Soft X-ray Laser Microscopy of Lipid Rafts towards GPCR-Based Drug Discovery Using Time-Resolved FRET Spectroscopy
Soft X-ray Laser Microscopy of Lipid Rafts towards GPCR-Based Drug Discovery Using Time-Resolved FRET Spectroscopy
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DOI：
10.3390/ph4030524
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发表时间：
2011-03-14
期刊：
影响因子：
4.6
通讯作者：
Kodama T
中科院分区：
文献类型：
--
作者：
Baba M;Kozasa T;Hamakubo T;Kuroda H;Masuda K;Yoneya S;Kodama T
许多参与G蛋白介导的信号转导的信号分子存在于脂筏中，并被认为在空间和时间上受到控制，影响神经递质受体和转运蛋白的效力和功效。这些微结构域作为一种信号平台，因此在膜受体介导的信号转导、癌症、免疫反应、神经传递、病毒感染和各种其他现象的表达中具有重要作用，这是由于根据细胞外刺激的特异性和有效的信号传导。然而，脂筏的真实的结构由于其体积小和缺乏足够复杂的观测系统，至今还没有被观测到。使用相干软X射线激光在水窗区域（2.3-4.4 nm）的软X射线显微镜应该被证明是一个最强大的工具，观察在活细胞中的几十纳米大小的脂筏的动态结构。我们研制了一种用于X射线显微镜的新型紧凑型强诱导等离子体高次谐波共振软X射线激光器。我们还开发了一个时间分辨的高灵敏度的荧光共振能量转移（FRET）系统，并确认蛋白质-蛋白质相互作用与配体耦合。同时使用这些新的工具来观察G蛋白偶联受体（GPCRs）在筏中的定位，已成为一个重要的和最佳的工具系统来分析通过筏作为信号平台的信号转导的动力学。可视化筏的新技术有望导致对这些动态的理解和靶向脂筏中GPCR的药物发现的创新发展。
Many signaling molecules involved in G protein-mediated signal transduction, which are present in the lipid rafts and believed to be controlled spatially and temporally, influence the potency and efficacy of neurotransmitter receptors and transporters. This has focus interest on lipid rafts and the notion that these microdomains acts as a kind of signaling platform and thus have an important role in the expression of membrane receptor-mediated signal transduction, cancer, immune responses, neurotransmission, viral infections and various other phenomena due to specific and efficient signaling according to extracellular stimuli. However, the real structure of lipid rafts has not been observed so far due to its small size and a lack of sufficiently sophisticated observation systems. A soft X-ray microscope using a coherent soft X-ray laser in the water window region (2.3–4.4 nm) should prove to be a most powerful tool to observe the dynamic structure of lipid rafts of several tens of nanometers in size in living cells. We have developed for the X-ray microscope a new compact soft X-ray laser using strongly induced plasma high harmonic resonance. We have also developed a time-resolved highly sensitive fluorescence resonance energy transfer (FRET) system and confirmed protein-protein interactions coupled with ligands. The simultaneous use of these new tools for observation of localization of G-protein coupled receptors (GPCRs) in rafts has become an important and optimum tool system to analyze the dynamics of signal transduction through rafts as signaling platform. New technology to visualize rafts is expected to lead to the understanding of those dynamics and innovative development of drug discovery that targets GPCRs localized in lipid rafts.