Multidrug Resistance-linked Transport Proteins

ABC转运蛋白，如P-糖蛋白（P-gp）、多药耐药相关蛋白（MRP 1）和米托蒽醌耐药蛋白（MXR，也称为乳腺癌耐药蛋白、BCRP或ABCP），作为ATP依赖性外排泵发挥作用，在大多数癌症的多药耐药发展中发挥重要作用。此外，MRP亚家族的一些其他成员（MRP 2 -5）也以缀合形式转运抗癌剂。因此，这些转运蛋白也可能有助于恶性细胞多药耐药性的发展。多药耐药相关的ABC转运蛋白可以识别和转运多种两亲性细胞毒性天然产物抗癌药物。我们的研究旨在了解多药耐药相关ABC转运蛋白（如P-gp和MRP 1）的作用机制。最近的研究与P-gp处理底物和ATP位点之间的相互作用和阐明ATP水解的催化循环。重组纯化P-gp水解ATP的动力学分析表明，ADP释放是催化循环中的限速步骤，底物通过调节ADP释放发挥作用。此外，我们提供的证据表明，ATP水解在一个单一的营业额的P-gp，一个在运输药物和其他影响构象变化，以重置下一个催化循环的转运蛋白的两个不同的角色。我们进一步利用钒酸盐（Vi）诱导的ADP捕获的P-gp的过渡态构象来解决ATP水解对核苷酸结合位点的影响的问题。我们发现，在第一次水解事件结束时，核苷酸（ATP或ADP）对P-gp的亲和力降低，与受损的底物结合一致。测定了整个催化循环中[a-32 P]-8-azidoADP的重复连续捕获和释放的动力学，并且我们还监测了每个捕获事件开始和结束时的底物结合。虽然两个水解事件维斯底物结合和易位的恢复具有不同的功能结果，但是它们对于核苷酸的掺入和释放都显示出相当的动力学性质，并且在钒酸盐存在下[a-32 P]-8-叠氮基ATP的Km是相同的。这些数据表明，这两个核苷酸结合结构域的行为对称，并在个别水解事件的ATP位点以随机的方式招募。此外，在任何给定的时间只有一个核苷酸位点水解ATP，并且该位点的构象变化急剧降低（>30倍）第二位点对ATP结合的亲和力。因此，阻断ATP与第二个位点的结合，而第一个位点处于催化构象，似乎是P-gp水解ATP的交替催化循环的基础。热力学参数的分析表明，100-115 kJ/mole的活化能所需的药物（底物）刺激的ATP水解的P-gp。我们证明了在ATP水解的存在或不存在下产生的P-gp的过渡态中间体的性质在功能上是不可区分的。然而，与通过ATP水解产生过渡态中间体时观察到的活化能相比，在不存在水解的情况下用ADP捕获P-gp需要约2.5倍的活化能。P-gp水解ATP催化循环的另一个独特之处在于，刺激稳态ATP水解以及通过ATP水解形成过渡态的底物在缺乏ATP水解的情况下抑制过渡态中间体的形成。因此，底物刺激的P-gp水解ATP似乎是一个矢量过程，这与其在药物转运中的生理作用一致。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。为了研究转运蛋白分子的结构域间相互作用，我们通过突变糖基化位点并在不同的细胞外位置插入Flag表位序列构建了许多野生型MRP 1的重组变体。我们用M2单克隆抗体检测了Flag-tag在不同构建体中的可及性，发现表位的可及性依赖于糖基化位点的利用。Flag表位在未糖基化的MRP 1中是可接近的，而在部分或完全糖基化的蛋白质中表位被完全掩蔽。Flag标记的非糖基化MRP 1变体沿着野生型蛋白在HEK 293和HeLa细胞中稳定表达。标记的MRP 1稳定转染子表现出与表达野生型MRP 1蛋白的那些相似的药物（柔红霉素、长春碱和VP-16）抗性谱。此外，我们已经选择了稳定的转染子，其通过逐步增加VP-16浓度的选择过表达标记有标记的MRP 1变体。这些高抗性的转染子将用于MRP 1的结构-功能分析。

ABC transporters such as P-glycoprotein (P-gp), the multidrug resistance-associated protein (MRP1), and the mitoxantrone-resistance protein (MXR, also known as breast cancer resistance protein, BCRP, or ABCP), which function as an ATP-dependent efflux pumps, play an important role in the development of multidrug resistance in most cancers. In addition, some of the other members of MRP subfamily (MRP2-5) also transport anticancer agents in a conjugated form. Thus, these transporters also may contribute to the development of multidrug resistance in malignant cells. Multidrug resistance-linked ABC transporters can recognize and transport a wide variety of amphipathic cytotoxic natural product anticancer drugs. Our studies are directed toward understanding the mechanism of action of the multidrug resistance-linked ABC transporters such as P-gp and MRP1. Recent studies with P-gp deal with the interaction between substrate and ATP sites and elucidation of the catalytic cycle of ATP hydrolysis. The kinetic analyses of ATP hydrolysis by reconstituted purified P-gp suggest that ADP release is the rate-limiting step in the catalytic cycle, and the substrates exert their effect by modulating ADP release. In addition, we provide evidence for two distinct roles for ATP hydrolysis in a single turnover of P-gp, one in the transport of drug and the other in effecting con-formational changes to reset the transporter for the next catalytic cycle. We have further exploited the vanadate (Vi)-induced ADP trapped transition-state conformation of P-gp to address the question of what are the effects of ATP hydrolysis on the nucleotide-binding site. We find that at the end of the first hydrolysis event there is a decrease in the affinity of nucleotide (ATP or ADP) for P-gp coincident with the impaired substrate