FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1974133264> ?p ?o ?g. }

• W1974133264 endingPage "12641" @default.
• W1974133264 startingPage "12632" @default.
• W1974133264 abstract "Silent information regulator 2 (Sir2) family of enzymes has been implicated in many cellular processes that include histone deacetylation, gene silencing, chromosomal stability, and aging. Yeast Sir2 and several homologues have been shown to be NAD+-dependent histone/protein deacetylases. Previously, it was demonstrated that the yeast enzymes catalyze a unique reaction mechanism in which the cleavage of NAD+ and the deacetylation of substrate are coupled with the formation of O-acetyl-ADP-ribose, a novel metabolite. We demonstrate that the production of O-acetyl-ADP-ribose is evolutionarily conserved among Sir2-like enzymes from yeast,Drosophila, and human. Also, endogenous yeast Sir2 complex from telomeres was shown to generate O-acetyl-ADP-ribose. By using a quantitative microinjection assay to examine the possible biological function(s) of this newly discovered metabolite, we demonstrate that O-acetyl-ADP-ribose causes a delay/block in oocyte maturation and results in a delay/block in embryo cell division in blastomeres. This effect was mimicked by injection of low nanomolar levels of active enzyme but not with a catalytically impaired mutant, indicating that the enzymatic activity is essential for the observed effects. In cell-free oocyte extracts, we demonstrate the existence of cellular enzymes that can efficiently utilize O-acetyl-ADP-ribose. Silent information regulator 2 (Sir2) family of enzymes has been implicated in many cellular processes that include histone deacetylation, gene silencing, chromosomal stability, and aging. Yeast Sir2 and several homologues have been shown to be NAD+-dependent histone/protein deacetylases. Previously, it was demonstrated that the yeast enzymes catalyze a unique reaction mechanism in which the cleavage of NAD+ and the deacetylation of substrate are coupled with the formation of O-acetyl-ADP-ribose, a novel metabolite. We demonstrate that the production of O-acetyl-ADP-ribose is evolutionarily conserved among Sir2-like enzymes from yeast,Drosophila, and human. Also, endogenous yeast Sir2 complex from telomeres was shown to generate O-acetyl-ADP-ribose. By using a quantitative microinjection assay to examine the possible biological function(s) of this newly discovered metabolite, we demonstrate that O-acetyl-ADP-ribose causes a delay/block in oocyte maturation and results in a delay/block in embryo cell division in blastomeres. This effect was mimicked by injection of low nanomolar levels of active enzyme but not with a catalytically impaired mutant, indicating that the enzymatic activity is essential for the observed effects. In cell-free oocyte extracts, we demonstrate the existence of cellular enzymes that can efficiently utilize O-acetyl-ADP-ribose. silent information regulator 2 isopropyl-β-d-thiogalactopyranoside dithiothreitol high pressure liquid chromatography phosphate-buffered saline germinal vesicle breakdown bovine serum albumin ARTKQTARKSTGGK(Ac)APRKQL 1-methyladenine Reversible protein acetylation is emerging as a major regulatory mechanism that has been implicated in a wide range of biological signaling. One of the best known examples of reversible acetylation occurs within the amino-terminal end of core histone proteins (1.Roth S.Y. Denu J.M. Allis C.D. Annu. Rev. Biochem. 2001; 70: 81-120Crossref PubMed Scopus (1624) Google Scholar). Histones are DNA-binding proteins that form the basic building blocks (nucleosomes) of chromatin. These histone amino-terminal “tails” are also the sites of other post-translational modifications that include phosphorylation, methylation, and ubiquitination (2.Marmorstein R. Nat. Rev. Mol. Cell. Biol. 2001; 2: 422-432Crossref PubMed Scopus (176) Google Scholar, 3.Cheung P. Allis C.D. Sassone-Corsi P. Cell. 2000; 103: 263-271Abstract Full Text Full Text PDF PubMed Scopus (828) Google Scholar, 4.Wu J. Grunstein M. Trends Biochem. Sci. 2000; 25: 619-623Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Acetylation of lysine residues on histones tails, catalyzed by histone acetyltransferases, generally correlates with transcriptional activation, whereas deacetylation of histone tails, by histone/protein deacetylases, correlates with transcriptional silencing (reviewed in Ref. 1.Roth S.Y. Denu J.M. Allis C.D. Annu. Rev. Biochem. 