ETHANOL EFFECTS ON PROTEOLYTIC SYSTEMS IN THE LIVER

这个应用程序是竞争性更新正在进行的研究酒精消费影响肝细胞蛋白水解途径的机制。 其主要假设是异常蛋白的降解是细胞存活的关键事件，并且乙醇介导的肝细胞中蛋白降解途径的改变可以显著影响乙醇氧化产物修饰的蛋白的破坏。 在具体目标1中，我们将测量来自对照和乙醇喂养大鼠的完整肝细胞中的天然溶菌酶、去氧酶修饰的溶菌酶和氧化的溶菌酶的催化活性，以确定乙醇给药是否改变这种外源性添加的修饰蛋白的降解速率。 在具体目标2中，我们将研究乙醇诱导的多催化蛋白酶（蛋白酶体）活性下降的性质，通过研究乙醇是否会导致亚基组成的变化，与辅助蛋白的关联，或在免疫沉淀酶上形成醛加合物。 此外，蛋白酶体将从对照和乙醇喂养大鼠的肝脏中纯化，以确定酶活性的这种变化是否稳定，从而提供乙醇诱导的变化的性质的一些指示。 在具体目标3中，我们将确定是否泛素化和/或泛素介导的蛋白质底物的蛋白水解是/是由蛋白质修饰由于乙醇衍生的代谢产物的影响。 本研究将利用一种异源的体外蛋白水解系统来分析泛素-蛋白质结合物的形成和降解。 我们还将用原代肝细胞进行实验，以确定醛加合物的形成是否可以抑制特定修饰蛋白的破坏，包括调节蛋白I-κ B-α。

This application is for compeptitive renewal of ongoing research into the mechanisms by which alcohol consumption affects proteolytic pathways in liver cells. It's major hypothesis is that the degradation of abberant proteins is a crucial event in cell survival and that ethanol-mediated alterations of the protein degradative pathways in liver cells can significantly affect the destruction of proteins modified by the products of ethanol oxidation. In Specific Aim 1, we will measure the catabolism of native, aldehyde-modified and oxidized lysozyme in intact hepatocytes from control and ethanol-fed rats in order to determine whether ethanol administration alters the degradation rate of this exogenously-added modified protein. In Specific Aim 2 we will examine the nature of the ethanol-induced decline in the activity of the multicatalytic proteinase (proteasome) by examining whether ethanol causes changes in subunit composition, associations with accessory proteins, or the formation of aldehyde adducts on the immunoprecipitated enzyme. In addition, the proteasome will be purified from livers of control and ethanol-fed rats to ascertain whether such alterations in enzyme activity are stable, thereby providing some indication of the nature of ethanol-induced alterations. In Specific Aim 3 we will determine whether ubiquitylation and or ubiquitin-mediated proteolysis of protein substrates is/are affected by protein modification due to ethanol-derived metabolic products. This aim will utilize a heterologous in vitro proteolytic system to analyze the formation and the degradation of ubiquitin-protein conjugates. We will also conduct experiments with primary hepatocytes to determine whether aldehyde adduct formation can inhibit the destruction of specific modified proteins, including the regulatory protein, I-kappa B-alpha.