ETHANOL EFFECTS ON PROTEOLYTIC SYSTEMS IN THE LIVER

这项应用是对正在进行的关于酒精消费影响肝细胞蛋白分解途径的机制的研究的综合性更新。它的主要假设是ababant蛋白的降解是细胞生存中的关键事件，乙醇介导的肝细胞蛋白质降解途径的改变可以显著影响乙醇氧化产物修饰的蛋白质的破坏。在具体目标1中，我们将测量正常大鼠和乙醇喂养大鼠完整肝细胞中天然、醛修饰和氧化溶菌酶的分解代谢，以确定乙醇处理是否改变了这种外源添加的修饰蛋白的降解率。在特定的目标2中，我们将通过检测乙醇是否引起亚单位组成的变化、与辅助蛋白的关联或免疫沉淀酶上醛加合物的形成来检验乙醇引起的多催化蛋白酶(蛋白酶体)活性下降的本质。此外，将从对照组和酒精喂养的大鼠的肝脏中纯化蛋白酶体，以确定这种酶活性的变化是否稳定，从而为乙醇诱导的变化的性质提供一些指示。在具体目标3中，我们将确定蛋白质底物的泛素化和或泛素介导的蛋白质分解是否受到乙醇衍生代谢产物的蛋白质修饰的影响。这一目标将利用一个异源的体外蛋白分解系统来分析泛素-蛋白质结合物的形成和降解。我们还将对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-kappa 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.