2001; 70: 81-120Crossref PubMed Scopus (1624) Google Scholar). There are three known classes of histone/protein deacetylases, which are classified by their similarity to yeast proteins. These classes include the Rpd3-like (class I), the Hda1-like (class II), and the Sir21-like (class III) deacetylases (reviewed in Ref. 5.Gray S.G. Ekstrom T.J. Exp. Cell Res. 2001; 262: 75-83Crossref PubMed Scopus (497) Google Scholar). Class I and class II deacetylases are commonly referred to as histone deacetylases. Unique among the deacetylases, Sir2-like (class III) deacetylases, or sirtuins, are NAD+-dependent and are insensitive to trichostastin-A, a potent inhibitor of the histone deacetylases (reviewed in Ref. 5.Gray S.G. Ekstrom T.J. Exp. Cell Res. 2001; 262: 75-83Crossref PubMed Scopus (497) Google Scholar). In yeast, Sir2 is required for silencing at telomeres (6.Aparicio O.M. Billington B.L. Gottschling D.E. Cell. 1991; 66: 1279-1287Abstract Full Text PDF PubMed Scopus (609) Google Scholar, 7.Strahl-Bolsinger S. Hecht A. Luo K. Grunstein M. Genes Dev. 1997; 11: 83-93Crossref PubMed Scopus (594) Google Scholar, 8.Gottschling D.E. Aparicio O.M. Billington B.L. Zakian V.A. Cell. 1990; 63: 751-762Abstract Full Text PDF PubMed Scopus (1137) Google Scholar), the mating-type loci (6.Aparicio O.M. Billington B.L. Gottschling D.E. Cell. 1991; 66: 1279-1287Abstract Full Text PDF PubMed Scopus (609) Google Scholar, 9.Rine J. Herskowitz I. Genetics. 1987; 116: 9-22Crossref PubMed Google Scholar), and the ribosomal DNA (10.Bryk M. Banerjee M. Murphy M. Knudsen K.E. Garfinkel D.J. Curcio M.J. Genes Dev. 1997; 11: 255-269Crossref PubMed Scopus (329) Google Scholar, 11.Fritze C.E. Verschueren K. Strich R. Easton Esposito R. EMBO J. 1997; 16: 6495-6509Crossref PubMed Scopus (240) Google Scholar, 12.Smith J.S. Boeke J.D. Genes Dev. 1997; 11: 241-254Crossref PubMed Scopus (507) Google Scholar, 13.Loo S. Rine J. Annu. Rev. Cell Dev. Biol. 1995; 11: 519-548Crossref PubMed Scopus (183) Google Scholar, 14.Shou W. Seol J.H. Shevchenko A. Baskerville C. Moazed D. Chen Z.W. Jang J. Charbonneau H. Deshaies R.J. Cell. 1999; 97: 233-244Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar). At the telomeres and mating-type loci, Sir2 is found in a multiprotein SIR complex with SIR3 and SIR4 (6.Aparicio O.M. Billington B.L. Gottschling D.E. Cell. 1991; 66: 1279-1287Abstract Full Text PDF PubMed Scopus (609) Google Scholar, 7.Strahl-Bolsinger S. Hecht A. Luo K. Grunstein M. Genes Dev. 1997; 11: 83-93Crossref PubMed Scopus (594) Google Scholar, 15.Hecht A. Laroche T. Strahl-Bolsinger S. Gasser S.M. Grunstein M. Cell. 1995; 80: 583-592Abstract Full Text PDF PubMed Scopus (697) Google Scholar, 16.Moazed D. Kistler A. Axelrod A. Rine J. Johnson A.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2186-2191Crossref PubMed Scopus (177) Google Scholar, 17.Moazed D. Mol. Cell. 2001; 8: 489-498Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). The SIR complex contributes to the stability and maintenance of the telomeric repeats (18.Palladino F. Laroche T. Gilson E. Axelrod A. Pillus L. Gasser S.M. Cell. 1993; 75: 543-555Abstract Full Text PDF PubMed Scopus (342) Google Scholar). At the rDNA, Sir2 is associated with Net1 and Cdc14, termed the RENT (regulator of nucleolar silencing andtelophase exit) complex (14.Shou W. Seol J.H. Shevchenko A. Baskerville C. Moazed D. Chen Z.W. Jang J. Charbonneau H. Deshaies R.J. Cell. 1999; 97: 233-244Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar, 19.Straight A.F. Shou W. Dowd G.J. Turck C.W. Deshaies R.J. Johnson A.D. Moazed D. Cell. 1999; 97: 245-256Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). The Sir2-mediated silencing at the rDNA appears to prevent or delay the formation of extrachromosomal rDNA circles, which have been shown to segregate to yeast mother cells and promote senescence (Ref. 20.Sinclair D.A. Guarente L. Cell. 1997; 91: 1033-1042Abstract Full Text Full Text PDF PubMed Scopus (1187) Google Scholar and reviewed in Refs. 21.Guarente L. Genes Dev. 2000; 14: 1021-1026Crossref PubMed Google Scholar and 22.Guarente L. Trends Genet. 