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，同时对刺激作出反应，并在分裂前使其大分子成分加倍。 细胞膜磷脂酰胆碱（PtdCho）的供应和调控机制是本研究的重点。 PtdCho是生物膜的主要结构构件，并且是培养细胞中其他两种主要磷脂的前体。CTP：磷酸胆碱胞苷酰转移酶（CCT）控制胆碱头基的生物合成，最近发现的第二种CCT亚型（CCT β）揭示了一种新的调控机制，将进行研究。 CCT α和CCT β是不同基因的产物，其翻译后修饰和细胞定位不同。 通过与脂质的相互作用调节其活性将进行生化表征，并在体内评价这种机制的重要性。 在靶向破坏鼠CCT β基因后，将评价CCT β对PtdCho生物合成的非冗余生理贡献。关于控制PtdCho合成所需的甘油二酯（DG）供应的过程知之甚少。 我们的工作假设是，脂肪酸和DG的合成调节生长因子，而膜PtdCho积累，调节主要由CCT活动，周期性波动与细胞周期。 将使用代谢放射性标记实验评价促有丝分裂刺激后DG的代谢来源和限制PtdCho合成条件下生物合成DG的命运。 细胞还保持膜PtdCho含量的设定点，并提出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，已经分离出两种CPT同工型的cDNA。 CPT将在昆虫细胞中异源表达后进行生物化学表征，并将在培养细胞中评价逆反应在控制膜PtdCho含量中的作用。 PtdCho合成的细胞位点将用特异性免疫试剂和激光扫描共聚焦免疫荧光显微镜测定。 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控制生长细胞中磷脂稳态的机制的理解。

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.