Reversible Protein Acetylation and Chromatin Function

可逆蛋白质乙酰化和染色质功能

• 批准号: 8005210
• 负责人: JOHN M DENU
• 金额: $ 12.32万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-14 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8005210
• 关键词: ADP ribosylation; Acetate-CoA Ligase; Address; Age; Aging; Apoptosis; Binding; Biological; Biological Assay; Biological Process; Cardiovascular system; Cell physiology; Cells; Cellular Stress; Chemistry; Chromatin; Cleaved cell; Coupled; DNA Repair; Deacetylation; Dependence; Detection; Development; Disease; Drug Delivery Systems; Enzymatic Biochemistry; Enzymes; Family; Gene Silencing; Generations; Genetic; Goals; Health Benefit; Histones; Human; In Vitro; Investigation; Ion Channel; Knowledge; Ligands; Longevity; Malignant Neoplasms; Mammalian Cell; Measures; Metabolism; Metalloproteases; Molecular; Mono(ADP-Ribose) Transferases; Mono-S; Nerve Degeneration; Neurologic; O-Acetyl-ADP-Ribose; Organism; Pathway interactions; Peptide Library; Phenotype; Plants; Play; Protein Acetylation; Protein Family; Proteins; Publishing; Reaction; Reporting; Resistance; Resveratrol; Role; Screening procedure; Second Messenger Systems; Sirtuins; Substrate Specificity; Testing; Yeasts; analog; base; design; genetically modified cells; improved; in vivo; inhibitor/antagonist; novel; nudix hydrolase; polyphenol; repaired; research study; second messenger; small molecule; sulfated glycoprotein 2; tissue/cell culture; tool

DESCRIPTION (provided by applicant): The Sir2 (or sirtuin) protein family plays critical roles in aging, DMA repair, apoptosis, resistance to cell stress, and metabolism. However, the molecular basis for the varied phenotypes has remained elusive. The majority of sirtuins catalyze a reaction in which the cleavage of NAD+ and histone/protein deacetylation are coupled to the formation of O-acetyl-ADP-ribose (OAADPr), a novel metabolite. Several reports suggest that some sirtuins are mono-ADP-ribosyltransferases, while others have suggested that these enzymes harbor both activities. Detailed mechanistic studies have yet to validate these reports. The dependence on NAD+ and the generation of a potential second messenger offer clues toward understanding their cellular functions. Here, we will focus on answering questions of molecular mechanism, filling major gaps in our understanding of these unique proteins. To address their molecular and biological functions, this proposal outlines a multi-disciplinary approach, involving chemistry, enzymology and genetics. In Aim 1, the catalytic pathways that yield protein deacetylation versus ADP-ribosylation will be elucidated. Aim 2 focuses on understanding how sirtuins recognize specific protein targets. To explore the biological functions of OAADPr, Aim 3 will involve the synthesis of nonhydrolyzable OAADPr analogs, followed by an examination of their ability to act as ligands, substrates and inhibitors against reported protein targets. The experiments in Aim 4 directly probe the cellular function of OAADPr. An assay for the quantitation of cellular OAADPr levels will be coupled with cell- based assays of genetically modified cells. The ability of OAADPr to promote gene silencing in yeast will be assessed. In mammalian cells, the ability of sirtuins and OAADPr to modulate the gating of the TRPM2 ion channel will be determined. Resveratrol, plant polyphenol known to harbor a variety of cardiovascular and neurological health benefits, was reported to be an activator of Sir2 enzymes. Here several hypotheses for the ability of resveratrol to "activate" SIRT1 will be examined in vivo and in vitro (Aim 2). Given the implications for cancer, aging and neurodegeneration, sirtuins have emerged as viable drug targets to treat these diseases. The knowledge gained from this study will provide the major steps towards the design and development of such small molecules.

描述（由申请人提供）：Sir2（或 Sirtuin）蛋白家族在衰老、DMA 修复、细胞凋亡、细胞应激抵抗和新陈代谢中发挥着关键作用。然而，不同表型的分子基础仍然难以捉摸。大多数 Sirtuins 催化 NAD+ 裂解和组蛋白/蛋白质脱乙酰化与 O-乙酰基-ADP-核糖 (OAADPr)（一种新型代谢物）的形成相结合的反应。一些报告表明，一些去乙酰化酶是单 ADP-核糖基转移酶，而其他报告则表明这些酶具有这两种活性。详细的机制研究尚未验证这些报告。对 NAD+ 的依赖和潜在第二信使的产生为理解其细胞功能提供了线索。在这里，我们将重点回答分子机制的问题，填补我们对这些独特蛋白质理解的主要空白。为了解决它们的分子和生物功能，该提案概述了一种多学科方法，涉及化学、酶学和遗传学。在目标 1 中，将阐明产生蛋白质脱乙酰化与 ADP-核糖基化的催化途径。目标 2 侧重于了解 Sirtuins 如何识别特定的蛋白质靶点。为了探索 OAADPr 的生物学功能，目标 3 将涉及不可水解的 OAADPr 类似物的合成，然后检查它们作为已报道蛋白质靶标的配体、底物和抑制剂的能力。目标 4 中的实验直接探测 OAADPr 的细胞功能。细胞 OAADPr 水平定量测定将与基于细胞的转基因细胞测定相结合。将评估 OAADPr 促进酵母基因沉默的能力。在哺乳动物细胞中，将确定sirtuins 和OAADPr 调节TRPM2 离子通道门控的能力。白藜芦醇是一种植物多酚，已知具有多种心血管和神经系统健康益处，据报道是 Sir2 酶的激活剂。这里将在体内和体外检查白藜芦醇“激活”SIRT1 能力的几种假设（目标 2）。考虑到对癌症、衰老和神经退行性疾病的影响，去乙酰化酶已成为治疗这些疾病的可行药物靶点。从这项研究中获得的知识将为此类小分子的设计和开发提供主要步骤。