Regulation of Phospholipid Synthesis

描述(由申请人提供)： 长期目标是确定调节磷脂酰胆碱(PtdCho)生物合成的生化和遗传机制。PtdCho的合成控制是细胞膜磷脂稳态的核心，而CTP：磷酸胆碱胞苷转移酶(CCT)是哺乳动物合成PtdCho的主要途径的关键调节因子。PtdCho是膜双层的主要结构成分，占细胞膜的50%到80%，是另外两种主要膜磷脂的前体。从历史上看，研究的重点是PtdCho合成的调节，因为它与膜生物发生有关，膜生物发生是所有细胞共同的基本过程。然而，PtdCho在动物中有许多组织特异性的作用，这些作用是哺乳动物生理学的基本组成部分，但不容易在细胞培养中进行分析。即将到来的研究计划将扩大到包括构建基因敲除小鼠模型，以研究特定CCT亚型在特定组织中PtdCho合成中的非多余作用，同时继续揭开脂类调节CCT的分子细节，这对所有细胞的膜稳态的普遍控制至关重要。 第一个目的是阐明CCT亚型在哺乳动物脂代谢调节中的独特功能。我们最近最重要的贡献是表征了CCT基因和酶在哺乳动物中的数量和分布。我们已经产生了一种CCTbeta2(-/-)小鼠品系，它表现出意想不到的表型，并拥有一个纯合子CCTpha[Flox]小鼠的繁育群体。我们正在组装一组转基因Cre重组酶小鼠品系，这将使我们能够确定CCT亚型的组织特异性作用，并确定CCTpha或CCTbeta是否可以在整个动物中消除。我们在确定特定CCT基因/蛋白在哺乳动物生理学中的功能方面处于有利地位。第二个目标集中在脂质调节CCT异构体的机制上。了解CCT如何感知脂质成分，对于评价这种酶在磷脂动态平衡中的作用至关重要。我们将批判性地检验曲率弹性应力由CCT螺旋结构域感应的假设，并评估阴离子脂类通过C-末端调节的作用。在体外表征的CCT调节域的功能也将被评估为细胞定位和活细胞中的膜结合。

DESCRIPTION (provided by applicant): The long-term goal is to define the biochemical and genetic mechanisms that regulate phosphatidylcholine (PtdCho) biosynthesis. The control of PtdCho production is central to membrane phospholipid homeostasis, and CTP:phosphocholine cytidylyltransferase (CCT) is a key regulator of the major route for PtdCho synthesis in mammals. PtdCho is the principal structural component of the membrane bilayer accounting for between 50 and 80% of the cellular membrane and is a precursor to the other two major membrane phospholipids. Historically, research has focused on the regulation of PtdCho synthesis as it relates to membrane biogenesis, a fundamental process common to all cells. However, there are many tissue-specific roles for PtdCho in animals that are essential components of mammalian physiology, but do not readily lend themselves to analysis in cell culture. The research plan for the coming grant period expands to encompass the construction of knockout mouse models to investigate the non-redundant roles of specific CCT isoforms in PtdCho synthesis in specialized tissues while continuing to unravel the molecular details of CCT regulation by lipids that is critical to the universal control of membrane homeostasis in all cells. The first aim will elucidate the unique functions of the CCT isoforms in the regulation of mammalian lipid metabolism. Our most significant recent contribution was the characterization of the number and distribution of CCT genes and enzymes in mammals. We have generated a CCTbeta2(-/-) mouse strain that exhibits unexpected phenotypes, and have a breeding colony of homozygous CCTalpha[flox] mice. We are assembling a collection of transgenic Cre recombinase mouse strains that will allow us to define the tissue-specific roles of CCT isoforms as well as determine if either CCTalpha or CCTbeta can be eliminated in the whole animal. We are in an excellent position to define the functions of specific CCT genes/proteins in mammalian physiology. The second aim focuses on the mechanisms by which lipids regulate CCT isoforms. Understanding how CCT senses lipid composition is central to appreciating the role of this enzyme in phospholipid homeostasis. We will critically test the hypothesis that curvature elastic stress is sensed by the CCT helical domain and evaluate the role of anionic lipid regulation via the C-terminus. The functions of the CCT regulatory domains characterized in vitro will also be evaluated for cellular localization and membrane binding in living cells.