Biochemical Analysis of Multidrug Resistance-linked Transport Proteins

我们的研究旨在了解多种亲脂性细胞毒性天然产物抗癌药物的多药转运体作为ATP依赖的外排泵的作用机制。这些转运蛋白在大多数癌症的多药耐药形成中起着重要作用。我们的主要目标包括阐明底物与多药物转运体相互作用的机制，以及来自ATP水解物的能量如何耦合到药物转运体。我们对从MDR1杆状病毒感染的昆虫细胞中纯化的人P-糖蛋白(P-gp)进行了生化鉴定，并通过金属亲和层析和凝胶过滤优化了获得纯度为95%的P-gp的条件。药物刺激的ATP水解、底物类似物的光亲和标记等性质与天然膜中观察到的相似。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1g粗膜蛋白中提取出约15-20 mg的纯蛋白，用于三维结构的解析。为了消除糖基化对产生二维和三维晶体的可能干扰，我们表征了杆状病毒和痘苗病毒表达系统中糖基化缺陷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。为了鉴定药物结合所涉及的残基(S)，生成了保留大部分标记的6 kDa多肽。我们已经证明该肽是由P-gp的C-末端区域的蛋白分解产生的。在广泛的胰酶或溴化氰处理P-gp的C-末端60 kDa片段后，用MALDI-MS对多肽进行分析表明，存在两个独特的哌唑嗪类似物标记的多肽，它们似乎起源于TM 11及其周围区域(残基935至969)。利用纯蛋白和酶联法，我们已经开始剖析P-gp催化循环中的各个步骤。与P-gp类似的多药耐药蛋白(MRP1)在肿瘤细胞多药耐药的形成中起重要作用。MRP1具有与P-gp相似的功能，因此，了解MRP1各个结构域的结构与功能关系将为这些ABC转运蛋白的作用机制提供信息。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MRP1，并计划通过构建包含MRP1和MDR1区域的嵌合体来评估MRP1各个结构域的结构-功能关系。-ABC运输蛋白，ATP水解，癌症，药物结合部位，药物运输，多药耐药，MRP1，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