2001; 17: 391-392Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Sir2 at the silenced rDNA is linked to its function in promoting longevity. Increased dosage of the Sir2 gene resulted in increased lifespan in yeast (23.Kaeberlein M. McVey M. Guarente L. Genes Dev. 1999; 13: 2570-2580Crossref PubMed Scopus (1781) Google Scholar) and Caenorhabditis elegans (24.Tissenbaum H.A. Guarente L. Nature. 2001; 410: 227-230Crossref PubMed Scopus (1590) Google Scholar). Besides silencing and lifespan extension, Sir2 has been implicated in other cellular processes including the repair of chromosomal double-strand breaks through nonhomologous end-joining (25.Tsukamoto Y. Kato J. Ikeda H. Nature. 1997; 388: 900-903Crossref PubMed Scopus (310) Google Scholar), cell cycle progression, and chromosome stability (26.Brachmann C.B. Sherman J.M. Devine S.E. Cameron E.E. Pillus L. Boeke J.D. Genes Dev. 1995; 9: 2888-2902Crossref PubMed Scopus (534) Google Scholar). However, despite the many biological processes implicating the involvement of Sir2, its molecular function has only recently been explored (reviewed in Refs. 21.Guarente L. Genes Dev. 2000; 14: 1021-1026Crossref PubMed Google Scholar and27.Moazed D. Curr. Opin. Cell Biol. 2001; 13: 232-238Crossref PubMed Scopus (150) Google Scholar, 28.Shore D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14030-14032Crossref PubMed Scopus (71) Google Scholar, 29.Gottschling D.E. Curr. Biol. 2000; 10: R708-R711Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The first indication of Sir2 function came from the studies of cobalamin biosynthesis in Salmonella typhimurium (30.Tsang A.W. Escalante-Semerena J.C. J. Biol. Chem. 1998; 273: 31788-31794Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). In this organism, CobB, a distant Sir2 homologue, was shown to compensate for the loss of the phosphoribosyltransferase CobT, suggesting a ribosyltransferase activity for Sir2. Initial examinations of Sir2 molecular function reported a weak ADP-ribosyltransferase activity (31.Frye R.A. Biochem. Biophys. Res. Commun. 1999; 260: 273-279Crossref PubMed Scopus (677) Google Scholar,32.Tanny J.C. Dowd G.J. Huang J. Hilz H. Moazed D. Cell. 1999; 99: 735-745Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Further examination, however, provided strong support for an NAD+-dependent deacetylase activity (33.Imai S. Armstrong C.M. Kaeberlein M. Guarente L. Nature. 2000; 403: 795-800Crossref PubMed Scopus (2817) Google Scholar, 34.Landry J. Sutton A. Tafrov S.T. Heller R.C. Stebbins J. Pillus L. Sternglanz R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5807-5811Crossref PubMed Scopus (820) Google Scholar, 35.Smith 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. A. 2000; 97: 6658-6663Crossref PubMed Scopus (624) Google Scholar, 36.Landry J. Slama J.T. Sternglanz R. Biochem. Biophys. Res. Commun. 2000; 278: 685-690Crossref PubMed Scopus (221) Google Scholar, 37.Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Crossref PubMed Scopus (500) Google Scholar, 38.Tanny J.C. Moazed D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 415-420Crossref PubMed Scopus (225) Google Scholar). The observation that histones at yeast silenced loci are hypoacetylated (39.Braunstein M. Rose A.B. Holmes S.G. Allis C.D. Broach J.R. Genes Dev. 1993; 7: 592-604Crossref PubMed Scopus (714) Google Scholar, 40.Braunstein M. Sobel R.E. Allis C.D. Turner B.M. Broach J.R. Mol. Cell. Biol. 1996; 16: 4349-4356Crossref PubMed Scopus (330) Google Scholar) has provided a satisfying link from Sir2 function to heterochromatin structure. However, that Sir2 homologues are found in bacteria and that the cellular localization differs among the various homologues would suggest that Sir2 enzymes have additional functions. Recent advances toward understanding the biological functions of the Sir2 family of enzymes included exploration of other potential substrates. These studies have focused on examination of known acetylated proteins as potential targets. For instance, it was recently demonstrated that Sir2 could negatively regulate the tumor suppressor p53 function in vitro and in vivo (41.Luo J. Nikolaev