Rejuvenating SIRT1 Activators
2013; Cell Press; Volume: 17; Issue: 5 Linguagem: Inglês
10.1016/j.cmet.2013.04.016
ISSN1932-7420
Autores Tópico(s)Biochemical effects in animals
ResumoWhether the red wine component resveratrol directly activates the NAD+-dependent protein deacetylase SIRT1 has been debated. A recent study by Hubbard et al., 2013Hubbard B.P. Gomes A.P. Dai H. Li J. Case A.W. Considine T. Riera T.V. Lee J.E. E. S.Y. Lamming D.W. et al.Science. 2013; 339: 1216-1219Crossref PubMed Scopus (417) Google Scholar strengthens the case that SIRT1-activating compounds (STACs) function as positive allosteric regulators of SIRT1 and thereby regulate mitochondrial function. Whether the red wine component resveratrol directly activates the NAD+-dependent protein deacetylase SIRT1 has been debated. A recent study by Hubbard et al., 2013Hubbard B.P. Gomes A.P. Dai H. Li J. Case A.W. Considine T. Riera T.V. Lee J.E. E. S.Y. Lamming D.W. et al.Science. 2013; 339: 1216-1219Crossref PubMed Scopus (417) Google Scholar strengthens the case that SIRT1-activating compounds (STACs) function as positive allosteric regulators of SIRT1 and thereby regulate mitochondrial function. A small molecule that effectively delays the onset of aging-associated diseases, such as neurodegeneration, diabetes, and cancer, has the potential to revolutionize modern medicine. One candidate molecule is resveratrol, an antioxidant present in red wine discovered to be a naturally occurring SIRT1 activator. Resveratrol extends life span in yeast, worms, and flies (Wood et al., 2004Wood J.G. Rogina B. Lavu S. Howitz K. Helfand S.L. Tatar M. Sinclair D. Nature. 2004; 430: 686-689Crossref PubMed Scopus (1483) Google Scholar) and improves metabolic parameters in aged mice and obese humans (Baur et al., 2006Baur 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. et al.Nature. 2006; 444: 337-342Crossref PubMed Scopus (3433) Google Scholar; Timmers et al., 2011Timmers S. Konings E. Bilet L. Houtkooper R.H. van de Weijer T. Goossens G.H. Hoeks J. van der Krieken S. Ryu D. Kersten S. et al.Cell Metab. 2011; 14: 612-622Abstract Full Text Full Text PDF PubMed Scopus (867) Google Scholar). Soon after the seminal report showing that resveratrol activates SIRT1, novel synthetic SIRT1 activators were identified and further developed to reduce off-target effects and improve their pharmacokinetic properties. However, a number of controversies arose regarding the mechanism of action of resveratrol and synthetic SIRT1-activating compounds (STACs). Of significant concern was the demonstration that STACs and resveratrol enhanced SIRT1 deacetylase activity toward peptide substrates covalently bound to a fluorophore but not toward native peptide substrates (Pacholec et al., 2010Pacholec M. Bleasdale J.E. Chrunyk B. Cunningham D. Flynn D. Garofalo R.S. Griffith D. Griffor M. Loulakis P. Pabst B. et al.J. Biol. Chem. 2010; 285: 8340-8351Crossref PubMed Scopus (704) Google Scholar). This discrepancy raised significant questions on the validity of resveratrol and STACs as direct SIRT1 activators in vivo. However, subsequent studies showed that STAC treatment offered remarkable protective benefits against cardiovascular and metabolic disease as well as locomotor dysfunction in aged mice, revealing some discrepancy between the in vitro and in vivo effects (Baur et al., 2012Baur J.A. Ungvari Z. Minor R.K. Le Couteur D.G. de Cabo R. Nat. Rev. Drug Discov. 2012; 11: 443-461Crossref PubMed Scopus (289) Google Scholar). To address this controversy, Sinclair and his team (who published the original resveratrol/SIRT1 story [Howitz et al., 2003Howitz 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. et al.Nature. 2003; 425: 191-196Crossref PubMed Scopus (2962) Google Scholar]), set out to re-evaluate their previous findings by testing a clever alternative hypothesis. Could the failure of STACs to deacetylate native, nontagged peptides in vitro be explained by a requirement for the structure mimicked by the fluorophore tag? In other words, was the bulky, hydrophobic fluorophore mimicking properties of endogenous substrates required for SIRT1 activation by STACs? Two independent assays, one enzymatic and the other mass-spectrometry based, were used to interrogate SIRT1 deacetylation kinetics. As correctly predicted, they found that the fluorophore lowered the Michaelis constant (Km) in response to STACs only when located directly adjacent to the acetylated lysine 9 of histone 3 (H3K9) at the +1 position. Furthermore, a fluorophore moiety at positions +1 and +6 could be substituted with naturally occurring bulky hydrophobic amino acids such as tryptophan, tyrosine, or phenylalanine, and still support SIRT1 activation by resveratrol. Importantly, STAC-mediated lowering of the peptide Km toward two endogenous SIRT1 