Alcoholic liver disease (ALD) is a significant source of morbidity and mortality in the United States. ALD is a progressive disease encompassing hepatic steatosis, steatohepatitis, fibrosis and cirrhosis. It is widely accepted the molecular mechanisms underlying ALD are multifactorial including compromised antioxidant systems and the overproduction of reactive oxidant species having the potential to modify hepatocellular lipids, proteins and nucleic acids. The oxidant-mediated modification of lipids, and the resulting modification of key proteins critical for hepatocellular homeostasis, are important mechanistic events contributing to initiation and/or progression of ALD. The working hypothesis underlying the proposed experiments is focused on the proposition that protein modification occurring as a consequence of lipid peroxidation, protein glycosylation or acetylation are significant events in the pathogenesis of ALD. During the previous funding period of this MERIT AWARD, significant progress was made in refinement and development of state-of-theart proteomic approaches for isolating, identifying and functionally characterizing proteins modified by the lipid peroxidative products 4-hydroxynonenai (4-HNE). Significant progress was also made in development of mouse and rat models of ALD to probe specific components of hepatic dysregulation associated with oxidative stress. In addition, the use of bioinformatic approaches to identify components of metabolic pathways and biologic systems impacted by alcohol-induced oxidative stress were developed. These combined experimental approaches will be used to test our working hypothesis in the following three specific aims: Experiments in Aiml will continue characterization of the functionai consequences of alcohol-induced changes in the hepatic proteome resulting from 4-HNE, glycsoylation or acetylation using our recentiy developed mouse models which displays predictable progression from steatosis to steatohepatitis. Studies in Aim 2 will identify the modified proteomes using our recentiy developed PPARa, GSTA4-4 double KO mice.
Aim 3 will explore the role of autoimmunity in progression of ALD by evaluating modified proteomes in B6.12957-Rag1 mice as well as the triple KO B6.12957-Rag1- PPARa-GSTA4-4 mice to aid in identification of mechanisms involved in the immune response. Collectively, the mechanistic information derived from these experiments will provide new insight into novel therapeutic strategies to attenuate ALD.

Public Health Relevance

Alcoholic liver disease (ALD) is a significant cause of morbidity and mortality in the United States. Given the prevalence of this disorder the resulting financial impact on our health care system is significant. There is general agreement among researchers who study this disease that it is a multifactorial and complex disease complicating discovery of effective therapeutic strategies for arresting initiation and progression ofthis syndrome. The studies proposed in this application have a high probability of identifying cellular mechanisms ofthis disorder which could lead to development of effective strategies and therapeutic agents for treatment of ALD.

National Institute of Health (NIH)
Method to Extend Research in Time (MERIT) Award (R37)
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No Study Section (in-house review) (NSS)
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Radaeva, Svetlana
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University of Colorado Denver
Schools of Pharmacy
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Galligan, James J; Fritz, Kristofer S; Backos, Donald S et al. (2014) Oxidative stress-mediated aldehyde adduction of GRP78 in a mouse model of alcoholic liver disease: functional independence of ATPase activity and chaperone function. Free Radic Biol Med 73:411-20
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Fritz, Kristofer S; Kellersberger, Katherine A; Gomez, Jose D et al. (2012) 4-HNE adduct stability characterized by collision-induced dissociation and electron transfer dissociation mass spectrometry. Chem Res Toxicol 25:965-70
Galligan, James J; Smathers, Rebecca L; Fritz, Kristofer S et al. (2012) Protein carbonylation in a murine model for early alcoholic liver disease. Chem Res Toxicol 25:1012-21
Fritz, Kristofer S; Galligan, James J; Hirschey, Matthew D et al. (2012) Mitochondrial acetylome analysis in a mouse model of alcohol-induced liver injury utilizing SIRT3 knockout mice. J Proteome Res 11:1633-43
Smathers, Rebecca L; Petersen, Dennis R (2011) The human fatty acid-binding protein family: evolutionary divergences and functions. Hum Genomics 5:170-91
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