FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2157860216> ?p ?o ?g. }

• W2157860216 endingPage "8351" @default.
• W2157860216 startingPage "8340" @default.
• W2157860216 abstract "Sirtuins catalyze NAD+-dependent protein deacetylation and are critical regulators of transcription, apoptosis, metabolism, and aging. There are seven human sirtuins (SIRT1–7), and SIRT1 has been implicated as a key mediator of the pathways downstream of calorie restriction that have been shown to delay the onset and reduce the incidence of age-related diseases such as type 2 diabetes. Increasing SIRT1 activity, either by transgenic overexpression of the Sirt1 gene in mice or by pharmacological activation by small molecule activators resveratrol and SRT1720, has shown beneficial effects in rodent models of type 2 diabetes, indicating that SIRT1 may represent an attractive therapeutic target. Herein, we have assessed purported SIRT1 activators by employing biochemical assays utilizing native substrates, including a p53-derived peptide substrate lacking a fluorophore as well as the purified native full-length protein substrates p53 and acetyl-CoA synthetase1. SRT1720, its structurally related compounds SRT2183 and SRT1460, and resveratrol do not lead to apparent activation of SIRT1 with native peptide or full-length protein substrates, whereas they do activate SIRT1 with peptide substrate containing a covalently attached fluorophore. Employing NMR, surface plasmon resonance, and isothermal calorimetry techniques, we provide evidence that these compounds directly interact with fluorophore-containing peptide substrates. Furthermore, we demonstrate that SRT1720 neither lowers plasma glucose nor improves mitochondrial capacity in mice fed a high fat diet. SRT1720, SRT2183, SRT1460, and resveratrol exhibit multiple off-target activities against receptors, enzymes, transporters, and ion channels. Taken together, we conclude that SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1. Sirtuins catalyze NAD+-dependent protein deacetylation and are critical regulators of transcription, apoptosis, metabolism, and aging. There are seven human sirtuins (SIRT1–7), and SIRT1 has been implicated as a key mediator of the pathways downstream of calorie restriction that have been shown to delay the onset and reduce the incidence of age-related diseases such as type 2 diabetes. Increasing SIRT1 activity, either by transgenic overexpression of the Sirt1 gene in mice or by pharmacological activation by small molecule activators resveratrol and SRT1720, has shown beneficial effects in rodent models of type 2 diabetes, indicating that SIRT1 may represent an attractive therapeutic target. Herein, we have assessed purported SIRT1 activators by employing biochemical assays utilizing native substrates, including a p53-derived peptide substrate lacking a fluorophore as well as the purified native full-length protein substrates p53 and acetyl-CoA synthetase1. SRT1720, its structurally related compounds SRT2183 and SRT1460, and resveratrol do not lead to apparent activation of SIRT1 with native peptide or full-length protein substrates, whereas they do activate SIRT1 with peptide substrate containing a covalently attached fluorophore. Employing NMR, surface plasmon resonance, and isothermal calorimetry techniques, we provide evidence that these compounds directly interact with fluorophore-containing peptide substrates. Furthermore, we demonstrate that SRT1720 neither lowers plasma glucose nor improves mitochondrial capacity in mice fed a high fat diet. SRT1720, SRT2183, SRT1460, and resveratrol exhibit multiple off-target activities against receptors, enzymes, transporters, and ion channels. Taken together, we conclude that SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1. Silent information regulator 2 (Sir2) 3The abbreviations used are: Sir2silent information regulator 2AceCSacetyl-CoA synthetaseHFDhigh fat dietNlenorleucineEX-5276-chloro-2,3,4,9-tetrahydro-1-H-carbazole-1-carboxamideTAMRA6-carboxytetramethylrhodamineDTTdithiothreitolELISAenzyme-linked immunosorbent assayITCisothermal titration calorimetryPEGpolyethylene glycolSPRsurface plasmon resonanceMSmass spectrometryLC-MSliquid chromatography-mass spectrometry. enzymes (or sirtuins) have recently emerged as central players in the regulation of critical metabolic pathways such as insulin secretion and lipid mobilization (1.Moynihan K.A. Grimm A.A. Plueger M.M. Bernal-Mizrachi E. Ford E. Cras-Méneur C. Permutt M.A. Imai S. Cell Metab. 