Accumulation of Isoaspartyl Residue-Bearing Proteins and Alcoholic Liver Disease

描述(申请人提供)：我们实验室最近的研究表明，在饮酒的大鼠肝脏中积累了异天冬氨酸残基承载蛋白，并通过补充甜菜碱来减弱这种积累。我们进一步报道，体内蛋白-L-异天冬氨酸甲基转移酶(PIMT)催化的修复活性的抑制(由乙醇诱导的肝细胞S-同型半胱氨酸水平升高)是含异天冬氨酸残基的蛋白质积累的原因。然而，这些非典型蛋白在酒精暴露后积累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水平的增加导致PIMT活性降低，从而导致含有异门冬氨酸残基的特定受损蛋白的积累。这些改变的蛋白(S)可能功能受损，可能改变肝细胞动力学，促进酒精性肝损伤的进展。此外，甜菜碱通过降低细胞内SAH水平来增强PIMT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。目前应用的目的是阐明PIMT催化的修复反应的改变在肝细胞SAH水平增加在导致肝细胞功能障碍的受损蛋白积累中的贡献，并确定甜菜碱的保护作用。具体地说，我们的重点将首先通过蛋白质组学的方法来鉴定一些独特的细胞结构和功能蛋白质，这些蛋白质在酒精摄入后容易受到异天冬氨酸的损害，这是我们实验室的一个新领域。在鉴定了这些蛋白质之后，我们将分析这些蛋白质的功能缺陷，并研究它们在酒精性肝病发病机制中的作用。我们还将尝试通过在没有乙醇干预的情况下通过特异性地影响肝细胞SAH水平来损害PIMT介导的蛋白质修复，然后确定受损蛋白质与肝细胞功能改变的关系。最后，我们还将研究甜菜碱在防止异天冬氨酸积聚从而治疗相关的功能和病理缺陷方面的作用。为实现这些目标，现提出以下具体目标：1)确定酒精摄入大鼠肝脏中最容易出现缺陷修复的PIMT底物，并描述甜菜碱在修复过程中的作用；2)确定通过实验上调肝细胞SAH水平是否也会导致异天门冬氨基相关蛋白的积累和酒精性肝损伤的发生，并研究甜菜碱的保护作用。 公共卫生相关性：这项应用的目的是确定异天冬氨酸倾向蛋白的积累机制和特性：它们在酒精性肝损伤的发生和发展中的作用以及甜菜碱的预防作用。

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.