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Acetylation-dependent ADP-ribosylation by Trypanosoma brucei Sir2

2007; Elsevier BV; Volume: 283; Issue: 9 Linguagem: Inglês

10.1074/jbc.m707613200

1083-351X

Terri M. Kowieski, Susan Lee, John M. Denu,

Sirtuins and Resveratrol in Medicine

Sirtuins are a highly conserved family of proteins implicated in diverse cellular processes such as gene silencing, aging, and metabolic regulation. Although many sirtuins catalyze a well characterized protein/histone deacetylation reaction, there are a number of reports that suggest protein ADP-ribosyltransferase activity. Here we explored the mechanisms of ADP-ribosylation using the Trypanosoma brucei Sir2 homologue TbSIR2rp1 as a model for sirtuins that reportedly display both activities. Steady-state kinetic analysis revealed a highly active histone deacetylase (kcat = 0.1 s–1, with Km values of 42 μm and for NAD+ and 65 μm for acetylated substrate). A series of biochemical assays revealed that TbSIR2rp1 ADP-ribosylation of protein/histone requires an acetylated substrate. The data are consistent with two distinct ADP-ribosylation pathways that involve an acetylated substrate, NAD+ and TbSIR2rp1 as follows: 1) a noncatalytic reaction between the deacetylation product O-acetyl-ADP-ribose (or its hydrolysis product ADP-ribose) and histones, and 2) a more efficient mechanism involving interception of an ADP-ribose-acetylpeptide-enzyme intermediate by a side-chain nucleophile from bound histone. However, the sum of both ADP-ribosylation reactions was ∼5 orders of magnitude slower than histone deacetylation under identical conditions. The biological implications of these results are discussed. Sirtuins are a highly conserved family of proteins implicated in diverse cellular processes such as gene silencing, aging, and metabolic regulation. Although many sirtuins catalyze a well characterized protein/histone deacetylation reaction, there are a number of reports that suggest protein ADP-ribosyltransferase activity. Here we explored the mechanisms of ADP-ribosylation using the Trypanosoma brucei Sir2 homologue TbSIR2rp1 as a model for sirtuins that reportedly display both activities. Steady-state kinetic analysis revealed a highly active histone deacetylase (kcat = 0.1 s–1, with Km values of 42 μm and for NAD+ and 65 μm for acetylated substrate). A series of biochemical assays revealed that TbSIR2rp1 ADP-ribosylation of protein/histone requires an acetylated substrate. The data are consistent with two distinct ADP-ribosylation pathways that involve an acetylated substrate, NAD+ and TbSIR2rp1 as follows: 1) a noncatalytic reaction between the deacetylation product O-acetyl-ADP-ribose (or its hydrolysis product ADP-ribose) and histones, and 2) a more efficient mechanism involving interception of an ADP-ribose-acetylpeptide-enzyme intermediate by a side-chain nucleophile from bound histone. However, the sum of both ADP-ribosylation reactions was ∼5 orders of magnitude slower than histone deacetylation under identical conditions. The biological implications of these results are discussed. Sir2 (Silent information regulator 2) NAD+-dependent enzymes (sirtuins) are found in all kingdoms of life and have been implicated in a variety of cellular processes such as gene silencing (1Tanny J.C. Dowd G.J. Huang J. Hilz H. Moazed D. Cell. 1999; 99: 735-745Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 2Gasser S.M. Cockell M.M. Gene (Amst.). 2001; 279: 1-16Crossref PubMed Scopus (229) Google Scholar, 3Rusche L.N. Kirchmaier A.L. Rine J. Annu. Rev. Biochem. 2003; 72: 481-516Crossref PubMed Scopus (595) Google Scholar), life span extension (4Tissenbaum H.A. Guarente L. Nature. 2001; 410: 227-230Crossref PubMed Scopus (1571) Google Scholar, 5Rogina B. Helfand S.L. Proc. Natl. Acad. Sci. U. S. 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Nature. 2004; 429: 771-776Crossref PubMed Scopus (1642) Google Scholar, 7Cohen H.Y. Miller C. Bitterman K.J. Wall N.R. Hekking B. Kessler B. Howitz K.T. Gorospe M. de Cabo R. Sinclair D.A. Science. 