Elucidating mechanisms of SIRT1 activation

阐明 SIRT1 激活机制

• 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
• 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

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.

项目摘要 在过去的75年中，对小分子抑制酶抑制的知识已经形成了 现代医学。但是，我们对如何通过小分子激活酶的理解要少得多 先进的。缺乏知识限制了我们可以开发的药物范围。 Sirtuins是NAD+的家庭 依赖的脱乙酰基酶被认为已经发展为增加生物的生存机会 不利条件。有七个哺乳动物Sirtuins，Sirt1-7。 SIRT1是最好的研究和坐标 中央过程包括DNA修复，脂肪酸和葡萄糖代谢，线粒体功能，低氧 反应，自噬和抗凋亡机制。 已经描述了超过100个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，并赋予潜水员的健康益处 包括保护II型糖尿病，肥胖，肝脂肪变性，炎症，心血管疾病和 神经变性。但是这些效果是由于直接SIRT1激活还是替代机制 被激烈辩论。 即使使用各种测定和不同的底物发现了Stacs，我们的初步结果 表明实际上存在一种常见的激活机制。我们已经确定了结构化激活 这些小分子结合的SIRT1的N末端中的域（AD），尤其是一个氨基酸 SIRT1在体外和细胞中激活100多个STAC所需的域（E230）。 在主细胞中代替SIRT1-E230，阻止白藜芦醇和合成性Stac的影响表明 直接激活是一种机制。我们还表明，与SIRT1底物相关的荧光团增强 在体外激活，因为它们模仿内源性底物中的疏水性氨基酸，例如PGC-1α， FOXO3A和EIF2A。鉴定激活共识目标序列（x6-k（ac）[y，w，f] -x5，x6- k（ac）x5- [y，w，f]）允许我们预测哪些底物将通过体内SIRT1激活调节。 在这项研究中，我们将利用这些新发现来机械和结构确定 SIRT1如何激活。我们将生成主要单元格，并使用SIRT1-E230K使用敲入小鼠 突变（小鼠中的E222K）确定哪种生物学作用是由于体内SIRT1激活引起的。 这些研究共同旨在提供有关蛋白质修饰的基本机理见解 酶识别细胞中的特定靶标以及如何在体内调节其酶活性。这会 提供有关复杂酶的工作方式以及如何针对小型酶的基本见解 分子。