Medical College of Wisconsin
ORCID: 0000-0001-6330-2768Publishes on Particle physics theoretical and experimental studies, High-Energy Particle Collisions Research, Sirtuins and Resveratrol in Medicine. 293 papers and 14.4k citations.
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Reflecting the myriad changes and advancements in the technologies involved in FTIR, particularly the development of diamond ATRs, this second edition of Fundamentals of Fourier Transform Infrared Spectroscopy has been extensively rewritten and expanded to include new topics and figures as well as updates of existing chapters. Designed for those ne
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. The silent information regulator 2 (Sir2) family of proteins (sirtuins) are NAD+-dependent histone/protein deacetylases that tightly couple the cleavage of NAD+ and deacetylation of protein substrates to form nicotinamide, the deacetylated product, and a novel metabolite, 2′-O-acetyl-ADP-ribose (OAADPr) 1The abbreviations used are: OAADPr, 2′-O-acetyl-ADP-ribose; AcH3, acetylated histone H3; AMC, 7-amino-4-methylcoumarin; Boc, butoxycarbonyl; HPLC, high performance liquid chromatography; MALDI MS, matrix-assisted laser desorption ionization mass spectrometry; R110, rhodamine 110. (1Imai S. Armstrong C.M. Kaeberlein M. Guarente L. Nature. 2000; 403: 795-800Crossref PubMed Scopus (2817) Google Scholar, 2Smith 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, 3Landry 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, 4Jackson M.D. Denu J.M. J. Biol. Chem. 2002; 277: 18535-18544Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 5Sauve A.A. Celic I. Avalos J. Deng H. Boeke J.D. Schramm V.L. Biochemistry. 2001; 40: 15456-15463Crossref PubMed Scopus (261) Google Scholar, 6Tanner 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, 7Tanny J.C. Moazed D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 415-420Crossref PubMed Scopus (225) Google Scholar). This family of proteins is evolutionarily conserved, with five homologs in yeast (ySir2 and HST1–4) and seven in humans (SIRT1–7) (8Frye R.A. Biochem. Biophys. Res. Commun. 1999; 260: 273-279Crossref PubMed Scopus (677) Google Scholar, 9Frye R.A. Biochem. Biophys. Res. Commun. 2000; 273: 793-798Crossref PubMed Scopus (1174) Google Scholar). The founding member of this family, ySir2, is essential for gene silencing at the three silent loci in yeast (10Aparicio O.M. Billington B.L. Gottschling D.E. Cell. 1991; 66: 1279-1287Abstract Full Text PDF PubMed Scopus (609) Google Scholar, 11Strahl-Bolsinger S. Hecht A. Luo K. Grunstein M. Genes Dev. 1997; 11: 83-93Crossref PubMed Scopus (594) Google Scholar, 12Gottschling D.E. Aparicio O.M. Billington B.L. Zakian V.A. Cell. 1990; 63: 751-762Abstract Full Text PDF PubMed Scopus (1137) Google Scholar, 13Rine J. Herskowitz I. Genetics. 1987; 116: 9-22Crossref PubMed Google Scholar, 14Shou 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, 15Shou W. Sakamoto K.M. Keener J. Morimoto K.W. Traverso E.E. Azzam R. Hoppe G.J. Feldman R.M. DeModena J. Moazed D. Charbonneau H. 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Science. 2002; 298: 2390-2392Crossref PubMed Scopus (478) Google Scholar), and life span extension (22Tissenbaum H.A. Guarente L. Nature. 2001; 410: 227-230Crossref PubMed Scopus (1590) Google Scholar, 23Kaeberlein M. McVey M. Guarente L. Genes Dev. 1999; 13: 2570-2580Crossref PubMed Scopus (1781) Google Scholar, 24Sinclair D.A. Guarente L. Cell. 1997; 91: 1033-1042Abstract Full Text Full Text PDF PubMed Scopus (1187) Google Scholar). SIRT1, the most extensively studied human homolog, mediates p53-dependent processes (25Luo 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, 26Vaziri 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 (2316) Google Scholar, 27Langley 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 (757) Google Scholar), transcription regulation (28Motta M.C. Divecha N. Lemieux M. Kamel C. Chen D. Gu W. Bultsma Y. McBurney M. Guarente L. Cell. 2004; 116: 551-563Abstract Full Text Full Text PDF PubMed Scopus (1203) Google Scholar, 29Brunet A. Sweeney L.B. Sturgill J.F. Chua K.F. Greer P.L. Lin Y. Tran H. Ross S.E. Mostoslavsky R. Cohen H.Y. Hu L.S. Cheng H.L. Jedrychowski M.P. Gygi S.P. Sinclair D.A. Alt F.W. Greenberg M.E. Science. 