The long term objective of this proposal is to investigate the antioxidant role of peroxiredoxin 6 (PRX6) in the liver and how the activity of this protein might be affected by oxidative stress due to chronic ethanol consumption. There are three proposed specific aims for this project. The first specific aim is to identify and characterize the location and biological effect of 4-hydroxynonenal and 4-oxononenal modification of recombinant PRX6. Adducts will be identified and characterized in vitro using liquid chromatography and tandem mass spectrometry. Molecular modeling will be used to investigate any conformational changes due to aldehyde modification and biochemical assays will be performed to assess biological significance of the modification. The second specific aim will evaluate the role of PRX6 in the progression of alcoholic liver disease using various rodent models. Wild type mice will be chronically fed an ethanol containing diet and the number of aldehyde-protein adducts will be assessed at various time points via two dimensional electrophoresis and Western blots. Also, PRX6 -/- knockout mice will be chronically fed an ethanol containing 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 liver due to this over-expression. The last specific aim is designed to evaluate any possible antioxidant compensation 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, protein expression via Western blotting and enzymatic activity will be assessed for the major cellular antioxidant proteins, i.e. catalase, glutathione peroxidase, and superoxide dismutase. Long-term, heavy alcohol use is a 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 is a known contributing factor. Therefore, by thoroughly investigating the role of PRX6 in the mechanism of ALD progression the scientific community can move a step closer to a better understanding of the disease and closer to designing treatment strategies for preventing the advancement of ALD.
