The overall working hypothesis of this proposal is that chronic alcohol consumption alters proteasome-dependent proteolytic activity of the liver. This decrease in activity contributes to the buildup of protein and impacts the degradation of modified proteins. Changes in the proteasome and/or ubiquitin dependent proteolysis could be due to a number of factors. These include changes in the proteasome itself, changes in the level and composition of two regulatory complexes of the proteasome, the 19S and PA28 regulators, changes in accessory proteins that are required for the function of the proteasome such as organelle and cytosol specific chaperones, changes in ubiquitin hydrolase activity, or changes in the susceptibility of proteins for degradation. It is possible that an inability to degrades modified protein permits CYP2E1-hydroxyethyl radical adducts to be presented on the cell surface. CYP2E1 has also been suggested to be a source of active oxygen species in the liver so that the failure to degrade the protein could exacerbate oxidative stress initiated by Kupffer cells. The goals of the research described in this proposal are to characterize the proteasome-dependent degradative pathway after acute and chronic ethanol treatment, characterize the effect of ethanol on the degradation of endoplasmic reticulum proteins, and characterize the fate of proteins that become covalently modified by products of lipid peroxidation and/or ethanol metabolism. This will be accomplished using a combination of cell lines, primary hepatocytes, and whole animal studies. CYP2E1 will be used a model for the degradation of an integral endoplasmic reticulum membrane protein. This enzyme also provides an excellent model to investigate the fate of a protein modified by ethanol metabolites. These goals will be attained with the following three specific aims: 1) Characterize the effect of ethanol on the activity of ubiquitin-dependent and independent degradation. Alteration in proteasome activity, location and interaction with the PA28nd 19S regulatory complexes will be monitored in livers from rats given ethanol intragastrically or in a Lieber-DeCarli liquid diet for 2, 4 and 6 weeks. 2) Determine whether the proteasome-dependent degradation of CYP2E1 requires the participation of ubiquitin, the 26S proteasome and/or specific chaperones. These studies will initially be conducted with CYP2E1 expressed in tet-HeLa cells. After the initial characterization with the transfected cell lines, similar analysis will be conducted with primary hepatocytes. 3) Characterize the fate of hydroxyethyl-modified CYP2E1 in cells in culture. These studies will determine whether cell surface expression is related to the original orientation of the enzyme in the endoplasmic reticulum.
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