RESUMO
Cyclin-dependent kinase 7 (CDK7) is a master regulatory kinase that drives cell cycle progression and stimulates expression of oncogenes in a myriad of cancers. Inhibitors of CDK7 (CDK7i) are currently in clinical trials; however, as with many cancer therapies, patients will most likely experience recurrent disease due to acquired resistance. Identifying targets underlying CDK7i resistance will facilitate prospective development of new therapies that can circumvent such resistance. Here we utilized triple-negative breast cancer as a model to discern mechanisms of resistance as it has been previously shown to be highly responsive to CDK7 inhibitors. After generating cell lines with acquired resistance, high-throughput RNA sequencing revealed significant upregulation of genes associated with efflux pumps and transforming growth factor-beta (TGF-ß) signaling pathways. Genetic silencing or pharmacological inhibition of ABCG2, an efflux pump associated with multidrug resistance, resensitized resistant cells to CDK7i, indicating a reliance on these transporters. Expression of activin A (INHBA), a member of the TGF-ß family of ligands, was also induced, whereas its intrinsic inhibitor, follistatin (FST), was repressed. In resistant cells, increased phosphorylation of SMAD3, a downstream mediator, confirmed an increase in activin signaling, and phosphorylated SMAD3 directly bound the ABCG2 promoter regulatory region. Finally, pharmacological inhibition of TGF-ß/activin receptors or genetic silencing of SMAD4, a transcriptional partner of SMAD3, reversed the upregulation of ABCG2 in resistant cells and phenocopied ABCG2 inhibition. This study reveals that inhibiting the TGF-ß/Activin-ABCG2 pathway is a potential avenue for preventing or overcoming resistance to CDK7 inhibitors.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/biossíntese , Quinases Ciclina-Dependentes/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Subunidades beta de Inibinas/metabolismo , Proteínas de Neoplasias/biossíntese , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Regulação para Cima/efeitos dos fármacos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Subunidades beta de Inibinas/genética , Proteínas de Neoplasias/genética , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Candida drug resistance 1 (Cdr1), a PDR subfamily ABC transporter mediates efflux of xenobiotics in Candida albicans. It is one of the prime factors contributing to multidrug resistance in the fungal pathogen. One hallmark of this transporter is its asymmetric nature, characterized by peculiar alterations in its nucleotide binding domains. As a consequence, there exists only one canonical ATP-binding site while the other is atypical. Here, we report suppressor analysis on the drug-susceptible transmembrane domain mutant V532D that identified the suppressor mutation W1038S, close to the D-loop of the non-catalytic ATP-binding site. Introduction of the W1038S mutation in the background of V532D mutant conferred resistance for most of the substrates to the latter. Such restoration is accompanied by a severe reduction of ATPase activity, of about 85%, while that of the V532D mutant is half-reduced. Conversely, alanine substitution of the highly conserved aspartate D1033A in that D-loop rendered cells selectively hyper-susceptible to miconazole without an impact on steady-state ATPase activity, suggesting altogether that ATP hydrolysis may not hold the key to restoration mechanism. Analysis of the ABCG5/ABCG8-based 3D-model of Cdr1p suggested that the W1038S substitution leads to the loss of hydrophobic interactions and H-bond with residues of the neighbor NBD1, in the non-catalytic ATP-binding site area. The compensatory effect within TMDs accounting for transport restoration in the V532D-W1038S variant may, therefore, be mainly due to an increase in NBDs mobility at the non-catalytic interface.
