Control of Sir2 by Nuclear NAD Salvage Pathways

描述(由申请人提供)：烟酰胺腺嘌呤二核苷酸(NAD)是许多氧化还原酶反应的关键辅因子，这些反应将氧化形式(NAD+)和还原形式(NADH)相互转化。一些酶反应也可以消耗NAD，破坏烟酰胺(NAM)和ADP-核糖部分之间的糖酵解键。例如，Sir2家族的NAD依赖的蛋白质脱乙酰酶(Sirtuins)将特定的乙酰赖氨酸侧链的脱乙酰化与NAD的水解结合在一起。重要的是，释放出来的NAM可以对脱乙酰基产生强烈的反馈抑制作用。酿酒酵母Sir2蛋白是一种组蛋白去乙酰基酶，是基因沉默和维持较长复制寿命所必需的。对哺乳动物sirtuins的大部分研究都集中在SIRT1蛋白上，这是一种与酵母Sir2最相似的人类sirtuin蛋白。SIRT1去乙酰化转录因子如P53、FOX03或NF-kappaB，这些转录因子通过抑制或促进细胞凋亡来决定细胞对特定细胞应激或细胞外信号的反应而存活。遗传学研究已经确定，在酵母中操纵核NAD挽救途径可以通过维持细胞内高NAD来调节Sir2介导的沉默和长寿活性 浓度和限制NAM浓度。综上所述，这些发现提出了一个有趣的可能性，即Sir2是细胞代谢状态(通过NAD浓度、NAD/NADH比率或NAM浓度)和特定蛋白质脱乙酰基之间的直接联系。因此，该研究项目的长期目标是确定NAD在细胞核中的挽救/合成如何随着细胞生长条件的变化而变化，以及这如何转化为酵母中的Sir2活性和人类细胞中的SIRT1活性的调节。对人类途径的分析将以从酵母系统中学到的信息为指导。第一个具体目标将使用遗传学方法进一步剖析酵母细胞如何响应和补偿细胞内和细胞外高浓度的NAM。第二个目标的实验主要集中在直接验证NAD合成/挽救和核内NAM清除对于酵母Sir2介导的沉默和长寿至关重要的假说。在第三个目标中，负责调节人类SIRT1的特定DNA合成或挽救途径将在已知SIRT1脱乙酰基酶靶标P53和NF-kappaB的背景下被识别和表征。总之，这些以酵母为灵感的实验将有助于确定癌症治疗或抗衰老干预的新潜在靶点。

DESCRIPTION (provided by applicant): Nicotinamide adenine dinucleotide (NAD) is a critical cofactor for numerous oxidoreductase reactions that interconvert the oxidized form (NAD+) and the reduced form (NADH). Several enzymatic reactions can also consume NAD, breaking the glycolytic bond between the nicotinamide (NAM) and ADP-ribose moieties. For example, the Sir2 family of NAD-dependent protein deacetylases (the Sirtuins) couple the deacetylation of specific acetyl-lysine side chains with NAD hydrolysis. Importantly, the liberated NAM can cause strong feedback inhibition of deacetylation. The Saccharomyces cerevisiae Sir2 protein is a histone deacetylase that is required for gene silencing and for maintaining a long replicative lifespan. Much of the research effort on mammalian Sirtuins has focused on the SIRT1 protein, which is the human Sirtuin most similar to yeast Sir2. SIRT1 deacetylates transcription factors such as p53, FOX03, or NF-KappaB, which helps decide cell survival in response to a particular cellular stress or extracellular signal, via the inhibition or potentiation of apoptosis. Genetic studies have determined that manipulations of a nuclear NAD salvage pathway in yeast can modulate Sir2-mediated silencing and longevity activity by maintaining high intracellular NAD concentration and by limiting the NAM concentration. Together these findings raise the intriguing possibility that Sir2 is a direct link between the cell metabolic status (via the NAD concentration, NAD/NADH ratio, or NAM concentration) and specific protein deacetylation. Therefore, the long-term goals of this research project are to determine how NAD salvage/synthesis in the nucleus changes in response to cellular growth conditions and how this translates into the regulation of Sir2 activity in yeast and SIRT1 activity in human cells. Analysis of the human pathways will be guided by information learned from the yeast system. The first specific aim will employ genetic methods to further dissect how yeast cells respond, to and compensate for, high intracellular and extracellular NAM concentrations. Experiments in the second aim are tightly focused on directly testing the hypothesis that NAD synthesis/salvage and NAM clearance in the nucleus is critical for yeast Sir2-mediated silencing and longevity. In the third aim, the specific DNA synthesis or salvage pathways responsible for regulating human SIRT1 will be identified and characterized in the context of the known SIRT1 deacetylase targets, p53 and NF-KappaB. Together these yeast-inspired experiments will help identify new potential targets for cancer therapeutics or anti-aging interventions.