Elucidating mechanisms of SIRT1 activation

阐明 SIRT1 激活机制

基本信息

批准号：
9315825
负责人：
DAVID A. SINCLAIR
金额：
$ 50.83万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-07-15 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9315825
关键词：
Acetylation Adult Amino Acid Substitution Amino Acids Animals Antibodies Apoptotic Autophagocytosis Binding Biological Biological Assay Biology Canis familiaris Cardiovascular Diseases Cells Chemicals Complex Consensus Consensus Sequence DNA Repair Deacetylase Deacetylation Deletion Mutation Deuterium Energy Intake Enzyme Activation Enzyme Inhibition Enzyme Inhibitor Drugs Enzymes Event Exercise Family Fatty Acids Fatty Liver Gene Expression Genetic Genetic Screening Glutamine Health Benefit High Fat Diet Human Hydrogen Hydrophobicity Hypoxia In Vitro Inflammation Knock-in Mouse Knowledge Laboratories Link Lysine Maps Mass Spectrum Analysis Measures Mediating Medicine Metabolic Metabolic Diseases Methods Mitochondria Modern Medicine Molecular Multienzyme Complexes Mus Muscle Cells Mutation N-terminal Nerve Degeneration Non-Insulin-Dependent Diabetes Mellitus Obesity Organism Pattern Pharmacology Phenotype Physiological Physiology Plants Play Positioning Attribute Process Proteins Proteome Proteomics Reaction Recording of previous events Reporting Resveratrol Role SIRT1 gene Sirtuins Site Structural Protein Structure TP53 gene Technology Testing Time Tissues Work comparative experimental study fluorophore glucose metabolism in vivo innovation insight insulin sensitivity muscle metabolism mutant new technology novel reconstitution response small molecule

项目摘要

Project Summary 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-1α, 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.

项目概要过去 75 年中获得的有关小分子酶抑制的知识已构成以下基础：然而，现代医学对小分子如何激活酶的了解却少之又少。这种知识的缺乏限制了我们可以开发的 NAD+- 药物的范围。依赖性脱乙酰酶被认为是为了增加生物体的生存机会而进化的哺乳动物有七种去乙酰化酶，SIRT1-7 是研究最多和协调的。中央过程，包括 DNA 修复、脂肪酸和葡萄糖代谢、线粒体功能、缺氧反应、自噬和抗凋亡机制。已描述了超过 100 种 SIRT1 激活分子 (STAC)，包括白藜芦醇和 SRT1720。这些分子对基因表达产生相似的影响，并赋予多种健康益处包括预防 II 型糖尿病、肥胖、肝脂肪变性、炎症、心血管疾病和但这些影响是由于 SIRT1 的直接激活还是其他机制所致。受到热烈争论。尽管 STAC 是通过各种测定和不同的底物发现的，但我们的初步结果表明实际上存在一种共同的激活机制我们已经确定了一种结构化的激活。 SIRT1 N 末端的结构域 (AD) 与这些小分子结合，特别是其中的一个氨基酸该结构域 (E230) 是体外和细胞内超过 100 个 STAC 激活 SIRT1 所必需的。在原代细胞中替代 SIRT1-E230 可阻断白藜芦醇和合成 STAC 的作用，表明我们还表明，与 SIRT1 底物连接的荧光团会增强直接激活。体外激活，因为它们模仿内源底物中的疏水性氨基酸，例如 PGC-1α， FOXO3a 和 eIF2a 激活共有靶序列 (X6-K(Ac)[Y,W,F]-X5、X6- K(Ac)X5-[Y,W,F]) 使我们能够预测哪些底物将在体内受到 SIRT1 激活的调节。在这项研究中，我们将利用这些新发现来确定机械和结构 SIRT1 是如何被激活的。我们将生成原代细胞并利用带有 SIRT1-E230K 的敲入小鼠。突变（小鼠中的 E222K）以确定哪些生物效应是由于体内 SIRT1 激活所致。总之，这些研究旨在提供关于蛋白质修饰如何发生的基本机制见解。酶识别细胞中的特定靶标以及如何在体内调节它们的酶活性。提供关于复杂酶如何工作以及它们如何被小分子靶向的基本见解分子。