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Chen X, Liu X, Gan X, Li S, Ma H, Zhang L, Wang P, Li Y, Huang T, Yang X, Fang L, Liang Y, Wu J, Chen T, Zhou Z, Liu X, Guo J. Differential regulation of phosphorylation, structure and stability of circadian clock protein FRQ isoforms. J Biol Chem 2023; 299:104597. [PMID: 36898580 PMCID: PMC10140173 DOI: 10.1016/j.jbc.2023.104597] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2022] [Revised: 01/20/2023] [Accepted: 01/23/2023] [Indexed: 03/12/2023] Open
Abstract
Neurospora crassa is an important model for circadian clock research. The Neurospora core circadian component FRQ protein has two isoforms, large FRQ (l-FRQ) and small FRQ (s-FRQ), of which l-FRQ bears an additional N-terminal 99-amino acid fragment. However, how the FRQ isoforms operate differentially in regulating the circadian clock remains elusive. Here, we show l-FRQ and s-FRQ play different roles in regulating the circadian negative feedback loop. Compared to s-FRQ, l-FRQ is less stable at three temperatures, and undergoes hypophosphorylation and faster degradation. The phosphorylation of the C-terminal l-FRQ 794-aa fragment was markedly higher than that of s-FRQ, suggesting the l-FRQ N-terminal 99-aa region may regulate phosphorylation of the entire FRQ protein. Quantitative label-free LC/MS analysis identified several peptides that were differentially phosphorylated between l-FRQ and s-FRQ, which were distributed in FRQ in an interlaced fashion. Furthermore, we identified two novel phosphorylation sites, S765 and T781; mutations S765A and T781A showed no significant effects on conidiation rhythmicity, although T781 conferred FRQ stability. These findings demonstrate that FRQ isoforms play differential roles in the circadian negative feedback loop and undergo different regulation of phosphorylation, structure, and stability. The l-FRQ N-terminal 99-aa region plays an important role in regulating the phosphorylation, stability, conformation, and function of the FRQ protein. As the FRQ circadian clock counterparts in other species also have isoforms or paralogues, these findings will also further our understanding of the underlying regulatory mechanisms of the circadian clock in other organisms based on the high conservation of circadian clocks in eukaryotes.
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Affiliation(s)
- Xianyun Chen
- State Key Laboratory of Biocontrol, Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China
| | - Xiaolan Liu
- State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
| | - Xihui Gan
- State Key Laboratory of Biocontrol, Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China
| | - Silin Li
- State Key Laboratory of Biocontrol, Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China
| | - Huan Ma
- State Key Laboratory of Biocontrol, Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China
| | - Lin Zhang
- State Key Laboratory of Biocontrol, Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China
| | - Peiliang Wang
- State Key Laboratory of Biocontrol, Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China
| | - Yunzhen Li
- State Key Laboratory of Biocontrol, Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China
| | - Tianyu Huang
- State Key Laboratory of Biocontrol, Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China
| | - Xiaolin Yang
- State Key Laboratory of Biocontrol, Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China
| | - Ling Fang
- Sun Yat-sen University Instrumental Analysis & Research Center, Sun Yat-sen University, Guangzhou 510006, China
| | - Yingying Liang
- State Key Laboratory of Biocontrol, Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China
| | - Jingjing Wu
- State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
| | - Tongyue Chen
- State Key Laboratory of Biocontrol, Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China
| | - Zengxuan Zhou
- State Key Laboratory of Biocontrol, Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China
| | - Xiao Liu
- State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
| | - Jinhu Guo
- State Key Laboratory of Biocontrol, Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China.
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Bonito CA, Ferreira RJ, Ferreira MJU, Gillet JP, Cordeiro MNDS, Dos Santos DJVA. Long-range communication between transmembrane- and nucleotide-binding domains does not depend on drug binding to mutant P-glycoprotein. J Biomol Struct Dyn 2023; 41:14428-14437. [PMID: 36858814 DOI: 10.1080/07391102.2023.2181633] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2022] [Accepted: 02/12/2023] [Indexed: 03/03/2023]
Abstract
In this study, the impact of four P-gp mutations (G185V, G830V, F978A and ΔF335) on drug-binding and efflux-related signal-transmission mechanism was comprehensively evaluated in the presence of ligands within the drug-binding pocket (DBP), experimentally related with changes in their drug efflux profiles. The severe repacking of the transmembrane helices (TMH), induced by mutations and exacerbated by the presence of ligands, indicates that P-gp is sensitive to perturbations in the transmembrane region. Alterations on drug-binding were also observed as a consequence of the TMH repacking, but were not always correlated with alterations on ligands binding mode and/or binding affinity. Finally, and although all P-gp variants holo systems showed considerable changes in the intracellular coupling helices/nucleotide-binding domain (ICH-NBD) interactions, they seem to be primarily induced by the mutation itself rather than by the presence of ligands within the DBP. The data further suggest that the changes in drug efflux experimentally reported are mostly related with changes on drug specificity rather than effects on signal-transmission mechanism. We also hypothesize that an increase in the drug-binding affinity may also be related with the decreased drug efflux, while minor changes in binding affinities are possibly related with the increased drug efflux observed in transfected cells.Communicated by Ramaswamy H. Sarma.
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Affiliation(s)
- Cátia A Bonito
- LAQV@REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Rua do Campo Alegre, Porto, Portugal
| | - Ricardo J Ferreira
- Red Glead Discovery AB, Medicon Village, Lund, Sweden
- Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisboa, Portugal
| | - Maria-José U Ferreira
- Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisboa, Portugal
| | - Jean-Pierre Gillet
- Laboratory of Molecular Cancer Biology, URPhyM, NARILIS, Faculty of Medicine, University of Namur, Namur, Belgium
| | - M Natália D S Cordeiro
- LAQV@REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Rua do Campo Alegre, Porto, Portugal
| | - Daniel J V A Dos Santos
- LAQV@REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Rua do Campo Alegre, Porto, Portugal
- Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisboa, Portugal
- CBIOS-Research Center for Biosciences & Health Technologies, Universidade Lusófona de Humanidades e Tecnologias, Lisbon, Portugal
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Mollazadeh S, Hadizadeh F, Ferreira RJ. Theoretical studies on 1,4-dihydropyridine derivatives as P-glycoprotein allosteric inhibitors: insights on symmetry and stereochemistry. J Biomol Struct Dyn 2020; 39:4752-4763. [DOI: 10.1080/07391102.2020.1780942] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Affiliation(s)
- Shirin Mollazadeh
- Department of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
- Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Farzin Hadizadeh
- Department of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
- Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Ricardo J. Ferreira
- Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisboa, Portugal
- Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden
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Bonito CA, Ferreira RJ, Ferreira MJU, Gillet JP, Cordeiro MNDS, Dos Santos DJVA. Theoretical insights on helix repacking as the origin of P-glycoprotein promiscuity. Sci Rep 2020; 10:9823. [PMID: 32555203 PMCID: PMC7300024 DOI: 10.1038/s41598-020-66587-5] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2020] [Accepted: 04/27/2020] [Indexed: 12/14/2022] Open
Abstract
P-glycoprotein (P-gp, ABCB1) overexpression is, currently, one of the most important multidrug resistance (MDR) mechanisms in tumor cells. Thus, modulating drug efflux by P-gp has become one of the most promising approaches to overcome MDR in cancer. Yet, more insights on the molecular basis of drug specificity and efflux-related signal transmission mechanism between the transmembrane domains (TMDs) and the nucleotide binding domains (NBDs) are needed to develop molecules with higher selectivity and efficacy. Starting from a murine P-gp crystallographic structure at the inward-facing conformation (PDB ID: 4Q9H), we evaluated the structural quality of the herein generated human P-gp homology model. This initial human P-gp model, in the presence of the “linker” and inserted in a suitable lipid bilayer, was refined through molecular dynamics simulations and thoroughly validated. The best human P-gp model was further used to study the effect of four single-point mutations located at the TMDs, experimentally related with changes in substrate specificity and drug-stimulated ATPase activity. Remarkably, each P-gp mutation is able to induce transmembrane α-helices (TMHs) repacking, affecting the drug-binding pocket volume and the drug-binding sites properties (e.g. volume, shape and polarity) finally compromising drug binding at the substrate binding sites. Furthermore, intracellular coupling helices (ICH) also play an important role since changes in the TMHs rearrangement are shown to have an impact in residue interactions at the ICH-NBD interfaces, suggesting that identified TMHs repacking affect TMD-NBD contacts and interfere with signal transmission from the TMDs to the NBDs.
