ETHANOL EFFECTS ON PROTEOLYTIC SYSTEMS IN THE LIVER

这项提议的假设是：1)慢性酒精消费 通过阻止溶酶体的加工和运输而损害溶酶体的生物发生 溶酶体水解酶，导致它们放置在其他细胞内或 (2)乙醇给药改变了泛素- 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。乙醇可能会有不同的影响 库普弗细胞中的蛋白酶体。2)乙醇给药会损害 肝细胞对乙醇修饰蛋白质的降解能力 新陈代谢。在特定目标1a中，将通过以下方式测量溶酶体的生物发生 组织蛋白酶L的炮制及区划研究 从对照组和乙醇喂养的大鼠分离出肝细胞。这种酶 沿着一条特定的路径穿过囊泡隔间 溶酶体，我们的目标是确定乙醇(S) 破坏了这一过程。组织蛋白酶L等水解酶的错误路由 可能会由于其强大的水解性而潜在地导致细胞损伤 容量。在特定的目标1b中，我们将使用亚细胞分离 免疫细胞化学和功能分析检测乙醇 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等介导溶酶体组装 水解酶作用于溶酶体。我们推测乙醇可能会改变 受体在细胞内的分布。在具体目标2中， 泛素-蛋白酶体途径的组成部分将被整体研究 肝脏以及对照组和乙醇的实质细胞和库普弗细胞- 饲养的大鼠接受随机和灌胃两种喂养方式。 这种蛋白分解途径在1)降解 改变的蛋白质；2)转录因子的激活 NfkappaB参与炎症反应的表达 回应。我们假设，虽然乙醇可能会下调 肝实质细胞中的蛋白酶体，其活性可能受到调节 在库普弗细胞中不同，因为后者起旁分泌作用 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的(即乙醛修饰的)和 溶菌酶的天然形式。

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.