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.
In Specific Aim 1, lysosome biogenesis will be measured by examining the processing, and compartmentalization of the protease, cathepsin L in hepatocytes isolated from control and ethanol-fed rats. This enzyme follows a specific pathway through vesicular compartments en route to the lysosome and the investigator's Aim 1 is to determine the step(s) at which this process is impaired by ethanol, because misrouting of cathepsin L and other hydrolases could potentially cause cell damage due to their potent hydrolytic capacities.
In Specific Aim 1 b they will examine whether ethanol influences the distribution of the mannose-6-phosphate receptor by subcellular fractionation immunocytochemistry and functional assay studies. This receptor mediates lysosome assembly by targeting cathepsin L and other hydrolases to the lysosome. The investigators postulate that ethanol may change its intracellular distribution as well as its ligand affinity for procathepsin L.
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 intra gastric feeding regimens. This nonlysosomal proteolytic pathway has a crucial role in: 1) the degradation of altered proteins; and 2) the activation of the transcription factor NFkappaB which initiates transcription of genes involved in the inflammatory and immune responses. The investigators postulate that while ethanol may down-regulate the proteasome in liver parenchymal cells, its activity may be regulated differentially in Kupffer cells, since they play a paracrine role in the pathogenesis of alcoholic liver disease.
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