substrates, peroxisome proliferator-activated receptor γ coactivator 1 α lysine 778 (PGC-1α-K778) and human forkhead box O3a protein lysine 290 (FOXO3a-K290), also depended on hydrophobic amino acids at positions +1 and +6 (PGC1-α) and +1 (FOXO3a). A screen for a peptide consensus motif incorporating these amino acids in proximity to a lysine revealed 400 matching sequences within the nuclear proteome. Five of these peptides were tested and it was found that their deacetylation by SIRT1 was enhanced by STACs. These results suggest an “assisted allosteric mechanism,” in which STACs activate SIRT1 only in the context of unique peptide substrates (Figure 1). Importantly, a mutagenesis screen further showed that substitution of glutamic acid 230 (Glu230) of SIRT1 with lysine or alanine attenuated resveratrol-induced SIRT1 enzymatic activity, both in fluorophore-tagged and native peptide sequences. Testing of a panel of 117 STACs confirmed the global requirement of Glu230 for allosteric activation of SIRT1 by members of this drug class, suggesting a shared mechanism. Finally, to translate these findings into mammalian physiology, primary myoblasts and primary embryonic fibroblasts from SIRT1 knockout mice were reconstituted with either wild-type or mutated SIRT1 (SIRT1-E222K, the murine equivalent of human SIRT1-E230K). Importantly, this single amino-acid substitution in SIRT1 completely suppressed the effect of resveratrol in promoting increased mitochondrial mass and ATP content. These experiments support the model that STACs activate SIRT1 directly and that some of the biological effects of resveratrol and STACs are strictly dependent on SIRT1 (Figure 1). Having re-established SIRT1 as an important target of resveratrol and STACs, the field can now focus again on key remaining questions. Are sirtuins valid and safe targets to extend health and life span? While controversy has also surrounded the role of sirtuin in aging, recent work brings renewed support for a role of sir2 in Drosophila aging (Banerjee et al., 2012Banerjee K.K. Ayyub C. Ali S.Z. Mandot V. Prasad N.G. Kolthur-Seetharam U. Cell Rep. 2012; 2: 1485-1491Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Moving forward, it will be important to determine which of the biological effects of resveratrol are mediated by SIRT1 and what other cellular targets mediate its SIRT1-independent effects. Given its small size (MW = 228 Da), it is quite likely that resveratrol regulates the activity of other cellular proteins in addition to SIRT1. Indeed, resveratrol binds to a hydrophobic pocket of the F1-ATPase and inhibits this key enzyme in mitochondrial respiration (Gledhill et al., 2007Gledhill J.R. Montgomery M.G. Leslie A.G.W. Walker J.E. Proc. Natl. Acad. Sci. USA. 2007; 104: 13632-13637Crossref PubMed Scopus (277) Google Scholar). Resveratrol also inhibits several phosphodiesterase (PDE) isoforms in vitro and shares several biological effects with the selective PDE4 inhibitor rolipram when administered to obese mice (Park et al., 2012Park S.-J. Ahmad F. Philp A. Baar K. Williams T. Luo H. Ke H. Rehmann H. Taussig R. Brown A.L. et al.Cell. 2012; 148: 421-433Abstract Full Text Full Text PDF PubMed Scopus (956) Google Scholar). In this study, resveratrol and rolipram are postulated to indirectly activate AMP-activated protein kinase (AMPK) via a cAMP/Epac1/CamKKβ axis. The canonical function of AMPK is to sense high AMP:ATP ratios during energetic stress. Several homeostatic responses, including an increase in mitochondrial biogenesis through PGC-1α, are initiated by AMPK signaling and promote metabolic health. Since AMPK is activated in conditions when ATP levels are reduced, this pathway may also be triggered through inhibition of F1-ATPase by resveratrol. The variety of cellular targets for resveratrol may explain its pleiotropic pharmacological effects and complicate the analysis of its effects on the complex trait of aging. The existence of a SIRT1 allosteric site distinct from the catalytic pocket also leads to the exciting hypothesis that endogenous metabolites or regulatory protein(s) may target this site. Candidate metabolites are likely to undergo dynamic changes during calorie restriction and feeding-fasting cycles. The discovery of a pharmacological equivalent of the fountain of youth still faces a long and uncertain path ahead. Nevertheless, the report by Hubbard et al., 2013Hubbard B.P. Gomes A.P. Dai H. Li J. Case A.W. Considine T. Riera T.V. Lee J.E. E. S.Y. Lamming D.W. et al.Science. 2013; 339: 1216-1219Crossref PubMed Scopus (417) Google Scholar brings renewed focus on the role of SIRT1 as an important target for calorie restriction mimetics and opens new avenues to further explore SIRT1 activation as a means to promote healthy aging.
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