Probing the role of cysteine sulfenylation in cell signaling

摘要 过氧化氢（H2 O2）是一种多功能氧化剂，在每个主要的细胞内介导许多生物功能。 器官系统H2 O2实现功能多样性的新兴分子途径是通过 特异性修饰蛋白质半胱氨酸残基以形成S-亚磺酰基半胱氨酸。这种翻译后 修饰，S-亚磺酰化，调节蛋白质活性和定位。尽管取得了相当大的进展， 单个蛋白质，生物化学，对特定H2 O2生成NADPH氧化酶的依赖性 (Nox)，并且在体内控制特定半胱氨酸残基的修饰的结构元件在很大程度上是 未知为了深入了解这些基本的生物学问题，敏感，验证和定量 化学蛋白质组学方法是必要的，但仍处于早期发展阶段。为此，在 在上一个资助期间，我们开发并实施了一种新的化学蛋白质组学方法。这个新 方法实现了S-亚磺酰半胱氨酸的特异性、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精确地定位了 在培养的哺乳动物细胞中778种蛋白质上的1，105种肽中的S-亚磺酰化位点。这些蛋白质组成 迄今为止报道的S-亚磺酰化蛋白质的最大数据集。在此更新申请中，我们建议使用 并将我们最先进的化学蛋白质组学平台扩展到三个主要目标：（1）定义 控制选择特定半胱氨酸残基和蛋白质用于S-亚磺酰化的分子决定簇， (2)阐明受S-亚磺酰化影响的功能网络和信号通路，以及（3） 鉴定控制蛋白质间苯酰化的酶系统。通过揭示内源性S- 小鼠肝、脑、肺和心脏的亚磺酰组蛋白质组学，并应用多种分析和 计算工具，生物化学和结构特性，支配特异性的S-亚磺酰化， 将定义vivo。生物功能和途径分析，结合定量化学计量 来自Nox敲除和转基因小鼠的S-亚磺酰基组的评估，将测试H2 O2特异性 在四个不同器官内和跨四个不同器官的信号级联中的功能调节。同时 获得内源性位点特异性、反应性半胱氨酸组和磷酸化蛋白质组将使 对互补性和协调性进行全面和全局性评价。调节的酶系统 将使用CRISPR序列特异性抑制或激活可能的候选物来鉴定亚甲基化。 总的来说，对蛋白质结构和功能通路的全面大规模研究将显著地 提高了我们对H2 O2介导生物学中S-亚磺酰化的认识。其中的分子成分 反过来，在快速发展的“氧化还原”领域， 生物学和医学”。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.