A Chemical‐Proteomic Platform to Investigate Cysteine S‐Nitrosation

Abstract

Cysteine residues on proteins play numerous functional roles as catalytic nucleophiles, redox‐active disulfides, or metal‐binding residues. Outside of their direct role in catalysis, cysteines can also regulate protein activity via posttranslational modification by endogenous electrophiles and reactive oxygen species. These modified cysteine residues do not conform to a conserved sequence or structural motif, rendering the global identification of regulatory cysteines a considerable challenge. We hypothesize that cysteines that are hypersensitive to different oxidants or nitrosating agents are likely to be enriched in functional residues that serve to modulate protein activity. To explore this hypothesis, we focused our initial efforts on investigating cysteine susceptibility to nitrosation. S‐nitrosation is a unique posttranslational modification that serves to temporally and spatially control protein activity and localization. In order to identify cysteine residues hypersensitive to nitrosation, we developed a chemical‐proteomic platform that ranks cysteines in the human proteome by sensitivity to a variety of nitric oxide donors. Our proteomic studies identified several known sites of nitrosation, as well as previously unannotated cysteines. We then proceeded to functionally characterize several of the unannotated cysteines using a variety of cell and molecular biology approaches. We show that many of these cysteines are located distal from the active site, and yet mediate essential protein functions such as substrate binding and localization. Overall, our studies aim to unearth novel functional cysteine residues in the proteome, with the long‐term goal of identifying new modes of protein regulation through reactive cysteines.