The long-range goal of this proposal is to determine the molecular mechanism and pathogenetic significance of Mallory body (MB) formation. Formation of MBs is implicated in progression of numerous liver diseases but most notably alcoholic liver disease (ALD) and nonalcoholic steatohepatitis (NASH). This phenomenon is evident only in man and mouse so research is limited only to those two species. Progress made by us has allowed identification of the MB as an aggresome analogous to the inclusions seen in various neurologic degenerative diseases such as Alzheimer's neurofibrillary tangles. We have established that the components of the MB are cytokeratins, native ubiquitin, the mutant ubiquitin (UBB+1), p62, heat shock proteins 70 and 90, tissue transglutaminase, tubulin and the proteasome 26S. In addition, we have demonstrated that the cytokeratins undergo beta sheet transformation and hyperphosphorylation, resembling the aggresome formation and that there is a tight association of UBB+1, a protein resulting from a frameshift mutation of ubiquitin, with MBs. Our working hypothesis is that MBs are indeed aggresomes triggered by UBB+1 and caused by inhibition of the proteasomal digestion of cytokeratins. The current proposal will determine the mechanism of cytokeratin aggresome/MB formation in vitro using newly developed tissue culture models in our laboratory. Two models will be used: 1) primary hepatocyte cultures from the drug primed mice that form MBs on day two of culture; 2) mouse hepatoma cell line in which cytokeratin aggresomes form in 24 h after treatment with a proteasome inhibitor. The time course and order of interactions between the MB components will be determined using fluorescent probes of UBB+1, C18 and p62 fused to GFP or RFP that are transduced by expression plasmids. Kinetic of co-localization and interactions of proteins in aggresomes will be studied by confocal fluorescence microscopy. A protein transporter Chariot will also be used to introduce large quantities of UBB+1, p62, and other components into the hepatocytes in vitro to promote the aggresome forming process in much shorter durations. This will allow real-time assessment of the sequence of events in the genesis of MBs and identification of the initialing factor and the elements involved during the progression to MB formation. This method will also be used to test whether and how hepatocytes from alcohol-fed mice are sensitized to MB formation triggered by introduction of different exogenous MB components. Lastly, we will test whether MB forming hepatocytes are vulnerable to cytotoxicity caused by TNFalpha, the effector molecule central to the pathogenesis of ALD. Once the mechanism and functional significance of MB formation are elucidated, interventions can be designed to prevent their formation and possibly associated diseases.
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