Chronic disorders and diseases cause profound changes in protein expression levels in affected organ and tissue systems, and proteomic analysis of protein expression can lead to elucidating the mechanisms underlying the disease. Alcoholic Liver Disease (ALD) is a major public health problem and cause for liver dysfunction, however, relatively little is known about the actual mechanisms for the development and progression of ALD. ALD has been associated with increased oxidative stress in the liver and has been shown to cause changes in the expression of proteins associated with antioxidant protection and metabolism. We used two-dimensional gel electrophoresis to characterize murine liver proteomes after 6 weeks exposure to ethanol. Protein changes were quantified using image analysis and protein spots showing significant changes in expression were identified using MALDI-TOF mass spectrometry. A total of 23 spots were identified as differentially expressed between ALD and control liver tissue. The identified proteins included peroxiredoxin 6 (PRXD6) and aldehyde dehydrogenase 2 (ALDH2); however, these proteins were down-regulated in ALD liver by 1.6 and 1.8-fold, respectively. This result is surprising as several studies have shown that chronic alcohol exposure reduces the functional forms of liver enzymes involved in the response to oxidative stress, including PRDX6 and ALDH2. Moreover, since acetaldehyde is a toxic metabolite generated from ethanol, enzymes involved in the further metabolism of acetaldehyde to acetate by ALDH2 were also expected to be up-regulated. Quantitative RT-PCR was used to determine the mRNA levels of these proteins in ALD and control liver tissue to ascertain if changes in the expression of PRXD6 and ALDH2 are seen at the transcriptional level as well. Our data show that PRDX6 and ALDH2 mRNA are either unchanged or even slightly up-regulated in ALD liver tissue, which indicates that changes in the expression of these proteins are post-transcriptionally regulated. Based on our data as well as previous reports suggesting that chronic alcohol exposure results in oxidatively modified proteins (e.g., sulfonation and carbonylation), we hypothesized that the decrease in expression of PRXD6 and ALDH2 in ALD is, in part, due to an increase in the degradation of oxidatively modified proteins.

A proteomic approach has been undertaken to profile the extent of protein carbonylation observed with oxidative stress in an in vitro cell culture model, and test our hypothesis on oxidative stress leading to increased protein turnover. Oxidative stress was induced in cultured HepG2 cells by exposure to hydrogen peroxide (H₂O₂). Protein degradation was inhibited using the molecule mg-132 which inhibits protein turnover through both the ubiquitin/proteasome and lysosomal pathways. Since ALD leads to fat accumulation (steatosis) in hepatocytes, an additional experimental group was included where HepG2 cells were exposed to fatty acids prior to peroxide exposure. Carbonylated proteins from oxidatively stressed HepG2 cells were enriched and separated from unmodified proteins using biotin-streptavidin affinity separation and the level of protein carbonylation quantified. The identities of the carbonylated proteins and the sites of carbonylation are being identified using LC-MS/MS mass spectrometry to develop a fundamental understanding of oxidative changes and protein turnover in ALD.

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