Chemical approaches for detecting S-nitrosothiols

描述（由申请人提供）：一氧化氮是一种参与许多生理和病理生理过程的细胞信号分子。一氧化氮对半胱氨酸残基的修饰，即S-亚硝化，改变了广谱蛋白质的功能。该反应代表了一种重要的翻译后修饰，其转导一氧化氮依赖性信号。然而，由于S-亚硝化产物S-亚硝基硫醇（RSNO）的不稳定性，生物样品中S-亚硝化的检测和定量仍然是一个挑战。我们的小组最近开发了一系列新的反应RSNO，其中包括双连接，还原连接，还原消除（形成脱氢丙氨酸）。这些反应选择性地靶向RSNO并将其转化为稳定/可检测的产物。基于对照实验，我们假设这些反应可以用来设计新的方法来检测RSNO。本课题主要研究以下四个方面的内容：（1）研究基于双连接的蛋白质S-亚硝化反应检测方法;（2）研究基于还原连接的蛋白质S-亚硝化反应检测方法;（3）研究S-亚硝基半胱氨酸生成脱氢丙氨酸的反应及其在蛋白质S-亚硝化反应检测中的应用;（4）验证新的RSNO检测方法在生物体系中的应用。这些研究将推进RSNOs的化学和生物学特性的理解，并允许在复杂系统中的S-亚硝化相关的一氧化氮信号转导的研究。 公共卫生相关性：蛋白质半胱氨酸残基的S-亚硝化在生理和病理生理学中具有重要作用，但S-亚硝化的检测仍是一个难题。本文提出的研究将为S-亚硝化的化学提供新的见解，并导致用于检测生物样品中S-亚硝化的新技术，这将对生物医学研究产生重大影响。

DESCRIPTION (provided by applicant): Nitric oxide is a cell-signaling molecule involved in a number of physiological and pathophysiological processes. Modification of cysteine residues by nitric oxide, i.e. S- nitrosation, changes the function of a broad spectrum of proteins. This reaction represents an important post-translational modification that transduces nitric oxide- dependent signals. However, the detection and quantification of S-nitrosation in biological samples remains a challenge because of the lability of the S-nitrosation products: S-nitrosothiols (RSNOs). Our group recently developed a series of new reactions for RSNOs, which include bis-ligation, reductive ligation, and reductive elimination (to form dehydroalanine). These reactions selectively target on RSNOs and convert them to stable/detectable products. Based on control experiments, we hypothesize these reactions can be used to design new methods for the detection of RSNOs. In this project, we plan to pursue the following four Specific Aims: (1) to study bis-ligation based methods for detecting protein S-nitrosation; (2) to study reductive ligation based methods for detecting protein S-nitrosation; (3) to study dehydroalanine formation from S-nitrosocysteines and the applications in the detection of protein S- nitrosation; and (4) to validate the applications of new RSNO detection methods in biological systems. These studies will advance the understanding of the chemical and biological properties of RSNOs and permit studies of S-nitrosation-related nitric oxide signal transducation in complex systems. PUBLIC HEALTH RELEVANCE: S-Nitrosation of protein cysteine residues plays important roles in physiology and pathophysiology, while the detection of S-nitrosation is still a challenge. The research proposed here will provide new insights into the chemistry of S-nitrosation and lead to new techniques for the detection of S-nitrosation in biological samples, which should have great impact on biomedical research.