Chemical Tools to Visualize Sirtuin Activity in Cells

描述（由申请人提供）：哺乳动物sirtuins （SIRT1-7）是NAD+依赖性脱乙酰酶，调节细胞对营养物质的适应性，并与低热量饮食的延寿和健康益处有关。不同的sirtuins在调节细胞生理方面发挥不同的作用，不同的同种异构体依赖于组织发挥作用。这些酶调节许多对适应低热量饮食很重要的过程，包括：脂肪生成、脂肪分解、线粒体生物生成、胰岛素分泌、脂肪酸氧化和抗逆性。重要的是，在低热量条件下，细胞中的sirtuins通常会上调。sirtuins实现其功能的机制仍然知之甚少，尽管它们的催化活性、丰度和定位很明显

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.