Defining Nuclear H2O2 Regulation by Covalent Regulators

项目摘要 代谢动态平衡的丧失是导致衰老和复制性衰老的主要原因。氧化还原的控制 平衡是维持新陈代谢平衡的关键。不适当的活性氧(ROS)水平 被认为是多种与衰老相关的疾病的重要因素，如代谢性疾病、癌症 和神经退行性疾病。在高水平上，ROS修饰核酸--这是一种重要的机制 这组反应性代谢产物导致基因组不稳定和复制性衰老。虽然已经有了 在了解不同的RO如何损害核酸方面投入了大量资金，令人惊讶的是，人们对此知之甚少 产生、解毒和感知核内RO的途径。这种知识鸿沟妨碍了我们的理解 核内ROS在生物衰老中所起的作用。破译核生物学的一个主要障碍 ROS是指不能以特定于细胞核的方式控制ROS水平，通过定义的机制 行动。小分子在生物突破中发挥了重要作用，通常调节生物过程。 达到即使是最先进的遗传模型也无法达到的特异性和精确度。目的 这一应用的目的是开发一套化学探测器，专门提高核ROS水平和 描述它们相应的蛋白质靶标。我们将通过将核定位的过氧化氢传感器 (HyPer7)用化学蛋白质组引导小分子筛选。我们以前曾使用过这些方法 为了确定一种能使线虫寿命延长约45%的小分子抑制物，并鉴定其目标蛋白， 为化学蛋白质组筛选方法在生物衰老研究中的应用提供支持。这里, 我们将利用以半胱氨酸为重点的小分子文库(5000+化学多样性 氯乙酰胺/丙烯酰胺)，以确定增加稳态核过氧化氢水平的共价探针。我们 鉴于半胱氨酸在蛋白质功能中的关键作用，以及识别共价抑制剂的能力，重点关注半胱氨酸 利用化学蛋白质组学让他们参与进来。270多个分子的初步筛选已经提供了9个 专门增加核内过氧化氢水平但不在其他隔间增加的化合物。我们随后将 利用化学蛋白质组学确定相应的蛋白质靶点并确定它们在调控中的重要性 核H_2O_2水平与复制衰老。本文提出的研究充分利用了一系列 最近开发的方法：基因编码的ROS报告和化学蛋白质组学，它们已经 以前是孤立使用的，要整合成一种有效的方法来识别控制 核过氧化氢水平。这些研究将使我们更深入地了解涉及的关键途径 并开发一套亟需的药理药物来研究ROS是如何在 不同的隔间塑造了生物衰老。

Project Summary Loss of metabolic homeostasis is through to contribute to aging and replicative senescence. Control of redox balance is critical for maintenance of metabolic homeostasis. Improper levels of reactive oxygen species (ROS) are thought to be an important contributor to multiple aging related diseases such as metabolic diseases, cancer and neurodegenerative disorders. At high levels, ROS modify nucleic acids–an important mechanism by which this group of reactive metabolites leads to genomic instability and replicative senescence. While there has been much investment into understanding how different ROS damage nucleic acids, surprisingly little is known about the pathways which generate, detoxify and sense nuclear ROS. This knowledge-gap hampers our understanding of the roles nuclear ROS plays during biological aging. A major obstacle to deciphering the biology of nuclear ROS is the inability to control the levels of ROS in a nucleus-specific manner, through defined mechanisms of action. Small molecules have been instrumental in biological breakthroughs often regulating biological processes at a level of specificity and precision not achievable with even the most advanced genetic models. The purpose of this application is to develop a suite of chemical probes that specifically increase nuclear ROS levels and characterize their corresponding protein targets. We will do so, by combining a nuclear-localized H2O2 sensor (HyPer7) with a chemical proteomic-guide small molecule screen. We have previously used these approaches to identify a small inhibitor that increases C elegans longevity by ~45% and characterized its target protein, providing support for the great utility of chemoproteomic screening approaches to study biological aging. Here, we will leverage a cysteine-focused small molecule library (5000+ chemically diverse chloroacetamide/acrylamides) to identify covalent probes that increase steady state nuclear H2O2 levels. We focus on cysteines given their critical role in protein function and the ability to identify covalent inhibitors that engage them using chemoproteomics. A preliminary screen of 270+ molecules has already furnished 9 compounds that specifically increase nuclear H2O2 levels but not at other compartments. We will subsequently use chemical proteomics to identify the corresponding protein target and determine their importance in regulating nuclear H2O2 levels and replicative senescence. The research proposed herein, takes full advantage of a series of recently developed methods: genetically encoded ROS reporters and chemical proteomics, which have previously been used in isolation, to be integrated into an effective approach to identify the pathways that control nuclear H2O2 levels. These studies will provide both a deeper understanding of the key pathways involved in nuclear ROS regulation and develop a much-needed suite of pharmacological agents to study how ROS in different compartments shapes biological aging.