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

摘要 所有有生命的生物体都需要外源食物/营养素来产生腺苷形式的能量 三磷酸(ATP)，是许多细胞功能所必需的，在线粒体中有效地产生 它们与活性氧物种(ROS)的产生和防御密切相关。一大早 饮酒后发生的事件是线粒体功能障碍，这在变化中是显而易见的 线粒体蛋白质组、呼吸缺陷和线粒体DNA(MtDNA)损伤。这些影响 乙醇在肝脏中占主导地位，是体内乙醇代谢的主要部位，促进两种细胞的凋亡 和坏死性细胞死亡，并有助于酒精性肝病(ALD)的发生或发展，导致 肝纤维化/肝硬变和癌症。和甲基化是一种翻译后修饰，它涉及到添加 SUMOS(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。我们最近证明了泛素 结合酶9(UBC9)是糖基化机制所需的唯一一种E2蛋白，在 灌胃喂养小鼠肝脏(IE)和肝硬变组织。我们还发现UBC9是磷酸化的，这是 与内毒素激活的Kupffer细胞中高水平的SUMO化活性相关，导致 炎症发展。此外，我们还阐明了苏木糖基化微粒体细胞色素的关键功能 P450 2E1(CYP2E1)在ALD中的表达，维持其酶活性和蛋白质的稳定性。很多细节 我们实验室已经开始研究SUMO化在ALD中的作用。然而，几个重要的 ALD中改变的机械性通路尚未被研究。为了探索它的作用， 在ALD中，用质谱仪(MS)在10天内鉴定SUMO化的蛋白质 乙醇-饲喂+1酒精(NIAAA)模型，其中使用SUMOS结合柱进行纯化 总肝脏中的Sumoylated蛋白质。有趣的是，我们发现乙醇引起糖基化的改变。 参与ATP合成的几种线粒体蛋白(ATP5B、SOD2、CLC25A5、HSPD1、FBP1、 、ATP代谢(ATP5A1、ATP5B、ATP5C1、ATP5F1、ATP5J2、ASPA8、AK3)和线粒体 疾病(UQCRC2、PC、HMGCS2、ECHS1、COX6B1、NDUFV3、COX6C)。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 比例可能正在调节这些蛋白质形成上述复合体的能力和/或调节它们的活性。 这项建议验证了酒精导致ALD患者线粒体功能障碍的新假设 呼吸链关键蛋白糖基化状态的调控及其分子机制探讨 机械装置。提出了两个具体的目标：1)探讨SUMO化在乙醇调节中的作用 ETC和ATP的产生，2)检测SUMO化在乙醇诱导的细胞色素P450 2 E1中的作用 维持线粒体氧稳态。如果成功完成，这些研究应该会提供 有关糖基化在肌萎缩侧索硬化症发生中的作用的高度新颖的信息，并可能提供新的 治疗策略，这是很高的

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