Biochemical Analysis of Multidrug Resistance-linked Tran

多药耐药性相关 Tran 的生化分析

• 批准号: 6762638
• 负责人: SURESH AMBUDKAR
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6762638
• 关键词: P glycoprotein; adenosine triphosphate; adenosinetriphosphatase; binding proteins; chemical kinetics; conformation; enzyme substrate complex; hydrolysis; intermolecular interaction; multidrug resistance; protein purification; protein structure function; site directed mutagenesis; thermodynamics; transfection; transport proteins

ABC transporters such as P-glycoprotein (Pgp), 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 Pgp and MRP1. Such studies will provide an insight into the role of these transporters in the development of multidrug resistance in cancers. In addition, since Pgp and MRPs are members of the ABC superfamily of transporters, these findings will also be applicable to understanding the mechanism and function of other members of this class of proteins, which have been associated with diseases in humans. Recent studies with Pgp deal with the elucidation of the catalytic cycle of ATP hydrolysis by Pgp, the biochemical basis for the action of modulators such as stipiamide, Disulfiram and curcumin and characterization of the catalytic cycle of MRP1. We assessed the role of conserved Glutamate residues in the Walker B domain of the two ATP sites (E556 and E1201, respectively) during the catalytic cycle of human Pgp. The mutant Pgps (E556Q, E556A, E1201Q, E1201A, E556/1201Q and E556/1201A) were characterized using a Vaccinia virus based expression system. Although steady-state ATP hydrolysis and drug transport activities were abrogated in both E556Q and E1201Q mutant Pgps, [a-32P]-8-azidoADP was trapped in the presence of vanadate (Vi) and the release of trapped [a-32P]-8-azidoADP occurred to a similar extent as in wild-type Pgp. This indicates that these mutations do not affect either the first hydrolysis event or the ADP release step. Similar results were also obtained when Glu residues were replaced with Ala (E556A and E1201A). Following the first hydrolysis event and release of [a-32P]-8-azidoADP, both E556Q and E1201Q mutant Pgps failed to undergo another cycle of Vi-induced [a-32P]-8-azidoADP trapping. Interestingly, the double mutants, E556/1201Q and E556/1201A trapped [a-32P]-8-azidoADP even in the absence of Vi and the occluded nucleotide was not released after incubation at 37?C for an extended period. In addition, the properties of transition state conformation of the double mutants generated in the absence of Vi were found to be similar to that of wild-type protein trapped in the presence of Vi (Pgp?[a-32P]-8-azido-ADP?Vi). Thus, in contrast to the single mutants, the double mutants appear to be defective in the ADP release step. These results suggest that E556 and E1201 residues in the Walker B domains may not be critical for the cleavage of the bond between the [g-P] and the [b-P] of ATP during hydrolysis but are essential for the second ATP hydrolysis step and completion of the catalytic cycle. The modulators such as stipiamide, disulfiram (a potent cysteine modifying agent) and curcumin (a natural phenolic compound, which is used in food preparation in South east Asia) reverse drug resistance by affection the function of Pgp. Disulfiram has duel effects-- it modifies the cysteine residues in ATP sites as well as interacts with the substrate-binding site(s). Curcumin was found to affect both the expression and function of Pgp. It is not yet clear how curcumin affects the expression level of Pgp but it seems to modulate the function by competing for the substrate-binding site. It remains to be seen whether curcumin is actually transported by Pgp. The multidrug resistance protein (MRP1) similar to Pgp plays an important role in the development of multidrug resistance in cancer cells. To investigate the mechanism of ATP hydrolysis by MRP1, we generated HEK293 transfected stably with the flag tagged MRP1. These stable transfectants exhibit resistance to daunorubicin, vinblastine and VP-16 similar to those expressing wild type MRP1 protein. Similar to Pgp, in MRP1 also, ADP can be trapped in the presence of vanadate or beryllium fluoride under hydrolysis and non-hydrolysis conditions. This trapped state shows reduced affinity for nucleotide. However unlike Pgp, the distribution of trapped 8azidoADP in both ATP sites is not similar suggesting that both ATP sites are asymmetric. It appears that the N-terminal ATP site in MRP1 has higher affinity for ATP than the C-terminal site.

