Chemical Proteomic Strategy to Investigate Cysteine Glutathionylation

摘要/摘要 包括过氧化氢(H_2O_2)在内的活性氧物种(ROS)是关键的信号分子， 调节不同的生物过程，包括参与组织修复、免疫反应和 癌症。ROS的中心分子靶标是形成各种硫醇氧化形式的蛋白质半胱氨酸残基， 包括S-谷胱甘肽基化的半胱氨酸，称为S-谷胱甘肽基化。这种蛋白S-谷胱甘肽的作用调节 蛋白质在许多信号通路中的活性。尽管在鉴定方面不断取得进展 谷胱甘肽基化蛋白：特定生物控制的谷胱甘肽半胱氨酸的鉴定 功能一直是具有挑战性的。为了深入了解全球半胱氨酸对谷胱甘肽的敏感性， 我们开发了一种名为可点击谷胱甘肽的化学蛋白质组学方法，使我们能够研究S- 谷胱甘肽基化。在这项提案中，我们将开发一种综合策略，结合我们的化学蛋白质组 具有功能生物学分析的平台以简化谷胱甘肽易感半胱氨酸的鉴定 控制细胞迁移的物质。首先，我们的目标是在哺乳动物细胞系中鉴定谷胱甘肽敏感的半胱氨酸。 在D-氨基酸氧化酶(DAAO)与D-ALA共同诱导的细胞迁移过程中，产生时空和 过氧化氢的大小受控水平。我们将利用我们的定量蛋白质组学和生物信息学分析 确定一组对谷胱甘肽高度敏感并在功能上与迁移相关的半胱氨酸。因为 关于本地化生产过氧化氢的重要性，该战略将扩展到使用本地化的DAAO/D-Ala 确定全球半胱氨酸的定位依赖的谷胱甘肽基化的系统。第二，我们的目标是确定 已识别的谷胱甘肽半胱氨酸在细胞迁移中的调节作用。在初步研究中，我们发现 PP2Cα、ARHGEF7和NISCH三种蛋白质中氧化还原活性谷胱甘肽基化的半胱氨酸含量增加 功能分析中的细胞迁移。我们将研究三种蛋白质及其谷胱甘肽的敏感性。 谷胱甘肽介导的下游信号通路。最后，我们将使用一种化学物质的组合 蛋白质组学、生物信息学和功能筛选分析寻找新的谷胱甘肽易感蛋白 调节细胞迁移的物质。

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.