Biochemical Analysis of Multidrug Resistance-linked Transport Proteins

我们的研究旨在了解多药转运体的作用机制，这些转运体作为atp依赖的外排泵，用于各种亲脂性细胞毒性天然产物抗癌药物。这些转运蛋白在大多数癌症的多药耐药发展中起着重要作用。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ATP水解产生的能量如何与药物转运耦合。对从感染mdr1杆状病毒的昆虫细胞中纯化得到的p -糖蛋白（P-gp）进行了生化表征，并采用金属亲和层析-凝胶过滤的方法优化了P-gp纯度为>95%的条件。其性质如药物刺激的ATP水解，与纯蛋白的底物类似物的光亲和标记类似于在天然膜中观察到的。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克粗膜蛋白中产生约15至20毫克的纯蛋白，用于三维结构的分辨率。为了消除糖基化对二维和三维晶体产生的可能干扰，我们对杆状病毒和牛痘病毒表达系统中糖基化缺陷的P-gp的特性进行了表征。基于光亲和标记的底物结合分析表明，P-gp上至少有两个由两半分子形成的不相同的位点。为了鉴定参与药物结合的残基，产生了保留大部分标签的6-kDa肽。我们已经证明这种肽是由P-gp的c端区域的蛋白质水解产生的。用MALDI-MS对P-gp的c端60 kDa片段进行胰蛋白酶或溴化氰处理后的肽分析表明，存在两个独特的prazosin类似物标记的肽，它们似乎起源于tm11及其周围的区域（残基935至969）。通过使用纯蛋白和酶联atp酶测定，我们已经开始剖析P-gp催化循环的各个步骤。与P-gp相似的多药耐药蛋白（MRP1）在癌细胞的多药耐药发展中起着重要作用。MRP1在功能上与P-gp相似，因此，了解MRP1各结构域的结构-功能关系将为这些ABC转运蛋白的作用机制提供信息。我们已经在基于牛痘病毒的瞬时表达系统中表达了功能性MRP1，并计划通过构建含有MRP1和MDR1区域的嵌合体来评估MRP1各结构域的结构-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，MRP1， p -糖蛋白，-既不是人类受试者也不是人类组织

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