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

烟酰胺核苷和 NAD：Sirtuins 和活性氧的调节

• 批准号: 8670002
• 负责人: ANTHONY A. SAUVE
• 金额: $ 32.21万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-05 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8670002
• 关键词: Address; Affect; Agonist; Animals; Apoptosis; Applications Grants; Applied Genetics; Behavior; Benchmarking; Biogenesis; Biological Process; Biology; Biotin; Cells; Chemistry; Clenbuterol; Complex; Coupling; Cyclic AMP; DNA Repair; Data; Deacetylase; Deacetylation; Dependence; Dependency; Detection; Development; Diet; Dyes; Enzymes; Event; Exhibits; Family; Forskolin; Glucagon; Gluconeogenesis; Goals; Health; Human; Image; Imagery; Insulin; Investigation; Kinetics; Label; Ligands; Mammalian Cell; Measurement; Measures; Mediating; Mediator of activation protein; Metabolism; Methods; Microscopy; Mitochondria; Modeling; Molecular; Monitor; NADH; Niacinamide; Nicotinic Acids; Nutritional; Oxygen; Pharmacodynamics; Physiology; Property; Protein Isoforms; Proteins; Regulation; Reporting; Research Personnel; Resistance; Resveratrol; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Sirtuins; Stimulus; Stress; Technology; Time; Transducers; Validation; adenylate kinase; base; design; enzyme activity; enzyme mechanism; extracellular; inhibitor/antagonist; innovative technologies; insulin signaling; interest; isonicotinamide; lipid biosynthesis; mutant; nicotinamide-beta-riboside; novel; pharmacophore; research study; response; signal processing; tool; vector

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.

描述（由申请人提供）：哺乳动物Sirtuins（SIRT1-7）是NAD+依赖性的脱乙酰基酶，可脱乙酰基细胞蛋白调节其靶蛋白。 Sirtuins被营养应激激活，以低热量饮食为例，被认为是适应细胞和人类生理学的重要信号传感器。了解如何在分子水平上调节sirtuins有很大的兴趣。这样的理解可以为他们更好地针对药理学调制提供机会。当前技术的关键限制是无法直接评估CEL中的Sirtuin活性。在定义的扰动的孔子下，在给定时间确定特定SIRTUIN在细胞中的活性的能力将显着加速对Sirtuin调节的研究。为了 例如，当前模型表明某些SIRTUIN受NAD+代谢的变化（包括NAD+或NAD+/NADH比）调节。由于无法直接测量细胞中的Sirtuin活性，因此不可能直接验证这一想法，而只能通过推断靶蛋白的推断。这种方法受到许多混杂影响。 Sirtuin领域中的另一个关键问题是胰岛素或胰高血糖素引发的信号通路的激活是否会影响Sirtuin活性。最近的数据表明，CAMP信号会增加SIRT1活性，但是该活性变化无法直接显示在细胞中。最后，该领域的关键问题与白藜芦醇等药理激活剂有关。白藜芦醇是否会导致SIRT1活性增加细胞？这个问题在Sirtuin Fied中至关重要，自2003年以来一直无法解决。该赠款的应用提出了开发突破性技术的发展，以通过基于活动的探针在细胞中成像细胞中的Sirtuin活性。该探针结合了一个可点击的组，该组可以将探针结合到染料中以提供可视化。我们的初步数据表明，该探针是可渗透的，探针标记Sirtuins需要酶机制，探针对细胞NAD+水平敏感，并且标记的时间依赖性可以用作细胞中SIRTUIN活性的度量。我们建议开发探测器并使用它来解决Sirtuin领域感兴趣的关键问题。为此，我们提出以下特定目标：在AIM 1中，我们将调查 SIRT1-7在细胞中标记的浓度和时间依赖性，从而确定针对每个SIRTUIN同工型的探针特性。在AIM 2中，我们将解决NAD+代谢在改变细胞中的SIRT1-7活性并解决关键问题的作用，例如NAD+水平和NAD+/NADH比在影响Sirtuin活性中的作用。在AIM 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对Sirtuin活性的影响。我们还将解决抑制剂和激活剂改变细胞中锡尔素蛋白的能力的能力。我们工具的更广泛可用性将加速其他研究人员对Sirtuins的研究，并提供刺激以扩展这些策略以研究其他关键信号过程。