PROTEIN CARBOXYL METHYLATION IN BRAIN

拟议研究的总体目标是了解 酶的功能，蛋白质羧甲基转移酶(PCMT)， 在哺乳动物的大脑中。最近的证据表明，PCMT 选择性地和化学计量学上只修饰蛋白质和 含有L-异天冬氨酸的多肽，即天冬氨酸 通过其侧链的β-羧基连接，而不是通过 典型的α-羧基连接。最可能的来源是 蛋白质中的异天冬氨酸是通过不稳定的自发脱酰胺化来实现的 天门冬酰胺部位。我们建议PCMT修饰脱酰胺化- 损坏的蛋白质，以促进其有效降解 或者修理。在检验这一假设时，我们的第一个目标将是 放射性标记异天冬氨酸的代谢去向研究 脑提取液中的多肽和蛋白质 支持或抑制PCMT活动。这应该可以让我们确定 如果它们以甲基化依赖的方式降解或修复。 我们的第二个目标是确定之前演示的 脑颗粒物中甲基化位点的丰度 (与其他组织相比)是由于 大脑中含有异天冬氨酸的蛋白质。我们的第三个目标是 比较两种主要异构体的部分氨基酸序列 脑PCMT以确定它们是否是不同的同工酶。我们的决赛 目的是克隆和测序牛脑PCMT基因。这 最终将使我们能够确定PCMT是否类似于 已知功能的蛋白质，将为 未来的研究旨在改变体内PCMT的活性水平。 拟议中的研究可能会为我们提供关于 脑细胞损伤的分子机制及酶促反应 处理这些问题的过程也在不断演变。

The overall goal of the proposed research is to understand the function of the enzyme, protein carboxyl methyltransferase (PCMT), in mammalian brain. Recent evidence indicates that PCMT selectively and stoichiometrically modifies only proteins and peptides which contain L-isoaspartate, i.e., aspartate which is coupled through its side-chain beta-carboxyl group, rather than via the typical alpha-carboxyl linkage. The most likely source of isoaspartate in proteins is via spontaneous deamidation of labile asparagine sites. We propose that PCMT modifies deamidation- damaged proteins in order to facilitate their efficient degradation or repair. In testing this hypothesis, our first goal will be to study the metabolic fate of radio-labeled isoaspartate-containing peptides and proteins in brain extracts under conditions which favor or suppress PCMT activity. This should allow us to determine if they are degraded or repaired in a methylation-dependent manner. Our second goal is determine if the previously demonstrated abundance of methylation sites in brain particulate fraction (compared with other tissues) is due to a relative enrichment in brain of isoaspartate-bearing proteins. Our third goal is to compare partial amino acid sequences of the two major isoforms of brain PCMT to determine if they are distinct isozymes. Our final goal is to clone and sequence the cDNA for bovine brain PCMT. This will ultimately allow us to determine if PCMT resembles any proteins of known function and will provide the foundation for future studies designed to alter levels of PCMT activity in vivo. The proposed studies may provide important new insights into the molecular mechanisms of cell damage in the brain and the enzymatic processes evolved to deal with them.