Ethanol Effects on Hepatic Proteolytic Systems

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DESCRIPTION (provided by applicant): Excessive ethanol consumption causes liver injury, resulting in steatosis (fatty liver), alcoholic hepatitis, fibrosis, and ultimately cirrhosis. Ethanol-induced liver injury is, in part, caused by oxidative stress, due to enhanced formation of oxidants. One oxidant, peroxynitrite (PN), a by-product of ethanol metabolism, is generated by the induction of nitric oxide synthase, generating nitric oxide, which reacts with superoxide to form PN. Peroxynitrite causes the nitration of proteins by forming 3-nitrotyrosine adducts on their tyrosine residues as well as S-nitroso groups on their cysteine residues. Such adducts can alter the biological activities of proteins. Degradation of proteins is also altered by the stress of oxidants on the lysosome and proteasome, the two principal proteolytic systems of the cell. The goal of my research is to explore how ethanol-induced oxidation and/or ethanol-elicited nitration, and S-nitrosylation of proteins affect the overall activity, and susceptibility of these modified proteins to degradation, and how these alterations may ultimately affect liver cell viability. We will use in vivo and in vitro methods to test our hypothesis that ethanol-induced oxidative stresscauses cell damage through formation of adducts on proteins by reactions of oxidants with proteins. In Aim 1, through the use of a model protein, hen egg lysozyme, we will determine how modification of this protein by peroxynitrite and/or peroxynitrite donors will affect protein function, structure and susceptibility to degradation by the proteasome in an isolated system. Next using a protein milieu from liver cells, which includes the proteasome, we will determine the effects of modification. In Aim 2 we will employ an animal model to determine how ethanol-induced oxidation and nitration causes alteration of proteins and determine what if any repair systems (such as rapid elimination of altered proteins by proteases) are able to help the cell control protein damage. Damage' i to proteins caused by alcohol consumption can cause damage to the cell leading to alcoholic fatty liver disease (AFLD). AFLD can reduce the ability of the liver to detoxify the body and cause further health issues. By learning how to help repair or facilitate removal of damaged proteins in the cell due to ethanol-induced oxidation it may be possible to enhance liver repair, and develop experimental and therapeutic strategies to minimize alcohol induced cell death.