Chemical Tools to Visualize Sirtuin Activity in Cells

可视化细胞中 Sirtuin 活性的化学工具

• 批准号: 8386178
• 负责人: ANTHONY A. SAUVE
• 金额: $ 29.58万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8386178
• 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; Screening procedure; 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; tool; tool development; trafficking; transcription factor

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. PUBLIC HEALTH RELEVANCE: The current proposal is designed to develop novel tools to visualize the activities, amounts and locations of sirtuins in cells. Sirtuins are important proteis that are implicated in providing the health benefits associated with low calorie diets. These tools are predicted to improve understanding of how sirtuins confer these health benefits.

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，可调节细胞对养分可用性的适应性，并与延长寿命和健康的低和健康益处有关。独特的Sirtuins在调节细胞生理学中起着不同的作用，而不同的同工型起着取决于组织的作用。这些酶调节许多对于适应低热量饮食的重要过程，包括：脂肪生成，掺杂，线粒体生物发生，胰岛素分泌，脂肪酸氧化和抗应激性。重要的是，通常通过低热量条件在细胞中上调Sirtuins。 Sirtuins实现其功能的机制仍然很少了解，尽管显然它们的催化活性，丰度和本地化 细胞内部对于它们的生物学功能至关重要。例如，Sirtuins自然被隔离； SIRT1，SIRT6和SIRT7是核的，而SIRT3，SIRT4和SIRT5是线粒体。 SIRT2是胞质的。为了应对各种生理条件，Sirtuins在细胞中重新定位以引起细胞生物学的变化。当前，没有工具可以同时定义催化活性，细胞中Sirtuins的丰富性和定位。这样的工具的开发极具挑战性，但如果可以开发出来，则肯定会为研究Sirtuins提供强大的新方法。该赠款提案通过提出一项计划来开发针对Sirtuins的同工型特异性探针的需求，这些探针可用于使用点击化学和光学显微镜方法可视化活动，丰度和定位。先前的工作已经确定，含有硫乙酰透析残基的小肽是Sirtuins的极好的一般抑制剂。这种抑制使用酶机制。这些硫代肽，如果与Alkynyl组或其他“可单击”组进行了适当的修饰，则形成稳定的硫代二聚体偶联物，可以通过“单击化学”与其他部分交联的sirtuins上。我们已经证明，这些可单击的硫乙酰透析肽的例子是可渗透细胞的，并形成稳定的复合物，以使细胞中的sirtuins形成稳定的复合物。通过已知的方法，建议将细胞中SIRTUIN活性位点形成的缀合物单击至染料，从而可以可视化细胞内Sirtuin活性，丰度和定位。完成 这些目标我们提供了以下特定目的：1）我们建议开发一组同工型特定的可单击的细胞渗透性硫乙酰基透析肽三肽，可用于成像Sirtuins I细胞。 2）我们建议使用同工型特异性硫乙烷衍生物开发活细胞成像，这些衍生物可用于可视化细胞中Sirtuin同工型的动态活性。我们将使用这些工具来解决对Sirtuin领域具有很高意义的生物学问题。通过实现目标，研究人员将有权以前所未有的特异性跟踪Sirtuin活动，以确定活细胞中的位置，丰度和活动。预计这些工具将加速研究，以阐明Sirtuins如何提供改善人类健康的适应性。 公共卫生相关性：当前的建议旨在开发新颖的工具来可视化细胞中Sirtuins的活动，数量和位置。 Sirtuins是重要的蛋白质，涉及提供与低热量饮食相关的健康益处。这些工具 预计可以提高对Sirtuins如何赋予这些健康益处的理解。