PROTEIN CARBOXYL METHYLATION IN BRAIN

描述(改编自申请人的摘要)：这项工作的总体目标 该项目是为了阐明蛋白质L-异天冬氨酸的生物学作用 甲基转移酶(PIMT)，一种富含 并催化非典型L-异天冬氨酸选择性甲基化 残留物。这些不寻常的结构的特征是一种异常的 天冬氨酸氨基残基与其 通过其侧链β-羧基的羧基侧翼邻居，而不是 而不是通过正常的阿尔法连锁。异天冬氨酸基位点被认为是 源于蛋白质在老化过程中的自发变化和/或 蛋白质合成中的错误。在体外，PIMT已被证明 通过将β-链转化为正常的α-链来催化异天冬氨酸修复 联系。这个延续应用程序的目标集中在两个方面 领域：几种看似重要的蛋白质的特性 细胞中PIMT作用的目标，并试图直接证明 PIMT在体内作为一种修复酶发挥作用。这将通过以下方式实现 解决4个具体目标。第一个将集中在组蛋白H2B，一种核蛋白 最近显示的蛋白质经历了选择性和戏剧性的增加 抑制大鼠PC12细胞PIMT活性时的异天冬氨酸含量。这个 第二个将涉及一个最近的提纯和表征 发现了高分子量甲基受体蛋白(HMAP)，即 只在大脑中发现，而且似乎含有异常高的 异天冬氨酸位点，以及对PIMT的高亲和力。第三个具体问题 目的是确定突触素-1，一种参与调节细胞周期的蛋白质 可利用突触小泡，在体内形成异天冬氨酸。决赛 目的是鉴定大肠杆菌中一种不寻常的富含异天冬氨酸的蛋白质 是大鼠脑PIMT的极好底物。把这些放在一起 调查应该提供对这一意义的重要新见解 正常细胞衰老和人类的蛋白质损伤和修复系统 疾病。

DESCRIPTION (Adapted from applicant's abstract): The overall goal of this project is to elucidate the biological role of protein L-isoaspartyl methyltransferase (PIMT), a protein methylating enzyme that is enriched in brain and catalyzes selective methylation of atypical L-isoaspartyl residues. These unusual structures are characterized by an abnormal isopeptide bond in which an aspartyl residue is linked to its carboxyl-flanking neighbor via its side-chain beta-carboxyl group, rather than through the normal alpha-linkage. Isoaspartyl sites are believed to arise from spontaneous alterations of proteins during aging and/or from errors in protein synthesis. In vitro, PIMT has been demonstrated to catalyze isoaspartate repair by converting beta-linkage to normal alpha linkages. The aims of this continuation application are focused on two areas: characterization of several proteins which appear to be important targets of PIMT action in cells, and an attempt to demonstrate directly that PIMT functions as a repair enzyme in vivo. This will be accomplished by addressing 4 specific aims. The first will focus on histone H2B, a nuclear protein recently shown to undergo a selective and dramatic increase in isoaspartate content when PIMT activity is inhibited in rat PC12 cells. The second will involve the purification and characterization of a recently discovered high molecular weight methyl acceptor protein (HMAP) that is found only in brain and appears to have an unusually high content of isoaspartyl sites, as well as a high affinity for PIMT. The third specific aim is to determine if synapsin-1, a protein involved in regulating the availability of synaptic vesicles, forms isoaspartate in vivo. The final aim is to characterize an unusual isoaspartate-rich protein in E. coli that serves as an excellent substrate for rat brain PIMT. Together these investigations should provide important new insights into the significance of protein damage and repair systems in normal cell aging and in human disease.