Molecular Basis of Novel Membrane Transport Mechanism

本研究的目的是通过研究ABC蛋白在膜转运中的功能以及它们的结构和病理生理作用来阐明新的膜转运机制的分子基础。在这项研究中，1）通过研究ABCA 3在患者和ABCA 3缺陷小鼠中的功能和病理生理学，我们确定ABCA 3在肺表面活性物质产生和板层体生物合成中至关重要，可能通过转运这些脂质作为底物。2）我们克隆了ABCA 2的全长cDNA，并显示ABCA 2在脑白色区域以高水平表达，主要在少突胶质细胞中。此外，我们使用ABCA 2缺陷小鼠表明，ABCA 2参与脑中鞘脂，特别是鞘磷脂和神经节苷脂GM 1的细胞内代谢。3）我们测量了纯化的ABCA 1的ATP水解活性，并证明它优先被磷脂酰胆碱刺激。此外，ABCG 1介导的 关于我们 胆固醇的流出依赖于细胞鞘磷脂水平，并与鞘磷脂的流出相关，表明ABCA 1和ABCG 1在HDL形成中的协同作用。4）ABC蛋白如何处理大量具有各种结构和分子量的疏水化合物或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起着重要作用，特别是通过MDR 1，当小分子药物与胆固醇结合时，胆固醇填充底物结合位点的空隙，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。使用Kir6.2缺陷小鼠，我们发现Kir6.2-含有KATP通道是至关重要的参与维持缺氧喘息和呼吸频率的抑制。此外，我们还发现含Kir6.1的KATP通道参与了脑内小动脉中神经活性调节的血流。6）建立了纯化人MDR 1蛋白的系统，该系统将用于获得晶体结构。此外，已经开发了基于GFP融合蛋白的定位来预测ABC蛋白结晶能力的系统。通过对19个ABC蛋白的筛选，获得了3个蛋白的晶体，其中2个蛋白适合于X射线晶体结构分析。建立了KATP通道（SUR 1-Kir 6.2复合物）的大规模制备和纯化方法，并获得了纯化和负染的SUR 1-Kir 6.2复合物的电镜照片。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