ETHANOL EFFECTS ON PROTEOLYTIC SYSTEMS IN THE LIVER

乙醇对肝脏蛋白水解系统的影响

• 批准号: 6509092
• 负责人: Terrence M. Donohue
• 金额: $ 8.62万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6509092
• 关键词: Kupffer's cell; RNase protection assay; alcoholic beverage consumption; carbohydrate receptor; cysteine endopeptidases; cytotoxicity; electrophoresis; enzyme linked immunosorbent assay; ethanol; gel mobility shift assay; hepatotoxin; immunocytochemistry; laboratory rat; liver metabolism; lysosomes; lysozyme; mannose 6 phosphate; nuclear factor kappa beta; proteasome; protein degradation; proteolysis; ubiquitin

The hypotheses of this proposal are: 1) chronic ethanol consumption impairs lysosome biogenesis by preventing processing and trafficking of lysosomal hydrolases, causing their placement at other intracellular or extracellular sites; (2) Ethanol administration alters the ubiquitin- proteasome pathway by inactivating the proteasome which could lead to accumulation of modified proteins. Ethanol may differentially influence the proteasome in Kupffer cells. 2) Ethanol administration impairs the capacity of hepatocytes to degrade proteins modified by ethanol metabolism. In Specific Aim 1a, Lysosome biogenesis will be measured by examining the processing and compartmentalizatin of cathepsin L in hepatocytes isolated from control and ethanol-fed rats. This enzymes follows a specific pathway through vesicular compartments en route to the lysosome and our aim is to determine the step(s) at which ethanol impairs this process. Misrouting of cathepsin L and other hydrolases could potentially cause cell damage due to their potent hydrolytic capacities. In Specific Aim 1b we will use subcellular fractionation immunocytochemistry and functional assays to examine whether ethanol influences the distribution of the mannose-6-phosphate receptor. This receptor mediates lysosome assembly by targeting cathepsin L and other hydrolases to the lysosome. We postulate that ethanol may change the receptor's intracellular distribution. In Specific Aim 2, the components of the ubiquitin-proteasome pathway will be examined in whole livers as well as parenchymal and Kupffer cells of control and ethanol- fed rats subjected to both ad lib and intragastric feeding specimens. This proteolytic pathway has a crucial role in 1) the degradation of altered proteins; and 2) the activation of the transcription factor NfkappaB which is involved in the expression of the inflammatory response. We postulate that while ethanol may down-regulate the proteasome in liver parenchymal cells, its activity may be regulated differentially in Kupffer cells, since the latter cells play a paracrine role in the pathogenesis of alcoholic liver disease. Specific Aims 3 and 4 will address third hypothesis by testing the capacity of hepatocytes and their extracts to degrade altered (i.e. aldehyde-modified) and native forms of lysozyme.

该提案的假设是：1）长期乙醇消耗 通过阻止加工和运输来损害溶酶体生物发生 溶酶体水解酶，导致它们位于其他细胞内或 细胞外位点； (2) 乙醇给药会改变泛素 蛋白酶体途径通过使蛋白酶体失活而导致 修饰蛋白质的积累。乙醇可能会产生不同的影响 库普弗细胞中的蛋白酶体。 2) 乙醇管理会损害 肝细胞降解乙醇修饰蛋白质的能力 代谢。在具体目标 1a 中，溶酶体生物发生将通过以下方式测量 检查组织蛋白酶 L 的加工和区室化 从对照大鼠和乙醇喂养大鼠中分离出肝细胞。这种酶 遵循通过囊泡区室的特定途径 溶酶体，我们的目标是确定乙醇的步骤 损害这个过程。组织蛋白酶 L 和其他水解酶的错误路由 由于其有效的水解作用，可能会导致细胞损伤 能力。在特定目标 1b 中，我们将使用亚细胞分级分离 免疫细胞化学和功能测定来检查乙醇是否 影响甘露糖-6-磷酸受体的分布。这 受体通过靶向组织蛋白酶 L 和其他物质介导溶酶体组装 水解酶进入溶酶体。我们假设乙醇可能会改变 受体的细胞内分布。在具体目标 2 中， 泛素-蛋白酶体途径的组成部分将被全面检查 肝脏以及对照和乙醇的实质细胞和枯否细胞- 对大鼠进行随意喂养和胃内喂养标本的喂养。 该蛋白水解途径在以下方面发挥着至关重要的作用：1) 改变蛋白质； 2）转录因子的激活 NfkappaB 参与炎症的表达 回复。我们假设虽然乙醇可能下调 肝实质细胞中的蛋白酶体，其活性可能受到调节 库普弗细胞有差异，因为后者发挥旁分泌作用 在酒精性肝病发病机制中的作用。具体目标 3 和 4将通过测试肝细胞的能力来解决第三个假设 及其提取物降解改变（即醛改性）和 溶菌酶的天然形式。