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Affiliation(s)
- Cátia A Bonito
- LAQV@REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Rua do Campo Alegre, 4169-007, Porto, Portugal
| | - Ricardo J Ferreira
- Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, 75124, Uppsala, Sweden
| | - Maria-José U Ferreira
- Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisboa, Portugal
| | - Jean-Pierre Gillet
- Laboratory of Molecular Cancer Biology, Molecular Physiology Research Unit-URPhyM, Namur Research Institute for Life Sciences (NARILIS), Faculty of Medicine, University of Namur, B-5000, Namur, Belgium
| | - M Natália D S Cordeiro
- LAQV@REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Rua do Campo Alegre, 4169-007, Porto, Portugal
| | - Daniel J V A Dos Santos
- LAQV@REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Rua do Campo Alegre, 4169-007, Porto, Portugal. .,Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisboa, Portugal.
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Ferreira RJ, Bonito CA, Ferreira MJU, dos Santos DJ. About P-glycoprotein: a new drugable domain is emerging from structural data. WILEY INTERDISCIPLINARY REVIEWS-COMPUTATIONAL MOLECULAR SCIENCE 2017. [DOI: 10.1002/wcms.1316] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Affiliation(s)
- Ricardo J. Ferreira
- Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy; Universidade de Lisboa; Lisboa Portugal
| | - Cátia A. Bonito
- Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy; Universidade de Lisboa; Lisboa Portugal
- LAQV@REQUIMTE/Department of Chemistry and Biochemistry, Faculty of Sciences; University of Porto; Porto Portugal
| | - Maria José U. Ferreira
- Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy; Universidade de Lisboa; Lisboa Portugal
| | - Daniel J.V.A. dos Santos
- Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy; Universidade de Lisboa; Lisboa Portugal
- LAQV@REQUIMTE/Department of Chemistry and Biochemistry, Faculty of Sciences; University of Porto; Porto Portugal
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Ferreira RJ, Ferreira MJU, dos Santos DJVA. Do adsorbed drugs onto P-glycoprotein influence its efflux capability? Phys Chem Chem Phys 2015; 17:22023-34. [PMID: 26235302 DOI: 10.1039/c5cp03216d] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
The membrane biophysical aspects by which multidrug resistance (MDR) relate to the ABC transporter function still remain largely unknown. Notwithstanding the central role that efflux pumps like P-glycoprotein have in MDR onset, experimental studies classified additionally the lipid micro-environment where P-gp is inserted as a determinant for the increased efflux capability demonstrated in MDR cell lines. Recently, a nonlinear model for drug-membrane interactions showed that, upon drug adsorption, long-range mechanical alterations are predicted to affect the P-gp ATPase function at external drug concentrations of ∼10-100 μM. However, our results also show that drug adsorption may also occur at P-gp nucleotide-binding domains where conformational changes drive the efflux cycle. Thus, we assessed the effect of drug adsorption to both protein-water and lipid-water interfaces by means of molecular dynamics simulations. The results show that free energies of adsorption are lower for modulators in both lipid/water and protein/water interfaces. Important differences in drug-protein interactions, protein dynamics and membrane biophysical characteristics were observed between the different classes. Therefore, we hypothesize that drug adsorption to the protein and lipid-water interface accounts for a complex network of events that affect the ability of transporters to efflux drugs.
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Affiliation(s)
- Ricardo J Ferreira
- Research Institute for Medicines (iMed.Ulisboa), Faculdade de Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, Lisboa, Portugal.
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Swartz D, Mok L, Botta S, Singh A, Altenberg G, Urbatsch I. Directed evolution of P-glycoprotein cysteines reveals site-specific, non-conservative substitutions that preserve multidrug resistance. Biosci Rep 2014; 34:e00116. [PMID: 24825346 PMCID: PMC4069687 DOI: 10.1042/bsr20140062] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2014] [Revised: 05/05/2014] [Accepted: 05/06/2014] [Indexed: 11/21/2022] Open
Abstract
Pgp (P-glycoprotein) is a prototype ABC (ATP-binding-cassette) transporter involved in multidrug resistance of cancer. We used directed evolution to replace six cytoplasmic Cys (cysteine) residues in Pgp with all 20 standard amino acids and selected for active mutants. From a pool of 75000 transformants for each block of three Cys, we identified multiple mutants that preserved drug resistance and yeast mating activity. The most frequent substitutions were glycine and serine for Cys427 (24 and 20%, respectively) and Cys1070 (37 and 25%) of the Walker A motifs in the NBDs (nucleotide-binding domains), Cys1223 in NBD2 (25 and 8%) and Cys638 in the linker region (24 and 16%), whereas close-by Cys669 tolerated glycine (16%) and alanine (14%), but not serine (absent). Cys1121 in NBD2 showed a clear preference for positively charged arginine (38%) suggesting a salt bridge with Glu269 in the ICL2 (intracellular loop 2) may stabilize domain interactions. In contrast, three Cys residues in transmembrane α-helices could be successfully replaced by alanine. The resulting CL (Cys-less) Pgp was fully active in yeast cells, and purified proteins displayed drug-stimulated ATPase activities indistinguishable from WT (wild-type) Pgp. Overall, directed evolution identified site-specific, non-conservative Cys substitutions that allowed building of a robust CL Pgp, an invaluable new tool for future functional and structural studies, and that may guide the construction of other CL proteins where alanine and serine have proven unsuccessful.
