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: 6168157
• 负责人: Terrence M. Donohue
• 金额: $ 8.13万
• 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/6168157
• 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.

该建议的假设是：1）慢性乙醇消费 通过阻止蛋白质的加工和运输， 溶酶体水解酶，导致其放置在其他细胞内或 （2）乙醇给药改变了泛素- 通过灭活蛋白酶体， 修饰蛋白质的积累。乙醇可能会对 枯否细胞中的蛋白酶体。2)乙醇给药会损害 肝细胞降解乙醇修饰蛋白质的能力 新陈代谢.在特定目标1a中，将通过以下方法测量溶酶体生物发生： 研究组织蛋白酶L加工和区室化 从对照和乙醇喂养的大鼠分离的肝细胞。这些酶 沿着一条特定的途径穿过囊泡隔室， 我们的目标是确定乙醇在溶酶体中的步骤， 阻碍了这一进程。组织蛋白酶L和其他水解酶的错误路由 可能会导致细胞损伤，因为它们具有强大的水解性， 能力。在特定目标1b中，我们将使用亚细胞分离 免疫细胞化学和功能测定，以检查乙醇是否 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和其他蛋白酶介导溶酶体组装 水解酶到溶酶体。我们假设乙醇可以改变 受体的细胞内分布。具体目标2 泛素-蛋白酶体途径的组成部分将在整体检查 肝脏以及对照组和乙醇组的实质和枯否细胞， 喂食大鼠，自由进食和胃内喂食标本。 这种蛋白水解途径在以下方面具有关键作用：1）降解 改变的蛋白质;和2）转录因子的激活 NfkappaB参与炎症因子的表达， 反应我们假设，虽然乙醇可以下调 肝实质细胞中的蛋白酶体，其活性可被调节 库普弗细胞中的差异，因为后者细胞发挥旁分泌作用， 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，以降解改变的（即去乙酰化修饰的）， 天然形式的溶菌酶