ABC转运蛋白，例如P-糖蛋白（PGP），多药抗性相关蛋白（MRP1）和Mitoxantrone耐药蛋白（MXR，称为乳腺癌抗癌蛋白，BCRP或ABCP），它们是ANP依赖于ANP依赖性Efflux Pumpers的重要作用，在大多数Cans can can中起着重要的作用。此外，MRP亚家族的其他一些成员（MRP2-5）也以共轭形式运输抗癌药。因此，这些转运蛋白还可能有助于恶性细胞中多药耐药性的发展。多药抗性与ABC转运蛋白可以识别并运输各种两亲性的细胞毒性天然产物抗癌药。我们的研究旨在理解与PGP和MRP1等多药抗性相关的ABC转运蛋白的作用机理。这样的研究将洞悉这些转运蛋白在癌症中多药耐药性发展中的作用。此外，由于PGP和MRP是转运蛋白的ABC超家族的成员，因此这些发现也适用于了解该类别蛋白质其他成员的机制和功能，这些蛋白质与人类疾病有关。 PGP的最新研究涉及PGP阐明ATP水解的催化周期，PGP是调节剂（例如stipiamide，disulfiram and Curcumin and Curcumin）作用的生化基础，以及MRP1催化循环的表征。我们评估了在人PGP的催化周期期间，在两个ATP位点（分别E556和E1201）的Walker B结构域中评估了保守的谷氨酸残基的作用。使用基于Vaccinia病毒的表达系统对突变PGP（E556Q，E556A，E1201Q，E1201A，E1201A，E556/1201Q和E556/1201A）进行了表征。尽管在E556Q和E1201Q突变PGP中废除了​​稳态的ATP水解和药物传输活性，但[A-32P] -8-AzidoAdp被困在钒酸盐（VI）的存在下，并且释放[A-32P] -8-氮杂量在Wild-ty-ty-ty-type pgp中发生了类似的范围。这表明这些突变不会影响第一个水解事件或ADP释放步骤。当将GLU残基替换为ALA（E556A和E1201A）时，也获得了类似的结果。在第一次水解事件和[A-32P] -8-AsidoAdoP的释放之后，E556Q和E1201Q突变体PGP都未能经历另一个VI诱导的[A-32P] -8-AzidoADP捕获的循环。有趣的是，即使在VI没有VI的情况下，双突变体E556/1201Q和E556/1201A被困[A-32p] -8-AsiDoADP，并且在37 c在37？c孵育后均未释放闭塞核苷酸。此外，发现在没有VI的情况下产生的双突变体的过渡状态构象的性质与在VI存在（PGP？[A-32P] -8-8-Azido-Adp？vi）存在下捕获的野生型蛋白相似。因此，与单个突变体相反，双突变体在ADP释放步骤中似乎有缺陷。这些结果表明，Walker B结构域中的E556和E1201残基可能对水解过程中ATP的[G-P]和[B-P]之间的键裂解可能并不重要，但对于第二个ATP水解步骤和催化循环的完成至关重要。 调节剂，例如stipiamide，dialfiram（一种有效的半胱氨酸修饰剂）和姜黄素（一种天然酚类化合物，用于在东南亚的食物制备中），通过感情的PGP功能反向耐药性。二硫仑具有决斗作用 - 它修饰了ATP部位中的半胱氨酸残基，并与底物结合位点相互作用。发现姜黄素会影响PGP的表达和功能。目前尚不清楚姜黄素如何影响PGP的表达水平，但似乎通过竞争底物结合位点来调节功能。姜黄素是否实际上是由PGP运输的，还有待观察。 类似于PGP的多药耐药蛋白（MRP1）在癌细胞中多药耐药性的发展中起重要作用。为了研究MRP1的ATP水解机理，我们用标记为标记的MRP1的HEK293稳定转染了HEK293。这些稳定的转染剂表现出对daunorubicin，vinblastine和VP-16的耐药性，类似于表达野生型MRP1蛋白的抗性。与PGP相似，在MRP1中，ADP在水解和非水解条件下可能会在钒酸盐或氟化铍的存在下被困。该捕获状态显示对核苷酸的亲和力降低。但是，与PGP不同，两个ATP位点中捕获的8azidoADP的分布并不相似，这表明两个ATP位点都是不对称的。看来MRP1中的N末端ATP位点对ATP的亲和力高于C端位点。