Mechanism of Human SIRT1 Activation by Resveratrol

The NAD+-dependent protein deacetylase family, Sir2 (or sirtuins), is important for many cellular processes including gene silencing, regulation of p53, fatty acid metabolism, cell cycle regulation, and life span extension. Resveratrol, a polyphenol found in wines and thought to harbor major health benefits, was reported to be an activator of Sir2 enzymes in vivo and in vitro. In addition, resveratrol was shown to increase life span in three model organisms through a Sir2-dependent pathway. Here, we investigated the molecular basis for Sir2 activation by resveratrol. Among the three enzymes tested (yeast Sir2, human SIRT1, and human SIRT2), only SIRT1 exhibited significant enzyme activation (∼8-fold) using the commercially available Fluor de Lys kit (BioMol). To examine the requirements for resveratrol activation of SIRT1, we synthesized three p53 acetylpeptide substrates either lacking a fluorophore or containing a 7-amino-4-methylcoumarin (p53-AMC) or rhodamine 110 (p53-R110). Although SIRT1 activation was independent of the acetylpeptide sequence, resveratrol activation was completely dependent on the presence of a covalently attached fluorophore. Substrate competition studies indicated that the fluorophore decreased the binding affinity of the peptide, and, in the presence of resveratrol, fluorophore-containing substrates bound more tightly to SIRT1. Using available crystal structures, a model of SIRT1 bound to p53-AMC peptide was constructed. Without resveratrol, the coumarin of p53-AMC peptide is solvent-exposed and makes no significant contacts with SIRT1. We propose that binding of resveratrol to SIRT1 promotes a conformational change that better accommodates the attached coumarin group. The NAD+-dependent protein deacetylase family, Sir2 (or sirtuins), is important for many cellular processes including gene silencing, regulation of p53, fatty acid metabolism, cell cycle regulation, and life span extension. Resveratrol, a polyphenol found in wines and thought to harbor major health benefits, was reported to be an activator of Sir2 enzymes in vivo and in vitro. In addition, resveratrol was shown to increase life span in three model organisms through a Sir2-dependent pathway. Here, we investigated the molecular basis for Sir2 activation by resveratrol. Among the three enzymes tested (yeast Sir2, human SIRT1, and human SIRT2), only SIRT1 exhibited significant enzyme activation (∼8-fold) using the commercially available Fluor de Lys kit (BioMol). To examine the requirements for resveratrol activation of SIRT1, we synthesized three p53 acetylpeptide substrates either lacking a fluorophore or containing a 7-amino-4-methylcoumarin (p53-AMC) or rhodamine 110 (p53-R110). Although SIRT1 activation was independent of the acetylpeptide sequence, resveratrol activation was completely dependent on the presence of a covalently attached fluorophore. Substrate competition studies indicated that the fluorophore decreased the binding affinity of the peptide, and, in the presence of resveratrol, fluorophore-containing substrates bound more tightly to SIRT1. Using available crystal structures, a model of SIRT1 bound to p53-AMC peptide was constructed. Without resveratrol, the coumarin of p53-AMC peptide is solvent-exposed and makes no significant contacts with SIRT1. We propose that binding of resveratrol to SIRT1 promotes a conformational change that better accommodates the attached coumarin group. 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Nature. 2004; 429: 771-776Crossref PubMed Scopus (1679) Google Scholar), and inhibition of NF-κB-dependent transcription (31Yeung F. Hoberg J.E. Ramsey C.S. Keller M.D. Jones D.R. Frye R.A. Mayo M.W. EMBO J. 2004; 23: 2369-2380Crossref PubMed Scopus (2224) Google Scholar). In yeast, resveratrol was shown to mimic Sir2-dependent life span extension during calorie restriction, although the effects of calorie restriction and resveratrol were not additive (35Howitz K.T. Bitterman K.J. Cohen H.Y. Lamming D.W. Lavu S. Wood J.G. Zipkin R.E. Chung P. Kisielewski A. Zhang L.L. Scherer B. Sinclair D.A. Nature. 