Blockage of drug resistance in vitro by disulfiram, a drug used to treat alcoholism.

BACKGROUND: P-glycoprotein (P-gp) pumps a wide range of cytotoxic drugs out of cells. Inhibiting maturation of P-gp would be a novel method for circumventing P-gp-mediated multidrug resistance, which complicates cancer chemotherapy and treatment of patients infected with human immunodeficiency virus. We examined the effect of disulfiram (Antabuse(TM)) on the maturation and activity of P-gp. METHODS: Embryonic kidney cells were transfected with a complementary DNA for the P-pg gene, and the effects of disulfiram on the sensitivity of the transfected cells to cytotoxic agents were determined. Enzyme assays were used to determine the effects of disulfiram on the verapamil-stimulated adenosine triphosphatase (ATPase) activity of P-gp. Disulfiram modifies cysteine residues, and mutant forms of P-gp that lack individual cysteines were used to determine whether particular cysteine residues mediate disulfiram's effects on P-gp activity. Maturation of recombinant P-gp was followed on immunoblots. RESULTS: Disulfiram increased the sensitivity of P-gp-transfected cells to vinblastine and colchicine and inhibited P-gp's verapamil-stimulated ATPase activity. Half-maximal inhibition of ATPase activity occurred at 13.5 microM disulfiram. Disulfiram (at 100 microM) inhibited a P-gp mutant by 43% (95% confidence interval [CI] = 37%-48%) when cysteine was present at position 431 only and by 72% (95% CI = 66%-77%) when cysteine was present at position 1074 only. Treatment of P-gp-transfected cells with 50 nM disulfiram blocked maturation of recombinant P-gp. CONCLUSIONS: Disulfiram can potentially reduce P-gp-mediated drug resistance by inhibiting P-gp activity (possibly via cysteine modification) and/or by blocking its maturation. These results suggest that disulfiram has the potential to increase the efficacy of drug therapies for cancer and acquired immunodeficiency syndrome.

Determining the structure and mechanism of the human multidrug resistance P-glycoprotein using cysteine-scanning mutagenesis and thiol-modification techniques.

The multidrug resistance P-glycoprotein is an ATP-dependent drug pump that extrudes a broad range of hydrophobic compounds out of cells. Its physiological role is likely to protect us from exogenous and endogenous toxins. The protein is important because it contributes to the phenomenon of multidrug resistance during AIDS and cancer chemotherapy. We have used cysteine-scanning mutagenesis and thiol-modification techniques to map the topology of the protein, show that both nucleotide-binding domains are essential for activity, examine packing of the transmembrane segments, map the drug-binding site, and show that there is cross-talk between the ATP-binding sites and the transmembrane segments.

Interaction of Escherichia coli F1-ATPase with dicyclohexylcarbodiimide-binding polypeptide.

Antibody raised against the N,N'-dicyclohexylcarbodiimide (DCCD)-binding polypeptide of Escherichia coli bound to the cytoplasmic surface of the cell membrane. A weak reaction was seen with everted vesicles of the thermophile PS3. Rat-liver mitochondrial membranes did not react with the antibody. Reaction of the isolated DCCD-binding polypeptide with the antibody was prevented by oxidation of methionine residues or cleavage of the polypeptide with cyanogen bromide. Modification of the arginine residues of the DCCD-binding polypeptide did not affect interaction with the antibody. Purified F1-ATPase of E. coli bound to the isolated DCCD-binding polypeptide as shown by solid-phase radioimmune assay. Binding involved the alpha and/or beta subunits of F1 and the arginine residues of the polar central region of the DCCD-binding polypeptide. Our results are consistent with a looped arrangement of the DCCD-binding polypeptide in the membrane in which the carboxyl- and amino-terminal regions of the molecule are at the periplasmic surface and the polar central region, interacting with F1, is at the cytoplasmic surface of the cell membrane.

Expression of rubella virus cDNA coding for the structural proteins.

