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.