2004; 303: 2011-2015Crossref PubMed Scopus (2667) Google Scholar, 30Muth V. Nadaud S. Grummt I. Voit R. EMBO J. 2001; 20: 1353-1362Crossref PubMed Scopus (177) Google Scholar, 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), muscle differentiation (32Fulco M. Schiltz R.L. Iezzi S. King M.T. Zhao P. Kashiwaya Y. Hoffman E. Veech R.L. Sartorelli V. Mol. 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Chem. 2003; Full Text Full Text PDF PubMed Scopus Google as of In addition, including and resveratrol, shown to SIRT1 (35Howitz K.T. Bitterman K.J. Cohen H.Y. Lamming D.W. Lavu S. Wood Chung P. A. Sinclair D.A. Nature. 2003; PubMed Scopus Google Scholar). the resveratrol was found to be the most activator of SIRT1 and to a of (35Howitz K.T. Bitterman K.J. Cohen H.Y. Lamming D.W. Lavu S. Wood Chung P. A. Sinclair D.A. Nature. 2003; PubMed Scopus Google Scholar). is a polyphenol found in and the to M. Chen Y. L. 2002; 8: PubMed Scopus Google Scholar, J. T. W. A. Cell Biol. 2001; PubMed Scopus Google Scholar, S. H. Biochem. Biophys. 2001; PubMed Scopus Google Scholar, M.A. M. J. 2001; PubMed Scopus Google Scholar, M.J. J. Biochem. Biophys. 2000; PubMed Scopus Google Scholar, L. L. S. Sci. 1999; PubMed Scopus Google Scholar, M.C. P. J. Chem. 1999; PubMed Scopus Google Scholar, A.A. A. Biol. 1999; PubMed Scopus Google Scholar), and J. J. Natl. 2004; PubMed Scopus Google Scholar, A. S. Y. Res. 2004; Google Scholar, Res. 2004; PubMed Scopus Google Scholar, 2003; 12: Google Scholar, V. 2003; 16: PubMed Scopus Google Scholar). shown to increase SIRT1 by as as the for acetylated and to a that of with no reported on the of the enzyme (35Howitz K.T. Bitterman K.J. Cohen H.Y. Lamming D.W. Lavu S. Wood Chung P. A. Sinclair D.A. Nature. 2003; PubMed Scopus Google Scholar). was shown to cellular processes such as axonal protection (34Araki T. Sasaki Y. Milbrandt J. Science. 2004; 305: 1010-1013Crossref PubMed Scopus (916) Google Scholar), (33Picard F. Kurtev M. Chung N. Topark-Ngarm A. Senawong T. Machado De Oliveira R. Leid M. McBurney M.W. Guarente L. Nature. 2004; 429: 771-776Crossref PubMed Scopus (1679) Google Scholar), and of transcription 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 resveratrol was shown to Sir2-dependent life span extension the of and resveratrol (35Howitz K.T. Bitterman K.J. Cohen H.Y. Lamming D.W. Lavu S. Wood Chung P. A. Sinclair D.A. Nature. 2003; PubMed Scopus Google Scholar). on yeast that Sir2 of by M. K.T. S. Biol. 2004; PubMed Scopus Google Scholar). life span in and through a Lavu S. K. M. Sinclair D. Nature. 2004; PubMed Scopus Google Scholar). The by resveratrol Sir2 enzymes was In the we to resveratrol SIRT1. that resveratrol is a activator of Sir2 enzymes and that activation the presence of a fluorophore covalently attached to the peptide To deacetylation and three p53 acetylpeptide substrates either lacking a fluorophore or containing either coumarin (p53-AMC) or rhodamine The used Fluor de a was in the We that the peptide is for resveratrol the of the fluorophore on the peptide is to the model of p53-AMC peptide bound to SIRT1 was to the of the fluorophore with the We propose that resveratrol binding to SIRT1 promotes conformational in the enzyme binding of the fluorophore. de peptide, Fluor de peptide, and Fluor de Lys from was from The p53 peptide was from the of from and or as and of the and of SIRT1 was a The was to an of to with for