Project Summary Oxidative stress is a byproduct of energy production necessary for all living organisms and caused by unregulated reactive oxygen/nitrogen/carbonyl species (ROS/RNS/RCS) among others. Nature has evolved the oxidative stress response (OSR) as a key component of metabolism that maintains cellular homeostasis by detoxifying and neutralizing aberrant reactive molecules. Spatiotemporally control of OSR is achieved through compartmentalization and redundancies that are coupled to create a redox balance to promote survival. Unbalanced OSR due to a defective or overactive capacity to resolve oxidative damage is associated with various human diseases. For example, chronic OSR is a hallmark of obesity, a global epidemic as well as a major risk factor for developing cardiovascular diseases, metabolic syndrome, and cancer. To better understand obesity, it is paramount that we elucidate the coordination of the OSR metabolon, defined here as the sequential antioxidant enzymes, biochemical reactions, and cellular compartments that maintain redox homeostasis. The proper regulation of and adaptive changes by OSR require rapid signaling taking place in the seconds-to- minute timeframe. Such dynamics must therefore require fast regulatory networks such as protein post- translational modifications (PTMs). Phosphorylation of serine (S) (~90%), threonine (T) (~9%), and tyrosine (Y) (~0.1-1%) residues are one of the many ways cells regulate pathways that maximize survival. Initial evaluation of the published phosphoproteome stratified by enzyme classification and pathway enrichment analysis indicates that, despite low intracellular stoichiometry, pY are enriched on antioxidant enzymes. However, the majority of pY sites on antioxidant enzymes are not functionally characterized. My overarching goal in this proposal is to gain network level insight into the pY directed regulation of antioxidant enzymes and the resulting dynamics of dysregulated OSR. I hypothesize that obesity-driven pY on multiple antioxidant enzymes modulates their catalytic activity to produce systemic changes in OSR. I will test this hypothesis by employing proteomics, metabolomics, structural analysis, and computational modeling. During the mentored phase of this application, I will predict the functional role of previously uncharacterized pY, validate predictions using in vivo as well as in vitro enzyme kinetic assays, and demonstrate pY-driven OSR dysregulation in an in vivo high-fat diet (HFD)- induced obesity mouse model. Through these interdisciplinary approaches, I aim to define systems of pY- modified enzymes that “tune” metabolic response to HFD, and evaluate differential regulation of OSR in a sex specific manner. Additionally, I will determine how altered dietary serine, glycine, or addition of small molecule antioxidants ameliorate HFD phenotypes, and the sex specific responses in the OSR metabolon that may be therapeutically relevant. This proposal and the outlined training...