Mechanism-based probes to image protein sulfenylation in live cells and in vivo

描述（由申请人提供）：蛋白质磺化描述了细胞氧化剂（如过氧化氢）对半胱氨酸的可逆翻译后修饰。生长因子刺激诱导过氧化氢爆发，瞬时氧化蛋白磷酸酶的亲核半胱氨酸和其他近端氧化还原活性硫醇。半胱氨酸氧化开始于半胱氨酸硫酸酯与过氧化氢的反应，过氧化氢形成高度不稳定的磺酸中间体。通过过氧化物进一步氧化会形成亚磺酸，这通常是不可逆的改性。小分子二medone（5,5-二甲基环己烷-1,3-二酮）作为共价陷阱，与亚磺酸反应形成稳定且不可逆的硫醚键。最近，一些研究小组报道了二美酮功能化类似物（叠氮化物、炔、生物素等）的发展，用于二美酮活性氧化蛋白的亲和纯化和蛋白质组学注释。尽管最近取得了进展，但没有用于活细胞或体内亚砜化成像的探针。我们的长期目标是开发新的方法来可视化蛋白质亚砜化在体内的作用，并应用这些工具来表征这种修饰在疾病中的功能意义。在此应用中，我建议开发基于机制的化学探针来分析细胞和体内的时空动力学。这些方法将引入新的方法，利用先进的比例荧光成像和新兴的MRI方法来可视化活细胞和组织中的蛋白质亚砜化。这些方法将通过预测显示氧化还原调节改变的模式生物进行验证，为哺乳动物系统的扩展研究提供途径。最后，开发这些方法将提供一个独特的培训环境，连接有机合成，生物化学和光谱学。在由初级和高级教师导师组成的委员会的监督下，这种广泛的培训方法将为我提供技能和培训，以实现我的长期目标，即在一所多元化的研究型大学担任学术职位。

DESCRIPTION (provided by applicant): Protein sulfenylation describes the reversible post-translational modification of cysteine by cellular oxidants, like hydrogen peroxide. Growth factor stimulation induces a burst of hydrogen peroxide, which transiently oxidizes the nucleophilic cysteine of protein phosphatases and other proximal redox active thiols. Cysteine oxidation begins with the reaction of the cysteine thiolate with hydrogen peroxide, which forms a highly unstable sulfenic acid intermediate. Further oxidation by peroxide leads to formation of sulfinic acids, which are generally irreversible modifications. The small molecule dimedone (5,5-dimethylcyclohexane-1,3-dione) acts as a covalent trap, reacting with sulfenic acids to form a stable and irreversible thioether linkage. Several groups have recently reported the development of functionalized analogues of dimedone (azide, alkyne, biotin, etc.) for affinity purification and proteomic annotation of dimedone-reactive oxidized proteins. Despite this recent progress, there are no probes for live-cell or in vivo imaging of sulfenylation. Our long-term goal is to develop new methods to visualize the role of protein sulfenylation in vivo, and apply these tools to characterize the functional significance of this modification in disease. In this application, I propose to develop mechanism- based chemical probes to analyze the spatiotemporal dynamics in cells and in vivo. These methods will introduce new methods for visualizing protein sulfenylation in live cells and tissues using advanced ratiometric fluorescent imaging and emerging MRI methods. These approaches will be validated using model organisms predicted to display altered redox regulation, providing a path for extended studies in mammalian systems. Finally, developing these methods will provide a unique training environment bridging organic synthesis, biochemistry, and spectroscopy. This broad training approach, supervised by a committee of junior and senior faculty mentors, will provide me with the skills and training to pursue my long-term goal advancing to an academic position at a diverse, research university.