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（MRP1）和米托蒽酮耐药蛋白（MXR，也称为乳腺癌耐药蛋白、BCRP或ABCP），作为atp依赖的外排泵，在大多数癌症的多药耐药发展中起重要作用。此外，MRP亚家族的一些其他成员（MRP2-5）也以缀合形式运输抗癌药物。因此，这些转运体也可能有助于恶性细胞中多药耐药的发展。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与多药耐药相关的ABC转运蛋白（如P-gp和MRP1）的作用机制。最近对P-gp的研究涉及底物与ATP位点之间的相互作用以及ATP水解催化循环的阐明。重组纯化P-gp水解ATP的动力学分析表明，ADP的释放是催化循环中的限速步骤，底物通过调节ADP的释放来发挥作用。此外，我们提供的证据表明，ATP水解在P-gp的单次翻转中有两个不同的作用，一个是在药物的运输中，另一个是在影响构象变化以重置转运体以进行下一个催化循环。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。我们发现，在第一次水解事件结束时，核苷酸（ATP或ADP）对P-gp的亲和力随着底物结合受损而降低。在整个催化循环中测定了[a-32P]-8-叠氮adp的捕获和释放的重复序列动力学，并在每个捕获事件的开始和结束时监测了底物结合。尽管这两种水解事件在底物结合和易位的恢复方面具有不同的功能结果，但它们在核苷酸的结合和释放方面表现出相似的动力学性质，并且在钒酸盐存在下[a-32P]-8-叠氮atp的Km是相同的。这些数据表明，这两个核苷酸结合区域的行为是对称的，并且在个别水解事件中，ATP位点以随机的方式被招募。此外，在任何给定的时间内，只有一个核苷酸位点能水解ATP，而该位点的构象变化会大大降低第二个位点对ATP结合的亲和力（>30倍）。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交替催化ATP水解循环的基础。热力学参数分析表明，P-gp在药物（底物）刺激下水解ATP需要100-115 kJ/mol的活化能。我们证明了在没有或存在ATP水解的情况下产生的P-gp过渡态中间体的性质在功能上是不可区分的。然而，在没有水解的情况下，与通过ATP水解产生过渡态中间体时相比，用ADP捕获P-gp所需的活化能要高2.5倍。P-gp催化ATP水解循环的另一个独特特征是，在没有ATP水解的情况下，刺激稳态ATP水解以及通过ATP水解形成过渡态的底物抑制了过渡态中间体的形成。因此，底物刺激下P-gp对ATP的水解似乎是一个载体过程，这与它在药物运输中的生理作用是一致的。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。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MRP1的重组变体。我们用M2单克隆抗体检测了不同构建体中Flag-tag的可及性，发现表位的可及性依赖于糖基化位点的利用。在未糖基化的MRP1中，Flag表位是可接近的，而在部分或完全糖基化的MRP1中，Flag表位是完全被掩盖的。flag标记的未糖基化MRP1变异体与野生型蛋白一起在HEK293和HeLa细胞中稳定表达。标记的MRP1稳定的转染物表现出与表达野生型MRP1蛋白相似的药物（柔红霉素，长春花碱和VP-16）抗性谱。此外，我们选择了稳定的转染物，通过逐步增加VP-16浓度的选择，过表达标记MRP1变异体。这些高抗性的转染物将用于MRP1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.