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Key Words
- abc transporter
- multidrug transporter
- non-conservative cysteine substitutions
- protein evolution site-saturation mutagenesis
- yeast drug resistance
- abc, atp-binding-cassette
- cftr, cystic fibrosis transmembrane conductance regulator
- cl, cys-less
- cp-mts, 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin
- ddm, n-dodecyl-β-d-maltopyranoside
- icl, intracellular loop
- nbd, nucleotide-binding domain
- pgp, p-glycoprotein
- sec, size exclusion chromatography
- tmd, transmembrane domain
- wt, wild-type
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Affiliation(s)
- Douglas J. Swartz
- *Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas, U.S.A
- †Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas, U.S.A
| | - Leo Mok
- *Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas, U.S.A
- †Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas, U.S.A
| | - Sri K. Botta
- *Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas, U.S.A
- †Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas, U.S.A
| | - Anukriti Singh
- *Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas, U.S.A
- †Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas, U.S.A
| | - Guillermo A. Altenberg
- †Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas, U.S.A
- ‡Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, Texas, U.S.A
| | - Ina L. Urbatsch
- *Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas, U.S.A
- †Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas, U.S.A
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Jirsa M, Bronský J, Dvořáková L, Šperl J, Šmajstrla V, Horák J, Nevoral J, Hřebíček M. ABCB4 mutations underlie hormonal cholestasis but not pediatric idiopathic gallstones. World J Gastroenterol 2014; 20:5867-5874. [PMID: 24914347 PMCID: PMC4024796 DOI: 10.3748/wjg.v20.i19.5867] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/23/2013] [Revised: 07/25/2013] [Accepted: 08/17/2013] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the contribution of ABCB4 mutations to pediatric idiopathic gallstone disease and the potential of hormonal contraceptives to prompt clinical manifestations of multidrug resistance protein 3 deficiency.
METHODS: Mutational analysis of ABCB4, screening for copy number variations by multiplex ligation-dependent probe amplification, genotyping for low expression allele c.1331T>C of ABCB11 and genotyping for variation c.55G>C in ABCG8 previously associated with cholesterol gallstones in adults was performed in 35 pediatric subjects with idiopathic gallstones who fulfilled the clinical criteria for low phospholipid-associated cholelithiasis syndrome (LPAC, OMIM #600803) and in 5 young females with suspected LPAC and their families (5 probands, 15 additional family members). The probands came to medical attention for contraceptive-associated intrahepatic cholestasis.
RESULTS: A possibly pathogenic variant of ABCB4 was found only in one of the 35 pediatric subjects with idiopathic cholesterol gallstones whereas 15 members of the studied 5 LPAC kindreds were confirmed and another one was highly suspected to carry predictably pathogenic mutations in ABCB4. Among these 16, however, none developed gallstones in childhood. In 5 index patients, all young females carrying at least one pathogenic mutation in one allele of ABCB4, manifestation of LPAC as intrahepatic cholestasis with elevated serum activity of gamma-glutamyltransferase was induced by hormonal contraceptives. Variants ABCB11 c.1331T>C and ABCG8 c.55G>C were not significantly overrepresented in the 35 examined patients with suspect LPAC.
CONCLUSION: Clinical criteria for LPAC syndrome caused by mutations in ABCB4 cannot be applied to pediatric patients with idiopathic gallstones. Sexual immaturity even prevents manifestation of LPAC.
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Mutations adjacent to the end of transmembrane helices 6 and 7 independently affect drug efflux capacity of yeast ABC transporter Pdr5p. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2014; 1838:932-9. [DOI: 10.1016/j.bbamem.2013.12.002] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/01/2013] [Revised: 11/19/2013] [Accepted: 12/04/2013] [Indexed: 12/22/2022]
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Dynamics of a bacterial multidrug ABC transporter in the inward- and outward-facing conformations. Proc Natl Acad Sci U S A 2012; 109:10832-6. [PMID: 22711831 DOI: 10.1073/pnas.1204067109] [Citation(s) in RCA: 88] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
The study of membrane proteins remains a challenging task, and approaches to unravel their dynamics are scarce. Here, we applied hydrogen/deuterium exchange (HDX) coupled to mass spectrometry to probe the motions of a bacterial multidrug ATP-binding cassette (ABC) transporter, BmrA, in the inward-facing (resting state) and outward-facing (ATP-bound) conformations. Trypsin digestion and global or local HDX support the transition between inward- and outward-facing conformations during the catalytic cycle of BmrA. However, in the resting state, peptides from the two intracellular domains, especially ICD2, show a much faster HDX than in the closed state. This shows that these two subdomains are very flexible in this conformation. Additionally, molecular dynamics simulations suggest a large fluctuation of the Cα positions from ICD2 residues in the inward-facing conformation of a related transporter, MsbA. These results highlight the unexpected flexibility of ABC exporters in the resting state and underline the power of HDX coupled to mass spectrometry to explore conformational changes and dynamics of large membrane proteins.
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Iram SH, Cole SPC. Expression and function of human MRP1 (ABCC1) is dependent on amino acids in cytoplasmic loop 5 and its interface with nucleotide binding domain 2. J Biol Chem 2010; 286:7202-13. [PMID: 21177244 DOI: 10.1074/jbc.m110.166959] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette transporter that effluxes drugs and organic anions across the plasma membrane. The 17 transmembrane helices of MRP1 are linked by extracellular and cytoplasmic loops (CLs), but their role in coupling the ATPase activity of MRP1 to the translocation of its substrates is poorly understood. Here we have examined the importance of CL5 by mutating eight conserved charged residues and the helix-disrupting Gly(511) in this region. Ala substitution of Lys(513), Lys(516), Glu(521), and Glu(535) markedly reduced MRP1 levels. Because three of these residues are predicted to lie at the interface of CL5 and the second nucleotide binding domain (NBD2), a critical role is indicated for this region in the plasma membrane expression of MRP1. Further support for this idea was obtained by mutating NBD2 amino acids His(1364) and Arg(1367) at the CL5 interface, which also resulted in reduced MRP1 levels. In contrast, mutation of Arg(501), Lys(503), Glu(507), Arg(532), and Gly(511) had no effect on MRP1 levels. Except for K503A, however, transport by these mutants was reduced by 50 to 75%, an effect largely attributable to reduced substrate binding and affinity. Studies with (32)P-labeled azido-ATP also indicated that whereas ATP binding by the G511I mutant was unchanged, vanadate-induced trapping of azido-ADP was reduced, indicating changes in the catalytic activity of MRP1. Together, these data demonstrate the multiple roles for CL5 in the membrane expression and function of MRP1.
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Affiliation(s)
- Surtaj H Iram
- Division of Cancer Biology and Genetics, Queen's University Cancer Research Institute, Kingston, Ontario K7L 3N6, Canada
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Do Cao MA, Crouzy S, Kim M, Becchi M, Cafiso DS, Di Pietro A, Jault JM. Probing the conformation of the resting state of a bacterial multidrug ABC transporter, BmrA, by a site-directed spin labeling approach. Protein Sci 2009; 18:1507-20. [PMID: 19479721 DOI: 10.1002/pro.141] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Previously published 3-D structures of a prototypic ATP-binding cassette (ABC) transporter, MsbA, have been recently corrected revealing large rigid-body motions possibly linked to its catalytic cycle. Here, a closely related multidrug bacterial ABC transporter, BmrA, was studied using site-directed spin labeling by focusing on a region connecting the transmembrane domain and the nucleotide-binding domain (NBD). Electron paramagnetic resonance (EPR) spectra of single spin-labeled cysteine mutants suggests that, in the resting state, this sub-domain essentially adopts a partially extended conformation, which is consistent with the crystal structures of MsbA and Sav1866. Interestingly, one of the single point mutants (Q333C) yielded an immobilized EPR spectrum that could arise from a direct interaction with a vicinal tyrosine residue. Inspection of different BmrA models pointed to Y408, within the NBD, as the putative interacting partner, and its mutation to a Phe residue indeed dramatically modified the EPR spectra of the spin labeled Q333C. Moreover, unlike the Y408F mutation, the Y408A mutation abolished both ATPase activity and drug transport of BmrA, suggesting that a nonpolar bulky residue is required at this position. The spatial proximity of Q333 and Y408 was also confirmed by formation of a disulfide bond when both Q333 and T407 (or S409) were replaced jointly by a cysteine residue. Overall, these results indicate that the two regions surrounding Q333 and Y408 are close together in the 3-D structure of BmrA and that residues within these two sub-domains are essential for proper functioning of this transporter.
