Reactive Species in Vascular Disease: Mechanisms of Injury

描述（由申请人提供）：一氧化氮（NO）是一种多功能自由基，在每个主要器官系统中介导许多生物功能。一个新兴的分子途径，通过NO实现功能多样性的蛋白质半胱氨酸残基的特异性修饰，形成S-亚硝基半胱氨酸。这种翻译后修饰，S-亚硝基化，影响蛋白质的功能和位置。尽管个体蛋白质有相当大的进展，但生物化学、对特定一氧化氮合酶（NOS）的依赖性以及控制体内特定半胱氨酸残基修饰的结构元件仍是未知的。此外，还没有进行探索由S-亚硝基化调节的蛋白质信号通路或相关蛋白质簇的全面研究。为了提供这些重要的生物学问题的见解，敏感的，有效的和定量的蛋白质组学方法是必要的，但目前还没有。为此，在上一个资助期间，我们开发并实施了一种新的基于质谱的蛋白质组学方法。该方法实现了对修饰半胱氨酸残基的特异性、高效性、互补性和选择性鉴定。目前，该方法的实施已经精确地定位了741个肽中的S-亚硝基化位点，这些肽独立地与小鼠肝脏、心脏、肺、脑和胸腺中的521个蛋白质相匹配。这些蛋白质构成了迄今为止最大的内源性S-亚硝基化蛋白质数据集。使用这种强大的新方法，我们建议：（1）定义的结构要素，管理的特异性S-亚硝基化，（2）阐明的功能网络和信号通路的影响S-亚硝基化和（3）确定是否S-亚硝基化代表eNOS和瘦素之间的分子联系，在调节肝脏脂质代谢。通过揭示小鼠肝、脑、肺和心脏的内源性S-亚硝基半胱氨酸蛋白质组，并应用多种分析和计算工具，将定义支配体内S-亚硝基化的特异性和选择性的结构特性。通过鉴定不表达S-亚硝基谷胱甘肽还原酶（GSNOR）（代谢S-亚硝基谷胱甘肽（GSNO）的酶）的小鼠的S-亚硝基半胱氨酸蛋白质组，我们将阐明控制体内GSNO介导的S-亚硝基化的结构元件。功能途径和网络分析结合来自内皮NOS（eNOS）、神经元NOS（nNOS）和GSNOR敲除小鼠的S-亚硝基蛋白质组的定量评估将测试四种不同器官内和跨四种不同器官的信号级联中NOS特异性功能调节。一个新的假说连接eNOS介导的S-亚硝基化与瘦素在调节肝脏脂质代谢将被探讨。总的来说，对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一氧化氮生物学中S-亚硝基化的认识。

DESCRIPTION (provided by applicant): Nitric oxide (NO) is a versatile free radical that mediates numerous biological functions within every major organ system. An emerging molecular pathway by which NO accomplish functional diversity is the specific modification of protein cysteine residues to form S-nitrosocysteine. This post-translational modification, S-nitrosylation, impacts protein function and location. Despite considerable advances with individual proteins, the biological chemistry, the dependency on specific nitric oxide synthases (NOS) and the structural elements that govern the modification of specific cysteine residues in vivo are vastly unknown. Moreover comprehensive studies exploring protein signaling pathways or interrelated protein clusters that are regulated by S-nitrosylation have not been performed. To provide insights for these important biological questions, sensitive, validated and quantitative proteomic approaches are needed but are not currently available. To this end, during the last funding period we developed and implemented a novel mass spectrometry-based proteomic approach. The new method achieved specific, efficient, complementary and selective identification of the modified cysteine residue. Currently implementation of the method has precisely pinpointed the site of S-nitrosylation in 741 peptides, which were independently matched to 521 proteins in mouse liver, heart, lung brain and thymus. These proteins constitute the largest datasets of endogenous S-nitrosylated proteins to date. Using this robust new methodology we propose to: (1) Define the structural elements that govern the specificity of S-nitrosylation, (2) Elucidate the functional networks and signaling pathways that are influenced by S-nitrosylation and (3) Determine if S-nitrosylation represents the molecular link between eNOS and leptin in the regulation of liver lipid metabolism. By uncovering the endogenous S-nitrosocysteine proteomes of mouse liver, brain, lung and heart and applying multiple analytical and computational tools, the structural properties that govern the specificity and selectivity of S-nitrosylation in vivo will be defined. By identifying the S-nitrosocysteine proteome of mice which do not express S-nitrosoglutathione reductase (GSNOR), the enzyme that metabolizes S-nitrosoglutathione (GSNO), we will elucidate the structural elements governing the in vivo GSNO-mediated S-nitrosylation. Functional pathway and network analyses in conjunction with quantitative assessment of S-nitrosoproteomes derived from endothelial NOS (eNOS), neuronal NOS (nNOS) and GSNOR null mice will test NOS specific functional regulation in signaling cascades within and across the four different organs. A novel hypothesis linking eNOS-mediated S-nitrosylation with leptin in the regulation of liver lipid metabolism will be explored. Overall the comprehensive large-scale study of protein structures and functional pathways will significantly improve our appreciation of S-nitrosylation in nitric oxide biology.