Increase in protein tyrosine nitration (PTN) has been associated with numerous diseases and mounting evidence suggests this modification could be a causative factor rather than just a general outcome of disease. The generation of PTN has been implicated in early alcohol-induced hepatitis in mice where inducible nitric oxide synthase (iNOS) and the nitric oxide-derived product, peroxynitrite (a potent agent that causes PTN in the cell), have been identified as major players in the development of this pathophysiological condition of the liver. In an attempt to identify potential hepatic PTN targets, preliminary proteomic studies were performed in our laboratory and the results suggest that histone proteins undergo selective nitration in primary human hepatocyte culture after acute ethanol exposure. Accordingly, the goal of this R21 project is to examine the impact of ethanol-induced PTN on the histone modification code by testing the hypotheses that 1) protein nitration serves as a novel histone modification that could result in subsequent alteration of gene transcription processes and 2) oxidative/nitrative modifications of enzymes that directly modulate histone modification levels could have an impact on various ethanol-induced epigenetic markers (for example, acetylation of histone H3 at Lys9).
In Specific Aim 1 of this proposal, we will characterize the process of ethanol-induced tyrosine nitration in histone H2A, H2B, H3, and H4 isoforms.
In Specific Aim 2, we will characterize the nuclear nitroproteome after ethanol exposure to identify nitrated proteins associated with altering the histone modification code (for example, HAT or HDAC enzymes). Additionally, our findings from in vitro studies will be validated in an in vivo animal model of chronic ethanol exposure. The results from this project will help shed light on the role of oxidative/nitrative stress in ethanol-induced alterations of the histone modification code and provide further evidence to the link of PTN and disease causation. Identification of nuclear nitration targets and enzyme processes that affect PTN levels will likely provide new information on potential targets for drug therapy in ethanol-induced tissue injury where PTN is linked to injury onset or severity.
The goal of this research is to understand in greater detail the molecular processes that significantly change the function of liver cells when exposed to alcohol. We will investigate a protein modification (termed nitration) that could provide new evidence on how ethanol-induced oxidative stress alters processes of gene expression through histone proteins. Characterization of histone nitration and other nuclear nitroproteins could likel lead to the identification of novel drug targets for treatment or prevention of alcoholic liver disease.
|Seeley, Kent W; Fertig, Alison R; Dufresne, Craig P et al. (2014) Evaluation of a method for nitrotyrosine site identification and relative quantitation using a stable isotope-labeled nitrated spike-in standard and high resolution fourier transform MS and MS/MS analysis. Int J Mol Sci 15:6265-85|