Chemoproteomic discovery and functional characterization of infection-induced oxidation sites

PROJECT SUMMARY/ABSTRACT Microbial infections account for nearly one-sixth of all human cancers. Oxidative stress generated by cancer- causing pathogens can drive tumor formation, yet very little is known about the cellular targets of oxidative stress that contribute to cancer development during infection. At the molecular level, one of the primary consequences of oxidative stress is the site-specific oxidation of proteins containing redox-sensitive cysteines. Cysteine oxidation can regulate important cellular functions such as proliferation, metabolism, and other tumorigenic processes. However, almost nothing is known about the cysteines that are oxidized during infection, in part because they are difficult to study. Unlike DNA oxidation, post-translational oxidative modifications cannot be detected by DNA sequencing, transcriptional profiling, or even conventional proteomic analyses. Consequently, methods traditionally used to study host–microbe interactions overlook signaling events that could potentially be targeted to prevent disease. To address this challenge, we have developed and validated a chemical proteomic strategy that can detect specific sites of oxidative modification on host proteins in infected cells. We use chemical probes to quantify proteome-wide changes in thiol reactivity, which decreases upon oxidation. Using this approach, we identified cysteines in several host proteins that become less reactive when human gastric cells are infected with the cancer-causing pathogen Helicobacter pylori. Notably, we discovered an infection-induced oxidation site on Cys219 of the host protease legumain that accelerates tumor growth. The proposed research program will extend these findings by (1) uncovering the molecular mechanisms by which infection-induced oxidation sites influence host protein function and localization, with the goal of defining cysteine oxidation as a new mechanism of cancer pathogenesis during H. pylori infection; and (2) leveraging our approach to identify specific oxidation sites that promote cancer signaling in vivo. Our long-term goal is to establish a platform for the discovery and functional characterization of oxidized cysteines in the context of infection. We will test three specific aims. In Aim 1, we will define the molecular mechanisms linking Cys219 inactivation to defective legumain processing and intracellular trafficking. In Aim 2, we will use metabolic labeling and nucleoside recoding chemistry to establish whether oxidation of the ribosomal protein uL14 on Cys125 inhibits translation. In Aim 3, we will develop a quantitative, tandem mass tagging-based approach to functionally map novel sites of cysteine oxidation induced by a cancer-promoting bacterial protein in vivo. By providing mechanistic insights into how specific oxidation sites affect tumorigenic processes, and discovering additional candidates for characterization, the overall impact of this proposal will be to build fundamental understanding of how reactive cysteines shape cancer signaling and thereby inform the development of new strategies for perturbing these pathways during infection.