2005; 2: 105-117Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar, 2.Starai V.J. Celic I. Cole R.N. Boeke J.D. Escalante-Semerena J.C. Science. 2002; 298: 2390-2392Crossref PubMed Scopus (473) Google Scholar, 3.Liang F. Kume S. Koya D. Nat. Rev. Endocrinol. 2009; 5: 367-373Crossref PubMed Scopus (290) Google Scholar). The seven members of the mammalian sirtuin family, SIRT1–7, each with distinct cellular localizations, act on their respective targets to effect a wide range of biological processes (4.Smith B.C. Hallows W.C. Denu J.M. Chem. Biol. 2008; 15: 1002-1013Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The sirtuins regulate their targets by modulating the activity of their partner proteins through reversible deacetylation (5.Sauve A.A. Wolberger C. Schramm V.L. Boeke J.D. Annu. Rev. Biochem. 2006; 75: 435-465Crossref PubMed Scopus (582) Google Scholar). The most widely studied sirtuin, SIRT1, is homologous to yeast Sir2, which was initially discovered as maintaining the acetylation state of histones in heterochromatin, thereby controlling gene expression. Since then, the discovery of numerous and diverse sirtuin substrates has implicated the sirtuins in metabolism, apoptosis, transcription, and cell survival (2.Starai V.J. Celic I. Cole R.N. Boeke J.D. Escalante-Semerena J.C. Science. 2002; 298: 2390-2392Crossref PubMed Scopus (473) Google Scholar, 6.Rusche L.N. Kirchmaier A.L. Rine J. Annu. Rev. Biochem. 2003; 72: 481-516Crossref PubMed Scopus (595) Google Scholar, 7.Gasser S.M. Cockell M.M. Gene. 2001; 279: 1-16Crossref PubMed Scopus (229) Google Scholar, 8.Dryden S.C. Nahhas F.A. Nowak J.E. Goustin A.S. Tainsky M.A. Mol. Cell Biol. 2003; 23: 3173-3185Crossref PubMed Scopus (409) Google Scholar, 9.Rogina B. Helfand S.L. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 15998-16003Crossref PubMed Scopus (1068) Google Scholar, 10.Tissenbaum H.A. Guarente L. Nature. 2001; 410: 227-230Crossref PubMed Scopus (1579) Google Scholar, 11.Vaziri H. Dessain S.K. Ng Eaton E. 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 (2297) Google Scholar, 12.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 (1891) Google Scholar, 13.Langley E. Pearson M. Faretta M. Bauer U.M. Frye R.A. Minucci S. Pelicci P.G. Kouzarides T. EMBO J. 2002; 21: 2383-2396Crossref PubMed Scopus (755) Google Scholar). The role of the Sir2 enzymes in mediating lifespan extension in yeast (14.Kaeberlein M. McVey M. Guarente L. Genes Dev. 1999; 13: 2570-2580Crossref PubMed Scopus (1765) Google Scholar), worms (10.Tissenbaum H.A. Guarente L. Nature. 2001; 410: 227-230Crossref PubMed Scopus (1579) Google Scholar), and flies (9.Rogina B. Helfand S.L. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 15998-16003Crossref PubMed Scopus (1068) Google Scholar) has led to dramatic interest in this class of enzymes as a potential means of extending longevity. Sir2 deacetylases catalyze the NAD+-dependent deacetylation of specific ϵ-amino-acetylated lysine residues from its protein substrates to form nicotinamide, the deacetylated product, and a unique metabolite, 2′-O-acetyl-ADP-ribose. The biochemical and kinetic mechanism of Sir2 deacetylases has been extensively studied (15.Borra M.T. Langer M.R. Slama J.T. Denu J.M. Biochemistry. 2004; 43: 9877-9887Crossref PubMed Scopus (190) Google Scholar, 16.Jackson M.D. Schmidt M.T. Oppenheimer N.J. Denu J.M. J. Biol. Chem. 2003; 278: 50985-50998Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 17.Sauve A.A. Schramm V.L. Curr. Med. Chem. 2004; 11: 807-826Crossref PubMed Scopus (68) Google Scholar, 18.Smith B.C. Denu J.M. J. Am. Chem. Soc. 2007; 129: 5802-5803Crossref PubMed Scopus (65) Google Scholar). silent information regulator 2 acetyl-CoA synthetase high fat diet norleucine 6-chloro-2,3,4,9-tetrahydro-1-H-carbazole-1-carboxamide 6-carboxytetramethylrhodamine dithiothreitol enzyme-linked immunosorbent assay isothermal titration calorimetry polyethylene glycol surface plasmon resonance mass spectrometry liquid chromatography-mass spectrometry. In vivo, SIRT1 has been shown to be activated by fasting and caloric restriction (19.Cohen 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 (1682) Google Scholar, 20.Heilbronn L.K. Civitarese A.E. Bogacka I. Smith S.R. Hulver M. Ravussin E. Obes. Res. 2005; 13: 574-581Crossref PubMed Scopus (127) Google Scholar). Caloric restriction extends lifespan and produces a metabolic profile desirable for treating diseases of aging such as type 2 diabetes (21.Facchini F.S. Hua N. Abbasi F. Reaven G.M. J. Clin. Endocrinol. Metab. 