Role of phospho UBC9 in alcohol-associated liver disease

摘要 酒精性肝病(ALD)是长期饮酒导致的后果 肝细胞损伤和肝脏炎症。酗酒会增加肠源性内毒素的移位 (脂多糖；内毒素)进入门静脉循环，并引起库普弗细胞激活，驻留巨噬细胞 在肝脏中，通过Toll样受体4(TLR4)信号，导致核调节因子kappaB(NF-κB) 激活、分泌炎性细胞因子，包括肿瘤坏死因子-α和产生 活性氧(ROS)。相扑是一种翻译后修饰，涉及相扑的添加。 (小泛素样修饰物)调节蛋白质的稳定性、活性和定位。几项研究已经 提示SUMO化与ROS之间有密切的关系。我们最近展示了泛素 结合酶9(UBC9)是SUMO化机制所需的唯一的E2蛋白，在 小鼠NIAAA模型和胃内模型。我们还发现UBC9是磷酸化的，这与 在内毒素激活的KCs中，高水平的SUMO化活性导致炎症。此外，我们还澄清了 糖基化微粒体细胞色素P450 2E1在阿尔茨海默病中的关键作用 活性和蛋白质稳定性。然而，在ALD中改变的几个重要的机械通路并没有 已经被调查过了。通过对脂多糖激活的KCs和NIAAA KCs的磷酸肽图谱的分析，我们发现UBC9 在两个丝氨酸残基(S2和S7)和一个酪氨酸残基(Y68)上被磷酸化。有趣的是，S2 在正常和激活的KCs中均发现S7 UBC9磷酸化事件，而Y68磷酸化 在激活的细胞中特异性地诱导。为了检测UBC9的磷酸化是否具有促炎作用 在ALD中KCs的作用，用质谱仪(MS)鉴定分离的KCs中的UBC9相互作用蛋白。 NIAAA KCs。交联蛋白质组学揭示了UBC9与多种炎症途径的相互作用。 此外，研究还发现，磷酸化的UBC9与NF-κB信号转导元件有良好的相互作用。CRISPR- 定向基因编辑UBC9的Y68和S2残基(但不是S7)可降低小鼠体内的炎性细胞因子 激活的KCs。这些数据为研究磷酸UBC9如何调节 炎症信号。这一建议验证了乙醇诱导KCs激活的新假设。 ALD通过调节UBC9的S生物活性及其可能影响的关键炎症反应信号 小路。提出了三个具体目标：1)研究UBC9的磷酸化如何影响其 2)研究UBC9 Y68磷酸化对酒精暴露的影响。 ALD中KCs与肝细胞之间的串扰，3)检测UBC9 Y68基因编辑对c-DNA的影响。 ALD中的SRC相互作用。如果成功完成，这些研究应该会提供非常新颖的信息 磷光体UBC9在ALD发展中的作用，并可能提供新的治疗策略，这是 高

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 role of phosphor UBC9 in the development of ALD and may provide novel therapeutic strategies, which is of high