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Affiliation(s)
- Marie-Ange Do Cao
- Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS-Université Lyon I and IFR 128, 7 passage du Vercors, 69367 Lyon Cedex 07, France
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Globisch C, Pajeva IK, Wiese M. Identification of putative binding sites of P-glycoprotein based on its homology model. ChemMedChem 2008; 3:280-95. [PMID: 18175303 DOI: 10.1002/cmdc.200700249] [Citation(s) in RCA: 68] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
A homology model of P-glycoprotein based on the crystal structure of the multidrug transporter Sav1866 is developed, incorporated into a membrane environment, and optimized. The resulting model is analyzed in relation to the functional state and potential binding sites. The comparison of modeled distances to distances reported in experimental studies between particular residues suggests that the model corresponds most closely to the first ATP hydrolysis step of the protein transport cycle. Comparison to the protein 3D structure confirms this suggestion. Using SiteID and Site Finder programs three membrane related binding regions are identified: a region at the interface between the membrane and cytosol and two regions located in the transmembrane domains. The regions contain binding pockets of different size, orientation, and amino acids. A binding pocket located inside the membrane cavity is also identified. The pockets are analyzed in relation to amino acids shown experimentally to influence the protein function. The results suggest that the protein has multiple binding sites and may bind and/or release substrates in multiple pathways.
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15
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Storm J, Modok S, O’Mara ML, Tieleman DP, Kerr ID, Callaghan R. Cytosolic Region of TM6 in P-Glycoprotein: Topographical Analysis and Functional Perturbation by Site Directed Labeling. Biochemistry 2008; 47:3615-24. [DOI: 10.1021/bi7023089] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Affiliation(s)
- Janet Storm
- Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, United Kingdom, Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada, and Centre for Biochemistry and Cell Biology, School of Biomedical Sciences, University of Nottingham, United Kingdom
| | - Szabolcs Modok
- Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, United Kingdom, Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada, and Centre for Biochemistry and Cell Biology, School of Biomedical Sciences, University of Nottingham, United Kingdom
| | - Megan L. O’Mara
- Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, United Kingdom, Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada, and Centre for Biochemistry and Cell Biology, School of Biomedical Sciences, University of Nottingham, United Kingdom
| | - D. Peter Tieleman
- Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, United Kingdom, Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada, and Centre for Biochemistry and Cell Biology, School of Biomedical Sciences, University of Nottingham, United Kingdom
| | - Ian D. Kerr
- Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, United Kingdom, Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada, and Centre for Biochemistry and Cell Biology, School of Biomedical Sciences, University of Nottingham, United Kingdom
| | - Richard Callaghan
- Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, United Kingdom, Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada, and Centre for Biochemistry and Cell Biology, School of Biomedical Sciences, University of Nottingham, United Kingdom
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16
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Rosmorduc O, Poupon R. Low phospholipid associated cholelithiasis: association with mutation in the MDR3/ABCB4 gene. Orphanet J Rare Dis 2007; 2:29. [PMID: 17562004 PMCID: PMC1910597 DOI: 10.1186/1750-1172-2-29] [Citation(s) in RCA: 92] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2007] [Accepted: 06/11/2007] [Indexed: 12/14/2022] Open
Abstract
Low phospholipid-associated cholelithiasis (LPAC) is characterized by the association of ABCB4 mutations and low biliary phospholipid concentration with symptomatic and recurring cholelithiasis. This syndrome is infrequent and corresponds to a peculiar small subgroup of patients with symptomatic gallstone disease. The patients with the LPAC syndrome present typically with the following main features: age less than 40 years at onset of symptoms, recurrence of biliary symptoms after cholecystectomy, intrahepatic hyperechoic foci or sludge or microlithiasis along the biliary tree. Defect in ABCB4 function causes the production of bile with low phospholipid content, increased lithogenicity and high detergent properties leading to bile duct luminal membrane injuries and resulting in cholestasis with increased serum gamma-glutamyltransferase (GGT) activity. Intrahepatic gallstones may be evidenced by ultrasonography (US), computing tomography (CT) abdominal scan or magnetic resonance cholangiopancreatography, intrahepatic hyperechogenic foci along the biliary tree may be evidenced by US, and hepatic bile composition (phospholipids) may be determined by duodenoscopy. In all cases where the ABCB4 genotyping confirms the diagnosis of LPAC syndrome in young adults, long-term curative or prophylactic therapy with ursodeoxycholic acid (UDCA) should be initiated early to prevent the occurrence or recurrence of the syndrome and its complications. Cholecystectomy is indicated in the case of symptomatic gallstones. Biliary drainage or partial hepatectomy may be indicated in the case of symptomatic intrahepatic bile duct dilatations filled with gallstones. Patients with end-stage liver disease may be candidates for liver transplantation.
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Affiliation(s)
- Olivier Rosmorduc
- Service d'Hépatologie, INSERM U 680, Centre de Référence de Maladies Rares et des Maladies Inflammatoires des Voies Biliaires; Hôpital Saint-Antoine, Assistance Publique-Hôpitaux de Paris; Faculté de Médecine Pierre et Marie Curie et Université Paris 6; Paris, France
| | - Raoul Poupon
- Service d'Hépatologie, INSERM U 680, Centre de Référence de Maladies Rares et des Maladies Inflammatoires des Voies Biliaires; Hôpital Saint-Antoine, Assistance Publique-Hôpitaux de Paris; Faculté de Médecine Pierre et Marie Curie et Université Paris 6; Paris, France
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17
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Shilling RA, Venter H, Velamakanni S, Bapna A, Woebking B, Shahi S, van Veen HW. New light on multidrug binding by an ATP-binding-cassette transporter. Trends Pharmacol Sci 2006; 27:195-203. [PMID: 16545467 DOI: 10.1016/j.tips.2006.02.008] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2005] [Revised: 11/23/2005] [Accepted: 02/22/2006] [Indexed: 11/26/2022]
Abstract
ATP-binding-cassette (ABC) multidrug transporters confer multidrug resistance to pathogenic microorganisms and human tumour cells by mediating the extrusion of structurally unrelated chemotherapeutic drugs from the cell. The molecular basis by which ABC multidrug transporters bind and transport drugs is far from clear. Genetic analyses during the past 14 years reveal that the replacement of many individual amino acids in mammalian multidrug resistance P-glycoproteins can affect cellular resistance to drugs, but these studies have failed to identify specific regions in the primary amino acid sequence that are part of a defined drug-binding pocket. The recent publication of an X-ray crystallographic structure of the bacterial P-glycoprotein homologue MsbA and an MsbA-based homology model of human P-glycoprotein creates an opportunity to compare the original mutagenesis data with the three-dimensional structures of transporters. Our comparisons reveal that mutations that alter specificity are present in three-dimensional 'hotspot' regions in the membrane domains of P-glycoprotein.