A cDNA clone encoding the precursor polypeptide (Mr 115,000) to the nucleocapsid C (Mr 30,000) and two envelope glycoproteins E1 (Mr 58,000) and E2 (Mr 42,000-47,000) of rubella virus was inserted into a simian virus 40-derived eukaryotic expression vector. When the plasmid was introduced into COS cells, three proteins were synthesized. The expressed proteins were antigenically similar and identical in size to the authentic structural proteins of rubella virus. Expression in the presence of tunicamycin confirmed that E1 and E2 are glycoproteins. Unglycosylated E1 and E2 had Mrs of about 53,000 and 30,000, respectively. The mobility of the nucleocapsid protein was unaffected by tunicamycin. The locations of the translation start and stop codons for synthesis of the precursor to the structural proteins of rubella virus were determined by in vitro and in vivo expression studies. It was found that the first AUG codon at the 5' end of the rubella virus 24S cDNA acts as a start codon for translation. The stop codon was found to be 3183 bp from the start codon.

Deletion of NH2- and COOH-terminal sequences destroys function of the Ca2+ ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum.

Deletion mutants of the Ca2+ ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum (SERCA1a) were constructed and expressed in COS-1 cells. The mutants were expressed at levels 7- to 15-fold lower than the wild-type and were inactive. In vitro transcription-translation-insertion experiments showed that deletion of transmembrane sequences M1 and M2, but not of M8, M9, M10 or the NH2-terminal 30 amino acids inhibited the stable insertion of the enzyme into the membrane. Thus there was no correlation between loss of function and membrane insertion. A signal sequence for membrane insertion may exist in M1 and M2.

Detection of antibodies to individual proteins of rubella virus.

Individual rubella virus structural polypeptides were electroeluted from SDS-polyacrylamide gels. The eluted polypeptides were used, without further purification, as antigens in ELISA assays for the detection of rubella-specific antibodies in patients' sera. This provided a more sensitive detection method than that involving classical serological assays such as HI or VN or that using immunoprecipitation. Antisera against individual viral polypeptides were raised in mice. No haemagglutination inhibition activity was observed in any of these sera and weak virus neutralizing activity was only detected with antiserum to the E1 protein. Antisera to either the E1 or E2(a,b) complex proteins cross-reacted with both the E1 and E2(a,b) complex proteins.

Recent progress in understanding the mechanism of P-glycoprotein-mediated drug efflux.

P-glycoprotein (P-gp) is an ATP-dependent drug pump that can transport a broad range of hydrophobic compounds out of the cell. The protein is clinically important because of its contribution to the phenomenon of multidrug resistance during AIDS/HIV and cancer chemotherapy. P-gp is a member of the ATP-binding cassette (ABC) family of proteins. It is a single polypeptide that contains two repeats joined by a linker region. Each repeat has a transmembrane domain consisting of six transmembrane segments followed by a hydrophilic domain containing the nucleotide-binding domain. In this mini-review, we discuss recent progress in determining the structure and mechanism of human P-glycoprotein.

Identification of residues within the drug-binding domain of the human multidrug resistance P-glycoprotein by cysteine-scanning mutagenesis and reaction with dibromobimane.

P-glycoprotein (P-gp) can transport a wide variety of cytotoxic compounds that have diverse structures. Therefore, the drug-binding domain of the human multidrug resistance P-gp likely consists of residues from multiple transmembrane (TM) segments. In this study, we completed cysteine-scanning mutagenesis of all the predicted TM segments of P-gp (TMs 1-5 and 7-10) and tested for inhibition by a thiol-reactive substrate (dibromobimane) to identify residues within the drug-binding domain. The activities of 189 mutants were analyzed. Verapamil-stimulated ATPase activities of seven mutants (Y118C and V125C (TM2), S222C (TM4), I306C (TM5), S766C (TM9), and I868C and G872C (TM10)) were inhibited by more than 50% by dibromobimane. The activities of mutants S222C (TM4), I306C (TM5), I868C (TM10), and G872C (TM10), but not that of mutants Y118C (TM2), V125C (TM2), and S776C (TM9), were protected from inhibition by dibromobimane by pretreatment with verapamil, vinblastine, or colchicine. These results and those from previous studies (Loo, T. W. and Clarke, D. M. (1997) J. Biol. Chem. 272, 31945-31948; Loo, T. W. and Clarke, D. M. (1999) J. Biol. Chem. 274, 35388-35392) indicate that the drug-binding domain of P-gp consists of residues in TMs 4, 5, 6, 10, 11, and 12.

