Chemical Proteomic Strategy to Investigate Cysteine Glutathionylation

研究半胱氨酸谷胱甘肽化的化学蛋白质组学策略

• 批准号: 10274516
• 负责人: Young-Hoon Ahn
• 金额: $ 29.37万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10274516
• 关键词: Advanced Malignant Neoplasm; Affect; Biochemical; Bioinformatics; Biological; Biological Assay; Biological Process; Biotin; Cell Line; Cells; Cessation of life; Chemicals; Cysteine; D-Amino Acid Dehydrogenase; Data; Detection; Development; Diagnostic; Disease; Economic Burden; Engineering; Event; Foundations; Glutathione; Guanosine Triphosphate Phosphohydrolases; Hydrogen Peroxide; Immune response; Impairment; In Situ; Isotope Labeling; Link; MCF7 cell; Malignant Neoplasms; Mammalian Cell; Maps; Mediating; Migration Assay; Molecular Target; Neoplasm Metastasis; Organelles; Oxidation-Reduction; PPP2CA gene; Phosphoric Monoester Hydrolases; Play; Population; Predisposition; Production; Protein S; Proteins; Proteomics; Reaction; Reactive Oxygen Species; Reporter; Research; Role; Signal Pathway; Signaling Molecule; Sulfhydryl Compounds; System; Therapeutic; Time; Work; base; cell motility; chronic wound; fluorophore; genetic regulatory protein; glutathione synthase; innovation; insight; migration; mortality; mutant; novel; oxidation; protein function; screening; spatiotemporal; tissue repair; wound; wound healing

Summary/Abstract The reactive oxygen species (ROS) including hydrogen peroxide (H2O2) are key signaling molecules that mediate diverse biological processes, including cell migration involved in tissue repair, immune response, and cancers. The central molecular targets of ROS are protein cysteine residues that form various thiol oxoforms, including S-glutathionylated cysteines, termed as S-glutathionylation. This protein S-glutathionylation regulates protein activity in a number of signaling pathways. Despite the continuing advance on identification of glutathionylated proteins, identification of the specific glutathionylated cysteines that control definite biological functions has been challenging. To provide the insights into the glutathionylation-susceptibility of global cysteines, we have developed a chemical proteomic approach, termed clickable glutathione, that enables to study S- glutathionylation. In this proposal, we will develop an integrative strategy combining our chemical proteomic platforms with functional biological analyses to streamline identification of glutathionylation-susceptible cysteines that control cell migration. First, we aim to identify glutathionylation-sensitive cysteines in mammalian cell lines during cell migration induced by D-amino acid oxidase (DAAO) with D-Ala, which produces spatiotemporal and magnitude-controlled levels of H2O2. We will use our quantitative proteomics and bioinformatic analyses to identify a group of cysteines highly susceptible to glutathionylation and functionally related to migration. Because of the importance of localized H2O2 production, the strategy will be extended to the use of localized DAAO/D-Ala systems to determine localization-dependent glutathionylation of global cysteines. Second, we aim to determine regulatory roles of the identified glutathionylated cysteines in cell migration. In preliminary studies, we identified the redox-active glutathionylated cysteines in three proteins, PP2Cα, ARHGEF7, and NISCH, which increase cell migration in functional analyses. We will investigate glutathionylation-susceptibility of three proteins and their downstream signaling pathways mediated by glutathionylation. Lastly, we will apply a combination of chemical proteomics, bioinformatics, and functional screening analyses to find new glutathionylation-susceptible proteins that regulate cell migration.

摘要/摘要 包括过氧化氢 (H2O2) 在内的活性氧 (ROS) 是关键信号分子， 介导多种生物过程，包括参与组织修复、免疫反应和 ROS的中心分子靶点是形成各种硫醇氧代形式的蛋白质半胱氨酸残基， 包括S-谷胱甘肽化的半胱氨酸，称为S-谷胱甘肽化，这种蛋白质S-谷胱甘肽化调节。 尽管在鉴定方面不断取得进展，但许多信号通路中的蛋白质活性。 谷胱甘肽化蛋白质，识别控制特定生物的特定谷胱甘肽化半胱氨酸 功能一直具有挑战性。 我们开发了一种化学蛋白质组学方法，称为可点击谷胱甘肽，能够研究 S- 在本提案中，我们将结合我们的化学蛋白质组学制定综合策略。 具有功能生物学分析的平台，可简化谷胱甘肽化敏感半胱氨酸的鉴定 首先，我们的目标是鉴定哺乳动物细胞系中谷胱甘肽敏感的半胱氨酸。 在 D-氨基酸氧化酶 (DAAO) 和 D-Ala 诱导的细胞迁移过程中，产生时空和 我们将使用我们的定量蛋白质组学和生物信息学分析来控制 H2O2 的水平。 鉴定一组对谷胱甘肽化高度敏感且与迁移功能相关的半胱氨酸。 鉴于本地化 H2O2 生产的重要性，该战略将扩展到本地化 DAAO/D-Ala 的使用 其次，我们的目标是确定全局半胱氨酸的定位依赖性谷胱甘肽化。 在初步研究中，我们确定了所鉴定的谷胱甘肽化半胱氨酸在细胞迁移中的调节作用。 PP2Cα、ARHGEF7 和 NISCH 三种蛋白质中具有氧化还原活性的谷胱甘肽化半胱氨酸，可增加 我们将研究三种蛋白质及其它们的谷胱甘肽化敏感性。 最后，我们将应用化学组合介导的下游信号通路。 蛋白质组学、生物信息学和功能筛选分析，以寻找新的谷胱甘肽化敏感蛋白 调节细胞迁移。