Assuntos
Proteínas Fúngicas , Proteínas de Membrana Transportadoras , Nucleotídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Triptofano/metabolismo , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Sítios de Ligação/genética , Candida albicans/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Domínios e Motivos de Interação entre Proteínas/genética , Estrutura Secundária de Proteína/genética , Triptofano/química , Triptofano/genéticaRESUMO
ABC multidrug transporters are key players in cancer multidrug resistance and in determining the ADME-Tox properties of drugs and xenobiotics. The most sensitive and specific detection of these transporters is based on functional assays. Assessment of the transporter-dependent reduction of cellular uptake of the fluorescent dyes, such as Hoechst 33342 (Ho) and more recently DyeCycle Violet (DCV), have been widely advocated for the characterization of both ABCB1 and ABCG2 multidrug transporters. Detailed comparison of these supravital DNA-binding dyes revealed that DCV is less toxic to ABCG2- and ABCB1-expressing cells than Ho. ATPase measurements imply that DCV and Ho are similarly handled by ABCB1, whereas ABCG2 seems to transport DVC more effectively. In addition, we have developed an image-based high content microscopy screening method for simultaneous in situ measurement of the cellular activity and expression of the ABCG2 multidrug transporter. We demonstrated the applicability of this method for identifying ABCG2-positive cells in heterogeneous cell population by a single dye uptake measurement. These results may promote multidrug transporter studies at a single cell level and allow the quantitative detection of clinically important drug-resistant sub-populations. © 2016 International Society for Advancement of Cytometry.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/isolamento & purificação , Análise de Célula Única/métodos , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/isolamento & purificação , Adenosina Trifosfatases/genética , Benzimidazóis/química , Linhagem Celular Tumoral , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Corantes Fluorescentes/química , Regulação Neoplásica da Expressão Gênica , Humanos , Especificidade por SubstratoRESUMO
Aspergillus fumigatus can cause severe fatal invasive aspergillosis in immunocompromised patients but is also found in the environment. A. fumigatus infections can be treated with antifungals agents among which azole and echinocandins. Resistance to the class of azoles has been reported not only from patient samples but also from environmental samples. Azole resistance mechanisms involve for most isolates alterations at the site of the azole target (cyp51A); however, a substantial number of isolates can also exhibit non-cyp51A-mediated mechanisms.We aimed here to identify novel A. fumigatus genes involved in azole resistance. For this purpose, we designed a functional complementation system of A. fumigatus cDNAs expressed in a Saccharomyces cerevisiae isolate lacking the ATP Binding Cassette (ABC) transporter PDR5 and that was therefore more azole-susceptible than the parent wild type. Several genes were recovered including two distinct ABC transporters (atrF, atrI) and a Major Facilitator transporter (mdrA), from which atrI (Afu3g07300) and mdrA (Afu1g13800) were not yet described. atrI mediated resistance to itraconazole and voriconazole, while atrF only to voriconazole in S. cerevisiae Gene inactivation of each transporter in A. fumigatus indicated that the transporters were involved in the basal level of azole susceptibility. The expression of the transporters was addressed in clinical and environmental isolates with several azole resistance profiles. Our results show that atrI and mdrA tended to be expressed at higher levels than atrF in normal growth conditions. atrF was upregulated in 2/4 of azole-resistant environmental isolates and was the only gene with a significant association between transporter expression and azole resistance. In conclusion, this work showed the potential of complementation to identify functional transporters. The identified transporters were suggested to participate in azole resistance of A. fumigatus; however, this hypothesis will need further approaches to be verified.
Assuntos
Antifúngicos/metabolismo , Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/enzimologia , Farmacorresistência Fúngica , Genes Fúngicos , Proteínas de Membrana Transportadoras/metabolismo , Aspergillus fumigatus/genética , Aspergillus fumigatus/isolamento & purificação , Azóis/metabolismo , Azóis/farmacologia , Microbiologia Ambiental , Expressão Gênica , Teste de Complementação Genética , Humanos , Proteínas de Membrana Transportadoras/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genéticaRESUMO
ß-Lactams are mainstream antibiotics that are indicated for the prophylaxis and treatment of bacterial infections. The AcrA-AcrD-TolC multidrug efflux system confers much stronger resistance on Escherichia coli to clinically relevant anionic ß-lactam antibiotics than the homologous AcrA-AcrB-TolC system. Using an extensive combination of chimeric analysis and site-directed mutagenesis, we searched for residues that determine the difference in ß-lactam specificity between AcrB and AcrD. We identified three crucial residues at the "proximal" (or access) substrate binding pocket. The simultaneous replacement of these residues in AcrB by those in AcrD (Q569R, I626R, and E673G) transferred the ß-lactam specificity of AcrD to AcrB. Our findings indicate for the first time that the difference in ß-lactam specificity between AcrB and AcrD relates to interactions of the antibiotic with residues in the proximal binding pocket.