Membrane topology of a cysteine-less mutant of human P-glycoprotein.

A human P-glycoprotein devoid of cysteine residues was constructed by site-directed mutagenesis for studying its topology. The cDNA for human P-glycoprotein-A52 in which codons for cysteines 137, 431, 717, 956, 1074, 1125, 1227, 1288, and 1304 were changed to Ala, was transfected into NIH 3T3 cells and analyzed with respect to its ability to confer resistance to various drugs. The cysteine-less P-glycoprotein-A52 retained the ability to confer resistance to vinblastine, colchicine, doxorubicin, and actinomycin D with only a small decrease in efficiency relative to wild-type enzyme. Cysteine residues were then reintroduced into predicted extracellular or cytoplasmic loops of the cysteine-less P-glycoprotein-A52, and the topology of the protein was determined using membrane-permeant and impermeant thiol-specific reagents. It was found that 8 of 15 cysteine residues introduced into P-glycoprotein-A52 could be biotinylated, when cells expressing the mutant P-glycoprotein were incubated with membrane-permeant biotin maleimide. Biotinylation of a cysteine residue placed in predicted extracellular loops between transmembrane segment (TM) 5 and TM6, TM7 and TM8, or TM11 and TM12 was blocked by pretreatment of the cells with a membrane-impermeant maleimide, suggesting that these residues have an extracellular location. By contrast, biotinylation of cysteine residues located in the predicted cytoplasmic loops between TM2 and TM3, TM4 and TM5, TM8 and TM9, or TM10 and TM11 were not blocked by pretreatment with membrane impermeant maleimide, suggesting that these residues were in the cytoplasm. These results are consistent with the model of P-glycoprotein, which predicts six transmembrane segments in each of the two homologous halves of the molecule.

Functional expression of human renal Na+/Ca2+ exchanger in insect cells.

The cDNA coding for the human renal isoform of the Na+/Ca2+ exchanger was cloned from a human embryonic kidney cell line (HEK 293) using a polymerase chain reaction strategy. It was found by sequence analysis that the cDNA encoded for a human kidney isoform of the Na+/Ca2+ exchanger. The kidney isoform is nearly identical to the human cardiac Na+/Ca2+ exchanger except for a segment of amino acids within the predicted cytoplasmic loop region of the molecule. Within this region, the kidney isoform contains a deletion of 36 amino acids, as well as a segment of 33 amino acids that is only 33% identical to the equivalent region of the cardiac exchanger. The cDNA was then tested for function by expression in insect cells. We constructed a recombinant baculovirus containing renal Na+/Ca2+ exchanger cDNA under control of the polyhedrin promoter. High levels of expression and Na+/Ca2+ exchange activity were found in Sf9 insect cells infected with the recombinant virus. Our results indicate that human kidney expresses a functional and alternatively spliced variant of the human cardiac Na+/Ca2+ exchanger.

Functional consequences of phenylalanine mutations in the predicted transmembrane domain of P-glycoprotein.

