Mouse Sir2 Homolog SIRT6 Is a Nuclear ADP-ribosyltransferase

Abstract

Members of the Sir2 family of NAD-dependent protein deacetylases regulate diverse cellular processes including aging, gene silencing, and cellular differentiation. Here, we report that the distant mammalian Sir2 homolog SIRT6 is a broadly expressed, predominantly nuclear protein. Northern analysis of embryonic samples and multiple adult tissues revealed mouse SIRT6 (mSIRT6) mRNA peaks at day E11, persisting into adulthood in all eight tissues examined. At the protein level, mSIRT6 was readily detectable in the same eight tissue types, with the highest levels in muscle, brain, and heart. Subcellular localization studies using both C- and N-terminal green fluorescent protein fusion proteins showed mSIRT6-green fluorescent protein to be a predominantly nuclear protein. Indirect immunofluorescence using antibodies to two different mSIRT6 epitopes confirmed that endogenous mSIRT6 is also largely nuclear. Consistent with previous findings, we did not observe any NAD+-dependent protein deacetylase activity in preparations of mSIRT6. However, purified recombinant mSIRT6 did catalyze the robust transfer of radiolabel from [32P]NAD to mSIRT6. Two highly conserved residues within the catalytic core of the protein were required for this reaction. This reaction is most likely mono-ADP-ribosylation because only the modified form of the protein was recognized by an antibody specific to mono-ADP-ribose. Surprisingly, we observed that the catalytic mechanism of this reaction is intra-molecular, with individual molecules of mSIRT6 directing their own modification. These results provide the first characterization of a Sir2 protein from phylogenetic class IV. Members of the Sir2 family of NAD-dependent protein deacetylases regulate diverse cellular processes including aging, gene silencing, and cellular differentiation. Here, we report that the distant mammalian Sir2 homolog SIRT6 is a broadly expressed, predominantly nuclear protein. Northern analysis of embryonic samples and multiple adult tissues revealed mouse SIRT6 (mSIRT6) mRNA peaks at day E11, persisting into adulthood in all eight tissues examined. At the protein level, mSIRT6 was readily detectable in the same eight tissue types, with the highest levels in muscle, brain, and heart. Subcellular localization studies using both C- and N-terminal green fluorescent protein fusion proteins showed mSIRT6-green fluorescent protein to be a predominantly nuclear protein. Indirect immunofluorescence using antibodies to two different mSIRT6 epitopes confirmed that endogenous mSIRT6 is also largely nuclear. Consistent with previous findings, we did not observe any NAD+-dependent protein deacetylase activity in preparations of mSIRT6. However, purified recombinant mSIRT6 did catalyze the robust transfer of radiolabel from [32P]NAD to mSIRT6. Two highly conserved residues within the catalytic core of the protein were required for this reaction. This reaction is most likely mono-ADP-ribosylation because only the modified form of the protein was recognized by an antibody specific to mono-ADP-ribose. Surprisingly, we observed that the catalytic mechanism of this reaction is intra-molecular, with individual molecules of mSIRT6 directing their own modification. These results provide the first characterization of a Sir2 protein from phylogenetic class IV. Members of the Sir2 family of enzymes, conserved from bacteria to man, utilize oxidized NAD+ as a cosubstrate in the deacetylation of a wide variety of proteins, thereby regulating diverse processes including aging, genomic silencing, recombination, cell fate, and metabolism (1Blander G. Guarente L. Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1299) Google Scholar). In yeast, the archetypal Sir2p localizes to chromatin sites at the telomeres, ribosomal DNA, and silent mating type loci, facilitating genomic silencing through the deacetylation of lysines within the histone H3 and H4 tails (2Gasser S.M. Cockell M.M. Gene (Amst.). 