Chemical Tools to Visualize Sirtuin Activity in Cells

可视化细胞中 Sirtuin 活性的化学工具

基本信息

批准号：
8497684
负责人：
ANTHONY A. SAUVE
金额：
$ 16.31万
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：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8497684
关键词：
ADP ribosylation Active Sites Address Affect Apoptosis Applications Grants Binding Biogenesis Biological Biological Assay Biological Process Cell physiology Cells Cellular biology Chemicals Chemistry Chromatin Complex Cytoskeletal Proteins Cytosol DNA Repair Data Deacetylase Deacetylation Development Diet Drug Metabolic Detoxication Dyes Enzymes Family Genotoxic Stress Gluconeogenesis Goals Health Health Benefit Human Image Imagery In Vitro Individual Libraries Life Link Location Lysine Mammals Mediating Metabolic Metabolism Methodology Methods Microscopy Mitochondria Monitor Nuclear Nuclear Protein Nutrient Optics Organelles Peptides Physiological Physiological Processes Physiology Play Process Protein Isoforms Proteins Reaction Reactive Oxygen Species Regulation Research Personnel Resistance Role Signal Transduction Signaling Protein Sir2-like Deacetylases Sirtuins Specificity Stress System Technology Testing Time Tissues Work base catalyst cellular imaging computerized data processing crosslink design empowered enzyme mechanism fatty acid oxidation imaging probe improved inhibitor/antagonist insulin secretion lipid biosynthesis novel novel strategies pharmacophore research study response screening tool tool development trafficking transcription factor

项目摘要

DESCRIPTION (provided by applicant): The mammalian sirtuins (SIRT1-7) are NAD+-dependent deacetylase enzymes that regulate adaptations of cells to nutrient availability and are linked to the life-extending and health-providing benefits of low calorie diets. The distinct sirtuins play different roles in regulating cell physiology, and distinct isoforms play roles that depend on tissue. These enzymes regulate numerous processes important for adaptation to low calorie diets including: adipogenesis, adipolysis, mitochondrial biogenesis, insulin secretion, fatty acid oxidation and stress resistance. Importantly, sirtuins are generally upregulated in cell by low calorie conditions. The mechanisms by which sirtuins achieve their functions are still poorly understood although it is apparent that their catalytic activity, abundance and localization within cells are crucial to their biological functions. For example, sirtuins are naturally compartmentalized; SIRT1, SIRT6 and SIRT7 are nuclear, whereas SIRT3, SIRT4 and SIRT5 are mitochondrial. SIRT2 is cytosolic. In response to various physiologic conditions, sirtuins relocalize in cells to cause changes in cell biology. There are currently no tools that can define in a simultaneous way, catalytic activity, abundance and localization of sirtuins in cells. The development of such tools is highly challenging, but would certainly provide a powerful new approach to studying sirtuins if they could be developed. This grant proposal addresses that need by presenting a plan to develop isoform-specific probes for sirtuins that can be used to visualize activity, abundance and localization using click chemistry and optical microscopy methods. Prior work has established that small peptides containing thioacetyllysine residues are excellent general inhibitors of sirtuins. This inhibition uses the enzyme mechanism. These thiopeptides, if appropriately modified with alkynyl groups or other "clickable" groups, form stable thioimidate conjugates on sirtuins that can be crosslinked to other moieties by "click chemistry". We have demonstrated that examples of these clickable thioacetyllysine peptides are cell permeable and form stable complexes to sirtuins in cells. By known methods, the conjugates formed on sirtuin active sites in cells are proposed to be clicked to dyes, thus allowing visualization of intracellular sirtuin activity, abundance and localization. To accomplish these goals we provide the following specific aims: 1) We propose to develop a set of isoform specific clickable cell permeable thioacetyllysine tripeptides that can be used to image sirtuins i cells. 2) We propose to develop live cell imaging using isoform-specific thioacetyllysine derivatives that can be used to visualize the dynamic activities of sirtuin isoforms in cells. We will use these tools to address several biological questions of high significance to the sirtuin field. With accomplishment of the aims, researchers will be empowered to track sirtuin activities with unprecedented specificity, to determine location, abundance and activity in live cells. These tools are predicted to accelerate studies to elucidate how sirtuins provide adaptations that improve human health.

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，可调节细胞对营养可用性的适应，并与低热量饮食的延长寿命和提供健康益处相关。不同的去乙酰化酶在调节细胞生理学中发挥不同的作用，不同的异构体发挥的作用取决于组织。这些酶调节许多对于适应低热量饮食很重要的过程，包括：脂肪生成、脂肪分解、线粒体生物合成、胰岛素分泌、脂肪酸氧化和应激抵抗。重要的是，细胞中的去乙酰化酶通常在低热量条件下上调。尽管很明显它们的催化活性、丰度和定位与去乙酰化酶实现其功能的机制仍然知之甚少。细胞内的物质对其生物学功能至关重要。例如，sirtuins 自然是区室化的； SIRT1、SIRT6 和 SIRT7 位于核内，而 SIRT3、SIRT4 和 SIRT5 位于线粒体内。 SIRT2 是胞质的。为了响应各种生理条件，去乙酰化酶在细胞中重新定位，引起细胞生物学的变化。目前还没有工具可以同时定义细胞中去乙酰化酶的催化活性、丰度和定位。此类工具的开发极具挑战性，但如果能够开发出来，肯定会为研究 Sirtuins 提供一种强大的新方法。该拨款提案通过提出一项计划来开发沉默调节蛋白同种型特异性探针，可用于使用点击化学和光学显微镜方法来可视化活性、丰度和定位，从而解决了这一需求。先前的工作已经证实，含有硫代乙酰基赖氨酸残基的小肽是优异的去乙酰化酶的通用抑制剂。这种抑制作用利用了酶机制。这些硫肽如果用炔基或其他“可点击”基团进行适当修饰，则会在sirtuins上形成稳定的硫代亚氨酸盐缀合物，该缀合物可以通过“点击化学”与其他部分交联。我们已经证明，这些可点击的硫代乙酰基赖氨酸肽的例子是细胞可渗透的，并且与细胞中的去乙酰化酶形成稳定的复合物。通过已知的方法，在细胞中的sirtuin活性位点上形成的缀合物被提议点击染料，从而允许细胞内sirtuin活性、丰度和定位的可视化。为了完成为了实现这些目标，我们提出了以下具体目标：1）我们建议开发一套异构体特异性可点击细胞渗透性硫代乙酰基赖氨酸三肽，可用于对 Sirtuins i 细胞进行成像。 2）我们建议使用异构体特异性硫代乙酰赖氨酸衍生物开发活细胞成像，可用于可视化细胞中sirtuin异构体的动态活性。我们将使用这些工具来解决几个对沉默调节蛋白领域具有重要意义的生物学问题。随着目标的实现，研究人员将能够以前所未有的特异性追踪沉默调节蛋白的活性，以确定活细胞中的位置、丰度和活性。预计这些工具将加速研究，以阐明沉默调节蛋白如何提供改善人类健康的适应性。