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)、多药耐药相关蛋白 (MRP1) 和米托蒽醌耐药蛋白（MXR，也称为乳腺癌耐药蛋白、BCRP 或 ABCP），其功能相当于 ATP-依赖性外排泵在大多数癌症的多药耐药性的发展中发挥着重要作用。此外，MRP 亚家族 (MRP2-5) 的一些其他成员也以缀合形式转运抗癌药物。因此，这些转运蛋白也可能有助于恶性细胞中多药耐药性的发展。多药耐药性相关的 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（例如 Pgp 和 MRP1）的作用机制。此类研究将深入了解这些转运蛋白在癌症多药耐药性发展中的作用。此外，由于Pgp和MRPs是ABC转运蛋白超家族的成员，这些发现也将适用于理解此类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。 最近对 Pgp 的研究涉及阐明 Pgp ATP 水解的催化循环、斯蒂吡酰胺、双硫仑和姜黄素等调节剂作用的生化基础以及 MRP1 催化循环的表征。我们评估了两个 ATP 位点（分别为 E556 和 E1201）的 Walker B 结构域中保守的谷氨酸残基在人 Pgp 催化循环中的作用。使用基于牛痘病毒的表达系统对突变体 Pgps（E556Q、E556A、E1201Q、E1201A、E556/1201Q 和 E556/1201A）进行表征。尽管 E556Q 和 E1201Q 突变体 Pgps 中的稳态 ATP 水解和药物转运活性均被消除，但 [a-32P]-8-叠氮基 ADP 在钒酸盐 (Vi) 存在的情况下被捕获，并释放被捕获的 [a-32P]- 8-叠氮基 ADP 的发生程度与野生型 Pgp 中相似。这表明这些突变不会影响第一次水解事件或 ADP 释放步骤。当 Glu 残基替换为 Ala 时，也获得了类似的结果（E556A 和 E1201A）。在第一次水解事件和 [a-32P]-8-叠氮基 ADP 释放之后，E556Q 和 E1201Q 突变体 Pgps 均未能经历 Vi 诱导的 [a-32P]-8-叠氮基 ADP 捕获的另一个循环。有趣的是，即使在没有 Vi 的情况下，双突变体 E556/1201Q 和 E556/1201A 也会捕获 [a-32P]-8-azidoADP，并且在 37℃ 下孵育较长时间后，封闭的核苷酸不会释放。此外，发现在不存在 Vi 的情况下产生的双突变体的过渡态构象特性与存在 Vi 时捕获的野生型蛋白相似 (Pgp?[a-32P]-8-azido- ADP？Vi）。因此，与单突变体相比，双突变体似乎在 ADP 释放步骤中存在缺陷。这些结果表明，Walker B 结构域中的 E556 和 E1201 残基对于水解过程中 ATP 的 [g-P] 和 [b-P] 之间的键断裂可能并不重要，但对于第二个 ATP 水解步骤和完成催化循环。 斯蒂吡酰胺、双硫仑（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，在东南亚用于食品制备）等调节剂通过影响 Pgp 的功能来逆转耐药性。双硫仑具有双重作用——它修饰 ATP 位点中的半胱氨酸残基并与底物结合位点相互作用。研究发现姜黄素会影响 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，但它似乎通过竞争底物结合位点来调节功能。姜黄素是否真的通过 Pgp 运输还有待观察。 与Pgp类似的多药耐药蛋白（MRP1）在癌细胞多药耐药的发展中发挥着重要作用。为了研究 MRP1 水解 ATP 的机制，我们生成了用标记 MRP1 稳定转染的 HEK293。这些稳定的转染子表现出对柔红霉素、长春花碱和 VP-16 的抗性，与表达野生型 MRP1 蛋白的转染子相似。与Pgp类似，在MRP1中，在水解和非水解条件下，在钒酸盐或氟化铍存在下，ADP也可以被捕获。这种捕获状态显示出对核苷酸的亲和力降低。然而，与 Pgp 不同的是，捕获的 8azidoADP 在两个 ATP 位点中的分布并不相似，表明两个 ATP 位点是不对称的。看来 MRP1 中的 N 端 ATP 位点比 C 端位点对 ATP 具有更高的亲和力。