A.Y. Imai S. Chen D. Su F. Shiloh A. Guarente L. Gu W. Cell. 2001; 107: 137-148Abstract Full Text Full Text PDF PubMed Scopus (1902) Google Scholar, 42.Vaziri H. Dessain S.K. Eaton E.N. Imai S.I. Frye R.A. Pandita T.K. Guarente L. Weinberg R.A. Cell. 2001; 107: 149-159Abstract Full Text Full Text PDF PubMed Scopus (2316) Google Scholar) by deacetylating a known acetylation regulatory site (43.Abraham J. Kelly J. Thibault P. Benchimol S. J. Mol. Biol. 2000; 295: 853-864Crossref PubMed Scopus (50) Google Scholar, 44.Sakaguchi K. Herrera J.E. Saito S. Miki T. Bustin M. Vassilev A. Anderson C.W. Appella E. Genes Dev. 1998; 12: 2831-2841Crossref PubMed Scopus (1025) Google Scholar). Similarly, others have shown (45.Muth V. Nadaud S. Grummt I. Voit R. EMBO J. 2001; 20: 1353-1362Crossref PubMed Scopus (177) Google Scholar) that mouse Sir2a is capable of deacetylating the TAFI68 component of the TATA box binding protein-containing factor, repressing RNA polymerase I transcription in vitro. In the current study, we have taken a distinct approach to probe the function of Sir2 enzymes. As has been shown for class I and class II deacetylases, acetate is the direct product of this simple hydrolytic reaction (5.Gray S.G. Ekstrom T.J. Exp. Cell Res. 2001; 262: 75-83Crossref PubMed Scopus (497) Google Scholar, 46.Ng H.H. Bird A. Trends Biochem. Sci. 2000; 25: 121-126Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Surprisingly, however, the yeast Sir2 and HST2 enzymes (class III) couple protein deacetylation to the formation of a novel product O-acetyl-ADP-ribose, using NAD+and liberating nicotinamide in the process (37.Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Crossref PubMed Scopus (500) Google Scholar, 38.Tanny J.C. Moazed D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 415-420Crossref PubMed Scopus (225) Google Scholar). Thus acetate is not the product of this unique NAD+-dependent reaction. This finding raises important biological questions concerning the function of sirtuins and the role of O-acetyl-ADP-ribose. It is not clear whether this tightly coupled reaction is conserved among higher eukaryotes such as human nor is it known whether endogenous yeast complexes can produce O-acetyl-ADP-ribose. If they can, does generating O-acetyl-ADP-ribose in the cell serve a biological purpose? Here we examine these important questions using detailed enzymatic analyses and a quantitative cell microinjection assay designed to evaluate effects on the cell cycle. We demonstrate that indeed the endogenous yeast telomeric Sir2 complex, the human homologue, SIRT2, and the Drosophila homologue, dSir2 generate O-acetyl-ADP-ribose via a conserved enzymatic reaction. By using the microinjection assay, we show that both O-acetyl-ADP-ribose and Sir2 enzymes, in a dose-dependent fashion, cause a delay/block in oocyte maturation and caused a cell cycle delay/block in embryo development. The plasmid containing the full-length, carboxyl-terminally histidine-tagged DrosophilaSir2 (dSir2) gene was obtained from J. Lundblad, B. Newman, and S. Smolik, Oregon Health and Sciences University. Unacetylated and monoacetylated H3 peptide, ARTKQTARKSTGGK(Ac)APRKQL (AcH3), corresponding to the 20 amino-terminal residues of histones H3 was purchased from the Protein Chemistry Core Lab at Baylor College of Medicine. [3H]Acetyl-Coenzyme A (1.88 Ci/mmol) was purchased from PerkinElmer Life Sciences. All other reagents were of highest quality available. The H187A SIRT2 mutant was generated using QuikChange (Stratagene), and the mutation was verified by DNA sequencing. The transformation of BL21DE3-competent cells with the plasmid containing HST2 as well as the expression and purification of HST2 were as described previously (37.Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Crossref PubMed Scopus (500) Google Scholar). The plasmid containing the dSir2 was transformed in BL21DE3 and were grown on a 2× YT media containing 100 mg/liter ampicillin and 20 mg/liter of chloramphenicol until an A600 nm of 0.6 was obtained prior to induction with isopropyl-β-d-thiogalactopyranoside (IPTG) for 6 h. Protein purification was performed using the same protocol as reported for the purification of HST2 (37.Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Crossref PubMed Scopus (500) Google Scholar). In short, the plasmid containing hSIRT2 or the H187A mutant was transformed into BL21DE3-competent cells. Cells were grown on 2× YT media with 100 mg/liter ampicillin until an A600of 0.6 was obtained prior to induction with IPTG for 3 h. Cell lysis was accomplished using a French pressure cell with buffer consisting of 50 mm NaH2PO4, pH 8.0, 300 mm NaCl, 10 mm imidazole, and 1 mm 2-mercaptoethanol. Clarified extract was incubated with nickel-nitrilotriacetic acid-agarose for 1 h at 4 °C. The mixture was loaded into a small column, washed with 50 mmNa2HPO4, pH 8.0, 300 mm NaCl, 20 mm imidazole, and 1 mm 2-mercaptoethanol. SIRT2 was eluted using a linear gradient of 20–250 mm imidazole in 50 mm Na2HPO4, pH 8.0, 300 mm NaCl, and 1 mm 2-mercaptoethanol. SDS-PAGE analysis was performed to confirm the presence and purity of SIRT2 in the fractions. Pooled samples were dialyzed in 50 mm Tris, pH 7.5, 10% glycerol, and 1 mm DTT and stored at −20 °C until use. The deacetylase assay employed takes advantage of the different elution profiles between substrates and products through reverse-phase high performance liquid chromatography (HPLC). Reactions were carried out with [3H]AcH3 or chicken core histones and NAD+ in 50 mm Tris (pH 7.5 at 37 °C) containing 1 mm DTT. HPLC-based assays were performed as described by Tanner et al. (37.Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Crossref PubMed Scopus (500) Google Scholar). Samples were individually injected into a Beckman System Gold HPLC using a Vydac Preparative C18 (1 × 25 cm) or a Beckman (0.46 × 15 cm) analytical C18 reverse-phase HPLC column. Upon injection, the system was run isocratically using solvent A (0.05% trifluoroacetic acid/H2O) for 1 min. The isocratic step was followed by a linear gradient of 0–20% solvent B (0.02% trifluoroacetic acid/acetonitrile) over 20 min to resolve the substrate and product peaks. Deacetylated and acetylated H3 peptides were eluted at ∼13 and 16% acetonitrile, respectively. O-Acetyl-ADP-ribose eluted at ∼5% acetonitrile. Elution of substrates and products was monitored by measuring the absorbance at 214 nm. Radioactivity of collected fractions was determined by scintillation counting. To determine the stoichiometry of the SIRT2 reaction, unacetylated H3 peptide was3H-acetylated specifically at Lys-14 using p300/CBP-associated factor. A total of 30 μm[2-3H3]acetyl-CoA (specific activity ∼4500–5000 cpm/pmol) and 200 μm H3 peptide were reacted in the presence of 14.5 μm of PCAF in 5 mm DTT, 50 mm Tris, pH 7.5, at 25 °C, for 1 h. The reaction was quenched with 1% trifluoroacetic acid and was purified by reversed phase chromatography using a Beckman Biosystem 510 with a Vydac C18 Small Pore preparative (1 × 25 cm) column (Vydac, Hesperia, CA) at a flow rate of 1 ml/min. The HPLC was run with 100% solvent A for 1 min followed by a 0–40% gradient of solvent B over 40 min. Lyophilized [3H]AcH3 was resuspended in 50 mm Tris, pH 7.5, and the concentration was determined using a standard curve generated by obtaining the absorbance of known concentrations of AcH3 at 220 nm. The specific activity of the [3H]AcH3 peptide stock was determined as cpm/μl and cpm/pmol using a liquid scintillation counter. A Beckman Biosys 510 HPLC system and a Vydac C18 (1.0 × 25 mm) small pore preparative column (Vydac, Hesperia, CA) were used for the purification of O-acetyl-ADP-ribose. Compounds from the enzymatic reaction were separated using a gradient system comprising of solvent A and solvent B using a constant flow rate of 4 ml/min. All mobile phases were filtered through a Millipore 0.20-μm nylon filter (Millipore Corp., Bedford, MA) prior to use. Upon injection of the sample (up to 1 ml), the HPLC was run isocratic in solvent A for 5.0 min followed by a linear gradient of 0–8% B over a 20-min period with the detector set at 260 nm. The gradient was then increased to 40% solvent B over a 20-min period. At 21 min into the run, the detector was switched to 214 nm to detect acetylated and deacetylated H3 peptide. The product derived from NAD+ had a retention time (Rt) of 15.5 min, while the acetylated and deacetylated H3 peptide had retention times of 28.1 and 