ABSTRACT Alcoholic associated liver disease (ALD) is a consequence of chronic alcohol consumption that leads to hepatocellular injury and liver inflammation. Alcohol abuse increases the translocation of gut-derived endotoxins (lipopolysaccharide; LPS) to the portal circulation and causes Kupffer cells activation, the resident macrophages in the liver, through Toll-like receptor 4 (TLR4) signaling, leading to nuclear regulatory factor kappa B (NF-κB) activation, secretion of inflammatory cytokines, including tumor necrosis factor alpha (TNF-α) and production of reactive oxygen species (ROS) . SUMOylation is a posttranslational modification that involves addition of SUMOs (small ubiquitin-like modifiers) modulating protein stability, activity and localization. Several studies have intimated a close relationship between SUMOylation and ROS. We recently demonstrated that ubiquitin Conjugating Enzyme 9 (UBC9), the sole E2 protein required by the SUMOylation machinery, is upregulated in murine NIAAA and Intragastric models. We also found that UBC9 is phosphorylated and this is correlated with high level of SUMOylation activity in LPS-activated KCs that leads to inflammation. In addition, we elucidated the key function of SUMOylated microsomal Cytochrome P450 2E1 (CYP2E1) in ALD that sustains its enzymatic activity and protein stability. However, several important mechanistic pathways that are altered in ALD have not been investigated yet. By phospho-peptide mapping of LPS-activated KCs and NIAAA KCs, we found that UBC9 was phosphorylated at two serine residues (S2 and S7) and one tyrosine residue (Y68). Interestingly, the S2 and S7 UBC9 phospho-events were found in both normal and activated KCs whereas Y68 phosphorylation was induced specifically in activated cells. In order to examine whether UBC9 phosphorylation has pro-inflammatory effects in KCs in ALD, Mass Spectrometry (MS) was performed to identify UBC9 interacting proteins in isolated- NIAAA KCs. Crosslinking proteomics revealed interaction of UBC9 with several inflammatory pathways. Furthermore, phospho-UBC9 was found to interact favorably with components of NF-κB signaling. CRISPR- directed gene editing of the Y68 and S2 residues (but not S7) of UBC9 lowered inflammatory cytokines in activated KCs. These data provide the rationale to examine how phospho-UBC9 regulates components of inflammatory signaling. This proposal tests the novel hypothesis that ethanol induces KCs activation in ALD by modulating UBC9’s biological activity and this may impact key inflammatory response signaling pathways. Three specific aims are proposed: 1) Examine how phosphorylation of UBC9 influences its biological function upon alcohol exposure, 2) Investigate the effect of UBC9 Y68 phosphorylation on the crosstalk between KCs and hepatocytes in ALD, 3) Examine the effects of UBC9 Y68 gene editing on c- SRC interaction in ALD. If successfully completed, these studies should provide highly novel information on the rol...