2001; 86: 3574-3578Crossref PubMed Scopus (405) Google Scholar, 22.Civitarese A.E. Carling S. Heilbronn L.K. Hulver M.H. Ukropcova B. Deutsch W.A. Smith S.R. Ravussin E. PLoS Med. 2007; 4: e76Crossref PubMed Scopus (612) Google Scholar, 23.Barzilai N. Banerjee S. Hawkins M. Chen W. Rossetti L. J. Clin. Invest. 1998; 101: 1353-1361Crossref PubMed Google Scholar). Pharmacological activation of SIRT1 by the small molecule activator resveratrol has been reported to improve insulin sensitivity, increase mitochondrial content, and prolong survival of mice fed a high fat, high calorie diet (24.Baur J.A. Pearson K.J. Price N.L. Jamieson H.A. Lerin C. Kalra A. Prabhu V.V. Allard J.S. Lopez-Lluch G. Lewis K. Pistell P.J. Poosala S. Becker K.G. Boss O. Gwinn D. Wang M. Ramaswamy S. Fishbein K.W. Spencer R.G. Lakatta E.G. Le Couteur D. Shaw R.J. Navas P. Puigserver P. Ingram D.K. de Cabo R. Sinclair D.A. Nature. 2006; 444: 337-342Crossref PubMed Scopus (3672) Google Scholar, 25.Lagouge M. Argmann C. Gerhart-Hines Z. Meziane H. Lerin C. Daussin F. Messadeq N. Milne J. Lambert P. Elliott P. Geny B. Laakso M. Puigserver P. Auwerx J. Cell. 2006; 127: 1109-1122Abstract Full Text Full Text PDF PubMed Scopus (3337) Google Scholar, 26.Milne J.C. Lambert P.D. Schenk S. Carney D.P. Smith J.J. Gagne D.J. Jin L. Boss O. Perni R.B. Vu C.B. Bemis J.E. Xie R. Disch J.S. Ng P.Y. Nunes J.J. Lynch A.V. Yang H. Galonek H. Israelian K. Choy W. Iffland A. Lavu S. Medvedik O. Sinclair D.A. Olefsky J.M. Jirousek M.R. Elliott P.J. Westphal C.H. Nature. 2007; 450: 712-716Crossref PubMed Scopus (1462) Google Scholar). Furthermore, SIRT1 activation by transgenic overexpression of the Sirt1 gene in mice has shown similar phenotypes (27.Banks A.S. Kon N. Knight C. Matsumoto M. Gutiérrez-Juárez R. Rossetti L. Gu W. Accili D. Cell Metab. 2008; 8: 333-341Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar), indicating that SIRT1 may represent an attractive therapeutic target for the treatment of type 2 diabetes. SRT1720, SRT2183, and SRT1460 were recently described by Sirtris Pharmaceuticals as SIRT1 activators (26.Milne J.C. Lambert P.D. Schenk S. Carney D.P. Smith J.J. Gagne D.J. Jin L. Boss O. Perni R.B. Vu C.B. Bemis J.E. Xie R. Disch J.S. Ng P.Y. Nunes J.J. Lynch A.V. Yang H. Galonek H. Israelian K. Choy W. Iffland A. Lavu S. Medvedik O. Sinclair D.A. Olefsky J.M. Jirousek M.R. Elliott P.J. Westphal C.H. Nature. 2007; 450: 712-716Crossref PubMed Scopus (1462) Google Scholar) (Fig. 1). They are structurally unrelated to resveratrol and were reported to activate SIRT1 with potencies 1,000-fold greater than resveratrol. These compounds were identified via a high throughput fluorescence polarization assay followed by potency optimization for in vitro enzyme activity using high throughput mass spectrometry (MS). They were shown to exhibit EC1.5 (the compound concentration required to increase enzyme activity by 50%) of 0.16–2.9 μm, with 296–781% activation (when compared with 201% for resveratrol). As with other plant polyphenols such as resveratrol, the mechanism of SIRT1 activation by the Sirtris series was shown to occur through increased binding affinity by decreasing the Km of SIRT1 for acetylated peptide substrate without affecting the Km for NAD+ or the Vmax. SRT1720, the most potent activator of the series, was reported to improve glucose homeostasis, increase insulin sensitivity, and increase mitochondrial function in rodent models of type 2 diabetes (26.Milne J.C. Lambert P.D. Schenk S. Carney D.P. Smith J.J. Gagne D.J. Jin L. Boss O. Perni R.B. Vu C.B. Bemis J.E. Xie R. Disch J.S. Ng P.Y. Nunes J.J. Lynch A.V. Yang H. Galonek H. Israelian K. Choy W. Iffland A. Lavu S. Medvedik O. Sinclair D.A. Olefsky J.M. Jirousek M.R. Elliott P.J. Westphal C.H. Nature. 