Developing chemical-proteomic tools to investigate cysteine oxidation

项目摘要 不同的细胞过程会产生活性氧物种(ROS)，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 动态平衡和ROS生成是包括癌症和年龄在内的疾病的特征- 伴生的退化。ROS的主要细胞靶标是形成亚磺酸的半胱氨酸硫醇， 亚磺酸、磺酸以及二硫化物。ROS的产生局限于特定的细胞 区域，而且是短暂的，有必要用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学策略，称为等TOP-ABPP，用于研究 不同的氧化半胱氨酸修饰。为了提供必要的时空读数，我们 将通过以下方式扩展我们目前的IsoTOP-ABB平台：(1)使用笼式半胱氨酸反应探针 它可以被光快速激活，用于半胱氨酸标记的时间控制；以及，(2)适应 TurboID邻近生物素化方法将我们的分析空间定位到 牢房。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；以及(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的释放。我们 目的确定NOX2附近的半胱氨酸氧化事件，并表征其作用 氧化事件对生长因子信号的影响。在早期的研究中，我们确定了一种氧化还原活性 脂肪酸结合蛋白FABP5中的二硫键，它是由EGF刺激 A431细胞。我们将研究二硫键的形成对脂质结合的影响。 FABP5的性质及生长因子介导的下游信号通路 刺激。 线粒体是高度氧化还原活性的细胞器，我们的目标是提供一种 选择性地综述线粒体功能障碍中的氧化靶点 在分析前丰富线粒体蛋白质。我们将应用我们优化的线粒体 一种检测线虫SOD-2突变体半胱氨酸氧化的方法 线粒体ROS和延长寿命。

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.