Accumulation of Isoaspartyl Residue-Bearing Proteins and Alcoholic Liver Disease

描述（由申请人提供）：我们实验室最近的研究表明，饮用酒精的大鼠肝脏中含有异天冬氨酸残留蛋白的积累，并且通过补充甜菜碱可以减少这种积累。我们进一步报道，体内抑制蛋白- l-异天冬氨酸甲基转移酶（PIMT）催化的修复活性（由乙醇诱导的肝细胞s -腺苷型同型半胱氨酸（SAH）水平升高引起）是导致含异天冬氨酸残基蛋白积累的原因。然而，这些在酒精暴露后积累的非典型蛋白质的身份及其在酒精性肝病进展中的作用尚不清楚。我们的工作假设是，乙醇诱导的肝细胞SAH水平升高导致pmt活性降低，从而导致携带异天冬氨酸残基的特定受损蛋白的积累。这些改变的蛋白质可能具有功能受损，从而改变肝细胞动力学并导致酒精性肝损伤的进展。此外，甜菜碱通过降低细胞内SAH水平增强ppimt催化的蛋白质修复反应，以防止受损蛋白质的积累和这种积累的功能后果。当前应用的目的是通过增加肝细胞SAH水平来描述pmt催化的修复反应的变化，这些变化在引起肝细胞功能障碍的受损蛋白的积累中，以及确定甜菜碱的保护作用。具体来说，我们的重点将首先通过蛋白质组学方法确定一些独特的细胞结构和功能蛋白质，这些蛋白质在乙醇消耗后易受异天冬氨酸损伤，这是我们实验室的一个新领域。在确定蛋白质后，我们将分析功能缺陷的蛋白质，并研究它们在酒精性肝病发病机制中的作用。我们还将尝试在没有乙醇干预的情况下通过特异性影响肝细胞SAH水平来损害pmt介导的蛋白质修复，然后确定与肝细胞功能改变相关的受损蛋白质的身份。最后，我们还将研究甜菜碱在预防异天冬氨酸积累中的作用，从而治疗相关的功能和病理缺陷。为了实现这一目标，我们提出了以下具体目标：1)确定饮用乙醇的大鼠肝脏中最容易发生缺陷修复的PIMT底物，并描述甜菜碱在修复过程正常化中的作用；2)确定通过实验提高肝细胞SAH水平（通过非乙醇给药的方法）是否也会导致异天冬氨酸易感蛋白的积累和酒精性肝损伤特征的发展，并研究甜菜碱的保护作用。

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.