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75年中获得的关于小分子抑制酶的知识已经形成了 现代医学然而，我们对酶如何被小分子激活的理解还远远不够 先进这种知识的缺乏限制了我们可以开发的药物的范围。Sirtuins是NAD+-家族 一种依赖性脱乙酰酶，被认为是为了增加生物体的生存机会而进化的 不利条件。哺乳动物有7种sirtuins，SIRT 1 -7。SIRT 1是最好的研究和协调 中枢过程包括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。 已经描述了超过100种SIRT 1激活分子（STAC），包括白藜芦醇和SRT 1720。 这些分子对基因表达产生类似的影响，并赋予多种健康益处 包括防止II型糖尿病、肥胖症、肝脂肪变性、炎症、心血管疾病， 神经变性但是这些效应是由于SIRT 1的直接激活还是其他机制 被激烈讨论。 尽管STAC是使用各种检测和不同底物发现的，但我们的初步结果表明， 表明实际上存在共同激活机制。我们发现了一种结构性激活 这些小分子结合的SIRT 1的N-末端的结构域（AD），并且特别是SIRT 1中的一个氨基酸， 该结构域（E230）是体外和细胞中超过100种STAC激活SIRT 1所需的。 在原代细胞中取代SIRT 1-E230可以阻断白藜芦醇和合成STAC的作用，这表明 直接激活是一种机制。我们还表明，与SIRT 1底物连接的荧光团增强了 体外活化，因为它们模拟内源性底物中的疏水氨基酸，如PGC-1α， FOXO 3a和eIF 2a。用于活化的共有靶序列（X6-K（Ac）[Y，W，F]-X5，X6-K（Ac）[Y，W，F]-X5）的鉴定 K（Ac）X5-[Y，W，F]）的研究使我们能够预测哪些底物将在体内被SIRT 1激活所调节。 在这项研究中，我们将利用这些新发现，以确定机制和结构， SIRT 1是如何被激活的我们将产生原代细胞并利用SIRT 1-E230 K敲入小鼠 突变（小鼠中的E222 K），以确定哪些生物学效应是由于体内SIRT 1激活。 总之，这些研究的目的是提供基本的机械见解如何蛋白质修饰 酶识别细胞中的特定靶标以及它们的酶活性如何在体内被调节。这将 提供了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。