Molecular Basis of Novel Membrane Transport Mechanism

新型膜传输机制的分子基础

• 批准号: 15GS0301
• 负责人: INAGAKI Nobuya
• 金额: $ 309.17万
• 依托单位: Kyoto University (2005-2007)Akita University (2003-2004)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Creative Scientific Research
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15GS0301/
• 关键词: gene; protein; lipid; ABC protein; membrane transport; ion channel

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

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。在本研究中，1)通过研究ABCA3在患者和ABCA3缺陷小鼠体内的功能和病理生理学，我们确定ABCA3在肺表面活性物质的产生和板层小体的生物形成中起关键作用，可能是通过运输这些脂质作为底物。2)我们克隆了ABCA2的全长cDNA，表明ABCA2在脑白质区域高水平表达，主要在少突胶质细胞中表达。此外，我们在ABCA2基因缺失的小鼠中发现，ABCA2参与了神经鞘磷脂的细胞内代谢，特别是神经鞘磷脂和神经节苷脂GM1。3)我们检测了纯化的ABCA1的ATP水解酶活性，并证明它优先被磷脂酰胆碱刺激。此外，Abcg1介导的…更多的胆固醇外流取决于细胞的鞘磷脂水平，并与鞘磷脂的外流相关，这表明ABCA1和Abcg1在高密度脂蛋白形成中的协同作用。4)ABC蛋白质如何处理大量不同结构和分子量的疏水化合物，或磷脂和胆固醇，其机制尚不清楚。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MDR1，当小分子药物与胆固醇结合时，胆固醇填充底物结合部位的空隙，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。利用KIR 6.2缺陷小鼠，我们发现含有Kir6.2的KATP通道在维持低氧性喘息和抑制呼吸频率中起重要作用。此外，我们还证明了含有Kir6.1的KATP通道参与了神经活动调节的脑内小动脉的血流。6)建立了用于获得晶体结构的人MDR1蛋白的纯化系统。此外，还开发了基于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种ABC蛋白质的筛选，我们得到了3种蛋白质的晶体，其中2种适合于X射线晶体结构分析。此外，我们还建立了KATP通道(SUR1-KIR 6.2复合体)的大规模制备和纯化方法，并获得了纯化后的负染SUR1-KIR 6.2复合体的电子显微镜。较少

项目成果

Role of Pex19p in the targeting of PMP70 to peroxisome

• DOI: 10.1016/j.bbamcr.2005.10.006
• 发表时间: 2005-12-15
• 期刊: BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH
• 影响因子: 5.1
• 作者: Kashiwayama, Y;Asahina, K;Imanaka, T
• 通讯作者: Imanaka, T

Effect of PCB-126 on intracellular accumulation and transepithelial transport of vinblastine in LLC-PK1 and its transformant cells expressing human P-glycoprotein.

PCB-126 对表达人 P-糖蛋白的 LLC-PK1 及其转化细胞中长春花碱的细胞内积累和跨上皮转运的影响。

• 发表时间: 2004
• 期刊: J.Vet.Med.Sci. 66
• 作者: Sasawatari;S.;et al.
• 通讯作者: et al.

構造解析に適した真核生物由来のABCトランスポーターのスクリーニング

适合结构分析的真核ABC转运蛋白的筛选

• 发表时间: 2008
• 作者: 崎山慶太;木村泰久;小段篤史;中津亨;植田和光;加藤博章
• 通讯作者: 加藤博章

Functional Analysis of lipid transporter ABCG1

脂质转运蛋白ABCG1的功能分析

• 发表时间: 2006
• 作者: Kobayashi;A.;Takanezawa;Y.;Hirata;T.;Shimizu;Y.;Kioka;N.;Arai;H.;Ueda;K.;Matsuo;M.
• 通讯作者: M.

Arp感受性カリウムチャネルを介するインスリン分泌機構

通过 Arp 敏感钾通道的胰岛素分泌机制

• 发表时间: 2006
• 作者: Rouhier;N.;稲垣 暢也
• 通讯作者: 稲垣 暢也