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: 10551830
• 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/10551830
• 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; 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.

项目摘要 各种细胞过程产生活性氧（ROS），例如超氧化物 和过氧化氢，它们是信号通路的关键介体。氧化还原失调 稳态和ROS产生是包括癌症和年龄在内的疾病的标志 相关变性。 ROS的主要细胞靶标是形成硫酸的半胱氨酸硫醇， 硫酸和磺酸以及二硫酸。 ROS的产生本地化为特定的细胞 区域，是短暂的，需要时空方法研究半胱氨酸氧化 事件。我们已经制定了一种称为同位素ABPP的化学蛋白质组策略，以研究 多种氧化半胱氨酸的修饰。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 可以通过光快速激活半胱氨酸标记的光； （2）适应 涡轮接近生物素化方法，将我们的分析定位到 细胞。我们将将这些时空读数应用于细胞中的两个ROS生成系统： （1）生长因素介导的NADPH氧化酶（NOX）的激活； （2）抑制 电子传输链（ETC）和线粒体超氧化物歧化酶。 与EGFR结合的表皮生长因子（EGF）通过NOX2激活ROS释放。我们 旨在确定邻近NOX2的半胱氨酸氧化事件并表征这些事件 生长因素信号传导上的氧化事件。在早期研究中，我们确定了氧化还原活性 脂肪酸结合蛋白Fabp5中的二硫键在EGF刺激时形成 A431细胞。我们将研究二硫键键形成对脂质结合的影响 FABP5的性能和下游信号通路，由生长因子介导 刺激。 线粒体是高度氧化还原活性细胞器，我们旨在提供一个 线粒体功能障碍期间氧化靶标的全面视图，有选择地 分析前富集线粒体蛋白。我们将应用优化的线粒体 在秀丽隐杆线虫SOD-2突变体中审问半胱氨酸氧化的方法 线粒体ROS和延长的寿命。