Knowledge gained over the past 75 years on enzyme inhibition by small molecules has formed the basis of modern medicine. However, our understanding of how enzymes can be activated by small molecules is far less advanced. This lack of knowledge limits the scope of medicines we can develop. Sirtuins are a family of NAD+- dependent deacetylases that are thought to have evolved to increase an organism's chances of surviving adverse conditions. There are seven mammalian sirtuins, SIRT1-7. SIRT1 is the best studied and coordinates central processes including DNA repair, fatty acid and glucose metabolism, mitochondrial function, hypoxic responses, autophagy, and anti-apoptotic mechanisms. Over 100 SIRT1 activating molecules (STACs), including resveratrol and SRT1720, have been described. These molecules produce similar effects on gene expression and impart a diverse array of health benefits including protection from type II diabetes, obesity, hepatic steatosis, inflammation, cardiovascular disease, and neurodegeneration. But whether these effects are due to direct SIRT1 activation or an alternative mechanism is hotly debated. Even though STACs were discovered using a variety of assays and different substrates, our preliminary results indicate that there is in fact a common mechanism of activation. We have identified a structured activation domain (AD) in the N-terminus of SIRT1 where these small molecules bind, and in particular one amino acid in this domain (E230) that is required for SIRT1 activation by over 100 STACs both in vitro and in cells. Substituting SIRT1-E230 in primary cells blocks the effects of resveratrol and synthetic STACs indicating that direct activation is a mechanism. We also show that fluorophores linked to SIRT1 substrates enhance activation in vitro because they mimic hydrophobic amino acids in endogenous substrates, such as PGC-1?, FOXO3a, and eIF2a. The identification of a consensus target sequence for activation (X6-K(Ac)[Y,W,F]-X5, X6- K(Ac)X5-[Y,W,F]) has allowed us to predict which substrates will be modulated by SIRT1 activation in vivo. In this study, we will take advantage of these new discoveries to determine mechanistically and structurally how SIRT1 is activated. We will generate primary cells and utilize knock-in mouse with the SIRT1-E230K mutation (E222K in mice) to determine which of the biological effects are due to SIRT1 activation in vivo. Together, these studies are aimed at providing fundamental mechanistic insights into how protein-modifying enzymes recognize specific targets in cells and how their enzymatic activity may be modulated in vivo. This will provide fundamental insights into how complex enzymes work and how they might be targeted by small molecules.
The sirtuins are some of the most mechanistically complicated enzymes known, playing important roles in the body's physiological responses to energy intake and exercise. But whether they can be directly activated by small molecules is hotly debated. This study will make use of a novel SIRT1 mutant we have discovered (hE230K/mE222K), which inhibits SIRT1 activation by small molecules. By assessing the effect of this mutation on SIRT1 structure and the effects of small molecule activators in primary cells and mice carrying this mutation, these studies will elucidate fundamental mechanisms of allosteric enzyme activation and resolve the debate as to whether sirtuins can be targeted directly for the treatment of type 2 diabetes and other metabolic diseases.
|Mitchell, Sarah J; Madrigal-Matute, Julio; Scheibye-Knudsen, Morten et al. (2016) Effects of Sex, Strain, and Energy Intake on Hallmarks of Aging in Mice. Cell Metab 23:1093-1112|
|Van Meter, Michael; Simon, Matthew; Tombline, Gregory et al. (2016) JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks. Cell Rep 16:2641-2650|
|Schultz, Michael B; Sinclair, David A (2016) Why NAD(+) Declines during Aging: It's Destroyed. Cell Metab 23:965-966|
|Wu, Lindsay E; Sinclair, David A (2016) Restoring stem cells - all you need is NAD(.). Cell Res 26:971-2|
|Bonkowski, Michael S; Sinclair, David A (2016) Slowing ageing by design: the rise of NAD(+) and sirtuin-activating compounds. Nat Rev Mol Cell Biol 17:679-690|