2007; 450: 712-716Crossref PubMed Scopus (1462) Google Scholar). The SIRT1 activation by the aforementioned Sirtris series was determined only with a non-native fluorophore-containing p53-derived peptide substrate in both fluorescence polarization and MS assays. Recently, SIRT1 activation by resveratrol was shown to be completely dependent on the presence of a covalently attached fluorophore in the fluorescent peptide substrate (28.Borra M.T. Smith B.C. Denu J.M. J. Biol. Chem. 2005; 280: 17187-17195Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar, 29.Kaeberlein M. McDonagh T. Heltweg B. Hixon J. Westman E.A. Caldwell S.D. Napper A. Curtis R. DiStefano P.S. Fields S. Bedalov A. Kennedy B.K. J. Biol. Chem. 2005; 280: 17038-17045Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar). These results suggested that the resveratrol-mediated in vivo effects may occur through a different molecular mechanism independent of direct SIRT1 activation and raised questions about the Sirtris series of compounds and their ability to activate human SIRT1 in vitro. In the present study, to avoid any potential artifacts associated with fluorescently labeled non-native substrates, we have investigated the ability of the Sirtris series and resveratrol in activating human SIRT1 using a native peptide substrate by direct detection and quantification methods such as HPLC. Furthermore, we tested the hypothesis that the fluorophore may mimic a hydrophobic residue/pocket on the native full-length protein substrates, and therefore, the full-length substrates will behave differently from a peptide substrate. We report the first assessment of these putative SIRT1 activators on two native full-length substrates, p53 and acetyl-CoA synthetase1 (AceCS1). Herein, we report that neither the Sirtris series nor resveratrol activate SIRT1 in these native systems. Through biophysical studies, we provide additional evidence that the Sirtris compounds interact directly with fluorophore-containing peptide substrates with micromolar binding affinities even in the absence of SIRT1. In contrast to the previous report (26.Milne J.C. Lambert P.D. Schenk S. Carney D.P. Smith J.J. Gagne D.J. Jin L. Boss O. Perni R.B. Vu C.B. Bemis J.E. Xie R. Disch J.S. Ng P.Y. Nunes J.J. Lynch A.V. Yang H. Galonek H. Israelian K. Choy W. Iffland A. Lavu S. Medvedik O. Sinclair D.A. Olefsky J.M. Jirousek M.R. Elliott P.J. Westphal C.H. Nature. 2007; 450: 712-716Crossref PubMed Scopus (1462) Google Scholar), we demonstrate that SRT1720 neither lowers plasma glucose nor improves mitochondrial capacity in mice fed a high fat diet. We further demonstrate that the Sirtris series and resveratrol are highly promiscuous as they interact with multiple unrelated targets including receptors, enzymes, ion channels, and transporters. Taken together, these data suggest that SRT1720, SRT2183, SRT1460, and resveratrol are not direct SIRT1 activators. SRT1720, SRT2183, and SRT1460 were synthesized according to published procedures (26.Milne J.C. Lambert P.D. Schenk S. Carney D.P. Smith J.J. Gagne D.J. Jin L. Boss O. Perni R.B. Vu C.B. Bemis J.E. Xie R. Disch J.S. Ng P.Y. Nunes J.J. Lynch A.V. Yang H. Galonek H. Israelian K. Choy W. Iffland A. Lavu S. Medvedik O. Sinclair D.A. Olefsky J.M. Jirousek M.R. Elliott P.J. Westphal C.H. Nature. 2007; 450: 712-716Crossref PubMed Scopus (1462) Google Scholar). EX-527 (6-chloro-2,3,4,9-tetrahydro-1-H-carbazole-1-carboxamide) and resveratrol were purchased from Tocris Biosciences and Sigma, respectively. All compounds were stored as dry powders at room temperature in a nitrogen box and dissolved in DMSO to prepare concentrated stock solutions. Tris acetate and Triton X-100 were obtained from Research Organics and Calbiochem, respectively. HPLC solvents were from J.T. Baker. The p53-derived peptides, TAMRA-p53 peptide and native p53 peptide, were custom-synthesized by CPC Scientific, whereas 2,3-TAMRA was purchased from Molecular Probes. The amino acid sequence of all peptides used in the present study is summarized in supplemental Table S1. All reagents used were the highest quality commercially available. Acetyl-CoA synthetase1 (AceCS1) and acetylated acetyl-CoA synthetase1 (Ac-AeCS1) were provided by Dr. John Denu (University of Wisconsin, Madison, WI). All other reagents were purchased from Sigma unless otherwise