2004; 305: 390-392Crossref PubMed Scopus (1663) Google Scholar); and increase glucose response to insulin (26Moynihan K.A. Grimm A.A. Plueger M.M. Bernal-Mizrachi E. Ford E. Cras-Meneur C. Permutt M.A. Imai S. Cell Metab. 2005; 2: 105-117Abstract Full Text Full Text PDF PubMed Scopus (541) Google Scholar). Although there is general consensus that many sirtuins affect biological pathways by catalyzing the NAD+-dependent deacetylation of target proteins, a number of reports have suggested that some sirtuins catalyze protein ADP-ribosylation, either exclusively or in conjunction with their inherent deacetylase activity. The initial idea that sirtuins are indeed enzymes and could mediate phosphoribosyl transfer came from work on Salmonella typhimurium CobB, a Sir2 homologue that could compensate for the loss of CobT, a phosphoribosyltransferase involved in cobalamin biosynthesis (27Tsang A.W. Escalante-Semerena J.C. J. Biol. Chem. 1998; 273: 31788-31794Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Subsequent studies reported the abilities of CobB and human SIRT2 to transfer radiolabel from [32P]NAD+ to bovine serum albumin (11Frye R.A. Biochem. Biophys. Res. Commun. 1999; 260: 273-279Crossref PubMed Scopus (658) Google Scholar). Another study reported the ability of ySir2 to transfer ADP-ribose to both bovine serum albumin and histones (1Tanny J.C. Dowd G.J. Huang J. Hilz H. Moazed D. Cell. 1999; 99: 735-745Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). These early reports suggested sirtuins possessed intrinsic ADP-ribosyltransferase activity. However, additional reports demonstrated that a majority of sirtuins were robust NAD+-dependent histone/protein deacetylases, coupling deacetylation to the formation of a novel metabolite, O-acetyl-ADP-ribose (OAADPr) 3The abbreviations used are:OAADPrO-acetyl-ADP-riboseGSTglutathione S-transferaseDTTdithiothreitolHPLChigh pressure liquid chromatographyPDEphosphodiesteraseADPrADP-riboseAcH2Aacetylated recombinant histone H2A.3The abbreviations used are:OAADPrO-acetyl-ADP-riboseGSTglutathione S-transferaseDTTdithiothreitolHPLChigh pressure liquid chromatographyPDEphosphodiesteraseADPrADP-riboseAcH2Aacetylated recombinant histone H2A. (28Imai S. Armstrong C.M. Kaeberlein M. Guarente L. Nature. 2000; 403: 795-800Crossref PubMed Scopus (2753) Google Scholar, 29Smith J.S. Brachmann C.B. Celic I. Kenna M.A. Muhammad S. Starai V.J. Avalos J.L. Escalante-Semerena J.C. Grubmeyer C. Wolberger C. Boeke J.D. Proc. Natl. Acad. Sci. U. S. 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Valenzuela D.M. Yancopoulos G.D. Karow M. Blander G. Wolberger C. Prolla T.A. Weindruch R. Alt F.W. Guarente L. Cell. 2006; 126: 941-954Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar). Among mammalian sirtuins with little or no detectable histone deacetylase activity, SIRT4 was shown to ADP-ribosylate and down-regulate glutamate dehydrogenase and has been implicated in insulin regulation (35Haigis M.C. Mostoslavsky R. Haigis K.M. Fahie K. Christodoulou D.C. Murphy A.J. Valenzuela D.M. Yancopoulos G.D. Karow M. Blander G. Wolberger C. Prolla T.A. Weindruch R. Alt F.W. Guarente L. Cell. 2006; 126: 941-954Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar, 36Ahuja N. Schwer B. Carobbio S. Waltregny D. North B.J. Castronovo V. Maechler P. Verdin E. J. Biol. Chem. 2007; 282: 33583-33592Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). Also, SIRT6 functions in DNA base-excision repair and can mediate auto-ADP-ribosylation (37Liszt G. Ford E. Kurtev M. Guarente L. J. Biol. Chem. 2005; 280: 21313-21320Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). O-acetyl-ADP-ribose glutathione S-transferase dithiothreitol high pressure liquid chromatography phosphodiesterase ADP-ribose acetylated recombinant histone H2A. O-acetyl-ADP-ribose glutathione S-transferase dithiothreitol high pressure liquid chromatography phosphodiesterase ADP-ribose acetylated recombinant histone H2A. Other studies report that sirtuins, including ySir2, Hst2, and Sir2 orthologues from the parasites Trypanosoma brucei and Plasmodium falciparum, possess both protein deacetylase and mono-ADP-ribosyltransferase activity (28Imai S. Armstrong C.M. Kaeberlein M. Guarente L. Nature. 