ETHANOL EFFECTS ON PROTEOLYTIC SYSTEMS IN THE LIVER

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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.