2001; 279: 1-16Crossref PubMed Scopus (229) Google Scholar). In this context, yeast Sir2p promotes mother cell longevity by repressing ribosomal DNA recombination and formation of toxic extra-chromosomal ribosomal DNA circles (3Hekimi S. Guarente L. Science. 2003; 299: 1351-1354Crossref PubMed Scopus (377) Google Scholar, 4Bitterman K.J. Medvedik O. Sinclair D.A. Microbiol. Mol. Biol. Rev. 2003; 67: 376-399Crossref PubMed Scopus (206) Google Scholar). Overexpression of Sir2p extends yeast life span (5Kaeberlein M. McVey M. Guarente L. Genes Dev. 1999; 13: 2570-2580Crossref PubMed Scopus (1758) Google Scholar), and Sir2p activity increases in response to caloric restriction (6Lin S.J. Ford E. Haigis M. Liszt G. Guarente L. Genes Dev. 2004; 18: 12-16Crossref PubMed Scopus (544) Google Scholar), contributing to the resultant extension of life span (7Lin S.J. Defossez P.A. Guarente L. Science. 2000; 289: 2126-2128Crossref PubMed Scopus (1477) Google Scholar). Intriguingly, Sir2 orthologs in worm and fly also promote longevity, although probably by different molecular mechanisms (8Rogina B. Helfand S.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15998-16003Crossref PubMed Scopus (1066) Google Scholar, 9Wood J.G. Rogina B. Lavu S. Howitz K. Helfand S.L. Tatar M. Sinclair D. Nature. 2004; 430: 686-689Crossref PubMed Scopus (1570) Google Scholar, 10Tissenbaum H.A. Guarente L. Nature. 2001; 410: 227-230Crossref PubMed Scopus (1577) Google Scholar). Mammalian genomes contain seven Sir2 homologs, termed sirtuins (SIRTs). Of these, SIRT1, orthologous to the yeast Sir2, is the best characterized and has the broadest substrate specificity (11North B.J. Verdin E. Genome Biol. 2004; 5: 224Crossref PubMed Scopus (438) Google Scholar). A nuclear protein, SIRT1 deacetylates several substrates in vivo, including MyoD, p53, and FOXO transcription factors, thereby affecting cell differentiation and survival under stress (11North B.J. Verdin E. Genome Biol. 2004; 5: 224Crossref PubMed Scopus (438) Google Scholar, 12Motta M.C. Divecha N. Lemieux M. Kamel C. Chen D. Gu W. Bultsma Y. McBurney M. Guarente L. Cell. 2004; 116: 551-563Abstract Full Text Full Text PDF PubMed Scopus (1192) Google Scholar, 13Brunet A. Sweeney L.B. Sturgill J.F. Chua K.F. Greer P.L. Lin Y. Tran H. Ross S.E. Mostoslavsky R. Cohen H.Y. Hu L.S. Cheng H.L. Jedrychowski M.P. Gygi S.P. Sinclair D.A. Alt F.W. Greenberg M.E. Science. 2004; 303: 2011-2015Crossref PubMed Scopus (2634) Google Scholar). Recently, mouse SIRT1 was reported to inhibit the mobilization of fatty acids in white adipose tissue by repressing PPARγ, linking Sir2 proteins to the physiology of caloric restriction in mammals (14Picard F. Kurtev M. Chung N. Topark-Ngarm A. Senawong T. Machado De Oliveira R. Leid M. McBurney M.W. Guarente L. Nature. 2004; 429: 771-776Crossref PubMed Scopus (1652) Google Scholar). Of the remaining SIRTs (SIRT2–7), an in vivo substrate has only been identified for the cytoplasmic SIRT2 (15North B.J. Marshall B.L. Borra M.T. Denu J.M. Verdin E. Mol. Cell. 2003; 11: 437-444Abstract Full Text Full Text PDF PubMed Scopus (1232) Google Scholar). This substrate, β-tubulin, is specifically deacetylated by SIRT2 (15North B.J. Marshall B.L. Borra M.T. Denu J.M. Verdin E. Mol. Cell. 2003; 11: 437-444Abstract Full Text Full Text PDF PubMed Scopus (1232) Google Scholar), although the biological consequences of this reaction are unclear. Yeast Sir2 and its orthologs, including mouse SIRT1 and bacterial CobB, catalyze the tightly coupled cleavage of NAD+ and protein deacetylation, producing nicotinamide and 2-O-acetyl-ADP-ribose reaction products (16Sauve A.A. Schramm V.L. Curr. Med. Chem. 2004; 11: 807-826Crossref PubMed Scopus (68) Google Scholar). Whereas Sir2 proteins are generally thought to be NAD-dependent protein deacetylases, most also display a less robust mono-ADP-ribosyltransferase activity in vitro (17Frye R.A. Biochem. Biophys. Res. Commun. 