Molecular Basis of Novel Membrane Transport Mechanism

本研究的目的是通过研究ABC蛋白在膜运输中的功能，以及它们的结构和病理生理作用，来阐明新型膜运输机制的分子基础。在本研究中1)通过研究ABCA3在患者和ABCA3缺陷小鼠中的功能和病理生理，我们确定ABCA3在肺表面活性物质的产生和板层体生物形成中起关键作用，可能通过将这些脂质作为底物运输。2)我们克隆了ABCA2的全长cDNA，发现ABCA2在脑白质区高水平表达，主要在少突胶质细胞中表达。此外，我们在使用ABCA2缺乏的小鼠时发现，ABCA2参与脑内鞘脂的细胞内代谢，特别是鞘磷脂和神经节脂苷GM1。3)我们测定了纯化的ABCA1的ATP水解活性，证明了它优先受到磷脂酰胆碱的刺激。此外，ABCG1介导的胆固醇的更多外排依赖于细胞鞘磷脂水平，并与鞘磷脂的外排相关，这表明ABCA1和ABCG1在HDL形成中的协同作用。4) ABC蛋白如何处理大量具有不同结构和分子量的疏水化合物，即磷脂和胆固醇的机制尚不清楚。我们证明了胆固醇在底物识别中发挥重要作用，特别是通过MDR1，当小药物与底物结合时，胆固醇会填补底物结合位点的空白，并提出了胆固醇填充模型。5)确定了调节型ABC蛋白SUR的功能和病理生理作用。在Kir6.2缺陷小鼠中，我们发现含有kir6.2的KATP通道在维持缺氧喘息和降低呼吸频率中起关键作用。此外，我们已经证明含有kir6.1的KATP通道参与了大脑小动脉中神经活动调节的血液流动。6)建立了纯化人MDR1蛋白的体系，该体系将用于获得晶体结构。此外，基于gfp融合蛋白的定位，预测ABC蛋白结晶能力的系统已经开发出来。通过对19个ABC蛋白的筛选，我们获得了3个蛋白的晶体，其中2个将适合分析X射线晶体结构。此外，我们建立了大规模制备和纯化KATP通道（SUR1-Kir 6.2复合物），并获得了纯化和负染色的SUR1-Kir 6.2复合物的电镜图。少

Our aim in this study is to clarify the molecular basis of novel membrane transport mechanisms by investigating not only functions of ABC proteins in membrane transport but also on their structures and pathophysiological roles. In this study1) By investigating the function and pathophysiology of ABCA3 in patients and ABCA3-deficient mice, we determined that ABCA3 is critical in pulmonary surfactant production and lamellar body biogenesis, probably by transporting these lipids as substrates.2) We cloned a full-length cDNA of ABCA2, and showed that ABCA2 is expressed at high levels in brain white matter regions, mainly in oligodendrocytes. In addition, we showed using ABCA2-defiocient mice, that ABCA2 is involved in the intracellular metabolism of sphingolipids, particularly sphingomyelin and gangliosides GM1, in the brain.3) We measured the ATP hydrolysis activity of purified ABCA1 and demonstrated that it is stimulated preferentially by phosphatidylcholine. In addition, ABCG1-mediated … More efflux of cholesterol is dependent on the cellular sphingomyelin level and correlates with the efflux of sphingomyelin, suggesting the cooperative roles of ABCA1 and ABCG1 in HDL formation.4) The mechanism how ABC proteins handle enormous numbers of hydrophobic compounds with various structures and molecular weights, or phospholipids and cholesterol, is not known. We demonstrated that cholesterol plays an important role in substrate recognition, especially by MDR1, where cholesterol fills the empty space in the substrate binding site when small drugs bind to it, and proposed the cholesterol fill-in model.5) The function and pathophysiological role of regulator-type ABC protein, SUR, are determined. Using Kir 6.2-deficient mice, we showed that Kir6.2-containing KATP channels are critically involved in the maintenance of hypoxic gasping and depression of respiratory frequency. Furthermore, we have shown that Kir6.1-containing KATP channels are involved in the neural activity-regulated blood flow in small arterioles in the brain.6) The system to purify human MDR1 protein which will be used in obtaining crystal structure has been established. In addition, the system to predict the ability of ABC proteins to crystallize based on the localization of the GFP-fused proteins has been developed. By screening 19 ABC proteins, we obtained the crystal of 3 proteins, 2 of which will be suitable to analyze the X ray crystal structure. Furthermore, we have established large-scale preparation and purification of KATP channel (SUR1-Kir 6.2 complex), and obtained the electron microscopy of purified and negatively stained SUR1-Kir 6.2 complex. Less