ER stress in ethanol-induced injury to hepatocytes

乙醇诱导的肝细胞损伤中的内质网应激

• 批准号: 8954727
• 负责人: Shengyun Fang
• 金额: $ 18.23万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8954727
• 关键词: Accounting; Alcohol consumption; Alcohol-Induced Disorders; Alcoholic Hepatitis; Alcoholic Liver Diseases; Alcohols; Arts; Biochemical; Biological; Biotin; Calnexin; Cells; Cellular biology; Cessation of life; Cirrhosis; Cytosol; Development; Dominant-Negative Mutation; Endoplasmic Reticulum; Endoplasmic Reticulum Degradation Pathway; Ethanol toxicity; Event; Fatty Liver; Genetic; Goals; HepG2; Hepatocyte; Human; Lead; Lectin; Liver; Mass Spectrum Analysis; Membrane; Modeling; Molecular; Molecular Chaperones; Mus; Pathogenesis; Pathway Analysis; Primary carcinoma of the liver cells; Proteasome Inhibition; Proteins; Proteomics; Reporting; Research; Role; Severities; Study models; Techniques; Therapeutic Intervention; Time; Transgenic Organisms; Tunicamycin; Zebrafish; alcohol exposure; base; endoplasmic reticulum stress; innovative technologies; insight; liver injury; mortality; neglect; new technology; novel; novel strategies; novel therapeutic intervention; overexpression; protein function; protein misfolding; public health relevance; ubiquitin-protein ligase

 DESCRIPTION (provided by applicant): Alcohol consumption causes alcoholic liver disease (ALD) that includes an array of liver injury ranging from fatty liver to cirrhosis. Alcohol account for 40-50% of all the death due to cirrhosis and is the most common cause of liver-related mortality. Endoplasmic reticulum (ER) stress, caused by accumulation of alcohol-damaged proteins in the ER, has recently been established as a significant mechanism contributing to ALD. However, two events that govern the severity of ER stress, including the spectrum of proteins damaged in the ER by alcohol and the intrinsic protective mechanisms that remove the damaged proteins, are not known. The goal of this application is to 1) identify alcohol-induced damage of ER proteins by a newly developed proteomic approach with an aim to uncover novel molecular understanding of ALD; 2) elucidate mechanisms that eliminate these damaged proteins and offer protection. Using primary hepatocytes as a model, alcohol- damaged ER proteins will be identified using the spatially restricted enzymatic tagging technique in combination with mass spectrometry. All proteins in the ER lumen will first be tagged with biotin. Based on the mechanism of ERAD, biotinylated damaged ER proteins will be recovered from the cytosol following proteasome inhibition and will be identified by mass spectrometry. The proteomic results will be validated by biochemical and cell biological analysis. The involvement of the damaged ER proteins in ALD development in addition to inducing ER stress will be assessed by pathway analysis. With this new and innovative technology, we will be able to systematically identify, for the first time, the ER proteins that are damaged by alcohol, which wil lead to a clearer molecular picture as to how alcohol causes ER stress and ALD. ERAD is the major defense against ER stress by facilitating degradation of misfolded or damaged proteins from the ER. Zebrafish is an emerging model for studying ALD. Our preliminary studies suggest that gp78, one of the major E3 ubiquitin ligases functioning in ERAD, protects against alcohol-induced ER stress. Accordingly, we will study ALD development in transgenic zebrafish lines specifically overexpressing wt gp78 and its dominant negative mutant, gp78R2m, in liver, which we have established recently. We will also validate the proteomic results obtained in hepatocytes in alcohol-treated zebrafish liver. Together, this study will, for the first time, idenify alcohol-damaged ER proteins and the protective role of gp78 against alcohol-induced ER stress and ALD. It will not only provide new insight into the molecular mechanisms of ALD pathogenesis and may also identify novel protective mechanisms for therapeutic intervention to ALD.

 描述（由申请人提供）：饮酒会导致酒精性肝病（ALD），包括从脂肪肝到肝硬化等一系列肝损伤。 40-50% 的死亡由肝硬化引起，是肝脏相关死亡的最常见原因。内质网 (ER) 应激是由内质网中酒精损伤蛋白的积累引起的，最近已被确定为一个重要的原因。然而，控制 ER 应激严重程度的两个事件，包括 ER 中被酒精破坏的蛋白质谱和去除受损蛋白质的内在保护机制，尚不清楚。 1) 通过新开发的蛋白质组学方法识别酒精引起的 ER 蛋白损伤，旨在揭示 ALD 的新分子理解；2) 使用原代肝细胞作为酒精损伤模型，阐明消除这些受损蛋白并提供保护的机制。 ER 蛋白将使用空间限制酶标记技术结合质谱法进行鉴定。基于 ERAD 的机制，所有蛋白都将被生物素标记。蛋白酶体抑制后，受损的 ER 蛋白将从细胞质中回收，并通过质谱法进行鉴定。除了诱导 ER 应激外，受损的 ER 蛋白还参与 ALD 发育。通过这种新的创新技术进行评估，我们将能够首次系统地识别被酒精破坏的 ER 蛋白，这将有助于我们更清楚地了解酒精如何导致 ER。 ERAD 是通过促进 ER 中错误折叠或受损蛋白质的降解来对抗 ER 应激的主要防御手段，我们的初步研究表明，gp78 是在 ERAD 中发挥作用的主要 E3 泛素连接酶。 ，防止酒精诱导的 ER 应激 因此，我们将研究转基因斑马鱼品系中特异过度表达 wt gp78 及其显性失活突变体的 ALD 发育。我们最近在肝脏中建立了 gp78R2m，我们还将验证酒精处理的斑马鱼肝脏中肝细胞的蛋白质组学结果，这项研究将首次鉴定酒精损伤的 ER 蛋白和 gp78 的保护作用。它不仅可以为 ALD 发病机制提供新的见解，还可以确定 ALD 治疗干预的新保护机制。