1999; 260: 273-279Crossref PubMed Scopus (665) Google Scholar, 18Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Crossref PubMed Scopus (494) Google Scholar, 19Tanny J.C. Dowd G.J. Huang J. Hilz H. Moazed D. Cell. 1999; 99: 735-745Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 20Tanny J.C. Moazed D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 415-420Crossref PubMed Scopus (224) Google Scholar). Mutations in phylogenetically conserved residues within the catalytic core of Sir2 and SIRT1 have failed to separate the two enzymatic activities (21Armstrong C.M. Kaeberlein M. Imai S.I. Guarente L. Mol. Biol. Cell. 2002; 13: 1427-1438Crossref PubMed Scopus (51) Google Scholar). The initial observation that ADP-ribosylation by Sir2 is stronger on acetylated substrates (22Imai S. Armstrong C.M. Kaeberlein M. Guarente L. Nature. 2000; 403: 795-800Crossref PubMed Scopus (2774) Google Scholar), combined with subsequent mechanistic studies (18Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Crossref PubMed Scopus (494) Google Scholar, 20Tanny J.C. Moazed D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 415-420Crossref PubMed Scopus (224) Google Scholar), has led to the conclusion that the deacetylase and phosphoribosyltransferase activities of Sir2 proteins are coupled (16Sauve A.A. Schramm V.L. Curr. Med. Chem. 2004; 11: 807-826Crossref PubMed Scopus (68) Google Scholar). Using sequence similarity, eukaryotic Sir2 genes have been divided into four broad phylogenetic classes (23Frye R.A. Biochem. Biophys. Res. Commun. 2000; 273: 793-798Crossref PubMed Scopus (1153) Google Scholar), known as classes I–IV (Fig. 1). SIRT1, SIRT2, and SIRT3 are class I, SIRT4 is class II, and SIRT5 falls within the predominantly prokaryotic class III. Finally, mammalian SIRT6 and SIRT7 are class IV sirtuins (23Frye R.A. Biochem. Biophys. Res. Commun. 2000; 273: 793-798Crossref PubMed Scopus (1153) Google Scholar). In vitro studies indicate that human SIRT1, SIRT2, SIRT3, and SIRT5 possess NAD-dependent histone deacetylase activity (15North B.J. Marshall B.L. Borra M.T. Denu J.M. Verdin E. Mol. Cell. 2003; 11: 437-444Abstract Full Text Full Text PDF PubMed Scopus (1232) Google Scholar), whereas SIRT4, SIRT6, and SIRT7 fail to deacetylate 3H-labeled acetylated histone H4 peptide (15North B.J. Marshall B.L. Borra M.T. Denu J.M. Verdin E. Mol. Cell. 2003; 11: 437-444Abstract Full Text Full Text PDF PubMed Scopus (1232) Google Scholar). The lack of detectable deacetylase activity in these SIRTs may result from their specificity for targets other than those tested, or it may indicate an enzymatic activity other than deacetylation inherent in these sirtuins. Mono-ADP-ribosylation is emerging as a common mechanism of reversible protein modification within mammalian cells. Originally described as the acting mechanism for specific bacterial toxins, ADP-ribosylation is typically performed by separate families of intra- and extracellular enzymes in vertebrates (24Corda D. Di Girolamo M. EMBO J. 2003; 22: 1953-1958Crossref PubMed Scopus (250) Google Scholar). Known targets of the intracellular class of enzymes include molecular chaperone GRP78/BiP, translational elongation factor 2, and β-subunit of heterotrimeric G-proteins (24Corda D. Di Girolamo M. EMBO J. 2003; 22: 1953-1958Crossref PubMed Scopus (250) Google Scholar). Extracellular mammalian ADP-ribosyltransferases, generally found in cells of the immune system, modify substrates important for the immune response, as well as integrin α7 and the antimicrobial peptide defensin (24Corda D. Di Girolamo M. EMBO J. 2003; 22: 1953-1958Crossref PubMed Scopus (250) Google Scholar). In most known cases, ADP-ribosylation of arginine residues results in reversible inactivation of the substrate protein (25Riese M.J. Goehring U.M. Ehrmantraut M.E. Moss J. Barbieri J.T. Aktories K. Schmidt G. J. Biol. Chem. 2002; 277: 12082-12088Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), although this modification may enhance certain enzymatic functions within the substrate (26Weng B. Thompson W.C. Kim H.J. Levine R.L. Moss J. J. Biol. Chem. 1999; 274: 31797-31803Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). In this report, we characterize the tissue-specific expression, subcellular localization, and