PROJECT SUMMARY/ABSTRACTThe ability for cells to detect and respond to metabolic cues is critical to maintaining homeostasis andperturbations in the sensing mechanisms that respond to oscillations in metabolic flux are the root cause of manydiseases including sepsis autoimmunity cancer and diabetes. There is mounting evidence that protein post-translational modifications (PTMs) are the critical sensors for these metabolic fluctuations and are oftendysregulated in disease. Currently we have a fundamental gap in our understanding of the compositionabundance and enzymatic control of PTMs and how they are altered in disease.My laboratory focuses on the identification and characterization of PTMs and how they are regulated in bothhealth and disease. To accomplish this goal we have developed sensitive methods to identify and quantify globalchanges in PTMs across a broad spectrum of biological samples. Using this approach we have identified anovel lysine PTM that is derived from a glycolytic by-product. These PTMs are elevated when glyoxalase 2(GLO2) is inhibited resulting in reduced glycolytic output and disrupted one-carbon metabolism. Our primarygoal is to establish the therapeutic efficacy of a GLO2 inhibition strategy for the treatment of metabolic disorders.My research program is dedicated to understanding four fundamental questions: 1) How does GLO2 controlone-carbon metabolism and cellular redox? GLO2 knockout cells have reduced glutathione and increasedoxidative stress. We will quantify the role of GLO2 in the regulation of de novo glutathione synthesis. In additionthe role of GLO2 in the regulation of antioxidant responses will be evaluated in a cellular model for oxidativestress and inflammatory signaling. 2) How are LactoylLys modifications regulated? We will employquantitative proteomics using CRISPR-Cas9 knockout cell lines of candidate proteins to identify enzymaticregulators of LactoylLys modifications in cells. 3) Is GLO2 a viable target for the treatment of glycolysis-dependent disease states? A xenograft model will be employed using GLO2 knockout cell lines to quantifyproliferation and metabolic regulation in vivo. This will determine the therapeutic feasibility of targeting GLO2 forthe treatment of disease. 4) Are LactoylLys modifications functional histone marks? We have identifiedhistones as targets for modification by LactoylLys modifications in unstimulated cells. The presence of thesePTMs basally suggests a putative role in transcriptional regulation. We will use proteomics to identify site-specificmodifications and putative `reader' domains for LactoylLys modifications in cells.Our primary goal is to establish the role of GLO2 and LactoylLys modifications in cell metabolism and chromatinbiology. This project will address a fundamental gap in our basic understanding of how cell metabolism isregulated. Understanding how these PTMs regulate homeostasis is a critical first step to understanding their rolein disease. Due to the far-reaching implications of this project and the broad applications for the treatment ofhighly glycolytic disease states this research program is an ideal fit for the ESI MIRA Award.