Efflux transporters in drug disposition during pregnancy.

Evidence-based dose selection of drugs in pregnant women has been lacking due to challenges in studying maternal-fetal pharmacokinetics. Hence, many drugs are administered off-label during pregnancy based on data obtained from non-pregnant women. During pregnancy, drug transporters play an important role in drug disposition along with known gestational age-dependent changes in physiology and drug-metabolizing enzymes. In this review, as Dr. Qingcheng Mao's former and current lab members, we summarize the collective contributions of Dr. Mao, who lost his life to cancer, focusing on the role of drug transporters in drug disposition during pregnancy. Dr. Mao and his team initiated their research by characterizing the structure of Breast Cancer Resistance Protein [BCRP, ATP-Binding Cassette (ABC) G2]. Subsequently, they have made significant contributions to the understanding of the role of BCRP and other transporters, particularly P-glycoprotein (P-gp/ABCB1), in the exposure of pregnant women and their fetuses to various drugs, including nitrofurantoin, glyburide, buprenorphine, bupropion, tetrahydrocannabinol, and their metabolites. This review also highlights the gestation- and pregnancy-dependent transporter expression at the blood-brain and blood-placenta barriers in mice. Significance Statement Dr. Qingcheng Mao and his team have made significant contributions to the investigation of the role of efflux transporters, especially P-glycoprotein and breast cancer resistance protein, in maternal-fetal exposure to many xenobiotics: nitrofurantoin, glyburide, buprenorphine, bupropion, tetrahydrocannabinol and their metabolites. Studies of individual compounds and the expression of transporters during gestation and pregnancy have improved the understanding of maternal-fetal pharmacokinetics.

A Bayesian population physiologically based pharmacokinetic absorption modeling approach to support generic drug development: application to bupropion hydrochloride oral dosage forms.

We propose a Bayesian population modeling and virtual bioequivalence assessment approach to establishing dissolution specifications for oral dosage forms. A generalizable semi-physiologically based pharmacokinetic absorption model with six gut segments and liver, connected to a two-compartment model of systemic disposition for bupropion hydrochloride oral dosage forms was developed. Prior information on model parameters for gut physiology, bupropion physicochemical properties, and drug product properties were obtained from the literature. The release of bupropion hydrochloride from immediate-, sustained- and extended-release oral dosage forms was described by a Weibull function. In vitro dissolution data were used to assign priors to the in vivo release properties of the three bupropion formulations. We applied global sensitivity analysis to identify the influential parameters for plasma bupropion concentrations and calibrated them. To quantify inter- and intra-individual variability, plasma concentration profiles in healthy volunteers that received the three dosage forms, each at two doses, were used. The calibrated model was in good agreement with both in vitro dissolution and in vivo exposure data. Markov Chain Monte Carlo samples from the joint posterior parameter distribution were used to simulate virtual crossover clinical trials for each formulation with distinct drug dissolution profiles. For each trial, an allowable range of dissolution parameters ("safe space") in which bioequivalence can be anticipated was established. These findings can be used to assure consistent product performance throughout the drug product life-cycle and to support manufacturing changes. Our framework provides a comprehensive approach to support decision-making in drug product development.

Characterization of iron walnut in different regions of China based on phytochemical composition.

Little is known about the phytochemical composition of iron walnuts. Differences in the geographical origin of iron walnuts associated with economic benefits should also be examined. In this study, the phytochemical composition (fatty acids, Vitamin E, total polyphenols and flavonoids, amino acids, and minerals) of iron walnuts in China was investigated. The results showed that there were significant differences (p < 0.05) in the phytochemical composition of iron walnut oils and flours from different regions. Positive (r > 0.5, p < 0.05) and negative (r < - 0.5, p < 0.05) correlations were found between amino acids/minerals and amino acids/oleic acid, with the highest correlation coefficient (r = 0.742, p < 0.05) between Cu and tyrosine. In addition, based on the 12 phytochemical fingerprints selected by random forest, a geographical-origin identification model for iron walnuts was established, with a corresponding correct classification rate of 96.6%. The top three phytochemical fingerprints for the geographical-origin identification of iron walnut were microelements, macroelements, and antioxidant composition, with contribution rates of 61.7%, 18.1%, and 9.9%, respectively.