Site-directed mutagenesis was used to investigate whether phenylalanine residues in predicted transmembrane sequences play essential roles in the function of human P-glycoprotein. Mutant cDNAs, in which codons for each of the 31 phenylalanine residues were changed to alanine, were expressed in mouse NIH 3T3 cells and analyzed with respect to their ability to confer resistance to various drugs. Mutation of either Phe-335 to Ala in transmembrane segment 6, or Phe-978 to Ala in transmembrane segment 12, drastically altered the drug resistance profile conferred by the mutant P-glycoprotein in transfected cells. Mutant Phe-335-->Ala conferred little resistance to vinblastine or actinomycin D but retained the ability to confer resistance to colchicine and adriamycin. The mutant also showed increased binding of azidopine, which could be inhibited by lower levels of vinblastine, relative to the wild-type enzyme. By contrast, mutant Phe-978-->Ala conferred little or no resistance to colchicine or adriamycin, while its ability to confer resistance to vinblastine or actinomycin D was retained. These results suggest that Phe-335 and Phe-978 play important roles in the recognition and transport of specific substrates by P-glycoprotein. Mutation of Phe-777 to Ala affected the biosynthesis of the transporter. Mutation of the other 28 phenylalanine residues yielded protein products with structural and functional characteristics that were indistinguishable from the wild-type enzyme.

Covalent modification of human P-glycoprotein mutants containing a single cysteine in either nucleotide-binding fold abolishes drug-stimulated ATPase activity.

The ATPase activity of P-glycoprotein is inactivated by N-ethylmaleimide (NEM), which is postulated to modify cysteine residues within either of the homology A consensus sequences for nucleotide binding (GNSGCGKS and GSSGCGKS, respectively) (Al-Shawi, M. K., Urbatsch, I. L., and Senior, A. E. (1994) J. Biol. Chem. 269, 8986-8992). To test this postulate as well as determine the contribution of either nucleotide-binding domain to function, a Cys-less mutant was constructed, and then a single cysteine residue was reintroduced back into each nucleotide-binding consensus sequence. We then tested the sensitivity of the ATPase activity of each mutant to covalent modification by NEM. It was found that covalent modification of a single cysteine residue within either nucleotide-binding consensus sequence (Cys-431 and Cys-1074, respectively) with NEM inhibited drug-stimulated ATPase activity of P-glycoprotein. The concentrations of NEM required for half-maximal inactivation of ATPase activity were 7 and 35 microM for mutants Cys-431 and Cys-1074, respectively. In both cases, inactivation of ATPase activity by NEM was prevented by ATP. These results suggest that both nucleotide-binding domains may need to bind ATP to couple drug binding to ATPase activity.

P-glycoprotein. Associations between domains and between domains and molecular chaperones.

P-glycoprotein consists of two homologous halves, each composed of a transmembrane domain and a nucleotide-binding domain. In order to understand how the domains interact in P-glycoprotein, we expressed each domain as a separate polypeptide and tested for associations using coimmunoprecipitation assays. We found that the interactions between the two halves of P-glycoprotein were mediated through associations between the two transmembrane domains as well as through the nucleotide-binding domains. In addition, the nucleotide-binding domain also associated with the transmembrane domain in each half of the molecule. By contrast, we could not detect any association either between the first nucleotide-binding domain and the second transmembrane domain, or between the second nucleotide-binding domain and the first transmembrane domain. We then tested whether individual domains associated with molecular chaperones, since biogenesis of P-glycoprotein appears to involve the chaperones calnexin and Hsc70. We found that calnexin associated only with the transmembrane domains, while Hsc70 associated only with the nucleotide-binding domains. These results suggest that noncovalent interaction between the domains of P-glycoprotein can contribute to structure and function of P-glycoprotein and that chaperones may participate in the folding of each domain.

Rapid purification of human P-glycoprotein mutants expressed transiently in HEK 293 cells by nickel-chelate chromatography and characterization of their drug-stimulated ATPase activities.

