Chemical Proteomic Methods to Investigate Cysteine Post-Translational Modifications

Abstract

Functional cysteine residues within proteins often serve as active sites for nucleophilic or redox catalysis, ligands for metal binding, structural disulfides, and are susceptible to modifications. Post-translational modifications on functional cysteine residues play various roles in regulating proteins, altering activity, localization, or cellular signaling. Such modifications arise endogenously in response to reactive oxygen species or reactive nitrogen species, respectively termed protein oxidation or protein S-nitrosation. These modifications play a role in numerous biological processes, and have implications in neurodegenerative disorders and cancers. Using chemical proteomic methods developed in our lab, specifically isotopic Tandem Orthogonal Proteolysis - Activity Based Protein Profiling (isoTOP-ABPP), we identify functional cysteines within a proteome and characterize proteins that undergo redox post-translational modifications on cysteines. I have particularly focused on S-nitrosation mediated by the novel transnitrosation donor S-nitroso-coenzyme A (CoA-SNO), as well as the functional characterization of nitrosation on specific target proteins involved in various metabolic processes: phosphofructokinase (PFKP), ATP citrate synthase (ACLY), and ornithine aminotransferase (OAT). In addition, I have studied the redox regulation of the human ribonuclease inhibitor protein (RI) to elucidate the mechanism of protein oxidation and inactivation. Lastly, I have used chemical proteomic tools to interrogate reactive cysteines in the apicomplexan parasite, Toxoplasma Gondii, for prioritization of targets for drug discovery.