Bacterial metabolism rescues the inhibition of intestinal drug absorption by food and drug additives.

Food and drug products contain diverse and abundant small-molecule additives (excipients) with unclear impacts on human physiology, drug safety, and response. Here, we evaluate their potential impact on intestinal drug absorption. By screening 136 unique compounds for inhibition of the key intestinal transporter OATP2B1 we identified and validated 24 potent OATP2B1 inhibitors, characterized by higher molecular weight and hydrophobicity compared to poor or noninhibitors. OATP2B1 inhibitors were also enriched for dyes, including 8 azo (R-N=N-R') dyes. Pharmacokinetic studies in mice confirmed that FD&C Red No. 40, a common azo dye excipient and a potent inhibitor of OATP2B1, decreased the plasma level of the OATP2B1 substrate fexofenadine, suggesting that FD&C Red No. 40 has the potential to block drug absorption through OATP2B1 inhibition in vivo. However, the gut microbiomes of multiple unrelated healthy individuals as well as diverse human gut bacterial isolates were capable of inactivating the identified azo dye excipients, producing metabolites that no longer inhibit OATP2B1 transport. These results support a beneficial role for the microbiome in limiting the unintended effects of food and drug additives in the intestine and provide a framework for the data-driven selection of excipients. Furthermore, the ubiquity and genetic diversity of gut bacterial azoreductases coupled to experiments in conventionally raised and gnotobiotic mice suggest that variations in gut microbial community structure may be less important to consider relative to the high concentrations of azo dyes in food products, which have the potential to saturate gut bacterial enzymatic activity.

Interactions of Oral Molecular Excipients with Breast Cancer Resistance Protein, BCRP.

Mechanistic-understanding-based selection of excipients may improve formulation development strategies for generic drug products and potentially accelerate their approval. Our study aimed at investigating the effects of molecular excipients present in orally administered FDA-approved drug products on the intestinal efflux transporter, BCRP (ABCG2), which plays a critical role in drug absorption with potential implications on drug safety and efficacy. We determined the interactions of 136 oral molecular excipients with BCRP in isolated membrane vesicles and identified 26 excipients as BCRP inhibitors with IC50 values less than 5 µM using 3H-cholecystokinin octapeptide (3H-CCK8). These BCRP inhibitors belonged to three functional categories of excipients: dyes, surfactants, and flavoring agents. Compared with noninhibitors, BCRP inhibitors had significantly higher molecular weights and SLogP values. The inhibitory effects of excipients identified in membrane vesicles were also evaluated in BCRP-overexpressing HEK293 cells at similar concentrations. Only 1 of the 26 inhibitors of BCRP identified in vesicles inhibited BCRP-mediated 3H-oxypurinol uptake by more than 50%, consistent with the notion that BCRP inhibition depends on transmembrane or intracellular availability of the inhibitors. Collectively, the results of this study provide new information on excipient selection during the development of drug products with active pharmaceutical ingredients that are BCRP substrates.

Risk assessment and prediction for toxic heavy metals in chestnut and growth soil from China.

BACKGROUND: Toxic heavy metals (THMs) cause severe environmental hazards and threaten human health through various consumption of food stuff. However, little is known of THMs in chestnuts. In this study, the risk assessment and prediction of THMs [lead (Pb), arsenic (As), chromium (Cr), cadmium (Cd) and mercury (Hg)] in chestnuts and growth soils from China were investigated. RESULTS: The main detected THMs in chestnuts and growth soils were As and Cd. The total pollution levels of the five THMs (Nemerow pollution indexes, NPIs) were 0.062 and 1.06, respectively. The dietary risks for children were higher than those of adults, especially short-term non-carcinogenic risk. The main combined risks from the relationships between THMs were Pb-Cr (r = 0.85, P < 0.01) in chestnuts and Pb-As (r = 0.59, P < 0.01) in growth soils. The risk source was found to be the uptake effect of THMs from soil to chestnut, with the highest bioaccumulation factors (BCFs) of Cd (0.254). Several comprehensive risk models were established with the highest coefficient of determination (R2 ) of 0.79. In addition, the main contribution rates of different soil parameters to comprehensive risk of THMs uptake were 49.8% (Cd), 23.4% (pH), 13.8% (Cr) and 13.0% (organic carbon). CONCLUSION: The total pollution levels of THMs fell outside of the safety domain in growth soils. Furthermore, more attention needs to be paid to Cd pollution owing to its low environment background value and high accumulation ability. Three main soil parameters (Cr content, pH, organic carbon) played important roles in the formations and accumulations of THMs in chestnuts. © 2019 Society of Chemical Industry.

