BIOCHEMICAL ANALYSIS OF MULTIDRUG RESISTANCE-LINKED TRANSPORT PROTEINS

我们的研究是针对了解的多药转运蛋白的作用机制，作为ATP依赖性外排泵的各种亲脂性细胞毒性天然产物抗癌剂。这些转运蛋白在大多数癌症的多药耐药性的发展中起重要作用。我们的主要目标包括阐明底物与多药转运蛋白相互作用的机制，以及ATP水解产生的能量如何与药物转运结合。我们对从MDR 1-杆状病毒感染的昆虫细胞中纯化的人P-糖蛋白（P-gp）进行了生物化学表征，并优化了通过金属亲和层析和凝胶过滤获得纯度>95%的P-gp的条件。药物刺激的ATP水解，光亲和标记与底物类似物的纯蛋白质的性质是类似的天然膜中观察到的。我们还优化了从杆状病毒表达系统中大规模纯化P-gp的条件。我们可以从1 g粗膜蛋白中产生约15至20 mg的纯蛋白，这些纯蛋白将用于三维结构的解析。为了消除糖基化对生成二维和三维晶体的可能干扰，我们已经表征了杆状病毒和牛痘病毒表达系统中糖基化缺陷的P-gp的性质。基于光亲和标记的底物结合分析表明，P-gp上至少有两个不同的位点由分子的两半形成。为了鉴定参与药物结合的残基，产生了保留大部分标记的6-kDa肽。我们已经表明，这种肽是从蛋白水解的C-末端区域的P-gp。在对P-gp的C-末端60 kDa片段进行广泛的胰蛋白酶或溴化氰处理后，通过MALDI-MS对肽进行分析，结果表明存在两种独特的哌唑嗪类似物标记肽，其似乎源自TM 11内和周围区域（残基935至969）。通过使用纯蛋白和酶联ATP酶测定，我们已经开始解剖P-gp催化循环中的各个步骤。多药耐药蛋白1（multidrug resistance protein，MRP 1）与P-gp相似，在肿瘤细胞多药耐药的发生、发展中起重要作用。MRP 1与P-gp具有功能相似性，因此，了解MRP 1各个结构域的结构-功能关系将提供有关这些ABC转运蛋白作用机制的信息。我们已经在基于痘苗病毒的瞬时表达系统中表达了功能性MRP 1，并计划通过构建含有MRP 1和MDR 1区域的嵌合体来评估MRP 1的各个结构域的结构-功能关系。- ABC转运蛋白，ATP水解，癌症，药物结合位点，药物转运，多药耐药性，MRP 1，P-糖蛋白，-非人类受试者或人类组织

Our studies are directed toward understanding the mechanism of action of the multidrug transporters that function as ATP-dependent efflux pumps for a variety of lipophilic cytotoxic natural product anticancer agents. These transporters play an important role in the development of multidrug resistance in most cancers. Our major goals include elucidation of the mechanism of substrate interaction with the multidrug transporters and how the energy from ATP hydrolysis is coupled to drug transport. We have biochemically characterized the human P-glycoprotein (P-gp) purified from MDR1-baculovirus infected insect cells and have optimized the conditions for obtaining >95% pure P-gp by using metal affinity chromatography followed by gel filtration. The properties such as drug-stimulated ATP hydrolysis, photoaffinity labeling with substrate analogs of the pure protein are similar to those observed in native membranes. We have also optimized conditions for the large-scale purification of P-gp from a baculovirus expression system. We can generate about 15 to 20 mg of pure protein from 1 g of crude membrane protein, which will be used for the resolution of three- dimensional structure. To eliminate the possible interference by glycosylation for the generation of two- and three-dimensional crystals, we have characterized the properties of glycosylation- deficient P-gp in baculovirus and vaccinia virus expression systems. The analysis of substrate binding based on photoaffinity labeling suggests that there are at least two non-identical sites on P-gp formed by both halves of the molecule. For the identification of the residue(s) involved in the drug binding, a 6-kDa peptide retaining most of label has been generated. We have shown that this peptide is generated from the proteolysis of the C-terminal region of P-gp. The analysis of peptides by MALDI-MS following extensive trypsin or cyanogen bromide treatment of the C-terminal 60 kDa fragment of P-gp indicates the presence of two unique prazosin analog labeled peptides which appear to originate from the region in and around TM 11 (residues 935 to 969). By using pure protein and enzyme-linked ATPase assay we have begun to dissect various steps in the catalytic cycle of P-gp. The multidrug resistance protein (MRP1) similar to P-gp plays in important role in the development of multidrug resistance in cancer cells. MRP1 exhibits functional similarities to P-gp and for this reason, understanding of the structure-function relationship of various domains of MRP1 will provide information about the mechanism of action of these ABC transporters. We have expressed functional MRP1 in a vaccinia virus based transient expression system and plan to assess the structure- function relationships of various domains of MRP1 by constructing chimeras containing MRP1 and MDR1 regions. - ABC Transport proteins, ATP hydrolysis, Cancer, Drug binding site , Drug transport, Multidrug resistance, MRP1, P-glycoprotein, - Neither Human Subjects nor Human Tissues