Probing the role of cysteine sulfenylation in cell signaling

探讨半胱氨酸磺酰化在细胞信号传导中的作用

基本信息

批准号：
9380891
负责人：
Kate Suzanne Carroll
金额：
$ 41.1万
依托单位：
SCRIPPS FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-01 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9380891
关键词：
Active Sites Back Biochemical Biochemistry Biological Biological Assay Biological Markers Biological Process Biology Brain Cells Chemicals Chemistry Clustered Regularly Interspaced Short Palindromic Repeats Code Collaborations Complement Cultured Cells Cysteine Data Set Dependency Detection Development Disease Drug Targeting Elements Ensure Enzymes Epidermal Growth Factor Receptor Evaluation Event Family Funding Genes Genetic Transcription Heart Hydrogen Peroxide Individual Isotopes Knock-out Knockout Mice Learning Liver Lung Malignant Neoplasms Mammalian Cell Maps Mediating Medicine Metabolic Diseases Methods Modification Molecular Monitor Mus NADPH Oxidase Neurodegenerative Disorders Organ Oxidants Oxidation-Reduction Oxidoreductase Pathway Analysis Pathway interactions Peptides Phosphorylation Phosphotransferases Physiological Post-Translational Protein Processing Preparation Process Property Protein Isoforms Protein S Protein Tyrosine Kinase Proteins Proteome Proteomics Reaction Regulation Reporting Repression Research Resources Role Signal Pathway Signal Transduction Site Site-Directed Mutagenesis Specificity Sulfenic Acids Sulfhydryl Compounds Sulfinic Acids System Testing Therapeutic Intervention Time Transgenic Mice Vitronectin Work adduct analytical tool biophysical properties body system chemoproteomics computerized tools cysteine sulfinic acid cysteinesulfenic acid design diazene expectation experimental study improved in vivo insight novel oxidation protein structure reaction rate scaffold stoichiometry therapeutic target tool

项目摘要

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.

抽象的过氧化氢（H2O2）是一种多功能氧化物，可介导每个主要的生物学功能器官系统。 H2O2实现功能多样性的新兴分子途径是通过蛋白质半胱氨酸的特异性修饰保留为形成S-磺苯基半胱氨酸。这个后翻译修饰，S-磺苯基化，调节蛋白质活性和定位。尽管有很大的进步单个蛋白质，生物学化学，对特定H2O2生成的NADPH氧化物的依赖性（NOX），以及控制特定半胱氨酸在体内修饰的结构元素广泛未知。为了了解这些基本生物学问题，敏感，验证和定量需要化学蛋白质组学方法，但仍处于发展的早期阶段。为此，在我们开发并实施了一种新型的化学蛋白质组学方法。这个新方法已经实现了S-硫硝基化半胱氨酸的特定，高效，完整和选择性识别活细胞中的残留物。当前，我们的化学蛋白质瘤方法的实施已精确地指出了培养的哺乳动物细胞中778种蛋白质中的1,105种肽中S-磺苯基的位点。这些蛋白质构成迄今为止，最大的S-硫二苯基化蛋白数据集。在此续订应用程序中，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以下三个主要目标：（1）定义控制特定半胱氨酸住所和蛋白质以s-磺苯基化的选择的分子决定剂，（2）阐明受s-磺酰化影响的功能网络和信号通路，以及（3）识别控制蛋白质脱硫基化的酶系统。通过发现内源S- 小鼠肝脏，脑，肺和心脏的磺苯基蛋白质组学，并应用多个分析性和计算工具，管理s-磺酰化特异性的生化和结构特性体内将定义。生物功能和途径分析，结合定量化学计量学评估源自NOX基因敲除和转基因小鼠的S-磺苯组，将测试H2O2特异性在四个不同器官内外的信号级联反应中的功能调节。同时获得内源地点特异性，反应性半结晶组和磷蛋白质组的获取将实现对完成和协调的全面评估。调节的酶系统将使用CRISPR序列特异性表示或可能候选者的激活来识别脱硫硝基化。总体而言，蛋白质结构和功能途径的全面大规模研究将显着提高我们对H2O2介导的生物学中S-磺苯基化的欣赏。这些分子成分途径可能代表氧化还原快速生长领域的新生物标志物和药物靶标生物学和医学”。该提案中提出的研究工具和方法也应提供在一系列物理和疾病过程中表征氧化还原网络的一般价值。