28.8 min, respectively.O-Acetyl-ADP-ribose was collected directly from the HPLC after passage through the detector, frozen at −80 °C, and lyophilized. Samples were stored in a desiccator under argon prior to use. Chicken core histones and the yeast telomeric complex were purified according to the protocol described previously (47.Ghidelli S. Donze D. Dhillon N. Kamakaka R.T. EMBO J. 2001; 20: 4522-4535Crossref PubMed Scopus (76) Google Scholar). A yeast strain carrying a His6-HA3 epitope tag at the amino terminus of the SIR2 gene under its own promoter, at its endogenous locus, was generated previously (yeast strain ROY 1515; see Ref.47.Ghidelli S. Donze D. Dhillon N. Kamakaka R.T. EMBO J. 2001; 20: 4522-4535Crossref PubMed Scopus (76) Google Scholar). The epitope-tagged Sir2p was functionally active in silencing assays (47.Ghidelli S. Donze D. Dhillon N. Kamakaka R.T. EMBO J. 2001; 20: 4522-4535Crossref PubMed Scopus (76) Google Scholar). Briefly, whole cell extract was loaded into an SP-Sepharose cation exchange column, and proteins were eluted with varying KCl concentrations. The Sir2-containing complex, which eluted with 350 mm KCl, was then loaded into a cobalt-TALON affinity column and eluted with increasing concentrations of imidazole. The Sir2-containing fraction was then subjected to an anion-exchange chromatography using Q-Sepharose column and eluted with increasing KCl concentrations. Fractions containing the telomeric complex were identified by the presence of Sir4, but not Net1, in a Western blot analysis. The fractions containing the telomeric complex were then subjected to gel filtration chromatography using Superose 6B column. The size of the telomeric complex was determined to be ∼800 kDa. For the final step in the purification, sample was loaded into a heparin-Sepharose column and eluted with a linear gradient of 0.15 to 1.0 m KCl. Asterina miniata gametes were collected, and microinjections were performed as described previously (48.Carroll D.J. Albay D.T. Terasaki M. Jaffe L.A. Foltz K.R. Dev. Biol. 1999; 206: 232-247Crossref PubMed Scopus (103) Google Scholar). Briefly, quantitative microinjection of A. miniata oocytes was performed at 16 °C, using mercury-filled micropipets (49.Hiramoto Y. Exp. Cell Res. 1962; 27: 416-426Crossref PubMed Scopus (158) Google Scholar, 50.Kiehart D.P. Methods Cell Biol. 1982; 25: 13-31Crossref PubMed Scopus (158) Google Scholar) (see the on-line information available at www/egg/uchc.edu/injection), allowing injection of precisely calibrated picoliter volumes into the oocytes and blastomeres. Injection volumes were 3% of the total cellular volume (for oocytes 93 pl, and for blastomeres 47 pl). Buffer alone, purified O-acetyl-ADP-ribose, ADP-ribose (Sigma), or purified recombinant enzyme (prepared as described above) was injected into immature oocytes or one blastomere of a two-cell stage embryo to a final concentration in the cytoplasm as indicated in the figure legends and tables. 1-Methyladenine was from Sigma and was used at 10 μm. Oocyte suspensions were centrifuged (280 × g) briefly, and the seawater was removed. Oocytes were resuspended in lysis buffer (PBS, pH 7.4, 15 mm disodium EGTA, 1% Triton X-100, 0.5 mmsodium vanadate, 1 mm sodium fluoride, 10 μmeach of aprotinin, leupeptin, and benzamidine, 50 μmphenylmethylsulfonyl fluoride) and lysed by passage through a 27.5-gauge needle on ice. The sample was centrifuged at 18,000 ×g at 4 °C for 20 min and the soluble fraction was collected and kept on ice. Protein concentration was determined using the Pierce BCA assay using BSA as a standard. Aliquots were snap frozen in liquid nitrogen and stored at −80 °C. Yeast Sir2 (ySir2) has been found associated with Sir3 and Sir4 in multiprotein complexes at the telomeres and the mating type loci (6.Aparicio O.M. Billington B.L. Gottschling D.E. Cell. 1991; 66: 1279-1287Abstract Full Text PDF PubMed Scopus (609) Google Scholar, 7.Strahl-Bolsinger S. Hecht A. Luo K. Grunstein M. Genes Dev. 1997; 11: 83-93Crossref PubMed Scopus (594) Google Scholar, 15.Hecht A. Laroche T. Strahl-Bolsinger S. Gasser S.M. Grunstein M. Cell. 1995; 80: 583-592Abstract Full Text PDF PubMed Scopus (697) Google Scholar, 16.Moazed D. Kistler A. Axelrod A. Rine J. Johnson A.