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MESH Headings
- ATP Binding Cassette Transporter, Subfamily B, Member 1/chemistry
- ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics
- ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism
- ATP-Binding Cassette Transporters/chemistry
- ATP-Binding Cassette Transporters/genetics
- ATP-Binding Cassette Transporters/metabolism
- Amino Acid Sequence
- Animals
- Antineoplastic Agents/metabolism
- Binding Sites/genetics
- Biological Transport
- Drug Resistance, Multiple
- Drug Resistance, Neoplasm
- Humans
- Models, Molecular
- Molecular Sequence Data
- Mutation
- Protein Structure, Tertiary
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Affiliation(s)
- Richard A Shilling
- Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, UK, CB2 1PD
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18
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Dalmas O, Orelle C, Foucher AE, Geourjon C, Crouzy S, Di Pietro A, Jault JM. The Q-loop Disengages from the First Intracellular Loop during the Catalytic Cycle of the Multidrug ABC Transporter BmrA. J Biol Chem 2005; 280:36857-64. [PMID: 16107340 DOI: 10.1074/jbc.m503266200] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The ATP-binding cassette is the most abundant family of transporters including many medically relevant members and gathers both importers and exporters involved in the transport of a wide variety of substrates. Although three high resolution three-dimensional structures have been obtained for a prototypic exporter, MsbA, two have been subjected to much criticism. Here, conformational changes of BmrA, a multidrug bacterial transporter structurally related to MsbA, have been studied. A three-dimensional model of BmrA, based on the "open" conformation of Escherichia coli MsbA, was probed by simultaneously introducing two cysteine residues, one in the first intracellular loop of the transmembrane domain and the other in the Q-loop of the nucleotide-binding domain (NBD). Intramolecular disulfide bonds could be created in the absence of any effectors, which prevented both drug transport and ATPase activity. Interestingly, addition of ATP/Mg plus vanadate strongly prevented this bond formation in a cysteine double mutant, whereas ATP/Mg alone was sufficient when the ATPase-inactive E504Q mutation was also introduced, in agreement with additional BmrA models where the ATP-binding sites are positioned at the NBD/NBD interface. Furthermore, cross-linking between the two cysteine residues could still be achieved in the presence of ATP/Mg plus vanadate when homobifunctional cross-linkers separated by more than 13 Angstrom were added. Altogether, these results give support to the existence, in the resting state, of a monomeric conformation of BmrA similar to that found within the open MsbA dimer and show that a large motion is required between intracellular loop 1 and the nucleotide-binding domain for the proper functioning of a multidrug ATP-binding cassette transporter.
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Affiliation(s)
- Olivier Dalmas
- Institut de Biologie et Chimie des Protéines, Unité Mixte de Recherche 5086 CNRS-UCBL1 and IFR 128, 7 Passage du Vercors, 69367 Lyon Cedex 07, France
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19
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Vethanayagam RR, Wang H, Gupta A, Zhang Y, Lewis F, Unadkat JD, Mao Q. Functional analysis of the human variants of breast cancer resistance protein: I206L, N590Y, and D620N. Drug Metab Dispos 2005; 33:697-705. [PMID: 15743976 DOI: 10.1124/dmd.105.003657] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Previous studies have shown that the V12M and Q141K variants of breast cancer resistance protein (BCRP) can affect expression and function of the transporter. In this study, the effects of the I206L, N590Y, and D620N variants on protein expression, plasma membrane localization, and transport activity of BCRP were investigated. Wild-type BCRP and the three variants were stably expressed in human embryonic kidney (HEK) cells. Confocal microscopy analysis showed that the three variants were predominantly routed to the plasma membrane of HEK cells. The expression level of I206L in the plasma membrane was approximately 45% of that of wild-type protein, whereas the N590Y and D620N levels were increased approximately 3.6-fold and 2.4-fold, respectively, as determined by immunoblotting. All three variants transported mitoxantrone, pheophorbide a, and BODIPY FL-prazosin. After normalization for differences in BCRP expression, I206L, N590Y, and D620N exhibited approximately 2-fold, 0.3-fold, and 0.5-fold wild-type efflux activities, respectively. The variants also conferred resistance to mitoxantrone and topotecan. Mitoxantrone and topotecan resistance by I206L and N590Y was approximately 2-fold and 0.3-fold of the wild-type BCRP resistance levels, respectively. Although D620N conferred a topotecan resistance similar to that of the wild-type protein, its level of mitoxantrone resistance was decreased by 50%. After normalization to BCRP expression levels, ATPase activities of I206L were not significantly different from those of wild-type protein, whereas N590Y and D620N exhibited approximately 30% and 50% of wild-type ATPase activities, respectively. These results suggest that I206L has the lowest protein expression and the highest activity, whereas N590Y and D620N display higher expression and lower activity, relative to wild-type BCRP.
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Affiliation(s)
- R Robert Vethanayagam
- Department of Pharmaceutics, School of Pharmacy, University of Washington, Box 357610, Seattle, WA 98195-7610, USA
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20
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Seigneuret M, Garnier-Suillerot A. A structural model for the open conformation of the mdr1 P-glycoprotein based on the MsbA crystal structure. J Biol Chem 2003; 278:30115-24. [PMID: 12777401 DOI: 10.1074/jbc.m302443200] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The validity of the structure of the Escherichia coli MsbA lipid transporter as a model from the mdr1 P-glycoprotein has been evaluated. Comparative sequence analyses, motif search and secondary structure prediction indicated that each of the two P-glycoprotein halves is structurally similar to the MsbA monomer and also suggested that the open dimer structure is valid for P-glycoprotein. Homology modeling was used to predict the structure of P-glycoprotein using MsbA as a template. The resulting modeled structure allowed a detailed study of the interactions between the intracellular domain and the nucleotide binding domain and suggested that these contacts are involved in mediating the coupling between nucleotide binding domain conformational changes and transmembrane helices reorientation during transport. In P-glycoprotein, the internal chamber open to the inner leaflet and the inner medium is significantly different in size and charge than in MsbA. These differences can be related to those of the transported substrates. Moreover an ensemble of 20 conserved aromatic residues appears to border the periphery of each side of the chamber in P-glycoprotein. These may be important for size selection and proper positioning of drugs for transport. The relevance of the modeled conformation to P-gp function is discussed.
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Affiliation(s)
- Michel Seigneuret
- Laboratoire de Physochimie Biomoléculaire et Cellulaire, UMR-CNRS 7033, Universitĕ Paris 6, France.