noted. An Agilent 1100 HPLC system was used in all HPLC experiments, and data were processed by the Agilent ChemStation software. The Escherichia coli codon-optimized cDNA for human SIRT1 was custom-synthesized at DNA2.0 (Menlo Park, CA) and cloned into a modified pET vector by using standard molecular biology techniques to generate a full-length human SIRT1 construct (amino acids 2–747) with an N-terminal His6 tag followed by a thrombin cleavage site. SIRT1 expression was accomplished in BL21gold (DE3) cells with induction at an A600 nm of 0.9 with a final concentration of isopropyl-1-thio-β-d-galactopyranoside at 50 μm for 16–20 h at 15 °C. SIRT1 was purified using a Ni2+ chelating column followed by size exclusion and anion exchange chromatography as described previously (26.Milne J.C. Lambert P.D. Schenk S. Carney D.P. Smith J.J. Gagne D.J. Jin L. Boss O. Perni R.B. Vu C.B. Bemis J.E. Xie R. Disch J.S. Ng P.Y. Nunes J.J. Lynch A.V. Yang H. Galonek H. Israelian K. Choy W. Iffland A. Lavu S. Medvedik O. Sinclair D.A. Olefsky J.M. Jirousek M.R. Elliott P.J. Westphal C.H. Nature. 2007; 450: 712-716Crossref PubMed Scopus (1462) Google Scholar). This procedure typically generated SIRT1 with ∼90% purity based on SDS-PAGE visualized by Coomassie Blue staining. Typically 3–5 mg of purified SIRT1 was obtained per liter of culture. The full-length p53 cDNA was cloned into the baculovirus vector pFastBac (Invitrogen) with a His6 tag at the N terminus, and p53 was expressed in Sf9 cells as described (30.Sun X.Z. Nguyen J. Momand J. Methods Mol. Biol. 2003; 234: 17-28PubMed Google Scholar). Sf9 cells expressing p53 from 2 liters of culture were lysed using a microfluidizer, and the resulting lysate was centrifuged at 100,000 × g for 40 min. The supernatant containing the soluble p53 fraction was purified by nickel-nitrilotriacetic acid-Sepharose FF column (GE Healthcare) followed by a Q Sepharose FF column (GE Healthcare). Purified p53 was acetylated by p300 as described (31.Wang Y.H. Tsay Y.G. Tan B.C. Lo W.Y. Lee S.C. J. Biol. Chem. 2003; 278: 25568-25576Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The acetylation reaction mixture (32 ml) contained 2.5 mg of purified p53, 38 μg of p300 acetylase (FLAG-tagged recombinant enzyme, Active Motif, Carlsbad, CA), 50 mm Tris-HCl, pH 8.0, 50 μm acetyl-CoA, 150 mm KCl, 1 mm DTT, 0.1 mm EDTA, and 10% glycerol. The reaction was incubated at 22 °C for 16 h. At the end of the reaction, p300 was removed by passing through an anti-FLAG M2 agarose column (Sigma-Aldrich), and the acetylated p53 (Ac-Lys382-p53) was dialyzed against a buffer containing 50 mm Tris-HCl, pH 8.0, 150 mm KCl, 10% glycerol, 0.5 mm EDTA, and 1 mm DTT. The final Ac-Lys382-p53 was confirmed to contain ∼30–70% acetylation at the Lys-382 residue by tryptic digest followed by LC-MS analysis. The purified and acetylated p53 (Ac-Lys382-p53) protein had purity greater than 98% based on SDS-PAGE visualized by Coomassie Blue staining. Typically 6 mg of purified Ac-Lys382-p53 was obtained per liter of culture. The SIRT1 reaction (100 μl) was carried out with 1 μm TAMRA-p53 peptide ((biotin-PEG3)-Ser-Thr-Ser-Ser-His-Ser-(Ac-Lys)-Nle-Ser-Thr-Glu-Gly-Cys(tetramethylrhodamine-5-maleimide)-Glu-Glu-NH2) (Nle stands for norleucine) (TAMRA Peptide 1 in supplemental Table S1) under the following standard SIRT1 reaction conditions: 50 mm Tris acetate, pH 7.4, 150 mm NaCl, 150 μm NAD+, 1 mm DTT, 0.01% Triton X-100, and 2% DMSO. Reactions were carried out with 5 nm SIRT1 at room temperature for 30 min, yielding ∼5% conversion of TAMRA-p53 peptide to product, allowing for the accurate measurement of ∼20-fold activation. For EC1.5 determination, resveratrol concentrations were varied at 0.01–600 μm, SRT2183 and SRT1460 were at 0.003–200 μm, and SRT1720 was at 0.005–200 μm. The reaction was quenched with trifluoroacetic acid to a final concentration of 2%, centrifuged at 16,000 × g for 10 min, and then transferred to glass HPLC vials. The HPLC separation of the acetylated TAMRA-p53 peptide substrate and deacetylated peptide product peaks was achieved using a Vydac C18, 5 μm, 50 × 4.6-mm (218TP5405) column with accompanying guard. Mobile phase A was 100% H2O + 0.1% trifluoroacetic acid, whereas mobile phase B was 100% CH3CN + 0.1% trifluoroacetic