26 research outputs found
A single active catalytic site is sufficient to promote transport in P-glycoprotein
P-glycoprotein (Pgp) is an ABC transporter responsible for
the ATP-dependent efflux of chemotherapeutic compounds from
multidrug resistant cancer cells. Better understanding of the
molecular mechanism of Pgp-mediated transport could promote
rational drug design to circumvent multidrug resistance. By
measuring drug binding affinity and reactivity to a
conformation-sensitive antibody we show here that nucleotide
binding drives Pgp from a high to a low substrate-affinity
state and this switch coincides with the flip from the
inward- to the outward-facing conformation. Furthermore, the
outward-facing conformation survives ATP hydrolysis: the
post-hydrolytic complex is stabilized by vanadate, and the
slow recovery from this state requires two functional
catalytic sites. The catalytically inactive double Walker A
mutant is stabilized in a high substrate affinity inward-open
conformation, but mutants with one intact catalytic center
preserve their ability to hydrolyze ATP and to promote drug
transport, suggesting that the two catalytic sites are
randomly recruited for ATP hydrolysis
Heterologous Overexpression and Mutagenesis of the Human Bile Salt Export Pump (ABCB11) Using DREAM (Directed REcombination-Assisted Mutagenesis)
Homologous recombination in Saccharomyces cerevisiae is a well-studied process. Here, we describe a yeast-recombination-based approach to construct and mutate plasmids containing the cDNA of the human bile salt export pump (BSEP) that has been shown to be unstable in E. coli. Using this approach, we constructed the necessary plasmids for a heterologous overexpression of BSEP in the yeast Pichia pastoris. We then applied a new site-directed mutagenesis method, DREAM (Directed REcombination-Assisted Mutagenesis) that completely bypasses E. coli by using S. cerevisiae as the plasmid host with high mutagenesis efficiency. Finally, we show how to apply this strategy to unstable non-yeast plasmids by rapidly turning an existing mammalian BSEP expression construct into a S. cerevisiae-compatible plasmid and analyzing the impact of a BSEP mutation in several mammalian cell lines
A Novel MDR1 GT1292-3TG (Cys431Leu) Genetic Variation and Its Effect on P-glycoprotein Biologic Functions
P-glycoprotein (P-gp) is a membrane-bound transporter protein that is encoded by the human multidrug resistance gene MDR1 (ABCB1). P-gp recognizes a wide range of xenobiotics, is pivotal in mediating cancer drug resistance, and plays an important role in limiting drug penetration across the blood–brain barrier. MDR1 genetic variation can lead to changes in P-gp function and may have implications on drug pharmacokinetics. We have identified a novel MDR1GT1292-3TG (Cys431Leu) genetic variation through systematic profiling of subjects with leukemia. The cellular and transport function of this variation was investigated with recombinant human embryonic kidney cells expressing MDR1. Compared with the wild type, MDR1GT1292-3TG recombinant cells exhibited a lower drug resistance phenotype for a panel of chemotherapeutic agents. When compared with wild type, MDR1GT1292-3TG recombinant cells exposed exhibited a 75% decrease in IC50 for doxorubicin (162.6 ± 17.4 to 37.9 ± 2.6 nM) and a 50% decrease in IC50 for paclitaxel (155.7 ± 27.5 to 87.7 ± 9.2 nM), vinblastine (128.0 ± 15.9 to 65.9 ± 5.1 nM), and vincristine (593.7 ± 61.8 to 307.3 ± 17.0 nM). The effects of the Cys431Leu variation, due to MDR1GT1292-3TG nucleotide transition, on P-gp-dependent intracellular substrate accumulation appeared to be substrate dependent where doxorubicin, vinblastine, and paclitaxel exhibit an increased accumulation (p < 0.05), while verapamil and Hoechst33342 exhibit a decreased intracellular concentration compared with wild type (p < 0.05). Collectively, these data suggest MDR1GT1292-3TG variation of P-gp may reduce drug resistance and that subjects with this genotype undergoing chemotherapy with drugs that are transported by P-gp could potentially be more responsive to therapy than those with MDR1 wild-type genotype