Nicotinamide Riboside and NAD+: Modulation of Sirtuins and Reactive Oxygen

描述(申请人提供)：哺乳动物sirtuins(SIRT1-7)是依赖NAD+的脱乙酰基酶，能使细胞蛋白脱乙酰化以调节其目标蛋白。Sirtuins被营养压力激活，例如低卡路里饮食，被认为是适应细胞和人类生理的重要信号转导。人们对了解sirtuins是如何在分子水平上进行调控有相当大的兴趣。这样的理解可以为更好地针对它们进行药物调节提供机会。当前技术的一个关键限制是无法直接评估CEL中的sirtuin活性。在特定的扰动条件下，能够在给定的时间确定细胞中特定sirtuin的活性，将大大加快对sirtuin调控的研究。为 例如，目前的模型表明，一些sirtuins受到NAD+代谢(包括NAD+或NAD+/NADH比率)变化的调节。因为sirtuin的活性不能在细胞中直接测量，所以不可能直接验证这一想法，只能通过目标蛋白的脱乙酰化来推断。这样的方法会产生许多令人困惑的影响。Sirtuin领域的另一个关键问题是，由胰岛素或胰升糖素启动的信号通路的激活是否会影响sirtuin的活性。最近的数据表明，cAMP信号增加了SIRT1的活性，但这种活性变化不能直接在细胞中显示。最后，该领域的关键问题与白藜芦醇等药理激活剂有关。白藜芦醇是否导致细胞内SIRT1活性增加？这一问题具有相当重要的现实意义，自2003年以来一直悬而未决。这项拨款申请提出了一项突破性技术的开发，能够通过基于活动的探针来成像细胞中的sirtuin活性。该探针包含一个可点击的基团，该基团使探针能够与染料结合以提供可视化。我们的初步数据表明，探针是细胞通透性的，sirtuins的探针标记需要酶机制，探针对细胞NAD+水平敏感，标记的时间相关性可以用作细胞中sirtuin活性的衡量标准。我们建议开发探头，并用它来解决Sirtuin领域感兴趣的关键问题。为此，我们提出了以下具体目标：在目标1中，我们将调查 SIRT1-7在细胞内标记的浓度和时间依赖性，从而提供针对每个sirtuin亚型的探针性质的确定。在目标2中，我们将讨论NAD+代谢在改变细胞内SIRT1-7活性中的作用，并解决关键问题，如NAD+水平和NAD+/NADH比率对SIRT1-7活性的影响。在目标3中，我们将筛选cAMP信号、AMP激酶信号以及胰岛素和胰升糖素的激动剂，以了解它们对sirtuin活性的影响。我们还将讨论抑制物和激活剂改变细胞中sirtuin活性的能力。我们工具的广泛使用将加快其他研究人员对sirtuins的研究，并为将这些策略扩展到研究其他关键信号过程提供刺激。

DESCRIPTION (provided by applicant): The mammalian sirtuins (SIRT1-7) are NAD+-dependent deacetylase enzymes that deacetylate cellular proteins to regulate their target proteins. Sirtuins are activated by nutritional stress, exemplified by low calorie diets, and are thought to be important signal transducers that adapt cells and human physiology. There is considerable interest in understanding how sirtuins are regulated on a molecular level. Such understanding could provide opportunities to better target them for pharmacologic modulation. A key limitation in current technology is the inability to directly assess sirtuin activity in the cel. The ability to determine the activity of a specific sirtuin in a cell at a given time, under conditons of a defined perturbation would significantly accelerate investigation into sirtuin regulation. For example, current models indicate that some sirtuins are regulated by changes in NAD+ metabolism (including NAD+ or NAD+/NADH ratio). Because sirtuin activity cannot be directly measured in cells, it is not possible to verify this idea directly, but only by inference through deacetylation of target proteins. Such an approach is subject to many confounding effects. Another key question in the sirtuin field is whether activation of signaling pathways initiated by insulin or glucagon affects sirtuin activity. Recent data suggests that cAMP signaling increases SIRT1 activity, but this activity change cannot be shown directly in cells. Finally, key questions in the field relate to pharmacological activators such as resveratrol. Does resveratrol cause SIRT1 activity to increase in cells? This question is of considerable importance in the sirtuin fied and has remained unresolved since 2003. This grant application proposes development of a breakthrough technology in being able to image sirtuin activity in cells via an activity-based probe. The probe incorporates a clickable group, which enables conjugation of the probe to a dye to provide visualization. Our preliminary data establishes that the probe is cell permeable, that probe labeling of sirtuins requires the enzyme mechanism, that probes are sensitive to cellular NAD+ levels, and that time-dependence of labeling can be used as a measure of sirtuin activity in cells. We propose to develop the probe and use it to address key questions of interest to the sirtuin field. To do so we propose the following specific aims: In Aim 1 we will investigate the concentration and time dependencies of labeling of SIRT1-7 in cells, thereby providing a determination of probe properties against each sirtuin isoform. In Aim 2, we will address the role of NAD+ metabolism in altering SIRT1-7 activities in cells and address key issues, such as effect of NAD+ level and NAD+/NADH ratio in affecting sirtuin activity. In Aim 3: we will screen agonists of cAMP signaling, AMP kinase signaling, and insulin and glucagon for their affects on sirtuin activity. We will also address the ability of inhibitors and activators to alter sirtuin acivities in cells. The wider availability of our tools will accelerate investigations of sirtuins by other researchers and provide stimulus to extend these strategies to study other key signaling processes.