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21
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Rosmorduc O, Hermelin B, Boelle PY, Parc R, Taboury J, Poupon R. ABCB4 gene mutation-associated cholelithiasis in adults. Gastroenterology 2003; 125:452-9. [PMID: 12891548 DOI: 10.1016/s0016-5085(03)00898-9] [Citation(s) in RCA: 178] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
BACKGROUND & AIMS We recently put forward arguments in favor of ABCB4 gene (adenosine triphosphate-binding cassette, subfamily B, member 4) defects as a risk factor for symptomatic cholelithiasis in adults. In this study, we characterized ABCB4 gene mutations in a series of patients with symptomatic cholelithiasis to determine the genetic basis and the clinical phenotype of ABCB4 gene mutation-associated cholelithiasis. METHODS We analyzed the entire ABCB4 gene coding sequences in a first group of 32 patients who had a clinical history compatible with the syndrome previously described, in a second group of 28 patients who presented with a classic gallstone disease that justified a cholecystectomy, and in a third group of 33 patients without a history of cholelithiasis. RESULTS We identified both heterozygous and homozygous ABCB4 gene point mutations in 18 of 32 (56%) patients who presented with clinical criteria of the syndrome, whereas no mutation was detected in the 2 other groups of patients (P < 0.001). Three independent clinical features were strongly associated with point mutations: recurrence of symptoms after cholecystectomy (odds ratio, 8.5); intrahepatic hyperechoic foci, intrahepatic sludge, or microlithiasis (odds ratio, 6.1); and age <40 years at the onset of symptoms (odds ratio, 3.0). ABCB4 gene point mutations were detected exclusively in the patients who showed 2 or 3 of these clinical features. CONCLUSIONS Our results show that ABCB4 gene mutations represent a major genetic risk factor in a symptomatic and recurring form of cholelithiasis in young adults.
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22
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Lam-Yuk-Tseung S, Govoni G, Forbes J, Gros P. Iron transport by Nramp2/DMT1: pH regulation of transport by 2 histidines in transmembrane domain 6. Blood 2003; 101:3699-707. [PMID: 12522007 DOI: 10.1182/blood-2002-07-2108] [Citation(s) in RCA: 93] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Mutations at natural resistance-associated macrophage protein 1 (Nramp1) impair phagocyte function and cause susceptibility to infections while mutations at Nramp2 (divalent metal transporter 1 [DMT1]) affect iron homeostasis and cause severe microcytic anemia. Structure-function relationships in the Nramp superfamily were studied by mutagenesis, followed by functional characterization in yeast and in mammalian cells. These studies identify 3 negatively charged and highly conserved residues in transmembrane domains (TM) 1, 4, and 7 as essential for cation transport by Nramp2/DMT1. The introduction of a charged residue (Gly185Arg) in TM4 found in the naturally occurring microcytic anemia mk (mouse) and Belgrade (rat) mutants is shown to cause a partial or complete loss of function in mammalian and yeast cells, respectively. A pair of mutation-sensitive and highly conserved histidines (His267, His272) was identified in TM6. Surprisingly, inactive His267 and His272 mutants could be rescued by lowering the pH of the transport assay. This indicates that His267/His272 are not directly involved in metal binding but, rather, play an important role in pH regulation of metal transport by Nramp proteins.
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Affiliation(s)
- Steven Lam-Yuk-Tseung
- Department of Biochemistry, McGill Cancer Center and Center for Host Resistance, McGill University, Montreal, QC, Canada
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23
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Cai J, Gros P. Overexpression, purification, and functional characterization of ATP-binding cassette transporters in the yeast, Pichia pastoris. BIOCHIMICA ET BIOPHYSICA ACTA 2003; 1610:63-76. [PMID: 12586381 DOI: 10.1016/s0005-2736(02)00718-6] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
The ATP-binding cassette (ABC) transporter superfamily is a large gene family that has been highly conserved throughout evolution. The physiological importance of these membrane transporters is highlighted by the large variety of substrates they transport, and by the observation that mutations in many of them cause heritable diseases in human. Likewise, overexpression of certain ABC transporters, such as P-glycoprotein and members of the multidrug resistance associated protein (MRP) family, is associated with multidrug resistance in various cells and organisms. Understanding the structure and molecular mechanisms of transport of the ABC transporters in normal tissues and their possibly altered function in human diseases requires large amounts of purified and active proteins. For this, efficient expression systems are needed. The methylotrophic yeast Pichia pastoris has proven to be an efficient and inexpensive experimental model for high-level expression of many proteins, including ABC transporters. In the present review, we will summarize recent advances on the use of this system for the expression, purification, and functional characterization of P-glycoprotein and two members of the MRP subfamily.
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Affiliation(s)
- Jie Cai
- Department of Biochemistry and McGill Cancer Center, McGill University, Montreal, Quebec, Canada
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24
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Falcón-Pérez JM, Martínez-Burgos M, Molano J, Mazón MJ, Eraso P. Domain interactions in the yeast ATP binding cassette transporter Ycf1p: intragenic suppressor analysis of mutations in the nucleotide binding domains. J Bacteriol 2001; 183:4761-70. [PMID: 11466279 PMCID: PMC99530 DOI: 10.1128/jb.183.16.4761-4770.2001] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC) transporter required for heavy metal and drug detoxification. Cluster analysis shows that Ycf1p is strongly related to the human multidrug-associated protein (MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may serve as an excellent model for the study of eukaryotic ABC transporter structure and function. Identifying intramolecular interactions in these transporters may help to elucidate energy transfer mechanisms during transport. To identify regions in Ycf1p that may interact to couple ATPase activity to substrate binding and/or movement across the membrane, we sought intragenic suppressors of ycf1 mutations that affect highly conserved residues presumably involved in ATP binding and/or hydrolysis. Thirteen intragenic second-site suppressors were identified for the D777N mutation which affects the invariant Asp residue in the Walker B motif of the first nucleotide binding domain (NBD1). Two of the suppressor mutations (V543I and F565L) are located in the first transmembrane domain (TMD1), nine (A1003V, A1021T, A1021V, N1027D, Q1107R, G1207D, G1207S, S1212L, and W1225C) are found within TMD2, one (S674L) is in NBD1, and another one (R1415G) is in NBD2, indicating either physical proximity or functional interactions between NBD1 and the other three domains. The original D777N mutant protein exhibits a strong defect in the apparent affinity for ATP and V(max) of transport. The phenotypic characterization of the suppressor mutants shows that suppression does not result from restoring these alterations but rather from a change in substrate specificity. We discuss the possible involvement of Asp777 in coupling ATPase activity to substrate binding and/or transport across the membrane.
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Affiliation(s)
- J M Falcón-Pérez
- Instituto de Investigaciones Biomédicas "Alberto Sols," CSIC-UAM, Madrid, Spain
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25
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Song J, Melera PW. Transmembrane domain (TM) 9 represents a novel site in P-glycoprotein that affects drug resistance and cooperates with TM6 to mediate [125I]iodoarylazidoprazosin labeling. Mol Pharmacol 2001; 60:254-61. [PMID: 11455011 DOI: 10.1124/mol.60.2.254] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
The multidrug resistant cell line DC-3F/ADII was obtained by stepwise selection for growth in actinomycin D (ActD). Compared with parental cells, it displays high resistance to ActD and vincristine and low resistance to colchicine and daunorubicin. These cells overexpress a form of P-glycoprotein (Pgp1) containing a double mutation, I837L and N839I, in transmembrane domain (TM) 9; when transfected into DC-3F, this mutation confers the DC-3F/ADII phenotype. We have shown previously that another cell line, DC-3F/ADX, also displays this phenotype and overexpresses a mutant form of Pgp1 containing a double mutation in TM6 (G338A, A339P). Hence, mutations in TM9 and TM6 are independently capable of conferring the same cross-resistance phenotype. The TM6 mutations inhibit the ability of cyclosporin A to reverse cross-resistance and to block labeling of the protein by [125I]iodoarylazidoprazosin (IAAP), whereas the TM9 mutations do not show similar effects. A chimeric protein containing both pairs of mutations confers twice the level of resistance to ActD than expected from the sum of the individual mutations, but it cannot be labeled to detectable levels with [125I]IAAP. Thus, TM9 represents a novel site that cooperates with TM6 to mediate drug resistance and [125I]IAAP labeling.
