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)构建了四环素/H+逆向转运蛋白TetA的完整半胱氨酸扫描突变体(B)，并通过定点化学修饰和双Cys突变体的氧化二硫交联形成研究了详细的拓扑结构。结果表明，TetA（B）呈圆形，分子中心有一个充满水的通道，膜中间有一个渗透屏障。（2）我们建立了利用荧光药物测量MRP2介导的药物流出的方法。然后，我们通过定点诱变的方法确定了其转运功能和抑制剂结合所必需的跨膜碱性残基。(3)我们首次成功获得了外源物输出体的晶体。现在，我们正在利用SPring8进行X射线晶体学分析。(4)我们发现了一种新的可能的血小板1-磷酸鞘氨醇输出蛋白，并且其完整的cDNA被克隆并在细胞系中表达。

项目成果

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,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”。

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)。

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

Iwaki,S.,Tamura,N.,Kimura-Someya,T.,Nada,S.,and Yamaguchi,A.: "Cysteine-scanning Mutagenesis of Transmembrane Segments 4 and 5 of the Tn10-encoded Metal-Tetracycline/H^+ Antiporter Reveals a Permeability Barrier in the Middle of a Transmembrane Water-fill

Iwaki,S.、Tamura,N.、Kimura-Someya,T.、Nada,S. 和 Yamaguchi,A.：“Tn10 编码的金属四环素/H^ 跨膜片段 4 和 5 的半胱氨酸扫描诱变”