It is important to characterize hepatosteatosis, the first stage of alcoholic liver disease (ALD), in order to identify therapies to reverse hepatosteatosis and/or prevent progression to more advanced disease stages. Lipid metabolism in hepatocytes is regulated by sterol regulatory element-binding protein-la (SREBP-1a) and peroxisome proliferator-activated receptor-alpha (PPARalpha), The lipid aldehydes 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA) are generated in response to chronic ethanol consumption, and are capable of covalently modifying proteins. The experiments proposed by this application are designed to test the general hypothesis that covalent modification of the transcription factors SREBP-1alpha and PPARalpha by 4-HNE and MDA alters binding affinity to their respective promoter sequences, as well as their rates of ubiquitin-dependent degradation, both of which may result in lipid accumulation in hepatocytes. Adducts will be identified by tandem mass spectrometry (MS/MS), and promoter binding activity of modified SREBP-1alpha and PPARalpha will be compared to native protein binding by electrophoretic mobility shift assays (EMSA). Ubiquitination and proteasomal degradation will be measured in a rabbit reticulocyte lysate (RRL) system.
|Stewart, Benjamin J; Roede, James R; Doorn, Jonathan A et al. (2009) Lipid aldehyde-mediated cross-linking of apolipoprotein B-100 inhibits secretion from HepG2 cells. Biochim Biophys Acta 1791:772-80|
|Stewart, Benjamin J; Doorn, Jonathan A; Petersen, Dennis R (2007) Residue-specific adduction of tubulin by 4-hydroxynonenal and 4-oxononenal causes cross-linking and inhibits polymerization. Chem Res Toxicol 20:1111-9|