A novel biological crosstalk between sumoylation and mitochondria dysfuntion in alcoholic liver disease

摘要 所有生物体都需要外源性食物/营养素以腺苷的形式产生能量 三磷酸（ATP），这是许多细胞功能所需的，并在线粒体中有效地产生 它们与活性氧（ROS）的产生和防御密切相关。早日 酒精消耗引起的线粒体功能障碍是一个很明显的变化， 线粒体蛋白质组、呼吸缺陷和线粒体DNA（mtDNA）损伤。的这些作用 乙醇在肝脏中是突出的，肝脏是体内乙醇代谢的主要部位， 和坏死细胞死亡，并有助于酒精诱导的肝脏疾病（ALD）的发作或进展， 肝纤维化/肝硬化和癌症。SUMO化是一种翻译后修饰，涉及添加 SUMO（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，在哺乳动物中上调。 灌胃给药小鼠肝脏（IE）和腹水组织。我们还发现UBC 9是磷酸化的， 与脂多糖激活的库普弗细胞中高水平的SUMO化活性相关， 炎症发展。此外，我们阐明了SUMO化微粒体细胞色素的关键功能， P450 2 E1（CYP 2 E1）在ALD中维持其酶活性和蛋白质稳定性。许多细节 本实验室已开始研究SUMO化在ALD中的作用。然而，一些重要的 尚未研究ALD中改变的机制途径。为了探讨 ALD中的SUMO化，进行质谱（MS）以鉴定10天内SUMO化的蛋白质。 乙醇进料+1次酒精狂欢（NIAAA）模型，其中SUMO结合柱用于纯化 来自整个肝脏的SUMO化蛋白质。有趣的是，我们发现乙醇诱导SUMO化的变化， 参与ATP合成的几种线粒体蛋白（ATP 5 B，SOD 2，CLC 25 A5，HSPD 1，FBP 1， CYCS）、ATP代谢（ATP 5A 1、ATP 5 B、ATP 5C 1、ATP 5 F1、ATP 5 J2、ASPA 8、AK 3）和线粒体 疾病（UQCRC 2、PC、HMGCS 2、ECHS 1、COX 6 B1、NDUFV 3、COX 6C）。这些发现可能表明， SUMO化在氧化磷酸化、电子传递链（ETC）和呼吸控制中的潜在作用 比率的调节可以是调节这些蛋白质形成上述复合物的能力和/或调节它们的活性。 该提议验证了乙醇诱导ALD中线粒体功能障碍的新假设 调节呼吸链关键蛋白的SUMO化状态，并探索其分子机制。 机制等本论文的主要目的有两个：1）探讨SUMO化在乙醇调控的细胞凋亡中的作用。 ETC和ATP的产生，2）检查SUMO化在乙醇诱导的CYP 2 E1中的作用， 维持线粒体氧稳态。如果顺利完成，这些研究将提供 关于SUMO化在ALD发展中的作用的高度新颖的信息，并可能提供新的 治疗策略，这是高

ABSTRACT All living organisms require exogenous foods/nutrients for producing energy in the form of Adenosine Triphosphate (ATP), which is needed for numerous cellular functions and is efficiently produced in mitochondria that are intimately involved in the generation of and defense against reactive oxygen species (ROS). An early event that occurs in response to alcohol consumption is mitochondrial dysfunction, which is evident in changes to the mitochondrial proteome, respiration defects and mitochondrial DNA (mtDNA) damage. These effects of ethanol are prominent in the liver, the major site of ethanol metabolism in the body, promoting both apoptotic and necrotic cell death and contribute to the onset or progression of alcohol-induced liver diseases (ALD), leading to hepatic fibrosis/cirrhosis and cancer. 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 SUMOylation machinery, is upregulated in Intragastric fed-mice liver (IE) and cirrhotic tissues. We also found that UBC9 is phosphorylated and this is correlated with high level of SUMOylation activity in lipopolysaccharides-activated Kupffer cells that leads to inflammation development. In addition, we elucidated the key function of SUMOylated microsomal Cytochrome P450 2E1 (CYP2E1) in ALD that sustains its enzymatic activity and protein stability. Much of the detailed investigation of the role of SUMOylation in ALD has been started in our laboratory. However, several important mechanistic pathways that are altered in ALD have not been investigated yet. In order to explore the role of SUMOylation in ALD, Mass Spectrometry (MS) was performed to identify SUMOylated proteins in 10-day ethanol-feeding+1 Binge ethanol (NIAAA) model, where SUMOs binding columns were used to purify SUMOylated proteins from total livers. Interestingly, we found that ethanol induces changes in SUMOylation state of several mitochondrial proteins involved in ATP synthesis (ATP5B, SOD2, CLC25A5, HSPD1, FBP1, CYCS), ATP metabolism (ATP5A1, ATP5B, ATP5C1, ATP5F1, ATP5J2, ASPA8, AK3), and mitochondria disorder (UQCRC2, PC, HMGCS2, ECHS1, COX6B1, NDUFV3, COX6C). These finding could suggest a potential role of SUMOylation in oxidative phosphorylation, electron transport chain (ETC) and respiratory control ratio may be modulating the ability of these proteins to form the complex above and/or regulating their activity. This proposal tests the novel hypothesis that ethanol induces mitochondrial dysfunction in ALD regulating the SUMOylation status of key respiratory chain proteins and explore the molecular mechanisms. Two specific aims are proposed: 1) Explore the role of SUMOylation in ethanol-modulated ETC and ATP production, 2) Examine the role of SUMOylation in ethanol-induced CYP2E1 for maintenance of mitochondrial oxygen homeostasis. If successfully completed, these studies should provide highly novel information on the role of SUMOylation in the development of ALD and may provide novel therapeutic strategies, which is of high