Developing chemical-proteomic tools to investigate cysteine oxidation

开发化学蛋白质组学工具来研究半胱氨酸氧化

• 批准号: 10318977
• 负责人: Eranthie Weerapana
• 金额: $ 61.82万
• 依托单位: BOSTON COLLEGE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10318977
• 关键词: Age; Binding; Biotinylation; Caenorhabditis elegans; Cell Proliferation; Cell physiology; Cells; Chemicals; Cysteine; Disease; Disulfides; Electron Transport; Epidermal Growth Factor; Epidermal Growth Factor Receptor; Event; FABP5 gene; Generations; Growth Factor; Homeostasis; Hydrogen Peroxide; Label; Light; Lipid Binding; Longevity; Malignant Neoplasms; Mediating; Mediator of activation protein; Methods; Mitochondria; Mitochondrial Proteins; Modification; NADPH Oxidase; Organelles; Oxidation-Reduction; Property; Proteins; Proteomics; Reactive Oxygen Species; Role; SOD2 gene; Signal Pathway; Signal Transduction; Sulfenic Acids; Sulfhydryl Compounds; Sulfinic Acids; Sulfonic Acids; Superoxides; System; bropirimine; cellular targeting; disulfide bond; fatty acid-binding proteins; mitochondrial dysfunction; mutant; oxidation; spatiotemporal; tool

Project Summary Various cellular processes generate reactive oxygen species (ROS) such as superoxide and hydrogen peroxide, which are key mediators of signaling pathways. Dysregulated redox homeostasis and ROS generation are hallmarks of diseases including cancer and age- associated degeneration. Primary cellular targets of ROS are cysteine thiols that form sulfenic, sulfinic, and sulfonic acids, as well as disulfides. ROS generation is localized to specific cellular regions, and are short-lived, necessitating spatiotemporal methods to study cysteine oxidation events. We have developed a chemical-proteomic strategy, termed isoTOP-ABPP, to study diverse oxidative cysteine modifications. To provide the necessary spatiotemporal readout, we will expand our current isoTOP-ABB platform by: (1) utilizing caged cysteine-reactive probes that can be activated rapidly by light for temporal control of cysteine labeling; and, (2) adapting the TurboID proximity biotinylation method to spatially localize our analysis to specific regions of the cell. We will apply these spatiotemporal readouts to two ROS-generating systems in the cell: (1) growth-factor mediated activation of NADPH oxidase (NOX); and, (2) inhibition of the electron transport chain (ETC) and mitochondrial superoxide dismutase. Epidermal growth factor (EGF) binding to EGFR, activates ROS release via NOX2. We aim to identify cysteine oxidation events proximal to NOX2 and characterize the role of these oxidation events on growth-factor signaling. In early studies, we identified a redox-active disulfide bond in a fatty-acid binding protein, FABP5, which is formed upon EGF stimulation of A431 cells. We will investigate the effects of disulfide-bond formation on the lipid-binding properties of FABP5, and downstream signaling pathways mediated by growth-factor stimulation. The mitochondria are highly redox-active organelles, and we aim to provide a comprehensive view of oxidation targets during mitochondrial dysfunction, by selectively enriching mitochondrial proteins prior to analysis. We will apply our optimized mitochondrial methods to interrogate cysteine oxidation in a C. elegans SOD-2 mutant with elevated mitochondrial ROS, and extended lifespan.

项目概要 各种细胞过程会产生活性氧 (ROS)，例如超氧化物 和过氧化氢，它们是信号通路的关键介质。氧化还原失调 体内平衡和ROS的产生是癌症和年龄等疾病的标志 相关的退化。 ROS 的主要细胞靶标是半胱氨酸硫醇，可形成硫， 亚磺酸和磺酸，以及二硫化物。 ROS 的产生局限于特定的细胞 区域，并且是短暂的，需要时空方法来研究半胱氨酸氧化 事件。我们开发了一种化学蛋白质组策略，称为 isoTOP-ABPP，来研究 多种氧化半胱氨酸修饰。为了提供必要的时空读数，我们 将通过以下方式扩展我们当前的 isoTOP-ABB 平台：(1) 利用笼式半胱氨酸反应探针 可以被光快速激活，用于半胱氨酸标记的时间控制； (2) 适应 TurboID 邻近生物素化方法可将我们的分析空间定位到特定区域 细胞。我们将把这些时空读数应用于细胞中的两个 ROS 生成系统： (1)生长因子介导的NADPH氧化酶(NOX)的激活； (2) 抑制 电子传递链（ETC）和线粒体超氧化物歧化酶。 表皮生长因子 (EGF) 与 EGFR 结合，通过 NOX2 激活 ROS 释放。我们 旨在识别接近 NOX2 的半胱氨酸氧化事件并表征这些事件的作用 氧化事件对生长因子信号传导的影响。在早期研究中，我们发现了一种具有氧化还原活性的 脂肪酸结合蛋白 FABP5 中的二硫键，是在 EGF 刺激下形成的 A431细胞。我们将研究二硫键形成对脂质结合的影响 FABP5 的特性以及生长因子介导的下游信号通路 刺激。 线粒体是具有高度氧化还原活性的细胞器，我们的目标是提供 通过选择性地全面了解线粒体功能障碍期间的氧化目标 在分析之前富集线粒体蛋白质。我们将应用我们优化的线粒体 询问线虫 SOD-2 突变体中半胱氨酸氧化的方法 线粒体ROS，并延长寿命。