2000; 403: 795-800Crossref PubMed Scopus (2753) Google Scholar, 31Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Crossref PubMed Scopus (492) Google Scholar, 38Garcia-Salcedo J.A. Gijon P. Nolan D.P. Tebabi P. Pays E. EMBO J. 2003; 22: 5851-5862Crossref PubMed Scopus (110) Google Scholar). T. brucei Sir2 orthologue TbSIR2rp1 localizes to the nucleus and plays a role in DNA repair and silencing in the insect and blood-stream stages of the parasite (38Garcia-Salcedo J.A. Gijon P. Nolan D.P. Tebabi P. Pays E. EMBO J. 2003; 22: 5851-5862Crossref PubMed Scopus (110) Google Scholar, 39Alsford S. Kawahara T. Isamah C. Horn D. Mol. Microbiol. 2007; 63: 724-736Crossref PubMed Scopus (97) Google Scholar). However, the mechanistic relationship between protein deacetylation and ADP-ribosylation has not been elucidated. Here we perform biochemical and kinetic studies to probe the mechanisms of ADP-ribosylation using sirtuin T. brucei TbSIR2rp1 as a model for sirtuins that display both deacetylase and ADP-ribosyltransferase activities. Steady-state kinetic analysis revealed a highly active histone deacetylase, but protein ADP-ribosyltransferase activity was ∼5 orders of magnitude lower. Moreover, protein/histone ADP-ribosylation by TbSIR2rp1 required an acetylated substrate. We propose that the dependence on acetylated substrate occurs through two distinct pathways. The first pathway involves a direct, noncatalytic reaction between the unique deacetylation product OAADPr (or its hydrolysis product ADPr) and histones. The second pathway is responsible for the majority of TbSIR2rp1-dependent ADPr transfer and involves a mechanism in which a side-chain nucleophile from bound histone attacks an intermediate from the catalytic pathway that normally leads to deacetylated protein and OAADPr. Plasmid Construction—T. brucei genomic DNA was generously provided by Dr. J. Bangs from University of Wisconsin, Madison, and used as a template for the subsequent PCR. TbSir2rp1 was amplified using primers 5′-GGGATCCATGACAGAACCGAAGTTAGCAACC-3′ and 5′-CCGCTCGAGACCCTCAACGACTTTTTC-3′ that introduced BamHI and XhoI, upstream and downstream recognition sites, respectively. PCR products were gel-purified, digested with BamHI and XhoI, and cloned into pGEX-KG (40Guan K.L. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1639) Google Scholar) upstream and inframe of a sequence encoding a glutathione S-transferase (GST) fusion protein. Using BamHI and HindIII, the TbSir2rp1 fragment was released and subcloned into pQE80 (Qiagen) downstream and in-frame of a sequence encoding an His6 tag. Eukaryotic expression plasmid pcDNA3.1 SirT1 was kindly provided by Dr. Eric Verdin from University of California, San Francisco, and used as a template for the following PCR. SirT1 was amplified using primers 5′-GCTTGGGATCCATGGCGGACGAG-3′ and 5′-CTCGAGTCGACATGATTTGTTTGATGGATAGTTCAT-3′ that introduced upstream BamHI and downstream SalI recognition sites. PCR fragments were gel-purified, digested with BamHI and SalI, and cloned into pQE80 (Qiagen) downstream and in-frame of a sequence encoding a His6 tag. All plasmids were sequence-verified. The SirT1 template contained a deletion of amino acid residues 6–84 resulting in a truncated form of bacterially expressed SIRT1. Protein Expression—Plasmids encoding TbSIR2rp1 and SIRT1 were transformed into Escherichia coli BL21DE3 and grown in 2× YT media containing 100 mg/liter of ampicillin at 37 °C until an A600 ∼ 0.7 was reached. Cultures were induced with 0.1 g/liter isopropyl β-d-thiogalactopyranoside for 4–6 h at room temperature. Harvested cells were lysed by sonication in 50 mm Tris (pH 8.0), 300 mm NaCl, 1.0 mm β-mercaptoethanol, and protease inhibitors (0.1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 5 μg/ml