Determination of benzo[a]pyrene in camellia oil via vortex-assisted extraction using the UPLC-FLD method.

In this paper, vortex-assisted extraction using the ultraperformance liquid chromatography analysis method was performed to determine benzo[a]pyrene in camellia oil. Optimum results were obtained when 0.5 g of oil sample was used followed by vortex-assisted extraction for 10 min with 25 mL of acetonitrile. Chromatographic separation was performed on an Agilent ZORBAX Eclipse Plus C18 column (2.1mm×100mm, particle size 1.8 µm). The optimum mobile phase comprised 70% acetone and 30% water. The detection limit of benzo[a]pyrene was 0.2 µg/kg. The recoveries were in the range of 81.0-97.0%. The proposed method was simple and fast, and it provided high throughput in the determination of benzo[a]pyrene in an oil matrix sample.

Pesticides in persimmons, jujubes and soil from China: Residue levels, risk assessment and relationship between fruits and soils.

Extreme and uncontrolled usage of pesticides produces a number of problems for vegetation and human health. In this study, the existence of organophosphates (OPs), organochlorines (OCs), pyrethroids (PYs) and fungicides (FUs) were investigated in persimmons/jujubes and their planted soils, which were collected from China. One OP (dimethoate), three OCs (DDT, quintozene and aldrin), six PYs (bifenthrin, fenpropathrin, cyhalothrin, cypermethrin, fenvalerate and deltamethrin) and two FUs (triadimefon and buprofezin) were found in 36.4% of persimmons and 70.8% of jujubes, with concentrations from 1.0 µg/kg to 2945.0 µg/kg. The most frequently detected pesticides in the two fruits were fenpropathrin in persimmons and cypermethrin in jujubes, with the detection frequencies of 30.0% and 22.7%, respectively. The residues of 4.5% (persimmon) and 25.0% (jujube) of samples were higher than the maximum residue limits (MRLs) of China. Compared with the fruits, more types of pesticides and higher residues were observed in their planted soils. The most frequently detected pesticides were HCH in persimmon soil and DDT in jujube soil, with the detection frequencies of 10.9% and 12.7%, respectively. For the tested samples, 39.1% of fruit samples and 63.0% of soil samples with multiple residues (containing more than two pesticides) were noted, even up to 8 residues in fruits and 14 residues in soils. Except for cyhalothrin, the other short-term risks for the tested pesticides in the fruits were below 10%, and the highest long-term risk was 14.13% for aldrin and dieldrin. There was no significant health risk for consumers via consumption of the two fruits.

Residue levels and risk assessment of pesticides in nuts of China.

