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: 6629529
• 负责人: Terrence M. Donohue
• 金额: $ 8.88万
• 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/6629529
• 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.

该提议的假设是：1）慢性乙醇消耗 通过防止加工和贩运来损害溶酶体生物发生 溶酶体水解酶，导致它们在其他细胞内或 细胞外部位； （2）乙醇给药改变了泛素 - 通过灭活蛋白酶体，可能导致蛋白酶体途径 修饰蛋白的积累。乙醇可能会差异影响 库普弗细胞中的蛋白酶体。 2）乙醇给药会损害 肝细胞降解乙醇修饰的蛋白质的能力 代谢。在特定的目标1a中，将通过 检查组织蛋白酶l的加工和隔离 从对照和乙醇喂养的大鼠中分离出来的肝细胞。这酶 遵循通过水泡隔室的特定途径 溶酶体和我们的目的是确定乙醇的步骤 损害这个过程。误用组织蛋白酶L和其他水解酶 由于其有效的水解，可能会导致细胞损伤 能力。在特定的目标1B中，我们将使用亚细胞分馏 免疫细胞化学和功能分析以检查乙醇是否是否 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 水解酶至溶酶体。我们假设乙醇可能会改变 受体的细胞内分布。在特定的目标2中 将全面检查泛素 - 蛋白酶体途径的组件 肝以及对照和乙醇的实质和kupffer细胞 饲养大鼠均受到AD LIB和胃内进食标本的约束。 该蛋白水解途径在1）降解中具有至关重要的作用 蛋白质改变； 2）转录因子的激活 NFKAPPAB参与炎症表达 回复。我们假设乙醇可能会下调 肝实质细胞中的蛋白酶体可以调节其活性 在库普弗细胞中有差异性，因为后一种细胞发挥了旁分泌 在酒精性肝病的发病机理中的作用。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 及其提取物以降解改变（即醛修饰）和 溶菌酶的天然形式。