aprotinin). Histidine-tagged proteins were purified over nickel-nitrilotriacetic acid resin (Qiagen), as described by the manufacturer, and eluted with a 0–250 mm imidazole gradient. TbSIR2rp1-GST cells were induced as described above, processed in 1× phosphate-buffered saline, and purified using glutathione-agarose resin (GE Healthcare) as described by the manufacturer. Purified proteins were dialyzed in the following storage buffer: 50 mm Tris-HCl (pH 7.5), 1.0 mm d l-dithiothreitol, and 10% glycerol. Histone Acetylation—Recombinant Xenopus laevis histones were expressed and purified from E. coli as described previously (41Luger K. Rechsteiner T.J. Richmond T.J. Methods Mol. Biol. 1999; 119: 1-16PubMed Google Scholar) and acetylated using the histone acetyltransferase complex, piccolo-NuA4 (picNuA4) (42Berndsen C.E. Albaugh B.N. Tan S. Denu J.M. Biochemistry. 2007; 46: 623-629Crossref PubMed Scopus (101) Google Scholar). Reactions containing 0.2 μm picNuA4, 5 mm DTT, 75 μm acetyl-CoA, and 300 μg recombinant histones in 50 mm Tris, 150 mm NaCl were incubated at 24 °C for 1 h. Histone acetyltransferases were then heat-inactivated by boiling at 95 °C for 20 min. picNuA4-acetylated histones (i.e. AcH2A) were used for all the experiments, unless otherwise indicated. Histones H2A and H4 were also chemically acetylated using acetic anhydride in 50 mm HEPES (pH 7.4) using 100× molar excess anhydride. Enzymatic and chemical acetylation were confirmed by electrospray ionization-mass spectrometry. Histone concentrations were determined by the BCA protein assay (Pierce) using bovine serum albumin as a standard or by molar extinction coefficients (ϵ276 nm, H2A = 4050m–1 cm–1, ϵ276 nm, H4 = 5400m–1 cm–1). Peptide Synthesis—Acetylated histone H3 11mer peptide corresponding to the residues around lysine 14 (AcH3, KSTGGK(ac)APRKQ) was synthesized at the University of Wisconsin Peptide Synthesis Facility. Mass Spectrometry—Mass spectrometry was performed at the University of Wisconsin Madison Biotechnology Center on an ABI 3200 Q-trap. Deacetylation Assays—Charcoal-binding deacetylase assay was used as described previously (43Borra M.T. Denu J.M. Methods Enzymol. 2004; 376: 171-187Crossref PubMed Scopus (40) Google Scholar) to determine the activity of the TbSIR2rp1 recombinant proteins. Saturation kinetics were performed with 5–750 μm [NAD+] with a fixed concentration of [3H]AcH3 (700 μm). A second set of saturation kinetic curves was performed with a fixed concentration of NAD+ (1 mm), with varying concentrations of [3H]AcH3 peptide from 10 μm to 1 mm. Reactions were incubated at 37 °C for 10 min and contained 0.7 μm TbSIR2rp1 in 1 mm DTT and 50 mm Tris-HCl (pH 7.5). Acetylated recombinant histone H2A (AcH2A) saturation curves were generated using a fixed concentration of NAD+ (500 μm) and varying concentrations of [3H]AcH2A (5–100 μm). All reactions were composed of 0.7 μm TbSIR2rp1, 10 mm DTT, 50 mm Tris-HCl (pH 8.8), and 150 mm NaCl. Reactions were incubated at 37 °C for 10 min and were performed under initial velocity conditions where product formed was linear with time. The deacetylation reaction was also monitored by an HPLC method, essentially as described (43Borra M.T. Denu J.M. Methods Enzymol. 2004; 376: 171-187Crossref PubMed Scopus (40) Google Scholar). In these assays, time points from a deacetylase reaction were quenched at 0, 2, 4, 8, and 10 min before being analyzed by HPLC. The initial deacetylation rates were calculated by comparing the nicotinamide peak area from each time point to a nicotinamide standard curve. Rates were calculated over the linear portion of the reactions. ADP-ribosylation Assays—Reactions, unless otherwise indicated, contained 3 μm recombinant Sir2 homologues in 50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 10 mm DTT, 25 μm unlabeled NAD+, and 1–10 μCi of [α-32P]NAD+ (1000 Ci mmol–1; GE Healthcare), for a total reaction volume of 100 μl. As specified, reactions contained 20 μg total (5 μg of each histone subunit) unacetylated or acetylated recombinant