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

基本信息

批准号：
8483680
负责人：
ANTHONY A. SAUVE
金额：
$ 32.2万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
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/8483680
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 computerized data processing 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 tool vector

项目摘要

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）：哺乳动物去乙酰化酶（SIRT1-7）是NAD+依赖性脱乙酰酶，其使细胞蛋白脱乙酰以调节其靶蛋白。 Sirtuins 会被营养应激（例如低热量饮食）激活，并且被认为是适应细胞和人体生理学的重要信号转导器。人们对了解去乙酰化酶如何在分子水平上受到调节非常感兴趣。这种理解可以为更好地针对它们进行药理调节提供机会。当前技术的一个关键限制是无法直接评估细胞中的去乙酰化酶活性。在给定的扰动条件下确定细胞中特定时间的去乙酰化酶活性的能力将显着加速对去乙酰化酶调节的研究。为了例如，目前的模型表明，一些去乙酰化酶受 NAD+ 代谢（包括 NAD+ 或 NAD+/NADH 比率）的变化调节。由于sirtuin活性无法在细胞中直接测量，因此无法直接验证这一想法，只能通过目标蛋白的脱乙酰作用来推断。这种方法会受到许多混杂效应的影响。去乙酰化酶领域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去乙酰化酶的活性。最近的数据表明 cAMP 信号传导会增加 SIRT1 活性，但这种活性变化不能直接在细胞中显示出来。最后，该领域的关键问题涉及白藜芦醇等药理激活剂。白藜芦醇会导致细胞中 SIRT1 活性增加吗？这个问题在 Sirtuin 领域非常重要，自 2003 年以来一直没有得到解决。这项拨款申请建议开发一项突破性技术，能够通过基于活性的探针对细胞中的 Sirtuin 活性进行成像。该探针包含一个可点击的基团，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，探针具有细胞渗透性，sirtuins 的探针标记需要酶机制，探针对细胞 NAD+ 水平敏感，并且标记的时间依赖性可用作细胞中 Sirtuin 活性的测量。我们建议开发该探针并用它来解决 Sirtuin 领域感兴趣的关键问题。为此，我们提出以下具体目标：在目标 1 中，我们将进行调查细胞中 SIRT1-7 标记的浓度和时间依赖性，从而确定每种 Sirtuin 异构体的探针特性。在目标 2 中，我们将探讨 NAD+ 代谢在改变细胞内 SIRT1-7 活性中的作用，并解决关键问题，例如 NAD+ 水平和 NAD+/NADH 比率对影响 Sirtuin 活性的影响。目标 3：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，了解它们对 Sirtuin 活性的影响。我们还将探讨抑制剂和激活剂改变细胞中沉默调节蛋白活性的能力。我们工具的更广泛可用性将加速其他研究人员对去乙酰化酶的研究，并为扩展这些策略以研究其他关键信号传导过程提供刺激。