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MESH Headings
- ATP Binding Cassette Transporter, Subfamily B, Member 1/chemistry
- ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics
- ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism
- Adenosine Triphosphatases/metabolism
- Animals
- Azides/metabolism
- Binding, Competitive
- Cells, Cultured
- Cricetinae
- Cyclosporine/pharmacology
- Drug Interactions
- Drug Resistance/physiology
- Drug Resistance, Multiple/physiology
- Iodine Radioisotopes
- Membrane Proteins/chemistry
- Membrane Proteins/genetics
- Membrane Proteins/metabolism
- Mutagenesis, Site-Directed
- Prazosin/analogs & derivatives
- Prazosin/metabolism
- Protein Structure, Tertiary
- Transfection
- Verapamil/pharmacology
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Affiliation(s)
- J Song
- Department of Biochemistry, University of Maryland School of Medicine, Baltimore, Maryland, USA
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26
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Gruol DJ, Bernd J, Phippard AE, Ojima I, Bernacki RJ. The use of a novel taxane-based P-glycoprotein inhibitor to identify mutations that alter the interaction of the protein with paclitaxel. Mol Pharmacol 2001; 60:104-13. [PMID: 11408605 DOI: 10.1124/mol.60.1.104] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Murine thymoma cell lines expressing mutated forms of the mdr1b P-glycoprotein were isolated using a novel taxane-based P-glycoprotein inhibitor tRA-96023 (SB-RA-31012). The selection strategy required resistance to a combination of tRA-96023 and colchicine. Five mutations were identified (N350I, I862F, L865F, L868W, and A933T) that reduce the capacity of tRA-96023 to inhibit P-glycoprotein-dependent drug resistance. These mutations also result in a loss of paclitaxel resistance ranging from 47 to 100%. Four mutations are located in the second half of the protein, within or near the proposed transmembrane segment (TMS) 10--11 regions. The fifth mutation (N350I) is within the first half of the protein, proximal (cytoplasmic) to TMS 6. The variant cell line expressing the L868W mutation was subjected to a second round of selection involving tRA-96023 and the toxic drug puromycin. This resulted in the isolation of a cell line expressing a P-glycoprotein with a double mutation. The additional mutation (N988D) is located within TMS 12 and conveys further decreases in resistance to paclitaxel and the capacity of tRA-96023 to inhibit drug resistance. Taken together, the results indicate a significant contribution by the TMS 10--12 portion of the protein to the recognition and transport of taxanes and give evidence that the cytoplasmic region proximal to TMS 6 also plays a role in taxane interactions with P-glycoproteins. Interestingly, mutations within TMS 6 and 12 were found to cause a partial loss of PSC-833 inhibitor activity, suggesting that these regions participate in the interactions with cyclosporin and its derivatives.
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Affiliation(s)
- D J Gruol
- The Sidney Kimmel Cancer Center, San Diego, California 92121, USA.
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27
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Hrycyna CA. Molecular genetic analysis and biochemical characterization of mammalian P-glycoproteins involved in multidrug resistance. Semin Cell Dev Biol 2001; 12:247-56. [PMID: 11428917 DOI: 10.1006/scdb.2000.0250] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
A variety of human cancers become resistant or are intrinsically resistant to treatment with conventional drug therapies. This phenomenon is due in large part to the overexpression of a 170 kDa plasma membrane ATP-dependent pump known as the multidrug resistance transporter or P-glycoprotein. P-glycoprotein is a member of the large ATP binding cassette (ABC) superfamily of membrane transporters. This review focuses on the use of structure-function analyses to elucidate further the mechanism of action of mammalian P-glycoproteins. Ultimately, a complete understanding of the mechanism is important for the development of novel strategies for the treatment of many human cancers.
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Affiliation(s)
- C A Hrycyna
- Department of Chemistry, Purdue University, 1393 Brown Building, West Lafayette, IN 47907-1393, USA.
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28
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Rosmorduc O, Hermelin B, Poupon R. MDR3 gene defect in adults with symptomatic intrahepatic and gallbladder cholesterol cholelithiasis. Gastroenterology 2001; 120:1459-67. [PMID: 11313316 DOI: 10.1053/gast.2001.23947] [Citation(s) in RCA: 199] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
BACKGROUND & AIMS Many studies indicate that gallstone susceptibility has genetic components. MDR3 is the phosphatidylcholine translocator across the hepatocyte canalicular membrane. Because phospholipids are a carrier and a solvent of cholesterol in hepatic bile, we hypothesized that a defect in the MDR3 gene could be the genetic basis for peculiar forms of cholesterol gallstone disease, in particular those associated with symptoms and cholestasis without evident common bile duct stone. METHODS We studied 6 adult patients with a peculiar form of cholelithiasis. MDR3 gene sequence was determined by reverse-transcription polymerase chain reaction amplification of mononuclear cell RNAs followed by direct sequencing. Hepatic bile was analyzed in 2 patients. RESULTS All patients shared the following features: at least 1 episode of biliary colic, pancreatitis, or cholangitis; biochemical evidence of chronic cholestasis; recurrence of symptoms after cholecystectomy; presence of echogenic material in the intrahepatic bile ducts; and prevention of recurrence by ursodeoxycholic acid therapy. Hepatic bile composition showed a high cholesterol/phospholipid ratio and cholesterol crystals. In all patients, we found MDR3 gene mutations involving a conserved amino acid region. CONCLUSIONS These preliminary observations suggest that MDR3 gene mutations represent a genetic factor involved in this peculiar form of cholesterol gallstone disease in adults. They require further studies to assess the prevalence of MDR3 gene defects in symptomatic and silent cholesterol gallstone disease.
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Affiliation(s)
- O Rosmorduc
- Service d'Hépato-gastroentérologie, Hôpital Saint-Antoine, Assistance publique-Hôpitaux de Paris, France
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Loo TW, Clarke DM. Identification of residues within the drug-binding domain of the human multidrug resistance P-glycoprotein by cysteine-scanning mutagenesis and reaction with dibromobimane. J Biol Chem 2000; 275:39272-8. [PMID: 11013259 DOI: 10.1074/jbc.m007741200] [Citation(s) in RCA: 99] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
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.