The pesticide residue levels of three nuts (chestnut, walnut, pinenut) collected from seven main producing areas of China were investigated. Twenty-nine pesticides, including organophosphates (OPs), organochlorines (OCs), pyrethroids (PYs) and two fungicides (triadimefon and buprofezin) were analyzed by gas chromatography (GC). Four OPs (acephate, dimethoate, chlorpyrifos and parathion-methyl) were found in 11.4% samples, with the concentrations of 19.0 µg kg(-1) to 74.0 µg kg(-1). Six OCs (DDT, HCH, endosulfan, quintozene, aldrin and dieldrin) were found in 18.2% samples, with the concentrations of 2.0 µg kg(-1) to 65.7 µg kg(-1). Among OCs, p,p-DDE and α-HCH were the dominant isomer for DDT and HCH. Five PYs (fenpropathrin, fenvalerate, cypermethrin, bifenthrin and cyhalothrin) were found in 15.9% samples, with the concentrations of 2.5 µg kg(-1) to 433.0 µg kg(-1). Fenpropathrin was the most frequently detected pesticide. In addition, triadimefon and buprofezin were detected only in two samples. For the tested nuts, 25.0% samples with multiple residues (containing more than two pesticides) were noted, even up to 9.1% samples with five pesticide residues. The residue of 15.9% samples was higher than the maximum residue limits (MRLs) of China. The short-term risks for the tested nuts were below 1.2%, and the highest long-term risk was 12.58%. The cumulative risk (cHI) for the tested pesticides were 8.43% (OPs), 0.42% (OCs), 12.82% (PYs) and 0.15% (fungicides), respectively. The total cHI was 21.82%. There was no significant health risk for consumers via nuts consumption.

The Fate of Organophosphorus Pesticides during Camellia Oil Production.

The purpose of this study was to investigate the fate of organophosphorus pesticides (OPs) during camellia oil production process, from camellia fruit to the final oil product. The results showed that the OPs were mainly distributed in the peel of camellia fruit, basically above 40% after the pesticide application of 7 d (P < 0.05). A small amount of OPs could enter into the seed and convert to crude camellia oil, with the concentration of 19.5 to 548.2 mg/L. In addition, metabolites of OPs (25.7 to 768.9 mg/L) could be detected in the crude camellia oil. Moreover, the refining process (degumming, deacidfying, bleaching) had a significant effect on the removal of OPs from the crude camellia oil (P < 0.05), and the effect was related to the octanol-water partition constant (LogP) of pesticide. The larger the LogP, the more stable the OPs were during refining process. The final refined camellia oil was found to have no detectable levels of OPs metabolite.

Predicting P-glycoprotein-mediated drug transport based on support vector machine and three-dimensional crystal structure of P-glycoprotein.

Human P-glycoprotein (P-gp) is an ATP-binding cassette multidrug transporter that confers resistance to a wide range of chemotherapeutic agents in cancer cells by active efflux of the drugs from cells. P-gp also plays a key role in limiting oral absorption and brain penetration and in facilitating biliary and renal elimination of structurally diverse drugs. Thus, identification of drugs or new molecular entities to be P-gp substrates is of vital importance for predicting the pharmacokinetics, efficacy, safety, or tissue levels of drugs or drug candidates. At present, publicly available, reliable in silico models predicting P-gp substrates are scarce. In this study, a support vector machine (SVM) method was developed to predict P-gp substrates and P-gp-substrate interactions, based on a training data set of 197 known P-gp substrates and non-substrates collected from the literature. We showed that the SVM method had a prediction accuracy of approximately 80% on an independent external validation data set of 32 compounds. A homology model of human P-gp based on the X-ray structure of mouse P-gp as a template has been constructed. We showed that molecular docking to the P-gp structures successfully predicted the geometry of P-gp-ligand complexes. Our SVM prediction and the molecular docking methods have been integrated into a free web server (http://pgp.althotas.com), which allows the users to predict whether a given compound is a P-gp substrate and how it binds to and interacts with P-gp. Utilization of such a web server may prove valuable for both rational drug design and screening.

Identification of proline residues in or near the transmembrane helices of the human breast cancer resistance protein (BCRP/ABCG2) that are important for transport activity and substrate specificity.

