SUMMARY While hydrogen peroxide has long been understood as a toxin used by the human immune system to kill infectious organisms, only recently has it become well accepted that it serves as a second messenger in eukaryotes, produced in response to growth factors, cytokines and immune system effectors and promoting or modulating downstream signal transduction pathways. Through insights contributed in part by the work of the PI, a family of cysteine-dependent, peroxide-reducing enzymes known as the peroxiredoxins (Prxs) have also emerged from relative obscurity to become widely recognized not just as one of the primary oxidant removal systems in almost all organisms, but also as key modulators of cell signaling pathways. PI Poole's work on the enzymology, biophysical attributes and structures of Prxs from a variety of organisms has contributed greatly to understanding the mechanism and regulation of this highly abundant family of enzymes. In 2003, PI Poole and collaborator Andy Karplus published a Science paper in which structural determinants of the sensitivity of Prxs toward peroxide-mediated hyperoxidation of the active site cysteine were identified. This led to our proposal of the “floodgate hypothesis” explaining the potential benefits of such a peroxide-mediated “off switch”; under conditions where peroxide levels begin to rise (e.g. NADPH oxidase activation), Prx inactivation would promote the local accumulation of peroxide near the source, allowing for the oxidation of alternative protein targets. Dr. Poole has also been at the forefront of developing chemical tools to evaluate protein oxidation in cells through targeting sulfenic acid (R-SOH), the direct product of peroxide-mediated oxidation. These probes are now commercially available and have been used widely by researchers studying redox regulation and signaling to evaluate protein oxidation with high spatiotemporal precision. PI Poole's lab used these tools to show that cancer-associated growth factors elicit “hot spots” of protein oxidation proximal to the internalized receptors, providing support for the floodgate hypothesis. Future studies proposed here will build upon our existing strengths and collaborations. Specifically, we propose to investigate the effects of additional posttranslational modifications, including nitration, acetylation and phosphorylation, on Prx structure and activity. We will also investigate the mechanism by which thioredoxin can regulate and be regulated by human Prxs. The interface of Prx function with the regulation of signal transduction pathways involving protein oxidation is another area with significant gaps; we plan to follow up on our data suggesting that Prx inactivation and rising peroxide levels are key to cell cycle regulation. Finally, a new area that we are currently investigating in collaboration with Sharon Campbell is the oxidation sensitivity of the cancer-causing G12C mutant of KRAS, which has the potential to severely limit the effectiv...