PROTEIN CARBOXYL METHYLATION IN BRAIN

拟议研究的总体目标是了解 酶的功能，蛋白质羧基甲基转移酶（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地仅修饰蛋白质， 含有L-异天冬氨酸的肽，即，天冬氨酸， 通过其侧链β-羧基偶联，而不是通过 典型的α-羧基键。 的最可能来源 蛋白质中的异天冬氨酸是通过不稳定的 天冬酰胺位点。 我们建议PCMT改变脱酰胺- 破坏蛋白质，以促进其有效降解 或修复。 在测试这个假设时，我们的第一个目标是 研究放射性标记的含异丙肾上腺素的 肽和蛋白质在脑提取物中的作用， 促进或抑制PCMT活性。 这样我们就能确定 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先前展示的 脑颗粒部分中甲基化位点的丰度 （与其他组织相比）是由于相对富集 大脑中的异戊二酸蛋白质。 我们的第三个目标是 比较两种主要同种型的部分氨基酸序列， 脑PCMT以确定它们是否是不同的同工酶。 我方最后 目的是克隆牛脑PCMT的cDNA并测序。 这 最终将使我们能够确定PCMT是否类似于任何 已知功能的蛋白质，并将为 未来的研究旨在改变体内PCMT活性水平。 拟议的研究可能会提供重要的新见解， 脑细胞损伤的分子机制和 进化出了处理它们的过程。

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.