Reversible Protein Acetylation and Chromatin Function

可逆蛋白质乙酰化和染色质功能

• 批准号: 7614396
• 负责人: JOHN M DENU
• 金额: $ 28.37万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7614396
• 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）：Sir 2（或sirtuin）蛋白家族在衰老、DMA修复、细胞凋亡、细胞应激抗性和代谢中起关键作用。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/蛋白质脱乙酰化偶联形成O-乙酰基-ADP-核糖（OAADPr）（一种新型代谢物）的反应。一些报道表明，一些sirtuins是单ADP核糖基转移酶，而另一些则表明这些酶具有两种活性。详细的机理研究尚未证实这些报告。对NAD+的依赖和潜在第二信使的产生为理解它们的细胞功能提供了线索。在这里，我们将专注于回答分子机制的问题，填补我们对这些独特蛋白质的理解的主要空白。为了解决其分子和生物学功能，该提案概述了一个多学科的方法，涉及化学，酶学和遗传学。在目标1中，将阐明产生蛋白质去乙酰化与ADP-核糖基化的催化途径。目标2的重点是了解sirtuins如何识别特定的蛋白质靶标。为了探索OAADPr的生物学功能，Aim 3将涉及合成不可水解的OAADPr类似物，然后检查它们作为配体，底物和抑制剂对报告的蛋白质靶点的能力。目的4中的实验直接探测OAADPr的细胞功能。用于定量细胞OAADPr水平的测定将与遗传修饰细胞的基于细胞的测定结合。将评估OAADPr促进酵母中基因沉默的能力。在哺乳动物细胞中，将测定沉默调节蛋白和OAADPr调节TRPM 2离子通道门控的能力。白藜芦醇是一种植物多酚，具有多种心血管和神经健康益处，据报道是Sir 2酶的激活剂。在这里，白藜芦醇“激活”SIRT 1的能力的几个假设将在体内和体外进行检查（目的2）。考虑到对癌症、衰老和神经变性的影响，sirtuins已经成为治疗这些疾病的可行药物靶标。从这项研究中获得的知识将为设计和开发这种小分子提供重要步骤。