PROTEIN CARBOXYL METHYLATION IN BRAIN

描述（改编自申请人摘要）：本研究的总体目标 项目是阐明蛋白质L-异戊酰的生物学作用 甲基转移酶（PIMT），一种富含甲基化酶的蛋白质甲基化酶， 脑和催化非典型的L-异戊烯基的选择性甲基化 残基 这些不寻常的结构的特征是 异肽键，其中乙酰基残基连接到其 羧基侧翼邻居通过其侧链β-羧基，而不是 而不是通过正常的α连接。 据信，异丙基位点 由衰老过程中蛋白质的自发改变和/或由 蛋白质合成的错误。 在体外，PIMT已被证明 通过将β-连接转化为正常α-连接来催化异天冬氨酸修复 联动 本延续申请的目的集中在两个方面 领域：几种蛋白质的表征，这似乎是重要的 细胞中PIMT作用的靶点，并试图直接证明， PIMT在体内起修复酶的作用。 这将通过 针对四个具体目标。 第一个将集中在组蛋白H2 B，一个核 蛋白质最近显示经历了选择性和戏剧性的增加， 在大鼠PC 12细胞中，当PIMT活性被抑制时，异天冬氨酸含量增加。 的 第二个将涉及最近的纯化和表征 发现了高分子量甲基受体蛋白（HMAP）， 只在大脑中发现，似乎有异常高的含量， 异丙基位点，以及对PIMT的高亲和力。 第三特定 目的是确定突触蛋白-1，一种参与调节 突触囊泡的可用性，在体内形成异天冬氨酸。 最终 目的是描述E.大肠杆菌， 作为大鼠脑PIMT的良好底物。 综合这些 调查应该提供重要的新见解的意义， 蛋白质损伤和修复系统在正常细胞老化和人类 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.