Chemical Tools to Visualize Sirtuin Activity in Cells

描述(申请人提供)：哺乳动物sirtuins(SIRT1-7)是依赖NAD+的脱乙酰酶，调节细胞对营养可获得性的适应，并与低热量饮食的延长寿命和提供健康的益处有关。不同的sirtuin在调节细胞生理方面发挥不同的作用，不同的异构体发挥着依赖于组织的作用。这些酶调节许多对适应低热量饮食非常重要的过程，包括：脂肪生成、脂肪分解、线粒体生物合成、胰岛素分泌、脂肪酸氧化和抗应激。重要的是，在低卡路里条件下，sirtuins通常在细胞内上调。Sirtuins实现其功能的机制仍然知之甚少，尽管很明显它们的催化活性、丰度和定位。 在细胞内对它们的生物学功能至关重要。例如，sirtuins是自然划分的；SIRT1、SIRT6和SIRT7是核的，而SIRT3、SIRT4和SIRT5是线粒体。SIRT2是胞质的。在不同的生理条件下，sirtuins在细胞内重新定位，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sirtuins的催化活性、丰度和定位。这类工具的开发极具挑战性，但如果能够开发，肯定会为研究sirtuins提供一种强有力的新方法。这项赠款提案通过提出一项计划来满足这一需求，该计划旨在开发针对sirtuins的异构体特异性探针，该探针可用于使用点击化学和光学显微镜方法可视化活性、丰度和定位。先前的工作已经证实，含有硫代乙酰赖氨酸残基的小肽是很好的sirtuins的一般抑制剂。这种抑制使用酶机制。如果用炔基或其他可点击的基团适当地修饰这些硫代多肽，就可以在sirtuins上形成稳定的硫代亚胺酸酯偶联物，该偶联物可以通过点击化学的方式与其他部分进行交联。我们已经证明了这些可点击的硫代乙酰赖氨酸肽的例子是细胞通透性的，并在细胞内与sirtuins形成稳定的络合物。通过已知的方法，在细胞中sirtuin活性部位形成的偶联物被建议点击到染料上，从而使细胞内sirtuin的活性、丰度和定位得以可视化。要完成 这些目标，我们提供了以下具体目标：1)我们建议开发一套异构体特异性点击细胞透性硫代乙酰赖氨酸三肽，可用于成像sirtuins I细胞。2)我们建议使用异构体特异的硫代乙酰赖氨酸衍生物开发活细胞成像，可以用来显示细胞中sirtuin异构体的动态活动。我们将使用这些工具来解决对Sirtuin领域具有高度重要性的几个生物学问题。随着这些目标的实现，研究人员将有权以前所未有的特异性追踪sirtuin的活动，确定活细胞中的位置、丰度和活性。这些工具预计将加速研究，以阐明sirtuins如何提供改善人类健康的适应。

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.