Accumulation of Isoaspartyl Residue-Bearing Proteins and Alcoholic Liver Disease

描述（由申请人提供）：我们实验室的最新研究表明，摄入酒精的大鼠肝脏中含有异戊酰残基的蛋白质会积累，补充甜菜碱可减弱这种积累。我们进一步报道了蛋白质-L-异戊酰甲基转移酶（PIMT）催化的修复活性（由乙醇诱导的肝细胞S-腺苷高半胱氨酸（SAH）水平升高引起）的体内抑制是含异戊酰残基的蛋白质积累的原因。然而，这些非典型蛋白质的身份，酒精暴露后积累和他们在酒精性肝病的进展中的作用是未知的。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活性的降低，从而导致具有异天冬氨酸残基的特定受损蛋白质的积累。这些改变的蛋白质可能具有受损的功能，可能改变肝细胞动力学并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。本申请的目的是通过增加肝细胞SAH水平来描述PIMT催化的修复反应的改变在导致肝细胞功能障碍的受损蛋白质积累中的贡献，以及确定甜菜碱的保护作用。具体来说，我们的重点将首先确定一些独特的细胞结构和功能蛋白质易受乙醇消耗后的蛋白质组学方法，这是一个新的领域，我们的实验室的异戊酰损伤。鉴定蛋白质后，我们将分析蛋白质的功能缺陷，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无乙醇干预的情况下特异性影响肝细胞SAH水平来损害PIMT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防止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。为了达到这一目的，我们提出了以下具体目标：（1）确定在摄入乙醇的大鼠肝脏中最易发生缺陷修复的PIMT底物，并阐明甜菜碱在使修复过程正常化中的作用; 2）确定是否实验性升高SAH的肝细胞水平（通过除乙醇给药以外的方法）也导致异戊酰倾向蛋白的积累和酒精性肝损伤特征的发展，并检查甜菜碱的保护作用。 公共卫生相关性：本申请的目的是确定积累的机制和异戊酰倾向蛋白的特性：它们在酒精性肝损伤的起始和进展中的作用以及通过甜菜碱给药的预防作用。

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.