PROJECT SUMMARY The goal of this Ruth L. Kirschstein NRSA F31 fellowship is to develop the expertise of the applicant in pathogenic mechanisms of alcohol-induced post-translational modifications (PTMs). Alcohol-associated liver disease (ALD) is a leading cause of preventable mortality worldwide causing approximately 5.9% of deaths every year, yet lacks preventative or regenerative therapies. The liver is the primary site of alcohol detoxification generating products that negatively impact hepatic biochemistry. A major consequence of alcohol metabolism is the induction of lysine acetylation, a metabolically induced PTM. One poorly characterized factor is the disruption to the network of cysteine thiols involved in the cysteine proteome, which is crucial to enzyme activity, protein structure, and signaling. Models of early-stage ALD have been applied to proteomic studies to better understand the downstream effects of alcohol metabolism and elucidate perturbed pathways. Using a 6-week Lieber-DeCarli (LD) model, we quantified thiol redox changes in the hepatic proteome employing a novel click-chemistry-based nHPLC-MS/MS assay. With the same model, we examined the impact of chronic alcohol consumption on hepatic lysine N-acetylation through quantitative analysis via acetyl-IP followed by nHPLC-MS/MS. Briefly, these studies identified that alcohol feeding induced an overall reduced cysteine proteome and increased lysine acetylation. Recent studies have identified a biophysical spatial association between lysine and cysteine amino acids leading to direct interactions. Lysine acetylation is amplified when cysteine residues are within 15 Å, referred to as a cysteine-lysine pair (CysLys). Our work suggests the alcohol-induced increase in N-acetylation is partially due to the acetylation of the more reactive cysteine thiol followed by transfer to a nearby lysine, resulting in an SàN acetyl transfer reaction. The proposed training plan will investigate this relationship in a chronic LD model by integrating acetyl- and thiol redox OMIC analyses to evaluate pathologically relevant protein targets. Therefore, the central hypothesis of this F31 application is that protein CysLys pairs are a mechanism for increased lysine acetylation and cysteine redox sensitivity during alcohol toxicity and contributes to the pathology of ALD. Specific aim 1 will characterize hepatic proteomic signatures impacted by Lys acetylation and Cys redox changes due to chronic alcohol metabolism using a multi-layered integrated analysis. Specific aim 2 will define regulatory mechanisms of SàN acetyl transfer in specific proteins critical to the progression of ALD. These aims will be interrogated utilizing a multidisciplinary approach that will provide a thorough understanding of lysine acetylation and thiol redox signaling by regulating protein structure. Completion of these specific aims will support the applicant in becoming an independent researcher in the field of alcohol-induced met...