Control of Sir2 by Nuclear NAD Salvage Pathways

性状（由申请方提供）：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，可使氧化形式（NAD+）和还原形式（NADH）相互转化。几种酶促反应也可以消耗NAD，破坏烟酰胺（NAM）和ADP-核糖部分之间的糖酵解键。例如，NAD依赖性蛋白质脱乙酰酶（Sirtuins）的Sir 2家族将特定乙酰基-赖氨酸侧链的脱乙酰化与NAD水解偶联。重要的是，释放的NAM可以引起脱乙酰化的强烈反馈抑制。酿酒酵母Sir 2蛋白是基因沉默和维持长复制寿命所需的组蛋白脱乙酰酶。对哺乳动物Sirtuins的大部分研究工作都集中在SIRT 1蛋白上，这是与酵母Sir 2最相似的人类Sirtuin。SIRT 1使转录因子如p53、FOX 03或NF-KappaB脱乙酰基，这有助于通过抑制或增强细胞凋亡来决定细胞存活以响应特定的细胞应激或细胞外信号。遗传学研究已经确定，在酵母中操纵核NAD补救途径可以通过维持高细胞内NAD水平来调节Sir 2介导的沉默和寿命活性 浓度和限制NAM浓度。总之，这些发现提出了一种有趣的可能性，即Sir 2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化之间的直接联系。因此，该研究项目的长期目标是确定细胞核中NAD的补救/合成如何响应细胞生长条件而变化，以及这如何转化为酵母中Sir 2活性和人类细胞中SIRT 1活性的调节。人类途径的分析将由从酵母系统中获得的信息指导。第一个具体目标将采用遗传方法进一步剖析酵母细胞如何响应和补偿高细胞内和细胞外NAM浓度。第二个目标的实验紧紧集中在直接测试细胞核中NAD合成/挽救和NAM清除对于酵母Sir 2介导的沉默和寿命至关重要的假设上。在第三个目标中，负责调节人SIRT 1的特异性DNA合成或补救途径将在已知的SIRT 1脱乙酰酶靶点p53和NF-κ B的背景下被鉴定和表征。这些酵母启发的实验将有助于确定癌症治疗或抗衰老干预的新潜在目标。

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.