Probing the role of cysteine sulfenylation in cell signaling

摘要 过氧化氢(H_2O_2)是一种多功能的氧化剂，在每一种主要的 器官系统。过氧化氢实现功能多样性的一个新的分子途径是通过 蛋白质半胱氨酸残基的特异性修饰形成S-亚磺基半胱氨酸。这个翻译后版本 修饰，S-磺化，调节蛋白质的活性和定位。尽管在以下方面取得了长足的进步 个体蛋白质、生物化学、对产生特定过氧化氢的NADPH氧化酶的依赖 (NOx)，以及控制体内特定半胱氨酸残基修饰的结构元素 未知。提供对这些敏感的、有效的和定量的基本生物学问题的见解 化学蛋白质组学方法是必要的，但仍处于发展的早期阶段。为此，在 在上一个资助期，我们开发并实施了一种新的化学蛋白质组学方法。这是一项新的 该方法实现了S磺化半胱氨酸的特异性、高效性、互补性和选择性鉴定 活细胞中的残留物。目前，我们的化学蛋白质组学方法的实施已经准确地定位了 培养哺乳动物细胞中1,105个肽对778个蛋白质的S-磺化位点。这些蛋白质构成 这是迄今为止报道的最大的S-磺化蛋白质数据集。在此续期申请中，我们建议使用 并扩展我们最先进的化学蛋白质组平台，以实现以下三个主要目标：(1)定义 控制S-磺化反应中特定半胱氨酸残基和蛋白质选择的分子决定因素， (2)阐明S磺化对功能网络和信号通路的影响；(3) 确定控制蛋白质去甲苯基化的酶系统(S)。通过揭开内生的S-- 小鼠肝、脑、肺和心脏的磺基蛋白质组学及其多重分析和应用 计算工具，控制S-亚磺化的特异性的生化和结构性质 VIVO将被定义。结合定量化学计量学的生物功能和途径分析 来自NOX基因敲除和转基因小鼠的S-磺酰体的评估，将检测H_2O_2特异性 信号的功能调节在四个不同的器官内和跨器官之间级联。同时进行 获得内源性位点特异的反应性半胱氨酸组和磷酸蛋白质组将使 对互补性和协调性进行全面和全球评价。调节的酶系统(S) 将使用CRISPR序列特异的抑制或激活可能的候选者来鉴定去甲苯化。 总体而言，对蛋白质结构和功能途径的全面大规模研究将显著 提高我们对过氧化氢介导的生物学中S-磺化反应的认识。这些物质的分子组成 在快速增长的氧化还原领域，通路可能反过来代表新的生物标志物和药物靶点 “生物和医学”。本提案中提出的研究工具和方法还应提供 在一系列生理和疾病过程中表征氧化还原网络的一般价值。

ABSTRACT Hydrogen peroxide (H2O2) is a versatile oxidant that mediates numerous biological functions within every major organ system. An emerging molecular pathway by which H2O2 accomplishes functional diversity is through the specific modification of protein cysteine residues to form S-sulfenylcysteine. This post-translational modification, S-sulfenylation, regulates protein activity and localization. Despite considerable advances with individual proteins, the biological chemistry, the dependency on specific H2O2-generating NADPH oxidases (Nox), and the structural elements that govern the modification of specific cysteine residues in vivo are vastly unknown. To provide insights into these fundamental biological questions, sensitive, validated, and quantitative chemical proteomic approaches are needed, but remain at an early stage of development. To this end, during the last funding period we developed and implemented a novel chemical proteomic approach. This new method has achieved specific, efficient, complementary and selective identification of S-sulfenylated cysteine residues in living cells. Currently, implementation of our chemoproteomic method has precisely pinpointed the site of S-sulfenylation in 1,105 peptides on 778 proteins in cultured mammalian cells. These proteins constitute the largest dataset of S-sulfenylated proteins reported to date. In this renewal application, we propose to use and expand our state-of-art chemical proteomic platform towards the three major objectives of: (1) defining the molecular determinants that govern the selection of specific cysteine resides and proteins for S-sulfenylation, (2) elucidating the functional networks and signaling pathways that are influenced by S-sulfenylation, and (3) identifying the enzyme system(s) that control protein desulfenylation. By uncovering the endogenous S- sulfenylome proteomics of mouse liver, brain, lung, and heart and applying multiple analytical and computational tools, the biochemical and structural properties that govern the specificity of S-sulfenylation in vivo will be defined. Biological functional and pathway analyses, in conjunction with quantitative stoichiometric assessment of S-sulfenylomes derived from Nox knockout and transgenic mice, will test H2O2-specific functional regulation in signaling cascades within and across the four different organs. Simultaneous acquisition of the endogenous site-specific, reactive cysteinome and phosphoproteome will enable comprehensive and global evaluation of complementation and coordination. Enzyme system(s) that regulate desulfenylation will be identified using CRISPR sequence-specific repression or activation of likely candidates. Overall, the comprehensive large-scale study of protein structures and functional pathways will significantly improve our appreciation of S-sulfenylation in H2O2-mediated biology. The molecular components of these pathways may, in turn, represent new biomarkers and drug targets in the rapidly growing fields of ‘redox biology and medicine’. The research tools and methods advanced in this proposal should also provide of general value for characterizing redox networks in a range of physiological and disease processes.