METHYLATION OF ATYPICAL PROTEIN ASPARTYL RESIDUES

非典型蛋白天冬酰残基的甲基化

• 批准号: 3288327
• 负责人: CLARE M O'CONNOR
• 金额: $ 17.9万
• 依托单位: WORCESTER FOUNDATION FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-08-30 至 1988-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3288327
• 关键词: S adenosylmethionine; asparagine; aspartate; chemical structure function; egg /ovum; enzyme substrate; high performance liquid chromatography; methylation; oogenesis; racemization; stereoisomer; tissue /cell culture

A protein methyltransferase with a specificity for structurally altered aspartyl residues is widely distributed in bacteria and eucaryotic cells. The intracellular concentration of the enzyme is similar in extracts from bacteria, amphibian oocytes, human erythrocytes and transformed mammalian cell lines, suggesting that the enzyme is of fundamental importance to cell function. The protein aspartyl residues which serve as the substrates for the enzyme have arisen by spontaneous racemization and deamidation-linked isomerization events, and it has been proposed that the methyltransferase may function in the correction or metabolism of these damaged sequences. We will be investigating specific functional roles for the methyltransferase using Xenopus laevis oocytes. In these experiments radiolabeled protein or peptides containing altered aspartyl residues will be injected into oocytes and the methylation-dependent processing of these substrates will be monitored using HPLC. Individual metabolites will be analyzed to determine the nature of the structural changes initiated by the methyl transfer reactions. We will also analyze the impact of protein methylation reactions on the rates of aspartyl residue racemization and asparaginyl residue deamidation in oocyte proteins. The presence of high concentrations of the methyltransferase in all cell types studied would seem to imply that racemization and deamidation events occur more frequently than previously anticipated. In these experiments Xenopus proteins will be pulse-labeled with either L-[C14] aspartic acid or L[14C] asparagine, and the production of derivatized aspartyl residues will be measured in the absence and presence of methyltransferase inhibitors. These rates will be compared to the rate of protein carboxyl methylation measured in parallel groups of oocytes microinjected with S-adenosyl-L-[methyl-H-3] methionine. This comparison will provide a measure of the efficiency of methylation-dependent processing reactions. Finally, we will undertake a thorough characterization of the endogenous substrates for the oocyte methyltransferase. Particular attention will be paid to the prevalent proteins which are stored for extended periods of time during oogenesis. These proteins include the ribosomal, cytoskeletal, karyoplasmic and mitochondrial proteins. In the course of these studies, we may also uncover other kinds of regulatory methyltransferases or methyltransferases which are restricted to intracellular organelles.

一种蛋白质甲基转移酶，对结构改变的 乙酰丙酮残基广泛分布于细菌和真核细胞中。 酶的细胞内浓度在来自 细菌、两栖动物卵母细胞、人红细胞和转化的哺乳动物 细胞系，这表明这种酶对细胞生长至关重要。 功能 蛋白质乙酰基残基作为底物， 这些酶通过自发外消旋和脱酰胺连接而产生 异构化事件，并且已经提出甲基转移酶 可能在这些受损序列的纠正或代谢中起作用。 我们将研究 使用非洲爪蟾卵母细胞的甲基转移酶。 在这些实验中 放射性标记的蛋白质或含有改变的N-乙酰基残基的肽将 被注射到卵母细胞中以及这些卵母细胞的甲基化依赖性加工 将使用HPLC监测底物。 单个代谢物将 分析，以确定结构性变化的性质所引发的 甲基转移反应 我们还将分析蛋白质甲基化反应对细胞的影响。 乙酰基残基外消旋化和天冬酰胺基残基脱酰胺化速率 在卵母细胞蛋白质中。 存在高浓度的 甲基转移酶在所有类型的细胞研究似乎意味着， 外消旋化和脱酰胺化事件比以前更频繁地发生 预期的。 在这些实验中，非洲爪蟾蛋白质将被脉冲标记 与L-[C14]天冬氨酸或L[14 C]天冬酰胺反应， 将在不存在和 甲基转移酶抑制剂的存在。 这些比率将与 蛋白质羧基甲基化的速率在平行组中测量， 用S-腺苷-L-[甲基-H-3]甲硫氨酸显微注射卵母细胞。 这 比较将提供一个衡量的效率， 甲基化依赖的加工反应。 最后，我们将对内生性的 卵母细胞甲基转移酶的底物。 将特别注意 支付给普遍存在的蛋白质，这些蛋白质被储存了很长一段时间， 卵发生的时间。 这些蛋白质包括核糖体，细胞骨架， 核质和线粒体蛋白。 在这些研究的过程中， 我们也可能发现其他类型的调节甲基转移酶， 甲基转移酶，其仅限于细胞内细胞器。