The long term objective of this proposal is to investigate the antioxidant role of peroxiredoxin 6 (PRX6) inthe liver and how the activity of this protein might be affected by oxidative stress due to chronic ethanolconsumption. There are three proposed specific aims for this project. The first specific aim is to identifyand characterize the location and biological effect of 4-hydroxynonenal and 4-oxononenal modification ofrecombinant PRX6. Adducts will be identified and characterized in vitro using liquid chromatography andtandem mass spectrometry. Molecular modeling will be used to investigate any conformational changesdue to aldehyde modification and biochemical assays will be performed to assess biological significance ofthe modification. The second specific aim will evaluate the role of PRX6 in the progression of alcoholicliver disease using various rodent models. Wild type mice will be chronically fed an ethanol containing dietand the number of aldehyde-protein adducts will be assessed at various time points via two dimensionalelectrophoresis and Western blots. Also, PRX6 -/- knockout mice will be chronically fed an ethanolcontaining diet in order to investigate whether or not PRX6 is an important antioxidant in the liver. Lastly,transgenic, PRX6 over-expressing mice will be used to evaluate the protective effects in the alcoholic liverdue to this over-expression. The last specific aim is designed to evaluate any possible antioxidantcompensation due to chronic ethanol consumption in the liver of wild type and PRX6 -/- knockout mice.Using isolated hepatocytes and liver homogenates, mRNA expression via quantitative RT-PCR, proteinexpression via Western blotting and enzymatic activity will be assessed for the major cellular antioxidantproteins, i.e. catalase, glutathione peroxidase, and superoxide dismutase. Long-term, heavy alcohol use isa leading cause of illness and death from liver diease in the United States. As such, alcoholic liver disease(ALD) represents a major public health concern. ALD is a multifactorial disease in which oxidative stress isa known contributing factor. Therefore, by thoroughly investigating the role of PRX6 in the mechanism ofALD progression the scientific community can move a step closer to a better understanding of the diseaseand closer to designing treatment strategies for preventing the advancement of ALD.