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2186-2191Crossref PubMed Scopus (177) Google Scholar) while existing with Net1 and Cdc14 (RENT complex) at the ribosomal DNA (14.Shou W. Seol J.H. Shevchenko A. Baskerville C. Moazed D. Chen Z.W. Jang J. Charbonneau H. Deshaies R.J. Cell. 1999; 97: 233-244Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar, 19.Straight A.F. Shou W. Dowd G.J. Turck C.W. Deshaies R.J. Johnson A.D. Moazed D. Cell. 1999; 97: 245-256Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). The functional significance of these different complexes is not known. As briefly discussed in the Introduction, recent evidence (33.Imai S. Armstrong C.M. Kaeberlein M. Guarente L. Nature. 2000; 403: 795-800Crossref PubMed Scopus (2817) Google Scholar, 34.Landry J. Sutton A. Tafrov S.T. Heller R.C. Stebbins J. Pillus L. Sternglanz R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5807-5811Crossref PubMed Scopus (820) Google Scholar, 35.Smith 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. A. 2000; 97: 6658-6663Crossref PubMed Scopus (624) Google Scholar, 36.Landry J. Slama J.T. Sternglanz R. Biochem. Biophys. Res. Commun. 2000; 278: 685-690Crossref PubMed Scopus (221) Google Scholar, 37.Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Crossref PubMed Scopus (500) Google Scholar, 38.Tanny J.C. Moazed D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 415-420Crossref PubMed Scopus (225) Google Scholar) indicates that Sir2 and other sirtuins are potent NAD+-dependent protein/histone deacetylases. By using recombinant bacterially expressed enzymes, analyses with ySir2 and an additional homologue in yeast, HST2, have shown that the enzymes utilize NAD+ and an acetylated substrate to carry out deacetylation and the coupled production of the novel compound, O-acetyl-ADP-ribose (37.Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Crossref PubMed Scopus (500) Google Scholar,38.Tanny J.C. Moazed D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 415-420Crossref PubMed Scopus (225) Google Scholar). The unique and unanticipated production of O-acetyl-ADP-ribose raises important questions about this conserved and relatively large family of proteins. Is the catalytic mechanism requiring NAD+ consumption and the production of O-acetyl-ADP-ribose evolutionarily conserved? Also it is not known whether Sir2 endogenous complexes catalyze this reaction (i.e. generation of O-acetyl-ADP-ribose), as only bacterially expressed recombinant proteins have been characterized to date. To address whether endogenous Sir2-containing complexes produce O-acetyl-ADP-ribose, yeast Sir2 telomeric complex was purified from yeast (47.Ghidelli S. Donze D. Dhillon N. Kamakaka R.T. EMBO J. 2001; 20: 4522-4535Crossref PubMed Scopus (76) Google Scholar) and utilized in an in vitrodeacetylation assay. Purification of Sir2-containing telomeric complex involved five chromatographic steps (see “Experimental Procedures” and Ref. 47.Ghidelli S. Donze D. Dhillon N. Kamakaka R.T. EMBO J. 2001; 20: 4522-4535Crossref PubMed Scopus (76) Google Scholar), resulting in an ∼800-kDa complex containing ∼4 major polypeptides as described previously (47.Ghidelli S. Donze D. Dhillon N. Kamakaka R.T. EMBO J. 2001; 20: 4522-4535Crossref PubMed Scopus (76) Google Scholar). Fractions containing the telomeric complex were identified by the presence of Sir4, but not Net1, using Western blot analysis. By using an HPLC-based (reversed phase) deacetylase assay, we have shown that recombinant yeast HST2 generates O-acetyl-ADP-ribose, which can be resolved from the various reactant/products and quantified (37.Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Crossref PubMed Scopus (500) Google Scholar). Here, by employing3H-acetylated chicken core histones as substrate, we demonstrated that the native yeast telomeric complex was able to produce O-acetyl-A" @default.
• W1974133264 created "2016-06-24" @default.
• W1974133264 creator A5013024193 @default.
• W1974133264 creator A5016155944 @default.
• W1974133264 creator A5025168089 @default.
• W1974133264 creator A5035545940 @default.
• W1974133264 creator A5042935023 @default.
• W1974133264 creator A5077817714 @default.
• W1974133264 creator A5087806100 @default.
