PROTEIN CARBOXYMETHYLATION IN BRAIN

拟议研究的总体目标是阐明 和蛋白质L-异戊酰的生理底物特异性 甲基转移酶（PIMT），一种在大脑和神经内分泌系统中富集的酶 组织中 PIMT将甲基从腺苷-L-甲硫氨酸转移到 非典型β-连接的乙酰基残基的游离羧基。 这些异肽键的形成已被证明是一个主要来源 在生理条件下自发的蛋白质损伤。 在体外， PIMT已被证明可以催化异肽键的转化 回到正常的联系，增加了支持的想法，它可能会修复 体内受损的蛋白质。 在进一步探索这一假设时，我们 第一个目标将是表征大鼠PC中PIMT的主要底物12 细胞，确定甲基化的位点，并确定是否如预测的那样， 根据修复假说，这种底物积累异天冬氨酸， PIMT活性受到抑制。 我们的第二个目标是使用基于PCR的 方法来确定有多少不同的同工酶PIMT存在， 确定它们在顺序上的不同，并确定它们是否都是 由单个基因的选择性剪接产生。 我们的第三个目标是 表征来自牛脑的30 kD蛋白，其可逆地结合至 PIMT Ⅱ型同工酶。 我们希望知道这种蛋白质如何与PIMT结合 II，其中它位于细胞中，并且如果它的序列与 任何先前表征的已知功能的蛋白质。 我们的最终目标是 将PIMT基因定位到人类染色体的特定区域。 如果 地图附近的任何遗传性疾病的病因不明，我们将分析 代表这些疾病的组织或细胞系，以确定 PIMT表达缺陷可能是其发病原因。 拟议的研究 应该提供一种酶的重要新信息， 在控制细胞自发性损伤中发挥关键作用 proteins.

The overall goal of the proposed research is to elucidate the function and physiological substrate specificity of protein L-isoaspartyl methyltransferase (PIMT), an enzyme enriched in brain and neuroendocrine tissues. PIMT transfers methyl groups from Sadenosyl-L-methionine onto the free carboxyl group of atypical beta-linked aspartyl residues. Formation of these isopeptide bonds has been shown to be a major source of spontaneous protein damage under physiological conditions. In vitro, PIMT has been shown to catalyze the conversion of the isopeptide linkage back to a normal linkage, adding support to the idea that it may repair damaged proteins in vivo. In exploring this hypothesis further, our first aim will be to characterize a major substrate for PIMT in rat PC12 cells, determine the site of methylation, and determine if, as predicted by the repair hypothesis, this substrate accumulates isoaspartate when PIMT activity is inhibited. Our second aim is to use a PCR-based approach to determine how many distinct isozymes of PIMT exist, to determine how they differ in sequence, and to determine if they are all generated by alternative splicing of a single gene. Our third aim is to characterize a 30 kD protein from cow brain that binds reversibly to the type II isozyme of PIMT. We wish to know how this protein binds to PIMT II, where it is localized in the cell, and if its sequence is similar to any previously characterized protein of known function. Our final aim is to map the PIMT gene to a defined region of a human chromosome. If it maps near any hereditary diseases of unknown etiology, we will assay tissues or cell lines representative of these diseases to determine if a defect in PIMT expression might be the cause. The proposed studies should provide important new information on an enzyme which appears to play a key role in the control of spontaneous damage to cellular proteins.