BIOCHEMICAL INVESTIGATION OF P-GLYCOPROTEIN

P-糖蛋白的生化研究

• 批准号: 6627186
• 负责人: ALAN E. SENIOR
• 金额: $ 29.51万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6627186
• 关键词: Ascomycetes; P glycoprotein; X ray crystallography; active transport; adenosinetriphosphatase; atomic force microscopy; chemical kinetics; enzyme activity; enzyme mechanism; fluorescent dye /probe; fungal proteins; glycoprotein structure; intermolecular interaction; liposomes; magnesium ion; molecular site; multidrug resistance; nucleotide analog; protein purification; protein reconstitution; protein structure function; site directed mutagenesis; structural biology

Multidrug-resistance is a situation encountered in cancer patients in which the tumor becomes resistant to a variety of cytotoxic anti-cancer chemotherapeutic agents. It often involves enhanced expression of P- glycoprotein (Pgp), a plasma membrane protein. Involvement of Pgp in resistance to anti-AIDS drugs is also strongly-indicated. Pgp consists of 1280 amino acids, arranged in two repeated halves, each of which contains six predicted transmembrane helices and one ATP-binding site. It acts in an ATP-dependent manner to exclude drugs and a wide range of other hydrophobic compounds from cells, displays substantial drug- stimulated ATPase activity, and is now widely-believed to act as an ATP- driven drug-efflux pump. A catalytic cycle involving alternating catalytic sites and a mechanism for coupling of ATP-hydrolysis to drug-transport, presented by our laboratory, has become widely-adopted as a working model. We recently made a breakthrough, namely the development of a large- scale method for preparation of pure, detergent-soluble, mouse and human Pgp, using Pichia. Not only wild-type but also mutant Pgp may now be obtained in quantity, facilitating a broader range of structural, biophysical and biochemical approaches. The aim of this proposal is to characterize structure and function of Pgp. Structure will be determined by electron-microscopy and X-ray crystallography. Catalytic mechanism will be studied by specific insertion of fluorescent probes to monitor nucleotide binding parameters and occupancy of catalytic sites, and by mutagenesis of critical catalytic site residues. Coupling of ATP hydrolysis to drug transport will be investigated. The two halves of Pgp will be purified separately and reconstituted, to facilitate understanding of interactions between catalytic sites and membrane domains. Basic knowledge of this kind will be invaluable in devising ways to disable P-glycoprotein and overcome drug-resistance in patients.

多重耐药性是癌症患者遇到的一种情况，其中肿瘤对多种细胞毒性抗癌化疗药物产生耐药性。它通常涉及 P-糖蛋白（Pgp）（一种质膜蛋白）表达的增强。还强烈表明 Pgp 参与抗艾滋病药物的耐药性。 Pgp 由 1280 个氨基酸组成，重复排列成两半，每一半包含 6 个预测的跨膜螺旋和 1 个 ATP 结合位点。它以 ATP 依赖性方式发挥作用，从细胞中排除药物和多种其他疏水性化合物，显示出大量的药物刺激的 ATP 酶活性，并且现在被广泛认为充当 ATP 驱动的药物流出泵。我们实验室提出的涉及交替催化位点的催化循环以及 ATP 水解与药物转运的耦合机制已被广泛采用作为工作模型。我们最近取得了突破，即开发了一种使用毕赤酵母大规模制备纯的、可溶于洗涤剂的小鼠和人 Pgp 的方法。现在不仅可以大量获得野生型 Pgp，还可以大量获得突变型 Pgp，从而促进更广泛的结构、生物物理和生化方法。该提案的目的是表征 Pgp 的结构和功能。结构将通过电子显微镜和X射线晶体学确定。将通过特异性插入荧光探针来监测核苷酸结合参数和催化位点的占用情况，以及通过关键催化位点残基的诱变来研究催化机制。将研究 ATP 水解与药物转运的耦合。 Pgp 的两半将分别纯化并重构，以促进理解催化位点和膜域之间的相互作用。此类基础知识对于设计使 P-糖蛋白失效和克服患者耐药性的方法非常宝贵。