Redox Regulation of O-GlcNAcylation Signaling in the Pathogenesis of Alcoholic Fatty Liver Disease

项目摘要 酒精性脂肪性肝病（AFLD）是慢性肝病的主要原因。在全球范围内，AFLD占0.9% 占总死亡率的0.6%和残疾调整寿命年的0.6%，仍然是全球范围内的公共卫生问题。 尽管AFLD对健康和经济产生了深远的影响，但由于缺乏 详细了解其发病机制和进展的决定因素。该项目的目标是 阐明氧化还原调节的肝脏O-GlcNAc信号在酒精诱导的肝损伤中的新作用， 从而鉴定针对AFLD的潜在的新的预防、诊断和/或治疗靶标。氧化 应激被认为在参与AFLD的起始和发展的许多途径中起中心作用。 肝脏谷胱甘肽（GSH）在维持细胞氧化还原稳态中起作用。有趣的是，慢性GSH 小鼠中谷氨酸-半胱氨酸连接酶修饰亚基（Gclm）基因整体破坏的缺陷 提供针对酒精诱导的脂肪变性的保护。分子和代谢组学研究表明， 低GSH肝保护作用与AMPK信号通路和NRF 2抗氧化剂的激活有关 反应和肝代谢的重新编程，有利于维持细胞的氧化还原， 代谢平衡多组学分析表明，转录后机制发挥了重要作用 在介导低谷胱甘肽引起的代谢适应乙醇暴露。蛋白质的O-GlcNAc化是一种 翻译后修饰（PTM）的普遍形式，其中单个O-连接的N-乙酰葡糖胺（O- GlcNAc）部分被添加到细胞核、细胞质和线粒体蛋白中。该过程由 一对O-GlcNAc循环酶，O-GlcNAc转移酶（OGT）和O-GlcNAc酶（OGA）。在肝脏中，O- 已显示GlcNAc信号传导充当营养和胁迫感测的重要机制， 随后调节肝细胞内稳态。异常的O-GlcNAc酰化与肝脏 胰岛素抵抗、脂肪肝和相关的纤维化。到目前为止，人们对O的作用知之甚少。 GlcNAc在AFLD发病机制中的作用我们的初步研究表明，在GSH缺陷型Gclm基因敲除小鼠中， 在肝脏中，慢性酒精摄入诱导了整体O-GlcNA化和相关的有益变化， 细胞通路和信号蛋白。该项目将扩大这些挑衅性的发现，并测试 预测由慢性氧化应激（由于GSH）驱动适应性O-GlcNAc信号传导的假说 缺乏）在肝脏中保护免受酒精诱导的肝损伤。我们建议（1）利用Gclm零 小鼠模型，以检查乙醇诱导的O-GlcNAc酰化和磷酸化修饰的变化， O-GlcNAc循环酶的肝蛋白质组和底物靶向，以及（2）评估功能性 人类HepaRG中乙醇相关应激反应中最佳候选蛋白-O-GlcNAcylation的影响 细胞这项研究将是第一次检查肝脏氧化还原状态和O- 酒精诱导的肝损伤背景下的GlcNAc酰化信号传导。

PROJECT SUMMARY Alcoholic fatty liver disease (AFLD) is a major cause of chronic liver disease. Globally, AFLD accounts for 0.9% of total mortality and 0.6% of disability-adjusted life years and remains a public health problem worldwide. Despite its profound health and economic impact, the AFLD management remains challenging due to the lack of detailed understanding of determinants of its pathogenesis and progression. The goal of this project is to elucidate a novel role of redox-modulated hepatic O-GlcNAc signaling in alcohol-induced liver injury, and thereby identify potential novel preventive, diagnostic and/or therapeutic targets against AFLD. Oxidative stress is implicated to play a central role in many pathways involved in the initiation and progression of AFLD. Hepatic glutathione (GSH) functions in maintaining cellular redox homeostasis. Intriguingly, chronic GSH deficiency in mice harboring a global disruption of the glutamate-cysteine ligase modifier subunit (Gclm) gene confers protection against alcohol-induced steatosis. Molecular and metabolomics studies indicate that the hepatoprotective effect of low GSH is linked to activations of AMPK signaling pathway and NRF2 antioxidant response, and reprogramming of hepatic metabolism that benefits the maintenance of cellular redox and metabolic homeostasis. Multiomics analyses imply that post-transcriptional mechanisms play a significant role in mediating low GSH-elicited metabolic adaptation upon ethanol exposure. O-GlcNAcylation of protein is a prevalent form of post-translational modification (PTM), where a single O-linked N-acetylglucosamine (O- GlcNAc) moiety is added to nuclear, cytoplasmic and mitochondrial proteins. This process is controlled by a pair of O-GlcNAc cycling enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). In the liver, O- GlcNAc signaling has been shown to serve as an important mechanism for nutrient and stress sensing and subsequent regulation of liver cellular homeostasis. Aberrant O-GlcNAcylation has been implicated in hepatic insulin resistance, fatty liver disease and associated fibrosis. To date, little is known about the role of O- GlcNAcylation in AFLD pathogenesis. Our preliminary studies show that, in GSH-deficient Gclm-null mouse livers, chronic-binge ethanol feeding induced beneficial changes in global O-GlcNAylation and associated cellular pathways and signaling proteins. This project will expand upon these provocative findings and test the hypothesis predicting that adaptive O-GlcNAc signaling driven by chronic oxidative stress (due to GSH deficiency) in the liver protects against alcohol-induced liver injury. We propose to (1) utilize the Gclm-null mouse model to examine ethanol-induced changes in O-GlcNAcylation and phosphorylation modifications of the liver proteome and substrate targeting of O-GlcNAc cycling enzymes, and (2) evaluate the functional impacts of top candidate protein-O-GlcNAcylations in ethanol-associated stress response in human HepaRG cells. The proposed study will be the first examination of in vivo interplay between hepatic redox status and O- GlcNAcylation signaling in the context of alcohol induced liver injury.