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Affiliation(s)
- T W Loo
- Canadian Institute for Health Research Group in Membrane Biology, Departments of Medicine and Biochemistry, University of Toronto, Toronto, Ontario, Canada
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Duraisingh MT, Roper C, Walliker D, Warhurst DC. Increased sensitivity to the antimalarials mefloquine and artemisinin is conferred by mutations in the pfmdr1 gene of Plasmodium falciparum. Mol Microbiol 2000; 36:955-61. [PMID: 10844681 DOI: 10.1046/j.1365-2958.2000.01914.x] [Citation(s) in RCA: 135] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
The declining efficacy of chloroquine and pyrimethamine/sulphadoxine in the treatment of human malaria has led to the use of newer antimalarials such as mefloquine and artemisinin. Sequence polymorphisms in the pfmdr1 gene, the gene encoding the plasmodial homologue of mammalian multidrug resistance transporters, have previously been linked to resistance to chloroquine in some, but not all, studies. In this study, we have used a genetic cross between the strains HB3 and 3D7 to study inheritance of sensitivity to the structurally unrelated drugs mefloquine and artemisinin, and to several other antimalarials. We find a complete allelic association between the HB3-like pfmdr1 allele and increased sensitivity to these drugs in the progeny. Different pfmdr1 sequence polymorphisms in other unrelated lines were also associated with increased sensitivity to these drugs. Our results indicate that the pfmdr1 gene is an important determinant of susceptibility to antimalarials, which has major implications for the future development of resistance.
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Affiliation(s)
- M T Duraisingh
- London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK
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31
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Loo TW, Clarke DM. Identification of residues in the drug-binding domain of human P-glycoprotein. Analysis of transmembrane segment 11 by cysteine-scanning mutagenesis and inhibition by dibromobimane. J Biol Chem 1999; 274:35388-92. [PMID: 10585407 DOI: 10.1074/jbc.274.50.35388] [Citation(s) in RCA: 94] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The drug-binding domain of the human multidrug resistance P-glycoprotein (P-gp) probably consists of residues from multiple transmembrane (TM) segments. In this study, we tested whether the amino acids in TM11 participate in binding drug substrates. Each residue in TM11 was initially altered by site-directed mutagenesis and assayed for drug-stimulated ATPase activity in the presence of verapamil, vinblastine, or colchicine. Mutants G939V, F942A, T945A, Q946A, A947L, Y953A, A954L, and G955V had altered drug-stimulated ATPase activities. Direct evidence for binding of drug substrate was then determined by cysteine-scanning mutagenesis of the residues in TM11 and inhibition of drug-stimulated ATPase activity by dibromobimane, a thiol-reactive substrate. Dibromobimane inhibited the drug-stimulated ATPase activities of two mutants, F942C and T945C, by more than 75%. These results suggest that residues Phe(942) and Thr(945) in TM11, together with residues previously identified in TM6 (Leu(339) and Ala(342)) and TM12 (Leu(975), Val(982), and Ala(985)) (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 31945-31948) form part of the drug-binding domain of P-gp.
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Affiliation(s)
- T W Loo
- Medical Research Council Group in Membrane Biology, Department of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada
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32
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van Veen HW, Konings WN. Structure and function of multidrug transporters. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 1999; 456:145-58. [PMID: 10549367 DOI: 10.1007/978-1-4615-4897-3_8] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Affiliation(s)
- H W van Veen
- Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands.
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Demmer A, Andreae S, Thole H, Tümmler B. Iodomycin and iodipine, a structural analogue of azidopine, bind to a common domain in hamster P-glycoprotein. EUROPEAN JOURNAL OF BIOCHEMISTRY 1999; 264:800-5. [PMID: 10491126 DOI: 10.1046/j.1432-1327.1999.00702.x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Both the overexpression of P-glycoprotein and the broad range of substrates of this ATP-binding cassette (ABC) transporter induce the phenomenon of multidrug resistance, one major cause of the failure of cancer chemotherapy in humans. This study reports that [125I]iodipine, a structural analogue of the 1,4-dihydropyridine azidopine, shares a common binding site with iodomycin, a Bolton-Hunter derivative of the anthracycline daunomycin. This binding site is different from that described for iodoarylazidoprazosin, which is presumed to share a common binding site with azidopine. Edman sequencing revealed that [125I]iodipine had photolabelled the same peptide as iodomycin and spans the primary sequence of hamster isoform pgp1 from amino acid 230 to amino acid 312.
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Affiliation(s)
- A Demmer
- Klinische Forschergruppe, Zentrum Biochemie, Medizinische Hochschule, Hannover, Germany.
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Vo QD, Gruol DJ. Identification of P-glycoprotein mutations causing a loss of steroid recognition and transport. J Biol Chem 1999; 274:20318-27. [PMID: 10400654 DOI: 10.1074/jbc.274.29.20318] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
P-glycoproteins transport a wide variety of hydrophobic compounds out of cells. While the diversity of transported molecules suggests a mechanism involving broad specificity, there is evidence of significant discrimination within given classes of molecules. One example of this behavior is transport of corticosteroids by the murine mdr1 P-glycoprotein. The presence of hydroxyl groups, associated with specific steroid carbon atoms, regulates the ability of corticosteroids to be transported. This specificity is demonstrated here by experiments measuring the ability of steroids to inhibit drug transport. The results indicate that a keto oxygen associated with the 3- and 20-carbon atoms, as well as a 17-carbon hydroxyl group, each acts to enhance steroidal P-glycoprotein inhibitory activity. Moreover, inhibitory steroids can be used for directed selection of variant cells, expressing mutated P-glycoproteins with a severely impaired ability to transport dexamethasone. The five mutations, reported here, are located within transmembrane domains 4-6, proximal to the cytoplasmic interface. The altered P-glycoproteins exhibit reduced capacity to be inhibited by specific steroids, suggesting decreased capacity to bind these molecules avidly. Studies comparing the relative inhibitory activity of a series of steroids indicate that these mutations alter recognition of the 17alpha-hydroxyl group and the 20-keto oxygen atom.
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Affiliation(s)
- Q D Vo
- Sidney Kimmel Cancer Center, San Diego, California 92121, USA
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Ambudkar SV, Dey S, Hrycyna CA, Ramachandra M, Pastan I, Gottesman MM. Biochemical, cellular, and pharmacological aspects of the multidrug transporter. Annu Rev Pharmacol Toxicol 1999; 39:361-98. [PMID: 10331089 DOI: 10.1146/annurev.pharmtox.39.1.361] [Citation(s) in RCA: 1505] [Impact Index Per Article: 57.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Considerable evidence has accumulated indicating that the multidrug transporter or P-glycoprotein plays a role in the development of simultaneous resistance to multiple cytotoxic drugs in cancer cells. In recent years, various approaches such as mutational analyses and biochemical and pharmacological characterization have yielded significant information about the relationship of structure and function of P-glycoprotein. However, there is still considerable controversy about the mechanism of action of this efflux pump and its function in normal cells. This review summarizes current research on the structure-function analysis of P-glycoprotein, its mechanism of action, and facts and speculations about its normal physiological role.
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MESH Headings
- ATP Binding Cassette Transporter, Subfamily B, Member 1/chemistry
- ATP Binding Cassette Transporter, Subfamily B, Member 1/drug effects
- ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics
- ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism
- Animals
- Cell Membrane/metabolism
- Drug Resistance, Multiple/genetics
- Drug Resistance, Neoplasm/genetics
- Humans
- Neoplasms/genetics
- Neoplasms/metabolism
- Protein Conformation
- Structure-Activity Relationship
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Affiliation(s)
- S V Ambudkar
- Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
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