acid. The following gradient program was used: 0–35% over 15 min followed by rapid ramp up to 100% B to wash and then 100% A to re-equilibrate the column. Flow rate was kept constant at 1.0 ml/min, column temperature was set at 25 °C, and UV detection was set at 220 nm. The retention times of the deacetylated peptide and acetylated TAMRA-p53 peptide were ∼9.1 and ∼9.3 min, respectively. The percentage of conversion was determined by dividing the peak area of the product over the sum of the peak areas of both substrate and product peptides. This value was periodically reevaluated by comparing with a deacetylated peptide standard curve. The average rate of conversion of SIRT1 samples run in the absence of compound was compared with those in the presence of compound to establish the percentage of activation value. GraphPad Prism was used to analyze the data and calculate EC1.5 values. The SIRT1 reaction was carried out under the SIRT1 standard reaction conditions as described above with 1 μm native p53 peptide (Ac-Lys-Lys-Gly-Gln-Ser-Thr-Ser-Arg-His-Lys-(Ac-Lys)-Leu-Met-Phe-Lys-Thr-Glu-Gly-NH2) (Native Peptide 3 in supplemental Table S1) and 2.5 nm SIRT1 at room temperature for 30 min, yielding ∼10% conversion of substrate to product, allowing for the accurate measurement of ∼10-fold activation. Reactions were quenched and processed the same as with the TAMRA-p53 peptide. The separation of acetylated peptide substrate and deacetylated peptide product peaks was achieved using the same column, buffers, and parameters described with the TAMRA-p53 peptide, except that the gradient was altered to: 0–20% over 15 min followed by rapid ramp up to 100% B to wash and then 100% A to re-equilibrate the column. The retention times of the deacetylated and acetylated peptides were ∼10.8 and 11.7 min, respectively. Calculation of the percentage of activation was carried out as described with TAMRA-p53 peptide. The Km values for native-p53 peptide and NAD+ were obtained by fitting the plots of rate versus substrate concentration to the Michaelis-Menten equation using KaleidaGraph. To test the effect of TAMRA on SIRT1 activity by putative activators, the same SIRT1 reactions were run with or without 1, 10, and 100 μm 2,3-TAMRA, each in the presence and absence of compounds. The SIRT1 assay mixture (50 μl) contained 150 mm Tris acetate, pH 7.4, 150 mm NaCl, 150 μm NAD+, 1 mm DTT, 0.014% Triton X-100, 3% DMSO, 100 nm Ac-Lys382-p53, and 10 nm SIRT1. The reactions were carried out in 96-well round bottom polypropylene plates (Corning, Lowell, MA). Putative activators were preincubated with SIRT1 at room temperature for 15 min, and the reactions were initiated by NAD+. The reaction mixtures were incubated at room temperature for 1 h, and the reactions were stopped by the addition of EX-527 to a final concentration of 100 μm. SIRT1 activity was measured by quantifying the levels of Ac-p53 by ELISA. The ELISA was carried out in a black MaxiSorp 96-well plate (Nunc, Rochester, NY) precoated with 0.2 μg of mouse anti-p53 antibody (Abcam, Cambridge, MA) by overnight incubation at 4 °C. Nonspecific protein binding was then blocked by block Solution (StartingBlock T20 (PBS), Thermo Scientific) for 30 min. Typically, the SIRT1 reaction mixtures were diluted 10-fold in a buffer containing 50 mm Tris, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 100 μm EX-527. Aliquots (5 μl) of diluted SIRT1 mixture were placed in each well containing 95 μl of the above buffer in the precoated plates and incubated for 2 h at 4 °C. After washing the plates with wash buffer (PBST 0.1% Tween 20, Sigma-Aldrich), the plates were incubated with 100 μl of either 1:500 acetyl-Lys382-p53 antibody (Cell Signaling, Beverly, MA) or 1:1000 rabbit anti-p53 (FL-393) antibody (Santa Cruz Biotechnology, Santa Cruz, CA.) in block solution for 1 h at room temperature. After an additional washing with wash buffer, the plates were incubated with 100 μl of 1:10000 goat anti-rabbit IgG-horseradish peroxidase conjugate (Jackson ImmunoResearch Laboratories, West Grove, PA) in block solution for 1 h at room temperature. After a final washing with wash buffer, 100 μl of SuperSignal ELISA Pico chemiluminescent substrate (Thermo Scientific) was added to the plate for development. The plates were read using a Victor2 