REGULATION OF MEMBRANE PHOSPHOLIPID SYNTHESIS

膜磷脂合成的调控

• 批准号: 2835564
• 负责人: SUZANNE JACKOWSKI
• 金额: $ 27.41万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2835564
• 关键词: acyltransferase; cell cycle; cell growth regulation; choline; confocal scanning microscopy; diacylglycerols; enzyme activity; enzyme mechanism; gene targeting; growth factor; immunofluorescence technique; isozymes; laboratory mouse; lipid biosynthesis; membrane activity; membrane biogenesis; membrane lipids; nucleotidyltransferase; phosphatidylcholines; phospholipids; phosphotransferases; transfection

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.

膜磷脂是细胞存活和增殖所必需的。细胞保持独特的膜特性，同时对刺激作出反应，并在分裂前加倍其大分子成分。细胞膜磷脂酰胆碱（PtdCho）含量的供给和调控机制是本研究的重点。PtdCho是生物膜的主要结构构件，是培养细胞中其他两种主要磷脂的前体。磷脂酰转移酶（CCT）控制胆碱头基团的生物合成，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。CCTalpha和CCTbeta是不同基因的产物，它们的翻译后修饰和细胞定位不同。通过与脂质相互作用对其活性的调节将被生物化学表征，并在体内评估这一机制的重要性。在靶向破坏小鼠CCTbeta基因后，将评估CCTbeta对PtdCho生物合成的非冗余生理贡献。对于控制PtdCho合成所需的二酰基甘油（DG）供应的过程知之甚少。我们的工作假设是脂肪酸和DG的合成受生长因子的调节，而膜PtdCho的积累主要受CCT活性的调节，并随细胞周期周期性波动。在有丝分裂刺激后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含量的设定值，并提出了实验来表征细胞对过量磷脂生产的反应。PtdCho生物合成途径的最后一步由胆碱磷酸转移酶（CPT）催化，目前已分离出两种CPT亚型的cdna。在昆虫细胞中异种表达后，将对CPT进行生化表征，并在培养细胞中评估逆反应在控制膜PtdCho含量中的作用。PtdCho合成的细胞位点将用特定的免疫试剂和激光扫描共聚焦免疫荧光显微镜确定。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控制磷脂稳态的机制的理解。