Molecular Basis and Physiological Roles of Xenobiotic Exporters

外源物质出口者的分子基础和生理作用

• 批准号: 10308029
• 负责人: YAMAGUCHI Akihito
• 金额: $ 20.35万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A).
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10308029/
• 关键词: xenobiotic exporter; drug resistance; site-directed mutagenesis; efflux; exporter; tetracycline; X-ray crystallography; post genome; 多剤耐性; アンチポーター; cMOAT; Acr; ABCトランスポーター; Tet(B)

(1) We constructed the complete set of cysteine-scanning mutants of tetracycline/H+ antiporter TetA (B) and investigated the detailed topology by means of site-directed chemical modification and oxidative disulfide cross-linking formation of double Cys mutants. As a result, it was revealed that TetA (B) forms a circular form and involves a water-filled channel at the center of the molecule with a permeability barrier in the middle of the membrane.(2) We established the method for measuring the drug efflux mediated by MRP2 by means of fluorescent drugs. Then, we determined the transmembrane basic residues essential for its transport function and the binding of the inhibitor by means of site-directed mutagenesis.(3) We first succeeded to obtain crystals of xenobiotic exporters. Now, we are undergoing the X-ray crystallographic analysis using SPring8.(4) We found a novel possible exporter for sphingosine-1-phosphate from platelete and its complete cDNA was cloned and expressed in a cell line.

（1）我们构建了四环素/H+抗抗植物TETA（B）的半胱氨酸扫描突变体，并通过位置定向的化学修饰和双Cys突变体的氧化化学修饰和氧化二硫化物交联的形成研究了详细的拓扑。结果，发现TETA（B）形成了圆形形式，并涉及分子中心的水通道，在膜中间具有渗透性屏障。（2）我们确定了测量方法的方法由MRP2通过荧光药物介导的药物外排。然后，我们确定了跨膜基本残基对其运输功能所必需的及其抑制剂的结合。现在，我们正在使用Spring8进行X射线晶体学分析。（4）我们发现了一种新型的从血小板的鞘氨氨酸1-磷酸的出口剂，其完整的cDNA被克隆并在细胞系中表达。

项目成果

Kimura,T.et al: "Roles of Conserved Arginine Residues in the Metal-Tetracycline/H^+ Antiporter of Escherichia coli." Biochemistry. 37. 5475-5480 (1998)

Kimura,T.et al：“保守精氨酸残基在大肠杆菌金属四环素/H^ 逆向转运蛋白中的作用”。

Tamura,N.,Konishi,S.,Iwaki,S.,Kimura-Someya,T.,Nada,S.,and Yamaguchi,A.: "Complete Cysteine-Scanning Mutagenesis and Site-Directed Chemical Modification of the Tn10-Encoded Metal-Tetracycline/H^+ Antiporter."J.Biol.Chem. 276(in press). (2001)

Tamura,N.、Konishi,S.、Iwaki,S.、Kimura-Someya,T.、Nada,S. 和 Yamaguchi,A.：“Tn10 编码的完整半胱氨酸扫描诱变和定点化学修饰

Kimura-Someya, T., Iwaki, S., and Yamaguchi, A.: "Site-directed Chemical Modification of Cysteinescanning Mutants as to Transmembrane Segment II and Its Flanking Regions of the Tn10-encoded Metal-Tetracycline/H^+ Antiporter Reveales a Transmembrane Water-

Kimura-Someya, T.、Iwaki, S. 和 Yamaguchi, A.：“对 Tn10 编码的金属四环素/H^ 反转运蛋白的跨膜片段 II 及其侧翼区域进行半胱氨酸扫描突变体的定点化学修饰，揭示了

山口明人,西野邦彦: "薬剤排出タンパク質とゲノム創薬への応用"バイオサイエンスとバイオインダストリー. 58. 11-16 (2000)

Akito Yamaguchi、Kunihiko Nishino：“药物外排蛋白及其在基因组药物发现中的应用”《生物科学与生物工业》58. 11-16 (2000)。

Akihito Yamaguchi and Kunihiko Nishino: "Drug Efflux Transporters and the Application to Pharmacogenomics"Bioscience and Bioindustry. Vol.58. 11-16 (2000)

Akihito Yamaguchi 和 Kunihiko Nishino：“药物外流转运蛋白及其在药物基因组学中的应用”生物科学和生物工业。