histones. Samples were incubated at 37 °C for 4 h or times indicated. Reactions that contained AcH3 peptide were incubated with TbSIR2rp1 at 37 °C for 10 min prior to addition of unacetylated recombinant histones. Unincorporated [32P]NAD+ was removed by trichloroacetic acid protein precipitation followed by three acetone washes. Precipitated proteins were resuspended in SDS loading buffer, boiled at 95 °C for 15 min, and resolved on an 18% SDS-polyacrylamide gel. Gels were stained with Coomassie Blue, dried, and exposed to film or PhosphorImaging screen. Quantification was performed by densitometry using lane-specific background subtraction and a standard curve of [32P]NAD+ with the same specific activity. Reactions containing [32P]OAADPr or biotin-NAD+, used in place of [32P]NAD+, were the same as specified above. Biotin-NAD+, 6-bio-17-NAD+, was synthesized as described previously (44Zhang J. Snyder S.H. Biochemistry. 1993; 32: 2228-2233Crossref PubMed Scopus (38) Google Scholar) and confirmed by mass spectrometry. Synthesis of [32P]OAADPr—[32P]OAADPr was synthesized and purified as reported previously (45Borra M.T. O'Neill F.J. Jackson M.D. Marshall B. Verdin E. Foltz K.R. Denu J.M. J. Biol. Chem. 2002; 277: 12632-12641Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The 32P-labeled product was derived from a stock of [α-32P]NAD+ with known concentration and specific activity to ensure identical specific activities among the reaction substrates. Snake Venom Phosphodiesterase Cleavage—The cleavage reaction was conducted as described (46Shah G.M. Poirier D. Duchaine C. Brochu G. Desnoyers S. Lagueux J. Verreault A. Hoflack J.C. Kirkland J.B. Poirier G.G. Anal. Biochem. 1995; 227: 1-13Crossref PubMed Scopus (161) Google Scholar). TbSIR2 Deacetylase Activity—T. brucei Sir2-related protein (TbSIR2rp1) was cloned from genomic DNA. Bacterial constructs expressing TbSIR2rp1 either as an His6 or a GST fusion protein were generated. A previous study of the T. brucei SIR2-related protein utilized a GST fusion protein (38Garcia-Salcedo J.A. Gijon P. Nolan D.P. Tebabi P. Pays E. EMBO J. 2003; 22: 5851-5862Crossref PubMed Scopus (110) Google Scholar). Although NAD+-dependent deacetylase activity of TbSIR2rp1-GST had been observed previously (38Garcia-Salcedo J.A. Gijon P. Nolan D.P. Tebabi P. Pays E. EMBO J. 2003; 22: 5851-5862Crossref PubMed Scopus (110) Google Scholar), a quantitative steady-state analysis has not been reported. To verify that both bacterial constructs generated enzymes with similar deacetylase activity, the steady-state kinetic parameters of both purified proteins, termed TbSIR2-His (predicted molecular mass of 38.5 kDa) and TbSIR2-GST (predicted molecular mass of 64.5 kDa), were determined (see Fig. 1, A and B). In these reactions, 0.7 μm enzyme was reacted with 700 μm acetylated histone H3 peptide (KSTGGK(ac)APRKQ) and varied [NAD+] (Fig. 1A) or 1 mm [NAD+] and varied [AcH3] (Fig. 1B). TbSIR2-His and TbSIR2-GST displayed almost indistinguishable deacetylase activity under varied [NAD+], with Km, kcat, and kcat/Km values of 42 ± 3.2 μm, 0.075 ± 0.001 s–1, and 1800 ± 139 m–1 s–1, and 45 ± 3.5 μm, 0.090 ± 0.002 s–1, and 2000 ± 200 m–1 s–1, respectively (Fig. 1A). Similarly, the steady-state kinetic parameters with varied [AcH3], yielded Km, kcat, and kcat/Km values of 82 ± 5.9 μm, 0.060 ± 0.001 s–1, and 730 ± 54 m–1 s–1 for TbSIR2-His and 46 ± 5.8 μm, 0.050 ± 0.001 s–1, and 1100 ± 141 m–1 s–1 for TbSIR2-GST (Fig. 1B). Most importantly, the kinetic values are comparable with those reported for other highly active sirtuin deacetylases (34North 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 (1225) Google Scholar, 47Borra M.T. Langer M.R. Slama J.T. Denu J.M. Biochemistry. 