Biochemical Analysis of Multidrug Resistance-linked Tran

1.阐明PGP对ATP水解酶的催化循环及保守基序在ATP结合盒中的作用：我们正在继续对PGP对ATP水解酶催化循环的研究。根据热力学和动力学性质，我们确定了PGP介导的ATPase反应的E-S和E-P稳定反应中间体。利用这个框架和Walker B的E556Q/E1201Q双突变体，我们可以准确地将转运底物结合位点上的高到低亲和力开关归因于E-S反应中间体的形成。重要的是，水解后的E-P状态对药物-底物的亲和力仍然很低，这表明形成E-S复合体的构象变化耦合到运输底物位置来做机械功。因此，在催化循环的一次循环中，E-S反应中间体的形成似乎为药物底物从脂质双层的内叶向外叶移动提供了初始动力。为了补充这些研究，我们使用数据挖掘、18K ABC结构域的序列比对和定点突变的方法来评估NBDS保守亚域中的关键残基在ATP结合和水解中的作用。我们最近描述了ABC的一个保守的亚域，我们将其命名为A-环(芳香族残基与ATP的腺嘌呤环相互作用)。这个芳香残基位于Walker A基序上游的25个残基，在85%的ABC蛋白中保守。生化和诱变研究表明，该残基对ATP结合至关重要。根据E171Q突变体MJ0796NBD的二聚体结构对PGP的NBD进行同源建模，结果表明酪氨酸(Y401或Y1044)残基通过α-α相互作用堆积在ATP的腺嘌呤碱基上。2.合成或天然产物调节剂的开发，以抑制多个ABC药物转运蛋白：包括我们在内的几个实验室的工作已经证实，ABCB1、ABCG2和ABCC1是MDR发生的主要贡献者，并且这些转运蛋白在底物特异性上显示出显著的重叠。为了开发抑制多种转运蛋白的调节剂(S)，我们从多种来源以及天然产物中筛选了合成化合物。我们发现，从姜黄粉中分离出来的姜黄素对所有三种转运蛋白都是一种强有力的调节剂。在许多国家，姜黄粉每天都被用作香料。有趣的是，姜黄素不是很有细胞毒性，也不是由ABCB1、C1或G2转运的。除了姜黄素，姜黄素的主要代谢物四羟基姜黄素也被发现调节这些转运蛋白的功能(这些研究是与泰国清迈大学的P.Limtrakul博士合作进行的)。此外，与美国国立卫生研究院苏珊·贝茨博士合作，我们证明了临床上用于治疗高血压的二氢吡啶是ABCG2的转运底物，类似于ABCB1。因此，ABCB1和G2可能是二氢吡啶类药物治疗高血压疗效和用途的重要决定因素。3.人PGP的三维结构的解析：PGP的三维结构的解析是与细胞生物学实验室的Di Xia博士合作进行的一个项目。目前，我们已经能够用1，2-二庚酰基-sn-甘油磷胆碱(DHPC)或十二烷基麦芽糖苷洗涤剂获得10-12 mg/ml的PgP。除野生型蛋白外，还纯化了包括E556Q/E1201Q双突变体在内的几个突变体，该突变体在三磷酸腺苷存在下被困在E-S预水解过渡态。同样，为了稳定野生型蛋白在E-S预水解过渡态的构象，我们开发了包括使用三磷酸腺苷-？？-S在内的条件，三磷酸腺苷是三磷酸腺苷的非水解性类似物。

1.Elucidation of the catalytic cycle of ATP hydrolysis by Pgp and role of conserved motifs in the ATP-binding cassette:We are continuing our studies on the catalytic cycle of ATP hydrolysis by Pgp. Based on the thermodynamic and kinetic properties we have identified the E-S and E-P stable reaction intermediates of the Pgp-mediated ATPase reaction. Using this defined framework and the Walker B E556Q/E1201Q double mutant, we can precisely attribute the high to low affinity switch in the transport substrate binding site to the formation of the E-S reaction intermediate. Importantly, the post-hydrolysis E-P state continues to have low affinity for drug-substrate, suggesting that conformational changes that form the E-S complex are coupled to the transport substrate site to do mechanical work. Thus, the formation of the E-S reaction intermediate during a single turnover of the catalytic cycle appears to provide the initial power stroke for movement of drug substrates from the inner leaflet to the outer leaflet of the lipid bilayer. To complement these studies, the data mining, sequence alignment of 18K ABC domains and site-directed mutagenesis approaches are used to assess the role of critical residues in the conserved subdomains in the NBDs in ATP-binding and hydrolysis. We recently described a conserved subdomain of the ABC, which we named the A-loop (Aromatic residue interacting with the Adenine ring of ATP). This aromatic residue, 25 residues upstream of the Walker A motif, is conserved in 85% of ABC proteins. Biochemical and mutagenesis studies demonstrate that this residue is critical for ATP-binding. Homology modeling of the NBDs of Pgp based on the structure of a dimer of the E171Q mutant MJ0796 NBD shows that the tyrosine (Y401 or Y1044) residue stacks against the adenine base of ATP through ?a-?a interactions. 2.Development of synthetic or natural product modulators that inhibit multiple ABC drug transporters:Work from several labs including ours has established that ABCB1, ABCG2 and ABCC1 are the major contributors to the development of MDR and these transporters exhibit significant overlap in substrate specificity. To develop modulator(s) that will inhibit multiple transporters, we screened synthetic compounds from several sources as well as natural products. We found that curcumin isolated from the turmeric powder, which is consumed daily as a spice in many countries, is a potent modulator of all three transporters. Interestingly, curcumin is not very cytotoxic and it is not transported by ABCB1, C1 or G2. In addition to curcumin, tetrahydroxy curcumin, which is a major metabolite of curcumin, was also found to modulate the function of these transporters (these studies were carried out in collaboration with Dr. P. Limtrakul, Chiang Mai University, Thailand). In addition, in collaboration with Dr. Susan Bates (NCI, NIH) we demonstrated that dihydropyridines, which are used in the clinic to treat hypertension, are transport substrates for ABCG2, similar to ABCB1. Thus, both ABCB1 and G2 might be an important determinant for the potency and use of dihydropyridines for the treatment of hypertension. 3.Resolution of three-dimensional structure of human Pgp:The resolution of the three-dimensional structure of Pgp is an ongoing project in collaboration with Dr. Di Xia in the Laboratory of Cell Biology. Currently, we have been able to obtain 10-12 mg pure Pgp/ml using either 1,2-diheptanoyl-sn-glycero-phosphocholine (DHPC) or dodecyl maltoside detergent. In addition to wild-type protein, several mutants including the E556Q/E1201Q double mutant, which is trapped in an E-S pre-hydrolysis transition-like state in the presence of ATP, have also been purified. Similarly, to stabilize the conformation of wild-type protein in the E-S pre-hydrolysis transition state, we have developed conditions including the use of ATP-??-S, which is a non-hydrolyzable analog of ATP.