2003; 425: 191-196Crossref PubMed Scopus (3202) Google Scholar). One study on yeast aging suggested that Sir2 acts independently of pathways mediated by calorie restriction (60Kaeberlein M. Kirkland K.T. Fields S. Kennedy B.K. PLoS Biol. 2004; 2: E296Crossref PubMed Scopus (368) Google Scholar). Resveratrol extended life span in Caenorhabditis elegans and Drosophila melanogaster through a Sir2-mediated process (36Wood J.G. Rogina B. Lavu S. Howitz K. Helfand S.L. Tatar M. Sinclair D. Nature. 2004; 430: 686-689Crossref PubMed Scopus (1592) Google Scholar). The mechanism by which resveratrol activates Sir2 enzymes was unknown. In the current study, we sought to elucidate how resveratrol activates SIRT1. Our results indicate that resveratrol is not a general activator of Sir2 enzymes and that activation requires the presence of a fluorophore covalently attached to the peptide substrate. To validate our findings, four different deacetylation assays were used, and three p53 acetylpeptide substrates either lacking a fluorophore or containing either coumarin (p53-AMC) or rhodamine (p53-R110) fluorophores were synthesized. The commonly used BioMol (Plymouth, PA) assay kit, Fluor de Lys, which employs a similar fluorescent detection method, was included in the analysis. We show that the peptide sequence is inconsequential for resveratrol activation, but the covalent attachment of the fluorophore on the peptide is necessary to produce the resveratrol-mediated activation. A model of p53-AMC peptide bound to SIRT1 was developed to visualize the interaction of the fluorophore with the enzyme. We propose that resveratrol binding to SIRT1 promotes conformational changes in the enzyme which allow tighter binding of the fluorophore. Materials—Fluor de Lys-SIRT1 peptide, Fluor de Lys-H4 AcK16 peptide, and Fluor de Lys Developer II 5 × concentrate, were purchased from BioMol. Resveratrol was purchased from Sigma. The p53 peptide was purchased from the University of Wisconsin-Madison Peptide Synthesis Center. All other reagents were purchased from Sigma and Fisher, or as otherwise noted, and were of the highest quality available. Expression and Purification of SIRT1—Histidine-tagged SIRT1 was cloned into a pQE80 vector (Qiagen, Valencia, CA). The plasmid was transformed into BL21DE3 cells, which were grown to an A600 of 0.6–0.8 prior to induction with isopropyl-β-d-thiogalactopyranoside for 3–8 h. Cells were harvested and stored at –20 °C until use. Cells were lysed using a French pressure cell in 50 mm Tris, pH 8, 300 mm NaCl with 1 mm β-mercaptoethanol, 0.1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 5 μg/ml aprotinin. Cell debris was removed by centrifugation. The supernatant was rocked with nickel-nitrilotriacetic acid resin for 1 h at 4 °C. The resin was then loaded onto a column and washed with 50 mm Tris, pH 8.0, 300 mm NaCl, and 1 mm β-mercaptoethanol. SIRT1 was eluted with a gradient of 0–200 mm imidazole in 50 mm Tris, pH 8.0, 300 mm NaCl, and 1 mm β-mercaptoethanol. SIRT1 fractions were pooled and lyophilized in 25 mm Tris, pH 7.5, 100 mm NaCl, 10% glycerol, and 5 mm dithiothreitol and stored at –20 °C prior to use. SIRT1 Deacetylase Assays—Four different deacetylase assays were used to assess resveratrol activation of SIRT1 in vitro: the Fluor de Lys fluorescence assay, coumarin and rhodamine-based fluorescence assays, charcoal binding assay, and high performance liquid chromatography (HPLC)-based assays. All the reactions were performed with 10% final dimethyl sulfoxide concentration at 37 °C. The Fluor de Lys fluorescence assay was performed as indicated in the BioMol product sheets. Briefly, assays were performed using Fluor de Lys-SIRT1 or Fluor de Lys-H4 AcK16 peptides, NAD+, and SIRT1, in the absence and presence of resveratrol in SIRT1 assay buffer (25 mm Tris-Cl, pH 8.0, 137 mm NaCl, 2.7 mm KCl, 1 mm MgCl2, 1 mg/ml bovine serum albumin, as indicated in the BioMol product sheets). The buffer, dimethyl sulfoxide, resveratrol, and SIRT1 were preincubated for 10 min. Reactions were initiated by the addition of 2 × concentrations of the Fluor de Lys peptide and NAD+. Prior to quenching the reaction, 2 mm nicotinamide was added to 1× Developer II in the histone deacetylase assay buffer (25 mm Tris, pH 8.0, 137 mm NaCl, 2.7 mm KCl, 1 mm MgCl2, as indicated in the BioMol product sheets). At each time point, 50 μl of the reaction was removed and mixed with 50 μl of the developer solution. The quenched samples were kept at 37 °C for 45 min prior to fluorescence reading. Fluorescence readings were obtained