and at using a cell in with and Cell was by The was with acid for at The was a and with and SIRT1 was with a of in and SIRT1 and in and and at to SIRT1 deacetylase used to resveratrol activation of SIRT1 in the Fluor de Lys coumarin and binding and high performance liquid the with at The Fluor de Lys was as indicated in the using Fluor de or Fluor de and SIRT1, in the and presence of resveratrol in SIRT1 as indicated in the The resveratrol, and SIRT1 for by the of 2 of the Fluor de Lys peptide and to the 2 was to in the histone deacetylase as indicated in the of the was and with of the The at for to using the with the to and the to to The p53-AMC and to that for the Fluor de Lys for the peptide, the was to and the to the binding and the to in the The enzyme was in a of resveratrol, and the SIRT1 to of the substrates to the the binding histone peptide was used as a of the Fluor de Lys of was M.T. Denu J.M. 2004; PubMed Scopus Google Scholar). for the binding at the was by the of containing in 2 at for The and the a containing the of the was by the Fluor de and in the is on the are used in the of and the M.T. Denu J.M. 2004; PubMed Scopus Google The with acid to a of a The substrates and using of of the was by the Fluor de and p53 with using the binding to resveratrol the binding of the Fluor de or p53 The binding was with of the Fluor de or p53 with and resveratrol. The and the of was as M.T. Denu J.M. 2004; PubMed Scopus Google Scholar). of p53-AMC and was with The was using to form an The was by using a with to in that was used of was in and with and in was for at and in The and with and the was by HPLC, with of in The product, was to a in MALDI for from L. and R. of was to in a to the p53-AMC MALDI for of SIRT1 and SIRT1 crystal was by using the in N. M.C. 1997; PubMed Scopus Google Scholar). The p53-AMC peptide was from the crystal of p53 bound to J.L. Celic I. Muhammad S. Boeke J.D. Wolberger C. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Using an was the of and the of the of p53 bound to The SIRT1 and the containing the p53-AMC peptide in N. M.C. 1997; PubMed Scopus Google using the p53-AMC peptide the SIRT1 The crystal of p53-AMC peptide bound to SIRT1 was used for SIRT1 Sir2 resveratrol Sir2 ySir2, human and human SIRT1 using the commercially available Fluor de as a peptide with and resveratrol. with the with nicotinamide, and the was to the SIRT1, and ySir2, deacetylation of the Fluor de peptide of the of increase in the presence and of resveratrol that resveratrol SIRT1 by with studies using this (35Howitz K.T. Bitterman K.J. Cohen H.Y. Lamming D.W. Lavu S. Wood Chung P. A. Sinclair D.A. Nature. 2003; PubMed Scopus Google Scholar). no significant activation was and with the Fluor de a of was to significant deacetylation of the that Fluor de peptide is a for resveratrol activation was for SIRT1, studies using this Fluor de as a resveratrol activation of SIRT1, an was The binding M.T. Denu J.M. 2004; PubMed Scopus Google was the as for the Fluor de Lys that the Fluor de peptide was by peptide, to the of the histone including and the acetylated In the binding the from the is to the of NAD+ to form is as M.T. Denu J.M. 2004; PubMed Scopus Google Scholar). The with and resveratrol, and the of and shown in the the of was in the presence and of resveratrol, that resveratrol no on the was as a and the binding was of the from the Fluor de Lys and the binding for resveratrol activation To resveratrol an increase in the deacetylated Fluor de peptide was in the SIRT1 with and resveratrol. The was and no with and resveratrol was that resveratrol increase the of the fluorophore. To resveratrol the SIRT1 resveratrol. resveratrol was with the The of increase was in the presence and of the resveratrol that the resveratrol activation from activation of the To examine the that resveratrol activation the of the Fluor de peptide, an was with the Fluor de In this was used as the at from the by HPLC, and of the and NAD+ was by and a of the that resveratrol SIRT1 by an of with the activation in the Fluor de Lys p53-AMC and for of the Fluor de Lys and kit are from of the Fluor de Lys are is most the Fluor de Lys is to D. F. D. A. Chem. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, J. N. A. Biochem. 2004; PubMed Scopus Google Scholar), of a cleavage the attached fluorophore and the The Fluor de is a peptide on the p53 sequence, with a fluorophore at the to the acetylated this in we synthesized fluorophore-containing p53 the p53 sequence, with either 7-amino-4-methylcoumarin (p53-AMC) or rhodamine 110 and and to the acetylated the synthesized p53-AMC peptide was tested as a for SIRT1, and to resveratrol activation was using p53-AMC peptide and NAD+ in the presence and of resveratrol. shown in the deacetylation of the p53-AMC The of resveratrol the of by This that only the p53-AMC peptide be used in the that to the Fluor de peptide, resveratrol synthesized p53-AMC peptide was used to for the of resveratrol the synthesized peptide was used as a in the SIRT1 The p53 covalently attached to rhodamine and the as the p53-AMC peptide The with peptide and in the presence and of resveratrol. shown in the rhodamine in the presence of SIRT1, deacetylation of the The of resveratrol in the the of by a significant The and Fluor de the to be deacetylated more by SIRT1 in the presence of resveratrol. The of Fluor de and for the that and Fluor de AcH3, resveratrol activation that the either the peptide or the for resveratrol To the of the p53 peptide was for the Fluor de was from The Fluor de is a peptide on the of histone with the acetylated to the fluorophore as Fluor de The Fluor de Lys was using Fluor de in the presence and of resveratrol. Using SIRT1, the deacetylation of the of the of increase that resveratrol SIRT1 by was in to the activation using Fluor de and p53-AMC The of the peptide, was for resveratrol To resveratrol activation the a p53 peptide with the as Fluor de and was used as a SIRT1 in the presence and of resveratrol. using p53 peptide and and as The of the presence of resveratrol no significant on the of that the fluorophore was to resveratrol To of the fluorophore is for resveratrol using the p53 peptide in the presence and of AMC, resveratrol, or and resveratrol the and by shown in the of deacetylation of the p53 peptide, the of resveratrol and AMC, or activation on the of that resveratrol activation on the presence of that is covalently as in the p53-AMC of the Fluor de and p53-AMC to was shown that resveratrol the for the Fluor de peptide the of the enzyme (35Howitz K.T. Bitterman K.J. Cohen H.Y. Lamming D.W. Lavu S. Wood Chung P. A. Sinclair D.A. Nature. 2003; PubMed Scopus Google Scholar). is that resveratrol binding of the fluorophore-containing peptide the of the To this a competition was in Fluor de was used as a in a SIRT1 as The was in the presence of a of and NAD+ with of Fluor de in the presence and of resveratrol. In this competition of Fluor de with in a in the of resveratrol binding of Fluor de peptide to SIRT1, was that the of The of was and the of the shown the Fluor de peptide with as a In the presence of resveratrol, a of was that the Fluor de peptide affinity for the enzyme in the presence of resveratrol. Substrate competition using the p53-AMC and p53 as binding of in the presence and of resveratrol. shown in the p53-AMC and p53 decreased the of the p53-AMC peptide in the of resveratrol was the that of the affinity with binding of the p53 no in the competition the p53 peptide was that resveratrol increase the binding of p53 peptide in the presence of resveratrol, the of p53-AMC to with was to a to that with the p53 peptide the competition is that resveratrol the binding affinity for the for lacking a