P-glycoprotein containing 10 tandem histidine residues at the COOH end of the molecule was transiently expressed in HEK 293 cells and purified by nickel-chelate chromatography. The purified protein had an apparent mass of 170 kDa, and its verapamil-stimulated ATPase activity in the presence of phospholipid was 1.2 mumol/min/mg of P-glycoprotein. We then characterized P-glycoprotein mutants that exhibited altered drug-resistant phenotypes and analyzed the contribution of the two nucleotide binding folds to drug-stimulated ATPase activity. Mutation of residues in either nucleotide binding fold abolished drug-stimulated ATPase activity. The pattern of drug-stimulated ATPase activities of mutants, which conferred increased relative resistance to colchicine (G141V, G185V, G830V) or decreased relative resistance to all drugs (F978A), correlated with their drug-resistant phenotypes. By contrast, the ATPase activity of mutant F335A was significantly higher than that of wild-type enzyme when assayed in the presence of verapamil (3.4-fold), colchicine (9.1-fold), or vinblastine (3.7-fold), even though it conferred little resistance to vinblastine in transfected cells. These results suggest that both nucleotide-binding domains must be intact to couple drug binding to ATPase activity and that the drug-stimulated ATPase activity profile of a mutant does not always correlate with its drug-resistant phenotype.

Functional consequences of proline mutations in the predicted transmembrane domain of P-glycoprotein.

Site-directed mutagenesis was used to investigate whether prolines in the predicted transmembrane domains play essential roles in the function of human P-glycoprotein. Mutant cDNAs in which codons for each of the 13 prolines were changed to alanine were expressed in mouse NIH 3T3 cells and analyzed with respect to their ability to confer resistance to various drugs. Mutations of either Pro223 in transmembrane segment 4 or Pro866 in transmembrane segment 10, drastically reduced the ability of the mutant proteins to confer resistance to colchicine, adriamycin, or actinomycin D, whereas the capacity to confer resistance to vinblastine was retained. These results strongly suggest that residues in putative transmembrane segments 4 and 10, which are found in identical positions when homologous, presumably duplicated, halves of the transporter are aligned, play important roles in recognition of colchicine, adriamycin, and actinomycin D. They may either interact to form a single drug-binding site or form part of two equivalent, but independent, drug-binding sites. The lack of detectable effect of either mutation on vinblastine transport, however, indicates that there are differences in the requirements for binding of various substrates to P-glycoprotein. Mutation of Pro709 in transmembrane segment 7 resulted in a protein unable to confer drug resistance. A change at this position was found to induce a structural aberration, since the major protein product observed in transfected cells had an apparent molecular weight of 150,000, whereas the wild-type enzyme had an apparent molecular weight of approximately 170,000. Mutation of the other 10 prolines yielded protein products with structural and functional characteristics indistinguishable from wild-type P-glycoprotein.

Inhibition of oxidative cross-linking between engineered cysteine residues at positions 332 in predicted transmembrane segments (TM) 6 and 975 in predicted TM12 of human P-glycoprotein by drug substrates.

Each homologous half of P-glycoprotein consists of a transmembrane domain with six potential transmembrane segments and an ATP-binding domain. Labeling studies with photoactive drug analogs show that labeling occurs within or close to predicted transmembrane segments (TM) 6 (residues 331-351) and TM12 (residues 974-994). To test if these segments are in near-proximity we generated 42 different P-glycoprotein mutants in which we re-introduced a pair of cysteine residues into a Cys-less P-glycoprotein, one within TM6 (residues 332-338) and one within TM12 (residues 975-980) and assayed for cross-linking between the cysteines. All the mutants retained verapamil-stimulated ATPase activity. We found that only the mutant containing Cys-332 and Cys-975 was cross-linked in the presence of oxidant as judged by its decreased mobility on SDS gels. Similar results were obtained when the same mutations were introduced into Cys-less NH2-terminal and COOH-terminal half-molecules of P-glycoprotein followed by coexpression and treatment with oxidant. Cross-linking between Cys-332 and Cys-975, however, was inhibited by verapamil or vinblastine but not by colchicine. These results suggest that residues Cys-332 and Cys-975, which occupy equivalent positions when TM6 and TM12 are aligned, are close to each other in the tertiary structure of P-glycoprotein.

The minimum functional unit of human P-glycoprotein appears to be a monomer.