in vitro enzymatic activity of mouse SIRT6. This investigation, the first detailed study of a class IV sirtuin, reveals mouse SIRT6 to be a widely expressed, predominantly nuclear protein with a robust auto-ADP-ribosyltransferase activity. Multiple Sequence Alignments—Sequences of mouse proteins SIRT1, SIRT4, SIRT5, and SIRT6, as well as yeast Sir2p, were obtained from the Proteome BioKnowledge® Library (proteome.incyte.com/control/tools/proteome). Core domains were aligned by ClustalW using software from the DNASTAR package. Plasmid Construction—IMAGE clone 1259892, which contains the mouse SIRT6 cDNA, was obtained from American Type Culture Collection. The mSIRT6 1The abbreviations used are: mouse green fluorescent green fluorescent sequence was by and into the and sites of and the and sites of and the mSIRT6 sequence was by and into the and sites of and from and and were using the to the was by the of the mSIRT6 to acids and it into with and Multiple Northern the SIRT6 was using the to the This was used to Multiple Northern to the The were and with the in the and were into cells and into or of and The cells were at to an of was by the of to a of for and the cells were by The cells the were in and by in The was at in a The was with of for at a and with The purified protein was with in The was by the of The protein were and This protein was to for antibody The cells the were in and of at for and in The was by and the was at for The was with of for at and a The was with and the protein was with protein were and to for antibody The antibodies were as of the proteins used to the antibodies were of to the of the of antibody was combined with of antibody and to the was with antibody and the antibody was with The was by the of of The antibodies were to a of this the antibody was to the and the antibody was to the SIRT6 mouse tissues from four of the were obtained as a from L. of these proteins were K. Google Scholar), of which were used for samples were on to and using in with antibody was in with antibody K. Google Scholar), a from H. was used at a in was used as a in multiple tissue antibodies were or antibodies was by with the and cells were in modified for on in were for with with and by a with were with and with for to a and in antibody was at a for and cells were for in was in and with for were with was performed on a were obtained using a and software using for cells were with of using to were for on were and as described of SIRT6 were with the mSIRT6 were performed as described for antibody fusion protein was in at for The proteins were purified with under The was from a bacterial clone proteins were purified by a with with for proteins was from bacteria of recombinant proteins were at ADP-ribosylation and of were performed as described (22Imai S. Armstrong C.M. Kaeberlein M. Guarente L. Nature. 2000; 403: 795-800Crossref PubMed Scopus (2774) Google Scholar), with were performed in a of recombinant SIRT6 in and of [32P]NAD also of core as both and recombinant SIRT6 protein, of protein was to all were purified from [32P]NAD using with a were to and subsequent were with and the and were aligned to that the were SIRT6. of were performed as described with the reaction was including of protein, of and of reaction products were by and of 2, and were on the same SIRT6 protein in were by with these was by and SIRT6 were was by on a A to was by of known of [32P]NAD to the of to in the reaction. This was used to of in SIRT6 of was divided by of SIRT6. This the of to SIRT6. mSIRT6 in and the of we from eight adult mouse tissues by Northern (Fig. Using a to the of of mSIRT6 mRNA was observed in tissue type (Fig. the of sequence analysis (23Frye R.A. Biochem. Biophys. Res. Commun. 