1420 multilabel plate reader (PerkinElmer Life Sciences) under luminescence setting. The standard curves for ELISA were linear at 1–30 ng of Ac-p53 and total p53. Western blot analyses were conducted using the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE) with Ac-Lys382-p53, and total p53 was detected on the same blot by using 1:1000 mouse anti-p53 antibody (Abcam, Cambridge, MA) and 1:1000 rabbit anti-acetyl-Lys382-p53 antibody (Santa Cruz Biotechnology). The secondary antibodies used were 1:5000 IR700 goat anti-mouse and 1:5000 IR800 goat anti-rabbit (LI-COR Biosciences). SIRT1 reactions were performed and quenched as described in the ELISA procedure, and 5 μl of the reaction mixture was loaded in each well of SDS-PAGE. Quantification of total p53 levels were used to normalize the reactions for SIRT1 activity. The Km measurements of SIRT1 for p53 were performed using Western and ELISA assays by varying concentrations of p53 from 50 to 850 nm (maximal concentration available). In the first step of the coupled enzyme reaction, the SIRT1 reaction was carried out using standard reaction conditions as described above with 150 nm acetylated acetyl-CoA synthetase 1 (Ac-AceCS1) and 10 nm SIRT1 at room temperature for 30 min, yielding ∼10% conversion of substrate to active AceCS1 product. This allowed for the accurate measurement of ∼10-fold activation. Reactions were quenched by nicotinamide to a final concentration of 5 mm. In the second step of the coupled reaction, saturating concentrations of AceCS1 substrates (2.5 mm ATP, 2 mm NaOAc, and 2 mm coenzyme A) were added. The AceCS1 reactions were incubated for 60 min, quenched by trifluoroacetic acid to a final concentration of 2%, centrifuged at 16,000 × g for 10 min, and then transferred to vials for HPLC analysis. Separation of AMP from ATP and other substrates and products was achieved using a SUPELCOSIL LC-18-T (Sigma, 3 μm, 150 × 4.6 mm) column with accompanying guard. Mobile phase A was 100 mm NH4OAc, pH 5.0, whereas mobile phase B was 80% A + 20% methanol. The following gradient program was used: 0% B for 10 min, 0–100% for 5 min followed by wash, and then 100% A to re-equilibrate the column. Flow rate was kept constant at 1.0 ml/min, column temperature was kept at 25 °C, and UV detection was set at 260 nm. The retention times of ATP and AMP were ∼4 and ∼8 min, respectively. Chromatograms were analyzed, and the peak area was converted to molar concentration of AMP using an AMP standard curve and then converted to the total amount of active enzyme AceCS1 product turned over by SIRT1 using the standard curves mentioned above. The average rate of conversion of SIRT1 samples run in the absence of compound was compared with those in the presence of compound to establish the percentage of activation values. The final concentrations of putative activators tested were 1, 3, 10, and 30 μm. EX-527 was tested at 100 μm. For the measurement of the Km value for Ac-AceCS1 with SIRT1, the SIRT1 reactions were performed by varying concentrations of Ac-AceCS1 at 50–750 nm (maximal concentration available). NMR chemical shift perturbation of the peptide substrates was used to monitor the molecular interaction of SRT1460 with the p53-derived peptide substrates in the absence of SIRT1 enzyme. NMR binding experiments were carried out with peptide substrates, which included the TAMRA-p53 peptide (TAMRA Peptide 1 in supplemental Table S1) and an additional pair of peptides with identical amino acid sequence and differing only in the TAMRA group (Ac-Glu-Glu-Lys-Gly-Gln-Ser-Thr-Ser-Ser-His-Ser-(Ac-Lys)-Nle-Ser-Thr-Glu-Gly-Lys(TAMRA)-Glu-Glu-NH2) and (Ac-Glu-Glu-Lys-Gly-Gln-Ser-Thr-Ser-Ser-His-Ser-(Ac-Lys)-Nle-Ser-Thr-Glu-Gly-Lys-Glu-Glu-NH2) (TAMRA Peptide 2 and Native Peptide 2 in supplemental Table S1). 1H NMR spectra were recorded on a 600-MHz Bruker" @default.
• W2157860216 created "2016-06-24" @default.
• W2157860216 creator A5000155476 @default.
• W2157860216 creator A5004218003 @default.
• W2157860216 creator A5009489554 @default.
• W2157860216 creator A5009558339 @default.
• W2157860216 creator A5013313835 @default.
• W2157860216 creator A5016609209 @default.
• W2157860216 creator A5020391614 @default.
• W2157860216 creator A5022096407 @default.
• W2157860216 creator A5029173526 @default.
