REGULATION OF MEMBRANE PHOSPHOLIPID SYNTHESIS

膜磷脂是细胞生存和增殖所必需的。细胞保持独特的膜特性，同时对刺激做出反应，并在分裂前将其大分子成分加倍。细胞膜磷脂酰胆碱(PtdCho)含量的供应和控制机制是本研究的重点。PtdCho是生物膜的主要结构构件，也是培养细胞中另外两种主要磷脂的前体。CTP：磷酸胆碱胞苷转移酶(CCT)控制胆碱头基的生物合成，最近发现的第二种CCT异构体(CCTbeta)揭示了一种新的调节机制，将被研究。CCTα和CCTbeta是不同基因的产物，它们在翻译后修饰和细胞定位上存在差异。通过与脂质的相互作用来调节它们的活性将被生化表征，并在体内评估这一机制的重要性。CCTbeta对PtdCho生物合成的非冗余生理贡献将在小鼠CCTbeta基因被靶向破坏后进行评估。关于控制合成PtdCho所需的二酰基甘油(DG)供应的过程，人们知之甚少。我们的工作假设是，脂肪酸和DG的合成受到生长因子的调节，而细胞膜PtdCho的积累主要受CCT活性的调节，随着细胞周期的变化而周期性波动。有丝分裂刺激后DG的代谢来源以及在限制PtdCho合成的条件下生物合成DG的去向将通过代谢放射性标记实验进行评估。细胞也维持膜PtdCho含量的设定点，并建议进行实验来表征细胞对过量磷脂产生的反应。PtdCho生物合成途径的最后一步是由胆碱磷酸转移酶(CPT)催化的，目前已分离出两种CPT亚型的cDNA。CPT将在昆虫细胞中异源表达后进行生化表征，并将在培养细胞中评估反向反应在控制细胞膜PtdCho含量方面的作用。合成PtdCHO的细胞部位(S)将用特定的免疫试剂和激光扫描共聚焦免疫荧光显微镜来确定。这项研究将促进我们对影响细胞膜合成和运输的因素以及生长细胞中磷脂动态平衡的控制机制的理解。

Membrane phospholipids are essential for cell survival and proliferation. Cells maintain unique membrane characteristics while responding to stimuli and doubling their macromolecular components prior to division. The mechanisms that supply and control the cellular membrane phosphatidylcholine (PtdCho) content are the focus of this research. PtdCho is the major structural building block of biological membranes and is the precursor to the other two major phospholipids in cultured cells. The CTP:phosphocholine cytidylyltransferase (CCT) controls the biosynthesis of the choline headgroup and the recent discovery of a second CCT isoform (CCTbeta) reveals a new regulatory mechanism that will be investigated. CCTalpha and CCTbeta are products of different genes and differ in their post-translational modification and cellular location. The regulation of their activity by interaction with lipids will be biochemically characterized and the importance of this mechanism evaluated in vivo. The non-redundant physiological contribution of CCTbeta to PtdCho biosynthesis will be evaluated following targeted disruption of the murine CCTbeta gene. Less is known about the processes that control the supply of the diacylglycerol (DG) required for PtdCho synthesis. Our working hypothesis is that fatty acid and DG synthesis are regulated by growth factors whereas membrane PtdCho accumulation, regulated largely by CCT activity, fluctuates periodically with the cell cycle. The metabolic origin of DG after mitogenic stimulation and the fate of biosynthetic DG under conditions of restricted PtdCho synthesis will be evaluated using metabolic radiolabeling experiments. Cells also maintain a setpoint for membrane PtdCho content and experiments are proposed to characterize the cellular response to excess phospholipid production. The final step in the PtdCho biosynthetic pathway is catalyzed by the choline phosphotransferase (CPT) and cDNAs for two CPT isoforms have been isolated. CPT will be biochemically characterized following heterologous expression in insect cells and the role of the reverse reaction in controlling membrane PtdCho content will be evaluated in cultured cells. The cellular site(s) for PtdCho synthesis will be determined with specific immunological reagents and laser-scanning confocal immunofluorescent microscopy. This research will advance our understanding of the factors that influence cell membrane synthesis and trafficking and then mechanisms that control phospholipid homeostasis in growing cells.