2004; 43: 9877-9887Crossref PubMed Scopus (189) Google Scholar), establishing TbSIR2rp1 as a bona fide protein deacetylase. Because no significant differences in activity were observed between the two constructs, subsequent experiments were performed with TbSIR2rp1-His, abbreviated TbSIR2, unless otherwise indicated. TbSIR2 Displays ADP-ribosyltransferase Activity—After characterizing TbSIR2 NAD+-dependent deacetylase activity, we explored the ability of the enzyme to catalyze protein ADP-ribosylation. In several reports, sirtuins incubated with [α-32P]NAD+ resulted in 32P-labeling of proteins (histones, bovine serum albumin, and GDH) or of sirtuins themselves (1Tanny J.C. Dowd G.J. Huang J. Hilz H. Moazed D. Cell. 1999; 99: 735-745Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 11Frye R.A. Biochem. Biophys. Res. Commun. 1999; 260: 273-279Crossref PubMed Scopus (658) Google Scholar, 35Haigis M.C. Mostoslavsky R. Haigis K.M. Fahie K. Christodoulou D.C. Murphy A.J. Valenzuela D.M. Yancopoulos G.D. Karow M. Blander G. Wolberger C. Prolla T.A. Weindruch R. Alt F.W. Guarente L. Cell. 2006; 126: 941-954Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar, 37Liszt G. Ford E. Kurtev M. Guarente L. J. Biol. Chem. 2005; 280: 21313-21320Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 38Garcia-Salcedo J.A. Gijon P. Nolan D.P. Tebabi P. Pays E. EMBO J. 2003; 22: 5851-5862Crossref PubMed Scopus (110) Google Scholar), suggesting that these sirtuins possess ADP-ribosyltransferase activity. To begin examining the requirements and mechanism(s) of ADP-ribosylation, we performed initial TbSIR2 assays under conditions similar to those that previously yielded 32P-labeling of histones (38Garcia-Salcedo J.A. Gijon P. Nolan D.P. Tebabi P. Pays E. EMBO J. 2003; 22: 5851-5862Crossref PubMed Scopus (110) Google Scholar). Consistent with published work (38Garcia-Salcedo J.A. Gijon P. Nolan D.P. Tebabi P. Pays E. EMBO J. 2003; 22: 5851-5862Crossref PubMed Scopus (110) Google Scholar), TbSIR2 was capable of transferring the radiolabel from [α-32P]NAD+ to calf thymus histones (see supplemental Fig. S1A, lane 4). However, because commercially available calf thymus histones are relatively heterogeneous preparations, we repeated the above experiment with purified, recombinant X. laevis histones (H3, H2A, H2B, and H4), which lack detectable post-translational modifications. In dramatic contrast, TbSIR2 had a much lower capacity to transfer the label from [32P]NAD+ to recombinant purified histones (see Fig. S1A, lane 5). Note that both preparations of histones showed a low level of nonspecific, background labeling with the [32P]NAD+ (Fig. S1A, lanes 1 and 2), also previously observed by Liszt et al. (37Liszt G. Ford E. Kurtev M. Guarente L. J. Biol. Chem. 2005; 280: 21313-21320Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). Native histone preparations, such as commercially available calf thymus histones, include various amounts of post-translational modifications, including acetylation. We hypothesized that the observed increase in 32P labeling (putative ADP-ribosylation) upon incubation with calf thymus histones versus purified recombinant histones may be due to endogenous acetylation of calf thymus preparations, creating a possible link between the NAD+-dependent deacetylation activity of sirtuins and the putative ADP-ribosyltransferase activity. Acetylated Substrate Required for TbSIR2 ADP-ribosylation—To test this hypothesis, we acetylated pure recombinant X. laevis histones with the acetyltransferase complex picNuA4 (42Berndsen C.E. Albaugh B.N. Tan S. Denu J.M. Biochemistry. 2007; 46: 623-629Crossref PubMed Scopus (101) Google Scholar), and we incubated these acetylated histones with TbSIR2 and [32P]NAD+ as described above (Fig. 2A). Both picNuA4-acetylated and unmodified recombinant histones were incubated with [32P]NAD+ in the absence (Fig. 2A, lanes 1 and 2 and lanes 3 and 4, respectively) or presence of TbSIR2 enzyme (lanes 6 and 7, respectively). Acetylation of the recombinant histones greatly increased the TbSIR2-mediated ADP-ribosylation (Fig. 2A, lane 6) over controls without TbSIR2 (Fig. 2A, lanes 1 and 2). Unmodified recombinant histones showed no TbSIR2-dependent increase in labeling compared with control reactions without TbSIR2 (Fig. 2A, lane 7 versus lanes 3 and 4)