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

University Of Michigan At Ann Arbor, Ann Arbor MI

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Abstract

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.