Accumulation of Isoaspartyl Residue-Bearing Proteins and Alcoholic Liver Disease

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DESCRIPTION (provided by applicant): Recent studies from our laboratory have demonstrated an accumulation of isoaspartyl residue-bearing proteins in the livers of rats consuming alcohol and an attenuation of this accumulation by betaine supplementation. We have further reported that the in vivo inhibition of protein-L-isoaspartyl methyltransferase (PIMT)-catalyzed repair activity (caused by the ethanol- induced elevations in hepatocellular S-adenosylhomocysteine (SAH) levels) is responsible for the accumulation of the isoaspartyl residue-containing proteins. However, the identity of these atypical proteins that accumulate following ethanol exposure and their role in the progression of alcoholic liver disease is not known. Our working hypothesis is that ethanol-induced increases in hepatocellular SAH levels cause a reduction in PIMT activity and thereby result in an accumulation of specific damaged proteins bearing isoaspartate residues. These altered protein(s) may have impaired functions that could alter hepatocellular dynamics and contribute to the progression of alcoholic liver injury. Additionally, betaine via lowering intracellular SAH levels enhances PIMT-catalyzed protein repair reactions to prevent the accumulation of damaged proteins and the functional consequences of this accumulation. The objectives of the current application are to delineate the contribution of alterations in PIMT-catalyzed repair reactions by increased hepatocellular SAH levels in the accumulation of damaged proteins that cause hepatocyte dysfunction as well as to determine the protective role of betaine. Specifically, our focus will be first to identify some of the unique cellular structural and functional proteins susceptible to isoaspartyl damage following ethanol consumption by a proteomic approach which is a new area for our laboratory. After identifying the proteins, we will analyze the proteins for functional deficits and investigate their role in the pathogenesis of alcoholic liver disease. We will also attempt to impair PIMT-mediated protein repair by specifically affecting hepatocellular SAH levels without ethanol intervention and then determine the identity of the damaged proteins in relation to the alterations in hepatocyte functioning. Finally, we will also examine the role of betaine in preventing isoaspartate accumulation and thereby treating the associated functional and pathological defects. To achieve the objectives, the following specific aims are proposed: 1) To identify the substrates of PIMT which are most susceptible to defective repair in the livers of rats consuming ethanol and to delineate the role of betaine to normalize the repair process; 2) To determine whether experimentally elevating the hepatocellular levels of SAH (by methods other than by ethanol administration) also results in the accumulation of isoaspartyl- prone proteins and the development of features of alcoholic liver injury and to examine the role of betaine protection. Public Health Relevance: The objectives of this application are to determine the mechanism of accumulation and the identity of the isoaspartyl-prone proteins: their role in the initiation and progression of alcoholic liver injury and prevention by betaine administration.