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.