BIOCHEMICAL INVESTIGATION OF P-GLYCOPROTEIN

多药耐药是肿瘤患者对多种细胞毒性抗癌化疗药物产生耐药性的一种情况。它通常涉及P-糖蛋白（Pgp）的表达增强，Pgp是一种质膜蛋白。Pgp在抗艾滋病药物耐药中也有参与。Pgp由1280个氨基酸组成，排列在两个重复的半部分，每个半部分包含六个预测的跨膜螺旋和一个atp结合位点。它以ATP依赖的方式将药物和各种其他疏水化合物排除在细胞外，显示出大量药物刺激的ATP酶活性，现在被广泛认为是ATP驱动的药物外排泵。我们实验室提出的催化循环涉及交替的催化位点和atp水解与药物运输耦合的机制，已被广泛采用为工作模型。我们最近取得了一个突破，即开发了一种大规模的方法来制备纯的、可洗涤的、小鼠和人用毕赤酵母Pgp。现在不仅可以大量获得野生型Pgp，也可以获得突变型Pgp，这为更广泛的结构、生物物理和生化方法提供了便利。本提案的目的是表征Pgp的结构和功能。结构将通过电子显微镜和x射线晶体学来确定。将通过特定的荧光探针插入来监测核苷酸结合参数和催化位点的占用，以及通过诱变关键催化位点残基来研究催化机制。将研究ATP水解与药物运输的偶联。Pgp的两半将分别纯化和重组，以促进对催化位点和膜结构域之间相互作用的理解。这方面的基本知识对于设计使p糖蛋白失活和克服患者耐药性的方法将是非常宝贵的。

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.