The human breast cancer resistance protein (BCRP/ABCG2) confers multidrug resistance and mediates the active efflux of drugs and xenobiotics. BCRP contains one nucleotide-binding domain (NBD) followed by one membrane-spanning domain (MSD). We investigated whether prolines in or near the transmembrane helices are essential for BCRP function. Six proline residues were substituted with alanine individually, and the mutants were stably expressed in Flp-In(TM)-293 cells at levels comparable to that of wild-type BCRP and predominantly localized on the plasma membrane of the cells. While P392A showed a significant reduction (35-50%) in the efflux activity of mitoxantrone, BODIPY-prazosin, and Hoechst 33342, P485A exhibited a significant decrease of approximately 70% in the efflux activity of only BODIPY-prazosin. Other mutants had no significant changes in the efflux activities of these substrates. Drug resistance profiles of the cells expressing the mutants correlated well with the efflux data. ATPase activity was not substantially affected for P392A or P485A compared to that of wild-type BCRP. These results strongly suggest Pro(392) and Pro(485) are important in determining the overall transport activity and substrate selectivity of BCRP, respectively. Prazosin differentially affected the binding of 5D3, a conformation-sensitive antibody, to wild-type BCRP, P392A, or P485A in a concentration-dependent manner. In contrast, mitoxantrone had no significant effect on 5D3 binding. Homology modeling indicates that Pro(392) may play an important role in the communication between the MSD and NBD as it is predicted to be located at the interface between the two functional domains, and Pro(485) induces flexible hinges that may be essential for the broad substrate specificity of BCRP.

ATP-binding cassette efflux transporters in human placenta.

Pregnant women are often complicated with diseases including viral or bacterial infections, epilepsy, hypertension, or pregnancy-induced conditions such as depression and gestational diabetes that require treatment with medication. In addition, substance abuse during pregnancy remains a major public health problem. Many drugs used by pregnant women are off label without the necessary dose, efficacy, and safety data required for rational dosing regimens of these drugs. Thus, a major concern arising from the widespread use of drugs by pregnant women is the transfer of drugs across the placental barrier, leading to potential toxicity to the developing fetus. Knowledge regarding the ATP-binding cassette (ABC) efflux transporters, which play an important role in drug transfer across the placental barrier, is absolutely critical for optimizing the therapeutic strategy to treat the mother while protecting the fetus during pregnancy. Such transporters include P-glycoprotein (P-gp, gene symbol ABCB1), the breast cancer resistance protein (BCRP, gene symbol ABCG2), and the multidrug resistance proteins (MRPs, gene symbol ABCCs). In this review, we summarize the current knowledge with respect to developmental expression and regulation, membrane localization, functional significance, and genetic polymorphisms of these ABC transporters in the placenta and their relevance to fetal drug exposure and toxicity.

Structure and function of the human breast cancer resistance protein (BCRP/ABCG2).

The human breast cancer resistance protein (BCRP/ABCG2) is the second member of the G subfamily of the large ATP-binding cassette (ABC) transporter superfamily. BCRP was initially discovered in multidrug resistant breast cancer cell lines where it confers resistance to chemotherapeutic agents such as mitoxantrone, topotecan and methotrexate by extruding these compounds out of the cell. BCRP is capable of transporting non-chemotherapy drugs and xenobiotiocs as well, including nitrofurantoin, prazosin, glyburide, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. BCRP is frequently detected at high levels in stem cells, likely providing xenobiotic protection. BCRP is also highly expressed in normal human tissues including the small intestine, liver, brain endothelium, and placenta. Therefore, BCRP has been increasingly recognized for its important role in the absorption, elimination, and tissue distribution of drugs and xenobiotics. At present, little is known about the transport mechanism of BCRP, particularly how it recognizes and transports a large number of structurally and chemically unrelated drugs and xenobiotics. Here, we review current knowledge of structure and function of this medically important ABC efflux drug transporter.

Transmembrane helices 1 and 6 of the human breast cancer resistance protein (BCRP/ABCG2): identification of polar residues important for drug transport.