2000; 273: 793-798Crossref PubMed Scopus (1153) Google Scholar), mSIRT6 was as a that this gene is not found in to the highest levels of mSIRT6 mRNA were in the brain, and with the observed in mSIRT6 levels we Northern of from four mouse embryonic from embryonic day to (Fig. SIRT6 was readily detectable in all embryonic a at the of mSIRT6 in embryonic and adult we to the of mSIRT6 protein. specific to mSIRT6 was used to of proteins from eight adult tissue (Fig. tissue samples from four individual were obtained and by in mSIRT6 protein was detectable in all tissue as a from Northern levels were in brain, and to protein Surprisingly, mSIRT6 protein was most in (Fig. the of its in this This observation indicate mSIRT6 in or an of in this tissue type to of in to observe the subcellular localization of we two fusion under the of the a and an N-terminal fusion were into cells on were for at which were and to of cells fusion showed localization of to the by cytoplasmic as by of (Fig. of the and Indirect of mSIRT6 endogenous levels of to two different mSIRT6 were used to cells by a cells were antibody and with and was the and in these samples (Fig. mSIRT6 showed predominantly in the (Fig. of which antibody was used in the In both cases, endogenous mSIRT6 to be from the immunofluorescence and localization studies revealed the same predominantly nuclear localization for mSIRT6. that the of mSIRT6 is found in the whereas a may be to the mSIRT6 the of from a the seven mammalian Sir2 homologs, only SIRT1, SIRT2, SIRT3, and SIRT5 have been to have NAD+-dependent protein deacetylase activity in vitro (15North B.J. Marshall B.L. Borra M.T. Denu J.M. Verdin E. Mol. Cell. 2003; 11: 437-444Abstract Full Text Full Text PDF PubMed Scopus (1232) Google Scholar). Consistent with we did not observe any protein deacetylase activity of mouse or human SIRT6 using a variety of and substrates not sirtuins also possess a mono-ADP-ribosyltransferase activity J.C. Dowd G.J. Huang J. Hilz H. Moazed D. Cell. 1999; 99: 735-745Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). to mSIRT6 catalyze the transfer of radiolabel from to an of activity. human SIRT1 and mSIRT6 were in E. and purified by of and mSIRT6 were with in the and of core (Fig. Whereas to mSIRT6 failed to (Fig. However, mSIRT6 the robust transfer of to of were in the reaction (Fig. purified from E. detectable activity under these not to transfer by mSIRT6 required the catalytic activity of the Two in highly conserved residues within the mSIRT6 core were by (Fig. 1). The at was to a to enzymatic activity in human and yeast Sir2 proteins (17Frye R.A. Biochem. Biophys. Res. Commun. 1999; 260: 273-279Crossref PubMed Scopus (665) Google Scholar, 19Tanny J.C. Dowd G.J. Huang J. Hilz H. Moazed D. Cell. 1999; 99: 735-745Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). In a separate the phylogenetically at was to is in the of the and not to be important Biol. 2001; PubMed Scopus Google Scholar). proteins were in E. and in the of as described was by were by to and to Whereas mSIRT6 radiolabel to transfer was in and (Fig. The of proteins on the was by using antibody to mSIRT6 (Fig. on this for two catalytic core we that mSIRT6 the transfer of radiolabel from [32P]NAD by an enzymatic of proteins with also by the of as observed in the of J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), be as ADP-ribosylation of specific J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, M. M. H. M. J. Biochem. PubMed Scopus Google Scholar). have observed levels of of proteins in the of reaction modification in core and Sir2 generally not of protein a at (Fig. as by of protein In in the of we observed of a of to of of mSIRT6 in the reaction (Fig. this and because of the in and of we that the transfer of radiolabel a robust enzymatic of mSIRT6 the that mSIRT6 the mono-ADP-ribosylation of we the reaction products from the transfer with an antibody specific to T. Hilz H. J. Biochem. PubMed Scopus Google Scholar). This antibody recognized a to the mSIRT6 with [32P]NAD (Fig. was at the in preparations from mSIRT6 (Fig. the specificity of the antibody for the modified form of mSIRT6. This that this modified form of mSIRT6 is Whereas results not the that the modification of mSIRT6 in E. the of in SIRT6 that mono-ADP-ribosylation on the catalytic activity of mSIRT6. confirmed mono-ADP-ribosylation as the of the modification of we to this modification was in an or mSIRT6 