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. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
1F31AA016710-01
Application #
7220207
Study Section
Health Services Research Review Subcommittee (AA)
Program Officer
Gentry, Thomas
Project Start
2006-12-01
Project End
2009-11-30
Budget Start
2006-12-01
Budget End
2007-11-30
Support Year
1
Fiscal Year
2006
Total Cost
$27,343
Indirect Cost
Name
University of Colorado Denver
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045
Backos, Donald S; Fritz, Kristofer S; Roede, James R et al. (2011) Posttranslational modification and regulation of glutamate-cysteine ligase by the ?,?-unsaturated aldehyde 4-hydroxy-2-nonenal. Free Radic Biol Med 50:14-26
Orlicky, David J; Roede, James R; Bales, Elise et al. (2011) Chronic ethanol consumption in mice alters hepatocyte lipid droplet properties. Alcohol Clin Exp Res 35:1020-33
Roede, James R; Orlicky, David J; Fisher, Aron B et al. (2009) Overexpression of peroxiredoxin 6 does not prevent ethanol-mediated oxidative stress and may play a role in hepatic lipid accumulation. J Pharmacol Exp Ther 330:79-88
Roede, James R; Carbone, David L; Doorn, Jonathan A et al. (2008) In vitro and in silico characterization of peroxiredoxin 6 modified by 4-hydroxynonenal and 4-oxononenal. Chem Res Toxicol 21:2289-99
Roede, James R; Stewart, Benjamin J; Petersen, Dennis R (2008) Decreased expression of peroxiredoxin 6 in a mouse model of ethanol consumption. Free Radic Biol Med 45:1551-8