Rejuvenating Sirtuins


So what is it about those so called life span extending enzymes? It was in the late 1990’s when the Silent Information Regulator 2 (SIR2) gene was shown to extend the life span of yeast (1), an effect that was reported in worms and flies shortly after (2, 3). Since then, SIR2-like genes (named sirtuins) were identified in many other species (4, 5) and spurred studies about their life span extending effects in mammals. The possibility of small-molecule sirtuin activators that would increase human health span (or even life span) captivated many researchers and dramatically increased attention towards this enzyme family. But what are sirtuins? And what has red wine got to do with them?

Sirtuins are a highly conserved class of NAD+-dependent lysine deacylases that belong to the family of histone deacetylases (HDACs). Seven different sirtuins are identified in mammals (sirtuin 1 to sirtuin 7) that mostly catalyse a deacetylation reaction using NAD+ as cofactor (6). As the NAD+/NADH ratio represents the energy state of a cell, sirtuins readily react to an altered metabolism and in doing so strongly influence and regulate the metabolic state of the cell (7). Thus, it is not surprising that the identification of the role of sirtuin in metabolic disorders and how sirtuins can be targeted respectively have received much interest. Most research focused on the first member of the sirtuin family, sirtuin 1, but also on sirtuin 3 that is localized in mitochondria. For instance, reduced levels of sirtuin 1 has been associated with type 2 diabetes in humans and mice (8, 9). Activation of these enzymes can therefore have favourable physiological effects and not surprisingly numerous sirtuin activators (STACs) have been described and synthesised. And here is where our short story begins.

Lucas 3-2-16 Picture 1

Figure 1 | Potential physiological benefits of STACs in the treatment of age associated diseases. Picture from (10).

It was in 2003, under Harvard investigator David Sinclair, when resveratrol was identified as sirtuin 1 activator (11). Resveratrol, which is a natural compound found in red wine reportedly increased DNA stability in yeast and extended their life span by (stunningly) 70 % through activation of Sir2 (as mentioned, the yeast sirtuin analogue). For wine enthusiasts this was delightful news, unfortunately, the biochemical assay used to identify resveratrol was shortly after called into question. The original study used an enzymatic assay that contained a fluorescently labelled peptide substrate (Fluor de Lys) and that demonstrated activation of sirtuin 1 by the red wine polyphenol. However, other studies reported activation only in the presence of covalently bound fluorophore on the substrate and not by resveratrol itself (12, 13). Not until 2013 this issue should be resolved and be demonstrated that sirtuin 1 can actually be activated by resveratrol (under certain conditions). In the meanwhile, Sirtris Pharmaceuticals, Inc. was founded in 2004 by David Sinclair and bought in 2008 by GlaxoSmithKline (GSK) for $720 million. This move of GSK seemed rather surprising as the development of sirtuin activators as drugs had no strong foundation and even GSK internal Scientists failed to substantiate Sirtris’s claims (14). Especially after the (pharma giant) Pfizer report in 2010 (15) that concluded that resveratrol, as well as other STACs such as SRT1720 do not directly activate sirtuin 1, schadenfreude on the part of pharma watchers about GSK’s investment can be pictured.

Now, do resveratrol and other STACs actually activate sirtuin 1? This controversy should be resolved in 2013 by two studies, one led by David Sinclair (16) and the other by Clemens Steegborn (17). Sinclair’s et al. hypothesis was that the bulky and hydrophobic fluorophore used in the enzymatic assay mimics endogenous substrates required for sirtuin 1 activation by STACs (16). In fact, the fluorophore covalently bound to the peptide lowered the peptide Michaelis constant Km in response to sirtuin 1 activation by STACs. Substitution of the fluorophore with naturally occurring hydrophobic amino acids still supported activation. Based on their results, Sinclair’s group suggested an “assisted allosteric mechanism” in which STACs activate sirtuin 1 only with unique peptide substrates and concluded that “allosteric activation of SIRT1 by STACs remains a viable therapeutic intervention strategy for many diseases associated with aging” (16). Conveniently, Sirtris Pharmaceuticals gets integrated into GSK’s R&D in the same year of the Sinclair’s publication (14).

Lucas 3-2-16 Picture 2

Figure 2 | Assisted allosteric activation by STACs requires a glutamic acid residue (Glu230) in proximity to the catalytic core of sirtuin 1 and a specific hydrophobic motif of the endogenous substrate such as found in peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α). In detail, deacetylation by sirtuin1 is dependent on the presence of hydrophobic amino acids at positions +1 and +6 relative to the acetylated lysine on PGC-1α.

