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.
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