Regulation of oxidative stress signaling by tyrosine phosphorylation of antioxidant enzymes

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 bydetoxifying and neutralizing aberrant reactive molecules. Spatiotemporally control of OSR is achieved throughcompartmentalization 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 withvarious human diseases. For example chronic OSR is a hallmark of obesity a global epidemic as well as amajor risk factor for developing cardiovascular diseases metabolic syndrome and cancer. To better understandobesity it is paramount that we elucidate the coordination of the OSR metabolon defined here as the sequentialantioxidant 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 evaluationof the published phosphoproteome stratified by enzyme classification and pathway enrichment analysis indicatesthat despite low intracellular stoichiometry pY are enriched on antioxidant enzymes. However the majority ofpY sites on antioxidant enzymes are not functionally characterized. My overarching goal in this proposal is togain network level insight into the pY directed regulation of antioxidant enzymes and the resulting dynamics ofdysregulated OSR. I hypothesize that obesity-driven pY on multiple antioxidant enzymes modulates theircatalytic activity to produce systemic changes in OSR. I will test this hypothesis by employing proteomicsmetabolomics structural analysis and computational modeling. During the mentored phase of this applicationI will predict the functional role of previously uncharacterized pY validate predictions using in vivo as well as invitro 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 sexspecific manner. Additionally I will determine how altered dietary serine glycine or addition of small moleculeantioxidants ameliorate HFD phenotypes and the sex specific responses in the OSR metabolon that may betherapeutically relevant. This proposal and the outlined training plan will equip me with the technical skillsscientific knowledge and professional training that will serve as the foundation to launch my research focusedon OSR regulation as an independent investigator.