using the CytoFluor series 4000 fluorometer (Perseptive Biosystems Inc., Framingham, MA), with the excitation wavelength set to 360 nm and the emission set to 460 nm (according to BioMol specification). The p53-AMC and p53-R110 fluorescence assays were carried out similarly to that described above for the Fluor de Lys fluorescence assay. However, for the p53-R110 peptide, the excitation wavelength was set to 490 nm and the emission set to 520 nm. For the charcoal binding assay and the HPLC assay, conditions similar to those in the fluorescence assay were used. The enzyme was preincubated in a mixture of dimethyl sulfoxide, resveratrol, and the SIRT1 assay buffer prior to addition of the substrates to initiate the reaction. For the charcoal binding assay, [3H]acetylated histone H3 peptide ([3H]AcH3) was used as a substrate instead of the Fluor de Lys peptide. Generation of [3H]AcH3 was described previously (61Borra M.T. Denu J.M. Methods Enzymol. 2004; 376: 171-187Crossref PubMed Scopus (41) Google Scholar). Briefly, for the charcoal binding assay, at each time point, the reaction was quenched by the addition of charcoal slurry containing activated charcoal in 2 m glycine, pH 9.5. Samples were incubated at 95 °C for 1 h. The samples were centrifuged and the supernatant transferred into a new microcentrifuge tubes containing charcoal slurry. After centrifugation, the radioactivity of the supernatant was determined by scintillation counting. For the HPLC assay, Fluor de Lys-SIRT1 and [14C]NAD+, in which the 14C label is located on the nicotinamide ring, are used in the reaction (synthesis of [14C]NAD+ and the HPLC assay were described previously (61Borra M.T. Denu J.M. Methods Enzymol. 2004; 376: 171-187Crossref PubMed Scopus (41) Google Scholar)). The reactions were quenched with trifluoroacetic acid to a final concentration of 1%. Time points were injected into a reversed phase, C18, small pore column (Vydac, Hesperia, CA). The substrates and products were resolved using increasing concentrations of acetonitrile. Radioactivity of the collected fractions was determined by scintillation counting. Competition Assay between the Fluor de Lys-SIRT1 and p53 Peptide with [3H]AcH3—Competition assays were performed using the charcoal binding assay to determine whether resveratrol enhances the binding of the Fluor de Lys-SIRT1, p53-AMC, or p53 4-mer peptides. The charcoal binding assay was carried out with 20 μm [3H]AcH3, 100 μm NAD+, varying concentrations of the Fluor de Lys-SIRT1, p53-AMC, or p53 4-mer peptides, with and without 200 μm resveratrol. The reactions were quenched, and the amount of O-[3H]AADPr product was quantified as described previously (61Borra M.T. Denu J.M. Methods Enzymol. 2004; 376: 171-187Crossref PubMed Scopus (41) Google Scholar). Synthesis of p53-AMC and p53-R110 Peptides (Figs.1and2)—Boc-Lys(Ac)-AMC (28 mg, 63 μmol; Bachem, Switzerland) was deprotected with 1:1 trifluoroacetic acid:CH2Cl2. The resulting clear oil was further dried using hexane to form an azeotrope. The product was purified by reversed phase HPLC using a C18, small pore preparative column (Vydac), eluting with methanol to yield crude H-Lys(Ac)-AMC in quantitative yield that was used without further purification. Ac-R(Pmc)-H(Trt)-K(Boc)-OH (55 mg, 43 μmol; University of Wisconsin-Madison Peptide Synthesis Center) was dissolved in N,N-dimethylformamide (0.22 ml) and preactivated with O-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate (21 mg, 55 μmol) and N-methyl morpholine (18 μl, 164 μmol). H-Lys(Ac)-AMC (28 mg, 63 μmol) dissolved in N,N-dimethylformamide (0.11 ml) was added, reacted for 9 h at room temperature, and dried in vacuo. The arginine, histidine, and lysine protecting groups were cleaved with trifluoroacetic acid, and the crude product was purified by reversed phase HPLC, eluting with increasing concentrations of acetonitrile in water. The product, Ac-Arg-His-Lys-Lys(Ac)-AMC, was lyophilized to yield a white flocculent power (30 mg, 37 μmol) in 59% overall yield. MALDI MS calculated for C38H56N12O8 (M+H+), 809.9; found, 809.8. Boc-Lys(Ac)2-R110 (a generous gift from L. Lavis and R. Raines, University of Wisconsin-Madison) was converted to p53-R110 in a manner similar to the p53-AMC peptide. MALDI MS calculated for C38H56 N12O8 (M+H+), 1597.8; found, 1597.8. Molecular Modeling of SIRT1 and