• W2157860216 creator A5029392488 @default.
• W2157860216 creator A5031952528 @default.
• W2157860216 creator A5037727619 @default.
• W2157860216 creator A5044927871 @default.
• W2157860216 creator A5053484501 @default.
• W2157860216 creator A5069582141 @default.
• W2157860216 creator A5075329460 @default.
• W2157860216 creator A5085977633 @default.
• W2157860216 date "2010-03-01" @default.
• W2157860216 modified "2023-10-11" @default.
• W2157860216 title "SRT1720, SRT2183, SRT1460, and Resveratrol Are Not Direct Activators of SIRT1" @default.
• W2157860216 cites W1576985941 @default.
• W2157860216 cites W1981194888 @default.
• W2157860216 cites W1983465246 @default.
• W2157860216 cites W1985308585 @default.
• W2157860216 cites W1986093216 @default.
• W2157860216 cites W1990931780 @default.
• W2157860216 cites W1995095187 @default.
• W2157860216 cites W1997145913 @default.
• W2157860216 cites W2001854860 @default.
• W2157860216 cites W2003155232 @default.
• W2157860216 cites W2004010498 @default.
• W2157860216 cites W2005014046 @default.
• W2157860216 cites W2007842741 @default.
• W2157860216 cites W2012753961 @default.
• W2157860216 cites W2018951168 @default.
• W2157860216 cites W2026029935 @default.
• W2157860216 cites W2027616087 @default.
• W2157860216 cites W2054447845 @default.
• W2157860216 cites W2058662941 @default.
• W2157860216 cites W2064352591 @default.
• W2157860216 cites W2069193571 @default.
• W2157860216 cites W2069196002 @default.
• W2157860216 cites W2074740541 @default.
• W2157860216 cites W2077834313 @default.
• W2157860216 cites W2080192652 @default.
• W2157860216 cites W2081774189 @default.
• W2157860216 cites W2083215899 @default.
• W2157860216 cites W2091457094 @default.
• W2157860216 cites W2102646072 @default.
• W2157860216 cites W2103416697 @default.
• W2157860216 cites W2105487944 @default.
• W2157860216 cites W2116350182 @default.
• W2157860216 cites W2124595934 @default.
• W2157860216 cites W2124604057 @default.
• W2157860216 cites W2125121177 @default.
• W2157860216 cites W2128023946 @default.
• W2157860216 cites W2143499920 @default.
• W2157860216 cites W2144495216 @default.
• W2157860216 cites W2144600435 @default.
• W2157860216 cites W2152390180 @default.
• W2157860216 cites W2153975084 @default.
• W2157860216 cites W2157649587 @default.
• W2157860216 cites W2168702014 @default.
• W2157860216 doi "https://doi.org/10.1074/jbc.m109.088682" @default.
• W2157860216 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2832984" @default.
• W2157860216 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/20061378" @default.
• W2157860216 hasPublicationYear "2010" @default.
• W2157860216 type Work @default.
• W2157860216 sameAs 2157860216 @default.
• W2157860216 citedByCount "818" @default.
• W2157860216 countsByYear W21578602162012 @default.
• W2157860216 countsByYear W21578602162013 @default.
• W2157860216 countsByYear W21578602162014 @default.
• W2157860216 countsByYear W21578602162015 @default.
• W2157860216 countsByYear W21578602162016 @default.
• W2157860216 countsByYear W21578602162017 @default.
• W2157860216 countsByYear W21578602162018 @default.
• W2157860216 countsByYear W21578602162019 @default.
• W2157860216 countsByYear W21578602162020 @default.
• W2157860216 countsByYear W21578602162021 @default.
• W2157860216 countsByYear W21578602162022 @default.
• W2157860216 countsByYear W21578602162023 @default.
• W2157860216 crossrefType "journal-article" @default.
• W2157860216 hasAuthorship W2157860216A5000155476 @default.
• W2157860216 hasAuthorship W2157860216A5004218003 @default.
• W2157860216 hasAuthorship W2157860216A5009489554 @default.
• W2157860216 hasAuthorship W2157860216A5009558339 @default.
• W2157860216 hasAuthorship W2157860216A5013313835 @default.
• W2157860216 hasAuthorship W2157860216A5016609209 @default.
• W2157860216 hasAuthorship W2157860216A5020391614 @default.
• W2157860216 hasAuthorship W2157860216A5022096407 @default.
• W2157860216 hasAuthorship W2157860216A5029173526 @default.
• W2157860216 hasAuthorship W2157860216A5029392488 @default.
• W2157860216 hasAuthorship W2157860216A5031952528 @default.
• W2157860216 hasAuthorship W2157860216A5037727619 @default.
• W2157860216 hasAuthorship W2157860216A5044927871 @default.

• next