The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics. In this study, we investigated the role of polar residues within or near the predicted transmembrane α-helices 1 and 6 of BCRP in drug transport. We substituted Asn(387), Gln(398), Asn(629), and Thr(642) with Ala, Thr(402) with Ala and Arg, and Tyr(645) with Phe, and the mutants were stably expressed in human embryonic kidney-293 or Flp-In-293 cells. Immunoblotting and confocal microscopy analysis revealed that all of the mutants were well expressed and predominantly targeted to the plasma membrane. While T402A and T402R showed a significant global reduction in the efflux of mitoxantrone, Hoechst 33342, and BODIPY-prazosin, N629A exhibited significantly increased efflux activities for all of the substrates. N387A and Q398A displayed significantly impaired efflux for mitoxantrone and Hoechst 33342, but not for BODIPY-prazosin. In contrast, T642A and Y645F showed a moderate reduction in Hoechst 33342 efflux only. Drug resistance profiles of human embryonic kidney-293 cells expressing the mutants generally correlated with the efflux data. Furthermore, N629A was associated with a marked increase, and N387A and T402A with a significant reduction, in BCRP ATPase activity. Mutations of some of the polar residues may cause conformational changes, as manifested by the altered binding of the 5D3 antibody to BCRP in the presence of prazosin. The inward-facing homology model of BCRP indicated that Thr(402) within transmembrane 1 may be important for helical interactions, and Asn(629) may be involved in BCRP-substrate interaction. In conclusion, we have demonstrated the functional importance of some of these polar residues in BCRP activity.

Fluorescence resonance energy transfer (FRET) analysis demonstrates dimer/oligomer formation of the human breast cancer resistance protein (BCRP/ABCG2) in intact cells.

The human breast cancer resistance protein (BCRP/ABCG2) is a half ATP-binding cassette (ABC) efflux transporter that plays an important role in drug resistance and disposition. Although BCRP is believed to function as a homodimer or homooligomer, this has not been demonstrated in vivo in intact cells. Therefore, in the present study, we investigated dimer/oligmer formation of BCRP in intact cells. Wild-type BCRP and the mutant C603A were attached to cyan or yellow fluorescence protein and expressed in HEK293 cells by transient transfection. Protein levels, cell surface expression, and efflux activities of wild-type and mutant BCRP were determined by immunoblotting, 5D3 antibody binding, and flow cytometric efflux assay, respectively. Dimer/oligomer formation of BCRP in intact cells was analyzed using fluorescence resonance energy transfer (FRET) microscopy. Wild-type BCRP and C603A were expressed in HEK293 cells at comparable levels. C603A was predominantly expressed in the plasma membrane as was wild-type protein. Furthermore, C603A retained the same mitoxantrone efflux activity and the ability of dimer/oligmer formation as wild-type BCRP. Finally, cross-linking experiments yielded data consistent with the FRET analysis. In conclusion, we have, for the first time, demonstrated that BCRP can form a dimer/oligomer in vivo in intact cells using the FRET technique. We have also shown that Cys(603) alone does not seem to be essential for dimer/oligomer formation of BCRP.

The human breast cancer resistance protein (BCRP/ABCG2) shows conformational changes with mitoxantrone.

BCRP/ABCG2 mediates efflux of drugs and xenobiotics. BCRP was expressed in Pichia pastoris, purified to > 90% homogeneity, and subjected to two-dimensional (2D) crystallization. The 2D crystals showed a p12(1) symmetry and projection maps were determined at 5 A resolution by cryo-electron microscopy. Two crystal forms with and without mitoxantrone were observed with unit cell dimensions of a = 55.4 A, b = 81.4 A, gamma = 89.8 degrees , and a = 57.3 A, b = 88.0 A, gamma = 89.7 degrees , respectively. The projection map without mitoxantrone revealed an asymmetric structure with ring-shaped density features probably corresponding to a bundle of transmembrane alpha helices, and appeared more open and less symmetric than the map with mitroxantrone. The open and closed inward-facing forms of BCRP were generated by homology modeling, representing the substrate-free and substrate-bound conformations in the absence of nucleotide, respectively. These models are consistent with the experimentally observed conformational change upon substrate binding.

Role of basic residues within or near the predicted transmembrane helix 2 of the human breast cancer resistance protein in drug transport.