• W1974133264 date "2002-04-01" @default.
• W1974133264 modified "2023-10-16" @default.
• W1974133264 title "Conserved Enzymatic Production and Biological Effect of O-Acetyl-ADP-ribose by Silent Information Regulator 2-like NAD+-dependent Deacetylases" @default.
• W1974133264 cites W1514546358 @default.
• W1974133264 cites W1536757970 @default.
• W1974133264 cites W1537218374 @default.
• W1974133264 cites W1601675369 @default.
• W1974133264 cites W1689569903 @default.
• W1974133264 cites W1689892651 @default.
• W1974133264 cites W1951075546 @default.
• W1974133264 cites W1963578989 @default.
• W1974133264 cites W1964442923 @default.
• W1974133264 cites W1966868505 @default.
• W1974133264 cites W1971966311 @default.
• W1974133264 cites W1973321484 @default.
• W1974133264 cites W1974890383 @default.
• W1974133264 cites W1985308585 @default.
• W1974133264 cites W1987514467 @default.
• W1974133264 cites W1989051882 @default.
• W1974133264 cites W1990778506 @default.
• W1974133264 cites W1992238166 @default.
• W1974133264 cites W1995111407 @default.
• W1974133264 cites W1995902306 @default.
• W1974133264 cites W1997836839 @default.
• W1974133264 cites W2008187459 @default.
• W1974133264 cites W2008890179 @default.
• W1974133264 cites W2012753961 @default.
• W1974133264 cites W2017525855 @default.
• W1974133264 cites W2019032966 @default.
• W1974133264 cites W2019237376 @default.
• W1974133264 cites W2019790163 @default.
• W1974133264 cites W2021309301 @default.
• W1974133264 cites W2021424485 @default.
• W1974133264 cites W2021432006 @default.
• W1974133264 cites W2026452617 @default.
• W1974133264 cites W2026571586 @default.
• W1974133264 cites W2026794578 @default.
• W1974133264 cites W2032794324 @default.
• W1974133264 cites W2033271918 @default.
• W1974133264 cites W2036347938 @default.
• W1974133264 cites W2038077039 @default.
• W1974133264 cites W2044347050 @default.
• W1974133264 cites W2044578262 @default.
• W1974133264 cites W2049094938 @default.
• W1974133264 cites W2050376414 @default.
• W1974133264 cites W2056480485 @default.
• W1974133264 cites W2056827933 @default.
• W1974133264 cites W2057654105 @default.
• W1974133264 cites W2058346978 @default.
• W1974133264 cites W2060990349 @default.
• W1974133264 cites W2067116751 @default.
• W1974133264 cites W2069974281 @default.
• W1974133264 cites W2070614630 @default.
• W1974133264 cites W2072307255 @default.
• W1974133264 cites W2080090703 @default.
• W1974133264 cites W2096796564 @default.
• W1974133264 cites W2097948853 @default.
• W1974133264 cites W2098812571 @default.
• W1974133264 cites W2100325403 @default.
• W1974133264 cites W2102635586 @default.
• W1974133264 cites W2104608706 @default.
• W1974133264 cites W2112937062 @default.
• W1974133264 cites W2114217399 @default.
• W1974133264 cites W2121392264 @default.
• W1974133264 cites W2121670160 @default.
• W1974133264 cites W2122000530 @default.
• W1974133264 cites W2122380132 @default.
• W1974133264 cites W2124595934 @default.
• W1974133264 cites W2131019865 @default.
• W1974133264 cites W2137901143 @default.
• W1974133264 cites W2142300924 @default.
• W1974133264 cites W2155245588 @default.
• W1974133264 cites W2157649587 @default.
• W1974133264 cites W2164715751 @default.
• W1974133264 cites W2185855551 @default.
• W1974133264 cites W2277644197 @default.
• W1974133264 cites W4233036449 @default.
• W1974133264 doi "https://doi.org/10.1074/jbc.m111830200" @default.
• W1974133264 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11812793" @default.
• W1974133264 hasPublicationYear "2002" @default.
• W1974133264 type Work @default.
• W1974133264 sameAs 1974133264 @default.
• W1974133264 citedByCount "146" @default.
• W1974133264 countsByYear W19741332642012 @default.
• W1974133264 countsByYear W19741332642013 @default.
• W1974133264 countsByYear W19741332642014 @default.
• W1974133264 countsByYear W19741332642015 @default.
• W1974133264 countsByYear W19741332642016 @default.
• W1974133264 countsByYear W19741332642017 @default.

• next