and two different and were used in a molecular for enzymatic activity. was purified from E. and in with and mSIRT6 in the of were purified as described and to as an in all (Fig. mSIRT6 as a (Fig. However, this was from the of both that was to those of the to revealed of protein in all not that the reaction by mSIRT6 is we provide the first characterization of a class IV sirtuin, mouse SIRT6. This a sequence to the yeast Sir2 a in and enzymatic activity. Consistent with findings, we failed to any NAD-dependent protein deacetylase activity in preparations of mSIRT6 in bacterial and mammalian cells. Surprisingly, mSIRT6 did a robust activity in that recombinant bacterial preparations protein. that mSIRT6 is a mono-ADP-ribosyltransferase for the the purified protein catalyze the transfer of radiolabel from a reaction also by other Sir2 family (17Frye R.A. Biochem. Biophys. Res. Commun. 1999; 260: 273-279Crossref PubMed Scopus (665) Google Scholar, 19Tanny J.C. Dowd G.J. Huang J. Hilz H. Moazed D. Cell. 1999; 99: 735-745Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, S. Armstrong C.M. Kaeberlein M. Guarente L. Nature. 2000; 403: 795-800Crossref PubMed Scopus (2774) Google and to be mono-ADP-ribosylation J.C. Dowd G.J. Huang J. Hilz H. Moazed D. Cell. 1999; 99: 735-745Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). this activity the catalytic of mSIRT6 because two different in phylogenetically residues to enzymatic activity the transfer of from to mSIRT6. only the modified form of mSIRT6 is recognized by an antibody specific to Finally, the transfer of from [32P]NAD to mSIRT6 is by an that SIRT6 may ADP-ribosylation as a to its activity. mSIRT6 in E. and with was to a of to of the of mSIRT6 in the reaction. This of ADP-ribosylation is the of the by which a of SIRT6 is only on a substrate failed to observe a from that the of residues on mSIRT6 is a ADP-ribosylation of SIRT6, results indicate a activity in recombinant protein SIRT6 is broadly at the and protein levels and adulthood in at eight different tissue types, broad the The SIRT6 protein is in and mSIRT6 protein is highest in muscle, mRNA levels in this tissue are to those of This indicate SIRT6 protein in with other tissue the of SIRT6 protein observed in may result from an of of SIRT6 mRNA in this tissue studies of mSIRT6 using and as well as antibodies to two different epitopes showed that of mSIRT6 was largely with only a in endogenous levels of SIRT6 not certain probably to the (Fig. The only other mouse class IV sirtuin, is to the Ford and L. that mSIRT6 and sequence have functions within the SIRT6, SIRT7 has not been to possess any in vitro protein deacetylase activity (15North B.J. Marshall B.L. Borra M.T. Denu J.M. Verdin E. Mol. Cell. 2003; 11: 437-444Abstract Full Text Full Text PDF PubMed Scopus (1232) Google Scholar). are of reversible in enzymatic activity. a is a protein as well as an (25Riese M.J. Goehring U.M. Ehrmantraut M.E. Moss J. Barbieri J.T. Aktories K. Schmidt G. J. Biol. Chem. 2002; 277: 12082-12088Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). at arginine observed in vitro and in vivo, protein activity (25Riese M.J. Goehring U.M. Ehrmantraut M.E. Moss J. Barbieri J.T. Aktories K. Schmidt G. J. Biol. Chem. 2002; 277: 12082-12088Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), a mechanism for In of the protein from to (26Weng B. Thompson W.C. Kim H.J. Levine R.L. Moss J. J. Biol. Chem. 1999; 274: 31797-31803Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), a mechanism for of activity. that SIRT6 regulate its own activity in vivo by ADP-ribosylation of specific residues required for activity. At this we have not identified the targets of SIRT6. class sirtuins including yeast Sir2 and SIRT1, SIRT6 to be highly specific in to catalyze ADP-ribosylation of SIRT6 molecules in The of auto-ADP-ribosyltransferase activity in preparations of recombinant mouse SIRT6 is the first report of enzymatic activity for any class IV to substrates by SIRT6. H. Hilz for the also of for the of mouse tissue protein Finally, we Haigis and for and