsu A. Oka S. Moss J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16687-16691Crossref PubMed Scopus (91) Google Scholar), though the activity was orders of magnitude slower than that observed for the NUDIX family (19Rafty L.A. Schmidt M.T. Perraud A.L. Scharenberg A.M. Denu J.M. J. Biol. Chem. 2002; 277: 47114-47122Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). In eukaryotic cell extracts at least two different cellular activities were shown to contribute to OAADPr deacetylation (19Rafty L.A. Schmidt M.T. Perraud A.L. Scharenberg A.M. Denu J.M. J. Biol. Chem. 2002; 277: 47114-47122Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), but the identities of these factors remain unknown.Here we report a direct functional connection between sirtuins and a family of macrodomain proteins. We demonstrate that proteins belonging to this distinct branch of macrodomains are OAADPr deacetylases that efficiently catalyze the hydrolysis of OAADPr to produce ADP-ribose and free acetate. This group comprises eukaryotic MacroD proteins (orthologues of human MacroD1 and MacroD2), bacterial YmdB proteins and sirtuin-linked macrodomain proteins from certain pathogenic bacteria and fungi. Sirtuin-linked macrodomains constitute a macrodomain subfamily that are either fusions with sirtuin proteins or are genetically coupled through the same operon.