CONTROL OF EUKARYOTIC MEMBRANE FUNCTION BY METHYLATION

这项工作的目的是了解 人体组织和组织中的酶促蛋白质羧甲基化反应 其他细胞。 我们建议继续我们对L-异天冬氨酸/D-天冬氨酸的研究 甲基转移酶(E.C.2.1.1.77)，催化修饰 含有这些不寻常残基的受损蛋白质。这种酶被发现 在所有被检查的哺乳动物组织的胞浆部分中 识别自发产生的改变的残留物 正L-天冬氨酸和L-天冬氨酸的外消旋、异构化和脱酰胺 陈化蛋白质中的天冬酰胺残留量。在模型系统中，队形 L-异天冬氨酸甲酯残基上的甲酯可导致它们的转化 对L-天冬氨酸残基的作用，并提示该酶具有修复功能 某些类型的共价对细胞内蛋白质的损伤和限制 它们在老化的细胞中积累。我们将结合使用 生物化学和分子生物学技术表征人类 该酶的基因及其多态性，以描绘这三个基因。 人酶的空间结构，并分析残基 负责受损蛋白质底物的结合和甲基化。 我们也有兴趣研究酶的过度生产的影响。 以及转基因小鼠的生产不足。因为它的结构 从细菌到人类细胞，酶都被保存得非常好， 我们还将利用基因可操纵的模型系统，包括 细菌和线虫温暖的秀丽线虫。这 甲基转移酶可以代表一类的首批成员之一 可以检查细胞蛋白质自发损伤的酶，以及它的 病理状态的破坏可能会降低它们的用处 寿命长，加速衰老进程。 我们最近发现的一类新的蛋白质羧甲基转移酶 提示这些酶可能也在调节 肿瘤形成过程中的细胞代谢包括细胞周期调控。在……里面 1993年，我们发现了一种主要的细胞蛋白磷酸酶(2A)， 它在通过逆转来调节酶活性方面起着至关重要的作用 各种蛋白激酶的作用，是一种特定的底物 新的C末端亮氨酸甲基转移酶。在这一授权期内，我们将 继续我们对这种C末端亮氨酸甲基化的研究 反应。我们将提纯和鉴定与这些相关的酶 来自哺乳动物组织和酵母的反应，并表征 潜在的脱甲基酶也是如此。从药物上控制的能力 这些修饰酶可能代表着治疗癌症和 其他疾病。最后，我们有兴趣寻找其他 细胞中可能发挥作用的新型蛋白质羧甲基转移酶 在新陈代谢或信号反应中的独特作用。

The objective of this work is to understand the physiological role of enzymatic protein carboxyl methylation reactions in human tissues and other cells. We propose to continue our studies of an L-isoaspartyl/D-aspartyl methyltransferase (E.C. 2.1.1.77) that catalyzes the modification of damaged proteins containing these unusual residues. This enzyme is found in the cytosolic fraction of all mammalian tissues examined and recognizes the altered residues that result from spontaneous racemization, isomerization, and deamidation of normal L-aspartyl and L- asparaginyl residues in aged proteins. In model systems, the formation of methyl esters at L-isoaspartyl residues can lead to their conversion to L-aspartyl residues and suggests that this enzyme functions to repair certain types of covalent damage to intracellular proteins and limit their accumulation in aging cells. We will use a combination of biochemical and molecular biological techniques to characterize the human gene for this enzyme and its polymorphisms, to delineate the three- dimensional structure of the human enzyme, and to analyze residues responsible for binding and methylation of damaged protein substrates. We are also interested in examining the effects of enzyme overproduction and underproduction in transgenic mice. Since the structure of this enzyme has been remarkably well conserved from bacteria to human cells, we will also utilize genetically-manipulatable model systems, including bacteria and the nematode warm Caenorhabditis elegans. This methyltransferase may represent one of the first members of a class of enzymes that can check spontaneous damage to cellular proteins, and its disruption in pathological conditions may both decrease their useful lifetime and contribute to accelerated aging processes. Our recent discovery of a new class of protein carboxyl methyltransferase suggests that these enzymes may also have roles in the regulation of cellular metabolism including cell cycle control in tumor formation. In 1993, we found that one of the major cellular protein phosphatases (2A), which plays an essential role in modulating enzyme activity by reversing the action of various protein kinases, is a specific substrate for a novel C-terminal leucine methyltransferase. In this grant period, we will continue our investigation of this C-terminal leucine methylation reaction. We will purify and characterize the enzymes involved in these reactions from both mammalian tissues and yeast, and characterize potential demethylases as well. The ability to pharmacologically control these modification enzymes may represent new therapies for cancer and other diseases. Finally, we are interested in searching for additional new types of protein carboxyl methyltransferases in cells that may play unique roles in metabolism or signaling reactions.