Activation of CTP:phosphocholine cytidyltransferase

描述(申请人提供)：酶CTP：磷胆碱 胞苷酰转移酶(CT)是生物合成胞苷的关键酶。 磷脂酰胆碱(PC)，哺乳动物细胞的主要脂质成分 膜。在细胞中，CT从可溶池转移到膜上 表面会导致酶的激活。先前的研究已经确定，CT来自 RAT是模块化的，包括几个功能域，包括催化 结构域和脂类结合域。然而，最近的研究已经将 线虫CT与21个氨基酸的脂质结合区 拉伸形成一个两亲性的阿尔法螺旋。拟议的研究 将从分子水平探讨CT的脂质活化机制 以秀丽线虫CT为模型酶。具体目标是1)确定 两亲性α螺旋的疏水氨基酸侧链 决定与细胞膜的相互作用，2)相关氨基酸 两亲性螺旋的组成对脂质专一性，以及3)决定 在不激活脂类的情况下抑制CT的机制。本研究 将利用定点突变来改变单个氨基酸， 促进识别符合以下条件的单个化学官能团 对CT的脂质激活至关重要。研究项目完成后， 使制定完善的监管机制的模式成为可能 CT家族的成员。对自然机制的详细了解 CT调控及其与CT相互作用的分子决定因素 激活脂质分子对于最终的发展是必不可少的 CT的合成抑制剂和可改变CT活性的治疗剂 活着。

DESCRIPTION (provided by applicant): The enzyme CTP:phosphocholine cytidylyltransferase (CT) is a critical enzyme for the biosynthesis of phosphatidylcholine (PC), a major lipid component of mammalian cellular membranes. In a cell, translocation of CT from a soluble pool to the membrane surface results in enzyme activation. Prior work has established that CT from rat is modular and comprises several functional domains, including a catalytic domain and a lipid-binding domain. Recent work, however, has localized the lipid-binding region of CT from the nematode C. elegans to a 21 amino acid stretch postulated to form an amphipathic alpha helix. The proposed research will investigate the mechanism of lipid activation of CT at a molecular level using C. elegans CT as a model enzyme. The specific aims are to 1) identify the hydrophobic amino acid side chains of the putative amphipathic alpha helix dictating interaction with cellular membranes, 2) correlate amino acid composition of the amphipathic helix to lipid specificity, and 3) determine the mechanism of inhibition of CT in the absence of activating lipids. This study will utilize site-directed mutagenesis to alter individual amino acids, facilitating identification of individual chemical functional groups that are critical for lipid activation of CT. Completion of the research project will enable development of a refined model of the mechanism of regulation of the members of the CT family. A detailed understanding of the natural mechanism of CT regulation and the molecular determinants for interaction of CT with activating lipid molecules is essential for the eventual development of synthetic inhibitors of CT and therapeutic agents that alter CT activity in vivo.