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

描述（由申请人提供）：蛋白亚磺酰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，其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醇盐与过氧化氢的反应，其形成高度不稳定的次磺酸中间体。通过过氧化物的进一步氧化导致形成亚磺酸，其通常是不可逆的修饰。小分子双甲酮（5，5-二甲基环己烷-1，3-二酮）作为共价陷阱，与次磺酸反应形成稳定和不可逆的硫醚键。几个小组最近报道了双甲酮的官能化类似物（叠氮化物、炔、生物素等）的开发。用于双甲酮反应性氧化蛋白的亲和纯化和蛋白质组学注释。尽管最近取得了这一进展，但没有用于亚磺酰化的活细胞或体内成像的探针。我们的长期目标是开发新的方法来可视化蛋白质磺酰化在体内的作用，并应用这些工具来表征这种修饰在疾病中的功能意义。在这个应用中，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，用于使用先进的比率荧光成像和新兴的MRI方法来可视化活细胞和组织中的蛋白质磺酰化。这些方法将使用预测显示改变氧化还原调节的模式生物进行验证，为在哺乳动物系统中进行扩展研究提供途径。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。这种广泛的培训方法，由初级和高级教师导师委员会监督，将为我提供技能和培训，以追求我的长期目标，在一所多元化的研究型大学担任学术职务。

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.