Reversible Protein Acetylation and Chromatin Function

描述(由申请人提供)：Sir2(或sirtuin)蛋白家族在衰老、DNA修复、细胞凋亡、抵抗细胞压力和新陈代谢中发挥关键作用。然而，不同表型的分子基础仍然难以捉摸。大多数sirtuins催化的反应是NAD+的裂解和组蛋白/蛋白质的脱乙酰化偶联形成新的代谢产物O-乙酰-ADP-核糖(OAADPr)。一些报告表明，一些sirtuins是单-ADP-核糖基转移酶，而另一些报告则表明，这些酶同时具有这两种活性。详细的机制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学功能，这项提案概述了一种涉及化学、酶学和遗传学的多学科方法。在目标1中，将阐明产生蛋白质脱乙酰化而不是ADP-核糖化的催化途径。目标2侧重于了解sirtuins如何识别特定的蛋白质靶标。为了探索OAADPr的生物学功能，目标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、底物和抑制蛋白靶标的能力。目的4中的实验直接探讨了OAADPr的细胞功能。细胞OAADPr水平的定量分析将与转基因细胞的基于细胞的分析相结合。将评估OAADPr在酵母中促进基因沉默的能力。在哺乳动物细胞中，sirtuins和OAADPr调节TRPM2离子通道门控的能力将被确定。白藜芦醇是一种植物多酚，已知具有多种心血管和神经健康益处，据报道是Sir2酶的激活剂。在这里，关于白藜芦醇“激活”SIRT1的能力的几个假说将在体内和体外进行检验(目标2)。鉴于sirtuins对癌症、衰老和神经退化的影响，sirtuins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。

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.