Studies of the adduction of hepatocellular proteins by 4-HNE in animal models of alcoholic liver disease: Systematic analysis of hepatocellular Erk 1/2 modulation and dysregulation of the Erk-Elk-AP1 signal transduction pathway

Chronic ethanol consumption results in an oxidative burden on the liver, with free radical initiators causing the peroxidation of membrane lipids and the production of reactive alpha/beta-unsaturated aldehydes including 4-hydroxynonenal (4-HNE). The hepatic accumulation of lipid aldehyde-protein adducts was demonstrated to be an early event in experimental alcoholic liver disease (ALD), facilitating the identification of novel protein targets for 4-HNE-adduction and elucidation of the biochemical consequence of 4-HNE-adduct formation with carbamoyl phosphate synthetase I (CPS-I). An additional target of 4-HNE modification identified was the extracellular-signal-regulated kinases 1 and 2 (Erk 1/2) that are implicated in many pathologic states including ALD. Sequence analyses of the Erk 1/2 proteins demonstrated these enzymes are rich in nucleophilic amino acids reactive toward 4-HNE (i.e.: His, Cys, Lys). Chronic ethanol ingestion was shown to ablate constitutive Erk 1/2 phosphorylation in rats, which corresponded with significant increases in 4-HNE-Erk adduct formation. 4-HNE adduction of Erk was confirmed in primary hepatocytes and in vitro, and adduction of Erk 1/2 was shown to inhibit kinase phosphorylation, activity, and nuclear localization in culture. Additionally, tryptic digest and mass spectral analysis of the purified, adducted Erk-2 identified a 4-HNE adduct at His 178 within the phosphorylation lip that correlated with adduction of inactive kinase monomers observed in vivo. Ultimately, adduction of Erk 1/2 by 4-HNE results in the negative modulation of constitutive signal transduction through the cFos and cJun AP-1 transcription factors. The modification of His178, which lies in the activation-lip of Erk proteins, reveals a mechanism for 4-HNE-mediated inhibition of the Erk-Elk-AP1 survival pathway in hepatocytes, corroborated by the comprehensive data presented in this dissertation. Because Erk 1/2 is central to cellular proliferation and survival, 4HNE-mediated inhibition of this kinase may impact a multitude of disease states related by oxidative stress and lipid-peroxidation, a notion that is supported by similar results obtained from hepatocytes challenged with iron ascorbate.