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

描述（由申请人提供）：哺乳动物沉默调节蛋白（SIRT 1 -7）是NAD+依赖性脱乙酰酶，可使细胞蛋白脱乙酰以调节其靶蛋白。Sirtuins被营养应激激活，例如低热量饮食，并且被认为是适应细胞和人体生理的重要信号转导子。人们对理解sirtuins在分子水平上是如何调节的有相当大的兴趣。这样的理解可以提供机会，以更好地靶向他们的药理学调制。当前技术的一个关键限制是不能直接评估细胞中的沉默调节蛋白活性。在特定扰动的条件下，在给定时间测定细胞中特定沉默调节蛋白的活性的能力将显著加速对沉默调节蛋白调节的研究。为 例如，目前的模型表明，一些sirtuins受NAD+代谢（包括NAD+或NAD+/NADH比率）变化的调节。由于sirtuin活性不能在细胞中直接测量，因此不可能直接验证这一想法，而只能通过靶蛋白的脱乙酰化进行推断。这种方法会产生许多混淆的影响。去乙酰化酶领域的另一个关键问题是，由胰岛素或胰高血糖素引发的信号通路的激活是否会影响去乙酰化酶的活性。最近的数据表明，cAMP信号增加SIRT 1活性，但这种活性变化不能直接在细胞中显示。最后，该领域的关键问题涉及药理学激活剂，如白藜芦醇。白藜芦醇是否会导致SIRT 1在细胞中的活性增加？这个问题在锡尔杜地区相当重要，自2003年以来一直没有得到解决。该授权申请提出了一项突破性技术的开发，该技术能够通过基于活性的探针对细胞中的sirtuin活性进行成像。探针结合了可点击基团，其使得探针能够与染料缀合以提供可视化。我们的初步数据确定，探针是细胞渗透性的，探针标记的sirtuins需要酶的机制，探针是敏感的细胞NAD+水平，和时间依赖性的标记可以被用来作为衡量sirtuin在细胞中的活性。我们建议开发的探头，并使用它来解决感兴趣的sirtuin领域的关键问题。为此，我们提出以下具体目标：在目标1中，我们将研究 细胞中SIRT 1 -7标记的浓度和时间依赖性，从而提供针对每种沉默调节蛋白同种型的探针性质的测定。在目标2中，我们将讨论NAD+代谢在改变细胞中SIRT 1 -7活性中的作用，并解决关键问题，如NAD+水平和NAD+/NADH比例在影响sirtuin活性中的作用。在目标3中：我们将筛选cAMP信号传导、AMP激酶信号传导以及胰岛素和胰高血糖素的激动剂，以了解它们对沉默调节蛋白活性的影响。我们还将讨论抑制剂和激活剂改变细胞中沉默调节蛋白活性的能力。我们的工具的更广泛的可用性将加速其他研究人员对sirtuins的研究，并为扩展这些策略以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.