This brings us up to date and with interest the progress of Sirtris Pharmaceutical within GSK will be followed, as well as the outcome of their clinical trials. Activation of sirtuins in the treatment of age associated and metabolic disorders such as type 2 diabetes by small molecules is a promising approach. Interestingly, the general public seems in this regard already one step ahead and identified these rejuvenating enzymes for themselves. Sirtuin gene activating diets praise a “revolutionary plan for health & weight loss” (see “the sirt food diet” by Aidan Goggins), however, if these diets activate sirtuins is questionable. Same counts for resveratrol in red wine that in given concentrations probably does not activate sirtuins in humans (18). Nevertheless, we (wine enthusiasts – the author of this blog included) may continue to believe so. Cheers!

Blog written by Lucas Kraft

References

  1. Kaeberlein, M., McVey, M., and Guarente, L. (1999) The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms. Genes Dev. 13, 2570–80
  2. Rogina, B., and Helfand, S. L. (2004) Sir2 mediates longevity in the fly through a pathway related to calorie restriction. Proc. Natl. Acad. Sci. U. S. A. 101, 15998–6003
  3. Tissenbaum, H. A., and Guarente, L. (2001) Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans. Nature. 410, 227–30
  4. Frye, R. A. (2000) Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins. Biochem. Biophys. Res. Commun. 273, 793–8
  5. Frye, R. A. (1999) Characterization of five human cDNAs with homology to the yeast SIR2 gene: Sir2-like proteins (sirtuins) metabolize NAD and may have protein ADP-ribosyltransferase activity. Biochem. Biophys. Res. Commun. 260, 273–9
  6. Hirschey, M. D. (2011) Old enzymes, new tricks: sirtuins are NAD(+)-dependent de-acylases. Cell Metab. 14, 718–9
  7. Zhong, L., and Mostoslavsky, R. (2011) Fine tuning our cellular factories: sirtuins in mitochondrial biology. Cell Metab. 13, 621–6
  8. Orimo, M., Minamino, T., Miyauchi, H., Tateno, K., Okada, S., Moriya, J., and Komuro, I. (2009) Protective role of SIRT1 in diabetic vascular dysfunction. Arterioscler. Thromb. Vasc. Biol. 29, 889–94
  9. Cardellini, M., Menghini, R., Martelli, E., Casagrande, V., Marino, A., Rizza, S., Porzio, O., Mauriello, A., Solini, A., Ippoliti, A., Lauro, R., Folli, F., and Federici, M. (2009) TIMP3 is reduced in atherosclerotic plaques from subjects with type 2 diabetes and increased by SirT1. Diabetes. 58, 2396–401
  10. Lavu, S., Boss, O., Elliott, P. J., and Lambert, P. D. (2008) Sirtuins — novel therapeutic targets to treat age-associated diseases. Nat. Rev. Drug Discov. 7, 841–853
  11. Howitz, 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., and Sinclair, D. A. (2003) Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature. 425, 191–6
  12. Borra, M. T., Smith, B. C., and Denu, J. M. (2005) Mechanism of human SIRT1 activation by resveratrol. J. Biol. Chem. 280, 17187–95
  13. 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., and Kennedy, B. K. (2005) Substrate-specific activation of sirtuins by resveratrol. J. Biol. Chem. 280, 17038–45
  14. Ledford, H. (2013) GSK absorbs controversial “longevity” company. [online] http://blogs.nature.com/news/2013/03/gsk-absorbs-controversial-longevity-company.html (Accessed January 20, 2016)
  15. Pacholec, M., Bleasdale, J. E., Chrunyk, B., Cunningham, D., Flynn, D., Garofalo, R. S., Griffith, D., Griffor, M., Loulakis, P., Pabst, B., Qiu, X., Stockman, B., Thanabal, V., Varghese, A., Ward, J., Withka, J., and Ahn, K. (2010) SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1. J. Biol. Chem. 285, 8340–51
  16. Hubbard, 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., Pentelute, B. L., Schuman, E. R., Stevens, L. A., Ling, A. J. Y., Armour, S. M., Michan, S., Zhao, H., Jiang, Y., Sweitzer, S. M., Blum, C. A., Disch, J. S., Ng, P. Y., Howitz, K. T., Rolo, A. P., Hamuro, Y., Moss, J., Perni, R. B., Ellis, J. L., Vlasuk, G. P., and Sinclair, D. A. (2013) Evidence for a common mechanism of SIRT1 regulation by allosteric activators. Science. 339, 1216–9
  17. Lakshminarasimhan, M., Rauh, D., Schutkowski, M., and Steegborn, C. (2013) Sirt1 activation by resveratrol is substrate sequence-selective. Aging (Albany. NY). 5, 151–4
  18. Corder, R., Crozier, A., and Kroon, P. A. (2003) Drinking your health? It’s too early to say. Nature. 426, 119–119
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