Several studies have demonstrated the presence of oligomers of P-glycoprotein in multidrug-resistant cells. The minimum functional unit of P-glycoprotein, however, is not known. In order to determine whether the functional unit is an oligomer, we tested for associations between P-glycoproteins containing either a histidine tag or the epitope tag for monoclonal antibody A52 at the COOH-terminal end of the molecule. Both tagged molecules were active and had indistinguishable drug resistance profiles. The tagged P-glycoproteins were expressed contemporaneously in HEK 293 cells, purified by nickel-chelate chromatography followed by immunoblot analysis. We found that P-glycoprotein-A52 did not copurify with functionally active P-glycoprotein-(His)10, even when the former was overexpressed relative to the histidine-tagged protein. Similar results were obtained with phosphorylation-deficient mutants of P-glycoprotein. By contrast, we could purify and reconstitute drug-stimulated ATPase activity when the half-molecules NH2-terminal half-(His)10/COOH-terminal half-A52 or NH2-terminal half-A52/COOH-terminal half-(His)10 were coexpressed in HEK 293 cells. These results suggest that nickel-chelate chromatography may be a suitable method for studying protein-protein interactions in membrane proteins and that the minimal functional unit of P-glycoprotein is likely to be a monomer.

Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators.

There is growing evidence that abnormal protein folding or trafficking (protein kinesis) leads to diseases. We have used P-glycoprotein as a model protein to develop strategies to overcome defects in protein kinesis. Misprocessed mutants of the human P-glycoprotein are retained in the endoplasmic reticulum as core-glycosylated biosynthetic intermediates and rapidly degraded. Synthesis of the mutant proteins in the presence of drug substrates or modulators such as capsaicin, cyclosporin, vinblastine, or verapamil, however, resulted in the appearance of a fully glycosylated and functional protein at the cell surface. These effects were dose-dependent and occurred within a few hours after the addition of substrate. The ability to facilitate processing of the misfolded mutants appeared to be independent of the cell lines used and location of the mutation. P-glycoproteins with mutations in transmembrane segments, extracellular or cytoplasmic loops, the nucleotide-binding domains, or the linker region were processed to the fully mature form in the presence of these substrates. These drug substrates or modulators acted as specific chemical chaperones for P-glycoprotein because they were ineffective on the deltaF508 mutant of cystic fibrosis transmembrane conductance regulator. Therefore, one possible strategy to prevent protein misfolding is to carry out synthesis in the presence of specific substrates or modulators of the protein.

Drug-stimulated ATPase activity of human P-glycoprotein is blocked by disulfide cross-linking between the nucleotide-binding sites.

P-glycoprotein (P-gp) is an ATP-dependent drug pump that contains two nucleotide-binding domains (NBDs). Disulfide cross-linking analysis was done to determine if the two NBDs are close to each other. Residues within or close to the Walker A (GNSGCGKS in NDB1 and GSSGCGKS in NBD2) sequences for nucleotide binding were replaced with cysteine, and the mutant P-gps were subjected to oxidative cross-linking. Cross-linking was detected in two mutants, G427C(NBD1)/Cys-1074(NBD2) and L439C(NBD1)/Cys-1074(NBD2), because the cross-linked proteins migrated slower in SDS gels. Mutants G427C(NBD1)/Cys-1074(NBD2) and L439C(NBD1)/Cys-1074(NBD2) retained 10% and 82%, respectively, of the drug-stimulated ATPase activity relative to that of Cys-less P-gp. The cross-linking properties of the more active mutant L439C(NBD1)/Cys-1074(NBD2) were then studied. Cross-linking was reversed by addition of dithiothreitol and could be prevented by pretreatment of the mutant with N-ethylmaleimide. Cross-linking was also inhibited by MgATP, but not by the verapamil. Oxidative cross-linking of mutant L439C(NBD1)/Cys-1074(NBD2) resulted in almost complete inhibition of drug-stimulated ATPase activity. More than 60% of the drug-stimulated ATPase activity, however, was recovered after treatment with dithiothreitol. The results indicate that the two predicted nucleotide-binding sites are close to each other and that cross-linking inhibits ATP hydrolysis.