Role of phospho UBC9 in alcohol-associated liver disease

摘要 酒精相关性肝病（ALD）是慢性饮酒的结果， 肝细胞损伤和肝脏炎症。酒精滥用增加肠源性内毒素的移位 （脂多糖; LPS）的门静脉循环，并导致枯否细胞激活，居民巨噬细胞 在肝脏中，通过Toll样受体4（TLR 4）信号传导，导致核调节因子κ B（NF-κB） 活化、分泌炎性细胞因子，包括肿瘤坏死因子α（TNF-α）和产生 活性氧（ROS）。SUMO化是一种翻译后修饰，涉及SUMO的添加 （小泛素样修饰物）调节蛋白质稳定性、活性和定位。几项研究 提示SUMO化与ROS之间存在密切关系。我们最近证明了泛素 结合酶9（UBC 9）是SUMO化机制所需的唯一E2蛋白，在E2受体中上调。 鼠NIAAA和胃内模型。我们还发现UBC 9是磷酸化的，这与 LPS激活的KC中高水平的SUMO化活性，导致炎症。此外，我们还阐明了 SUMO化的微粒体细胞色素P450 2 E1（CYP 2 E1）在ALD中的关键功能是维持其酶活性， 活性和蛋白质稳定性。然而，在ALD中改变的几个重要的机制途径并没有改变。 已经被调查过了。通过对LPS激活的KCs和NIAAA KCs的磷酸肽图谱分析，我们发现UBC 9 在两个丝氨酸残基（S2和S7）和一个酪氨酸残基（Y 68）处磷酸化。有趣的是，S2 和S7 UBC 9磷酸化事件在正常和活化的KCs中均被发现，而Y 68磷酸化事件在正常和活化的KCs中均被发现。 在活化细胞中特异性诱导。为了检查UBC 9磷酸化是否具有促炎作用， 在ALD中KC中的作用，进行质谱（MS）以鉴定分离的- NIAAA KCs。交联蛋白质组学揭示了UBC 9与几种炎症途径的相互作用。 此外，发现磷酸化UBC 9与NF-κB信号传导的组分有利地相互作用。CRISPR- UBC 9的Y 68和S2残基（但不是S7）的定向基因编辑降低了炎症细胞因子， 激活KC。这些数据提供了研究磷酸-UBC 9如何调节细胞内的组分的基本原理。 炎症信号该提议测试了乙醇诱导KCs活化的新假设， ALD通过调节UBC 9的生物活性，这可能会影响关键的炎症反应信号 途径。提出了三个具体的目标：1）研究UBC 9的磷酸化如何影响其表达。 2）研究UBC 9 Y 68磷酸化对酒精暴露后的生物学功能的影响， ALD中KC和肝细胞之间的串扰，3）检查UBC 9 Y 68基因编辑对c- ALD中的SRC相互作用。如果成功完成，这些研究将提供高度新颖的信息， 磷UBC 9在ALD发展中的作用，并可能提供新的治疗策略， 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