The long-range goal of this project is to establish the mechanistic role(s) of aldehydic products of lipid peroxidation (4-hydroxynonenal [4-HNE] and malondialdehyde [MDA] in alcoholic liver disease (ALD). It is becoming more apparent that these chemically reactive aldehydes have the potential to interfere with a spectrum of cellular functions ranging from enzyme inhibition to initiation of gene expression. It is our hypothesis that when produced in excess as a result of alcohol-induced oxidative stress, 4-HNE and MDA interact with hepatocellular proteins resulting in altered protein function, impaired protein degradation and the dysfunctional expression of genes involved in various fibrotic processes. This hypothesis will be evaluated it three specific aims using liver tissue and cells obtained from rats and mice administered alcohol by intragastric infusion.
In Specific Aim 1, we will continue our characterization of hepatic aldehyde-adducted proteins isolated from rats treated chronically with alcohol. For these studies, the proteins will be immunoisolated, and subjected to matrix assisted laser desorption ionization-time of flight (MALDI-TOF) analysis for amino acid sequence analysis and identification of specific adducted amino acid residues. Experiments proposed in Specific Aim 2 will investigate the potential of 4-HNE and MDA to diminish the trans-activation of NFKB complex in hepatic stellate cells, resulting in down-regulation of IL-6 expression. The specific mechanistic events in NF-KB signaling pathway will be evaluated in isolated hepatocytes and stellate cells, including phosphorylation and proteolysis of 1KB proteins, phosphorylation of p65 and PKAc. We will also examine the role of IL-6 in ALD using transgenic IL-6 knockout mice.
In Specific Aim 3 we will evaluate how the interactions of 4-HNE and MDA with proteins and the multicatalytic proteinase/proteosome (MCP) alters protein degradation. These experiments will also employ immunoprecipitation/immunoblotting procedures as well as MALDI-TOF analysis to investigate degradation of adducted model proteins using in vitro systems and to identify the molecular targets of adduction involved in impaired hepatic protein degradation by the MCP. These studies will greatly enhance our understanding of the mechanistic role(s) of 4-HNE and MDA in alcohol-induced liver damage. Identification of susceptible cellular targets and specific mechanisms could provide information leading to the development of effective therapeutic interventions for altering specific events involved in ALD.
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