p53-AMC—The SIRT1 crystal structure was built by homology modeling using the SWISS-MODEL function in Swiss PDB viewer version 3.7 (62Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9641) Google Scholar). The p53-AMC peptide was modeled from the crystal structure of p53 bound to Sir2-Af2 (63Avalos J.L. Celic I. Muhammad S. Cosgrove M.S. Boeke J.D. Wolberger C. Mol. Cell. 2002; 10: 523-535Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Using Sybyl (version 6.8, Tripos, Inc., St. Louis, MO), an amide bond was built between the amino group of AMC and the main chain carboxyl group of the acetyllysine residue of p53 bound to Sir2-Af2. The built SIRT1 structure and the Sir2-Af2 structure containing the modeled p53-AMC peptide were aligned in Swiss PDB viewer version 3.7 (62Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9641) Google Scholar) using sequence homology thereby docking the p53-AMC peptide into the SIRT1 structure. The resulting crystal structure of p53-AMC peptide bound to SIRT1 was used for all subsequent analysis. Resveratrol Activates SIRT1 but Not Other Sir2 Homologs—To determine whether resveratrol activated Sir2 homologs, ySir2, human SIRT2, and human SIRT1 were analyzed using the commercially available Fluor de Lys-SIRT1 as a peptide substrate, with and without 200 μm resveratrol. Samples were quenched with the Developer II solution with added 2mm nicotinamide, and the fluorescence was measured according to the manufacturer's protocol. With SIRT1, SIRT2, and ySir2, fluorescence increased over time, indicating deacetylation of the Fluor de Lys-SIRT1 peptide (Fig. 3). Comparison of the rate of fluorescence increase in the presence and absence of resveratrol showed that resveratrol activated SIRT1 by 8-fold (Fig. 3A), consistent with previous studies using this assay (35Howitz K.T. Bitterman K.J. Cohen H.Y. Lamming D.W. Lavu S. Wood J.G. Zipkin R.E. Chung P. Kisielewski A. Zhang L.L. Scherer B. Sinclair D.A. Nature. 2003; 425: 191-196Crossref PubMed Scopus (3202) Google Scholar). However, no significant activation was observed when SIRT2 (Fig. 3B) and ySir2 (Fig. 3C) were assayed with the Fluor de Lys-SIRT1 peptide. Moreover, a higher concentration of ySir2 was required to observe significant deacetylation of the substrate, indicating that Fluor de Lys-SIRT1 peptide is a poor substrate for ySir2. Because resveratrol activation was specific for SIRT1, subsequent mechanistic studies were performed using this enzyme. Resveratrol Activation Requires Fluor de Lys-SIRT1 as a Substrate—To verify resveratrol activation of SIRT1, an alternative assay was employed. The charcoal binding assay (61Borra M.T. Denu J.M. Methods Enzymol. 2004; 376: 171-187Crossref PubMed Scopus (41) Google Scholar) was carried out under the same conditions as those for the Fluor de Lys fluorescence assay, except that the Fluor de Lys-SIRT1 peptide was replaced by [3H]AcH3 peptide, which corresponds to the sequence of the histone H3 N-terminal tail including and surrounding the acetylated lysine 14 (sequence: KSTGG([3H]AcK)APRKQ). In the charcoal binding assay, the [3H]acetyl group from the [3H]AcH3 is transferred to the ADP-ribose portion of NAD+ to form O-[3H]AADPr, which is quantified as described previously (61Borra M.T. Denu J.M. Methods Enzymol. 2004; 376: 171-187Crossref PubMed Scopus (41) Google Scholar). The assays were carried out with and without 200 μm resveratrol, and the concentrations of product formed were quantified and plotted versus time. As shown in Fig. 4A, the product formed increased over time; however, the rate of product formation was identical (within error) in the presence and absence of resveratrol, indicating that resveratrol had no effect on the enzymatic activity when [3H]AcH3 was employed as a substrate and when the charcoal binding assay was used. Because of the conflicting results from the Fluor de Lys fluorescence assay and the charcoal binding assay, other possible explanations for resveratrol activation were examined. To determine whether resveratrol induces an increase in product fluorescence, the deacetylated Fluor de Lys-SIRT1 peptide was incubated in the SIRT1 assay buffer with and without resveratrol. The fluorescence was measured, and no difference with and without resveratrol was observed (data not shown), indicating that resveratrol does not increase the fluorescenc