fluorophore. To the resveratrol binding of SIRT1 for Fluor de peptide, the for Fluor de and p53 with and resveratrol, from in the presence of of p53 or Fluor de as at of in the presence and of resveratrol. Using the binding and for the the is a and the to the on the the as R.A. to and Scholar). The for the Fluor de peptide and in the and presence of resveratrol, the p53 peptide, the and in the and presence of resveratrol, The binding of Fluor de peptide in the presence of resveratrol that the resveratrol activation is the of the affinity of the p53-AMC and Fluor de The of a resveratrol with p53 peptide that the covalently attached fluorophore in the of is to the binding affinity by resveratrol. that resveratrol a conformational change in SIRT1 better accommodates the binding of coumarin or peptide as in for the the of the coumarin in the p53-AMC peptide for resveratrol we a model of activation on the available no crystal of SIRT1 was a model was using the in N. M.C. 1997; PubMed Scopus Google Scholar). The crystal of a p53 peptide bound to J.L. Celic I. Muhammad S. Boeke J.D. Wolberger C. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google to a model of the p53-AMC peptide a SIRT1 by the using that the presence of coumarin at the of the p53 peptide is for and the of resveratrol to for this by the binding of the p53-AMC the crystal the coumarin is solvent-exposed to resveratrol binding In this the of coumarin are in the on the of the In addition, the coumarin significant contacts to the enzyme that This for binding affinity for p53-AMC with the p53 peptide activation of SIRT1 to be a binding as indicated by the decreased and decreased (35Howitz K.T. Bitterman K.J. Cohen H.Y. Lamming D.W. Lavu S. Wood Chung P. A. Sinclair D.A. Nature. 2003; PubMed Scopus Google Scholar). We that resveratrol to SIRT1 and a protein change the coumarin of bound p53-AMC This a binding to the attached coumarin in the binding of p53-AMC that with coumarin are and are in either or The presence of on that to fluorophore The binding for resveratrol and a is that resveratrol to SIRT1 in the the coumarin binding and with the coumarin The that resveratrol the peptide, of resveratrol with the rhodamine and coumarin are resveratrol to an from the coumarin binding This be the novel extension of SIRT1. binding to an conformational in SIRT1 the coumarin of p53-AMC peptide to more human SIRT1 Sir2 homologs (yeast Sir2 and human Although activation was independent of peptide sequence, SIRT1 activation a fluorophore-containing In the of resveratrol, the fluorophore binding of the peptide to the We propose that the binding of resveratrol to SIRT1 a conformational change in the enzyme fluorophore binding in the of the peptide for the of resveratrol as an in vivo activator of Sir2 homologs from a of In resveratrol activation to be for SIRT1, to the studies yeast Sir2 and resveratrol to cellular processes, such as life span increase and gene at this is the activation of SIRT1 by resveratrol an of SIRT1. as a to SIRT1 and an SIRT1. SIRT1 for a acetylated or a affinity for this SIRT1 is reported to harbor no on J. E. T. M. Guarente L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), SIRT1 the of an activator to of this and
The emergence of audio and video data types in databases will require new information retrieval methods adapted to the specific characteristics and needs of these data types. An effective and natural way of querying a musical audio database is by humming the tune of a song. In this paper, a system for querying an audio database by humming is described along with a scheme for representing the melodic information in a song as relative pitch changes. Relevant difficulties involved with tracking pitch are enumerated, along with the approach we followed, and the performance results of system indicating its effectiveness are presented.