Establishing Glyoxalase 2 as a Viable Target for the Treatment of Disease

The ability for cells to detect and respond to metabolic cues is critical to maintaining homeostasis, and perturbations in the sensing mechanisms that respond to oscillations in metabolic flux are the root cause of many diseases, including sepsis, autoimmunity, cancer, and diabetes. Protein post-translational modifications (PTMs) serve as these sensing mechanisms, facilitating alterations in enzyme function and/or localization. As a result, alterations in PTMs often drive disease, removing the breaks on homeostatic metabolic signaling to serve the nutritional needs of the diseased cell. Currently, we have a fundamental gap in our understanding of the composition, abundance, and enzymatic control of PTMs and how they are altered in both health and disease. My lab is dedicated to unraveling fundamental questions in PTM biology. To accomplish this, we developed sensitive LC-MS/MS-based methods to identify and quantify global changes in PTMs across a broad spectrum of biological samples. Using this approach, we identified lactoylLys as a novel PTM that dictates glycolytic flux and inflammatory signaling. These PTMs are driven through non-enzymatic reactions from acylglutathione intermediates that are controlled by glyoxalase 2 (GLO2). In cells lacking GLO2, these PTMs are significantly elevated, altering cell fate. Expanding on the success of the previous funding period, our primary goal for this renewal is to further define the role of GLO2 in the homeostatic regulation of Lys acylations. My research program is dedicated to understanding three fundamental questions: 1) What is the substrate profile for GLO2 and how does this dictate Lys acylation? We will determine the acylglutathione substrates for GLO2 using in vitro-based enzyme kinetic assays. In addition, we will determine the regulation of these substrates in cells and elucidate the primary transport mechanism into the mitochondria. Lastly, we will quantify the PTM profile across subcellular compartments in both wild-type and GLO2 knockout cells. 2) Is the glyoxalase cycle a metabolic liability in glycolysis-reliant disease states? Using informed decisions from our cell inventory of glyoxalase function, we will generate a panel of GLO1 and GLO2 knockout cell lines. Cell growth will be monitored on an Incucycte Live-Cell Analysis system to quantify proliferation and metabolic regulation. We will also evaluate Glo1 as a therapeutic target to reduce inflammatory signaling. 3) Is our view of the histone code too small? To date, we are capable of quantifying >100 PTMs using our robust LC-MS/MS-based assay. Using this approach, we will quantify the histone PTM profile in tissues collected from wild-type and Glo2 knockout mice. We will also screen eraser enzymes for their full spectrum of substrates in cells. Our primary goal is to re-define our understanding of how PTMs are regulated and how these processes go awry in disease. This project will address a fundamental gap in our basic understanding of how cell metabolism and PTMs are regulated. Due to the far-reaching implications of this project in the context of disease, this research program continues to be an ideal fit for the R35 MIRA Award. Project Number: 1R35GM161300-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: James Galligan | Institution: UNIVERSITY OF ARIZONA, TUCSON, AZ | Award Amount: $407,323 | Activity Code: R35 | Study Section: Special Emphasis Panel[ZRG1 MBBC-A (57)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11258647