The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics out of cells. In this study, we investigated the role of five basic residues within or near transmembrane (TM) 2 of BCRP in transport activity. Lys(452), Lys(453), His(457), Arg(465), and Lys(473) were replaced with Ala or Asp. K452A, K453D, H457A, R465A, and K473A were stably expressed in human embryonic kidney (HEK) cells, and their plasma membrane expression and transport activities were examined. All of the mutants were expressed predominantly on the plasma membrane of HEK cells. After normalization to BCRP levels, the activities of K452A and H457A in effluxing mitoxantrone, boron-dipyrromethene-prazosin, and Hoechst33342 were increased approximately 2- to 6-fold compared with those of wild-type BCRP, whereas the activities of K453D and R465A were decreased by 40 to 60%. Likewise, K452A and H457A conferred increased resistance to mitoxantrone and 7-ethyl-10-hydroxy-camptothecin (SN-38), and K453D and R465A exhibited lower resistance. The transport activities and drug-resistance profiles of K473A were not changed. These mutations also differentially affected BCRP ATPase activities with a 2- to 4-fold increase in V(max)/K(m) for K452A and H457A and a 40 to 70% decrease for K453D and R465A. These mutations may induce conformational changes as manifested by the altered binding of the 5D3 antibody to BCRP in the presence of prazosin and altered trypsin digestion. Molecular modeling and docking calculations indicated that His(457) and Arg(465) might be directly involved in substrate binding. In conclusion, we have identified several basic residues within or near TM2 that may be important for interaction of substrates with BCRP.

Membrane topology of the human breast cancer resistance protein (BCRP/ABCG2) determined by epitope insertion and immunofluorescence.

The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and organic anions across the plasma membrane. Hydropathy analysis suggests that BCRP consists of a nucleotide-binding domain (residues approximately 1-395) and a membrane-spanning domain (MSD) (residues approximately 396-655); however, its exact topology structure remains unknown. In this study, we determined the topology structure of BCRP by inserting hemagglutinin (HA) tags in its predicted hydrophilic regions of the MSD. HA-tagged BCRP mutants were expressed in HEK cells and tested for their ability to efflux mitoxantrone and BODIPY-prazosin. Polarity of the inserted tags with respect to the plasma membrane was determined by immunofluorescence. All of the mutants were expressed at levels comparable to wild-type BCRP as revealed by immunoblotting with specific antibodies against BCRP and the HA tag. Insertions at residues 423, 454, 462, 499, 529, 532, and 651 produced functional mutants, whereas insertions at residues 560, 594, and 623 resulted in mutants with significantly reduced activity and insertions at residues 387, 420, 474, and 502 completely abrogated the activity. HA tags inserted at residues 387, 474, 529, 532, 560, and 651 were localized intracellularly, whereas those inserted at residues 420, 423, 454, 499, 502, 594, and 623 revealed an extracellular location. Residue 462 was localized in a transmembrane (TM) segment. These results provide the first direct experimental evidence in support of a 6-TM model for BCRP with the amino and carboxyl termini of the MSD located intracellularly. These data may have important implications for understanding the transport mechanism of BCRP.

Effects of site-directed mutation on the function of the chloroplast ATP synthase epsilon subunit.

The previous work in our lab showed that the spinach chloroplast ATP synthase epsilon mutant with 3 amino acid residues deleted from the N-terminus had much lower ability to inhibit ATP hydrolysis and block proton leakage in comparison to a mutant with 1 or 2 residues deleted from the N-terminus. The present study aimed at determining whether there is special importance in the structure and function of the N-terminal third residue of the chloroplast epsilon subunit. The leucine residue at the N-terminal third site (Leu3) of the spinach chloroplast epsilon subunit was replaced with Ile, Phe, Thr, Arg, Glu or Pro by site-directed mutagenesis, forming mutants epsilonL3I, epsilonL3F, epsilonL3T, epsilonL3R, epsilonL3E and epsilonL3P, respectively. These epsilon variants all showed lower abilities to inhibit ATP hydrolysis and to block proton leakage, as compared to the wild type epsilon subunit (epsilonWT). The abilities of mutants epsilonL3I and epsilonL3F to restore the ATP synthesis activity of reconstituted membranes were higher than those of epsilonWT, but the abilities of the other epsilon variants were lower than that of epsilonWT. These results indicate that the hydrophobic and neutral characteristics of Leu3 of the chloroplast epsilon subunit are very important for its ability to inhibit ATP hydrolysis and block proton leakage, and for the ATP synthesis ability of ATP synthase.