binding. The kinetics of repeating succession of trapping and release of [a-32P]-8-azidoADP through an entire catalytic cycle was determined, and we also monitored the substrate binding at the beginning and end of each trapping event. Though the two hydrolysis events have different functional outcomes vis-a-vis the recovery of substrate binding and translocation, they show comparable kinetic properties for both incorporation and release of nucleotide and the Km for [a-32P]-8-azidoATP in the presence of vanadate is identical. These data demonstrate that both nucleotide-binding domains behave symmetrically, and during individual hydrolysis events the ATP sites are recruited in a random manner. Furthermore, only one nucleotide site hydrolyzes ATP at any given time and the conformational change in this site that drastically decreases (>30-fold) the affinity of the second site for the ATP-binding. Thus, the blocking of ATP binding to the second site, while the first one is in catalytic conformation, appears to be the basis for the alternate catalytic cycle of ATP hydrolysis by P-gp. Analyses of thermodynamic parameters indicate that 100-115 kJ/mole energy of activation is required for the drug (substrate)-stimulated ATP hydrolysis by P-gp. We demonstrated that the properties of the transition state intermediate of P-gp generated in the absence or presence of ATP hydrolysis is functionally indistinguishable. However, the trapping of P-gp with ADP in the absence of hydrolysis requires ~2.5-fold higher energy of activation compared with that observed when the transition state intermediate is generated through hydrolysis of ATP. Another unique feature of catalytic cycle of ATP hydrolysis by P-gp is that the substrates that stimulate steady-state ATP hydrolysis as well as the formation of transition state through hydrolysis of ATP, inhibit the formation of transition state intermediate in the absence of ATP hydrolysis. Thus the substrate-stimulated hydrolysis of ATP by P-gp appears to be a vectorial process, and this is consistent with its physiological role in drug transport. The multidrug resistance protein (MRP1) similar to P-gp plays an important role in the development of multidrug resistance in cancer cells. To investigate the interdomain interactions of the transporter molecule, we constructed numerous recombinant variants of wild-type MRP1 by mutating the glycosylation sites and inserting the Flag epitope sequence in different extracellular positions. We detected the accessibility of the Flag-tag in the different constructs by M2 monoclonal antibody and found that the accessibility of the epitope was dependent on the utilization of the glycosylation sites. The Flag epitope was accessible in unglycosylated MRP1, whereas the epitope was completely masked in the partially or fully-glycosylated protein. The Flag-tagged unglycosylated MRP1 variants along with the wild-type protein were stably expressed in both HEK293 and HeLa cells. The flag-tagged MRP1 stable transfectants exhibit drug (daunorubicin, vinblastine and VP-16) resistance profile similar to those expressing wild-type MRP1 protein. In addition, we have selected stable transfectants, which overexpress flag-tagged MRP1 variants by selection with step-wise increase in the concentration of VP-16. These highly resistant transfectants will be used for the structure-function analysis of MRP1.