METHYLATION OF ATYPICAL PROTEIN ASPARTYL RESIDUES

非典型蛋白天冬酰残基的甲基化

• 批准号: 3288328
• 负责人: CLARE M O'CONNOR
• 金额: $ 15.37万
• 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/3288328
• 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.

一种结构改变专一性的蛋白质甲基转移酶 天冬氨酸残留物广泛存在于细菌和真核细胞中。 这种酶在细胞内的浓度与其提取物中的浓度相似 细菌、两栖类卵母细胞、人类红细胞和转化的哺乳动物 细胞系，这表明该酶对细胞具有基本的重要性 功能。天冬氨酸作为底物的蛋白质天冬氨酸残基 这些酶是通过自发消旋和脱酰胺化而产生 异构化事件，并已提出甲基转移酶 可能在这些受损序列的纠正或新陈代谢中发挥作用。 我们将调查特定的职能角色 非洲爪哇卵母细胞甲基转移酶的研究在这些实验中 含有改变的天冬氨酸残基的放射性标记蛋白质或多肽将 被注射到卵母细胞中，以及这些细胞的甲基化依赖的处理 底物将使用高效液相色谱法进行监测。单独的代谢物将是 分析以确定由 甲基转移反应。 我们还将分析蛋白质甲基化反应对 天冬氨酸残基消旋速率和天冬酰胺残基脱酰胺速率 在卵母细胞蛋白中。高浓度化学物质的存在 所有被研究的细胞类型中的甲基转移酶似乎暗示 消旋化和去酰胺化事件比以前更频繁地发生 已经预料到了。在这些实验中，非洲爪哇的蛋白质将被脉冲标记 用L-[C14]天冬氨酸或L[14C]天冬酰胺制备 衍生天冬氨酸残留量将在没有和 甲基转移酶抑制剂的存在。这些利率将与 在平行分组中测量的蛋白质羧甲基化速率 卵母细胞显微注射S-腺苷-L-[甲基-H-3]蛋氨酸。这 比较将提供一个衡量效率的方法 甲基化依赖的加工反应。 最后，我们将对内生性进行彻底的表征 卵母细胞甲基转移酶的底物。我们将特别注意 支付给流行的蛋白质，这些蛋白质储存的时间较长 卵子发生的时间。这些蛋白质包括核糖体、细胞骨架、 核质和线粒体蛋白。在这些研究过程中， 我们还可能发现其他类型的调节性甲基转移酶或 仅限于细胞内细胞器的甲基转移酶。