Assuntos
Farmacorresistência Bacteriana Múltipla , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , beta-Lactamas/química , Antibacterianos/química , Sítios de Ligação , Proteínas de Transporte/metabolismo , Membrana Celular/efeitos dos fármacos , Cristalografia por Raios X , Escherichia coli/metabolismo , Mutagênese Sítio-Dirigida , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica , Transporte Proteico , Especificidade por SubstratoRESUMO
EmrE, a small multidrug resistance transporter, serves as an ideal model to study coupling between multidrug recognition and protein function. EmrE has a single small binding pocket that must accommodate the full range of diverse substrates recognized by this transporter. We have studied a series of tetrahedral compounds, as well as several planar substrates, to examine multidrug recognition and transport by EmrE. Here we show that even within this limited series, the rate of interconversion between the inward- and outward-facing states of EmrE varies over 3 orders of magnitude. Thus, the identity of the bound substrate controls the rate of this critical step in the transport process. The binding affinity also varies over a similar range and is correlated with substrate hydrophobicity within the tetrahedral substrate series. Substrate identity influences both the ground-state and transition-state energies for the conformational exchange process, highlighting the coupling between substrate binding and transport required for alternating access antiport.
Assuntos
Antiporters/metabolismo , Membrana Celular/metabolismo , Farmacorresistência Bacteriana Múltipla , Proteínas de Escherichia coli/metabolismo , Escherichia coli , Antiporters/química , Antiporters/genética , Transporte Biológico , Membrana Celular/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Cinética , Ligantes , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
In parasites, ATP-binding cassette (ABC) transporters represent an important family of proteins related to drug resistance and other biological activities. Resistance of leishmanial parasites to therapeutic drugs continues to escalate in developing countries, and in many instances, it is due to overexpressed ABC efflux pumps. Progressively adapted baicalein (BLN)-resistant parasites (pB(25)R) show overexpression of a novel ABC transporter, which was classified as ABCC2 or Leishmania donovani multidrug resistance protein 2 (LdMRP2). The protein is primarily localized in the flagellar pocket region and in internal vesicles. Overexpressed LdABCC2 confers substantial BLN resistance to the parasites by rapid drug efflux. The BLN-resistant promastigotes when transformed into amastigotes in macrophage cells cannot be cured by treatment of macrophages with BLN. Amastigote resistance is concomitant with the overexpression of macrophage MRP2 transporter. Reporter analysis and site-directed mutagenesis assays demonstrated that antioxidant response element 1 is activated upon infection. The expression of this phase II detoxifying gene is regulated by NFE2-related factor 2 (Nrf2)-mediated antioxidant response element activation. In view of the fact that the signaling pathway of phosphoinositol 3-kinase controls microfilament rearrangement and translocation of actin-associated proteins, the current study correlates with the intricate pathway of phosphoinositol 3-kinase-mediated nuclear translocation of Nrf2, which activates MRP2 expression in macrophages upon infection by the parasites. In contrast, phalloidin, an agent that prevents depolymerization of actin filaments, inhibits Nrf2 translocation and Mrp2 gene activation by pB(25)R infection. Taken together, these results provide insight into the mechanisms by which resistant clinical isolates of L. donovani induce intracellular events relevant to drug resistance.
Assuntos
Morte Celular/efeitos dos fármacos , Flavonas/farmacologia , Leishmania donovani/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Leishmania donovani/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Proteína 2 Associada à Farmacorresistência Múltipla , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Membrane transporters belonging to the multidrug and toxic compound extrusion family mediate the efflux of unrelated pharmaceuticals from the interior of the cell in organisms ranging from bacteria to human. These proteins are thought to fall into two classes that couple substrate efflux to the influx of either Na(+) or H(+). We studied the energetics of drug extrusion by NorM from Vibrio cholerae in proteoliposomes in which purified NorM protein was functionally reconstituted in an inside-out orientation. We establish that NorM simultaneously couples to the sodium-motive force and proton-motive force, and biochemically identify protein regions and residues that play important roles in Na(+) or H(+) binding. As the positions of protons are not available in current medium and high-resolution crystal structures of multidrug and toxic compound extrusion transporters, our findings add a previously unrecognized parameter to mechanistic models based of these structures.
Assuntos
Antiporters/metabolismo , Proteínas de Bactérias/metabolismo , Prótons , Sódio/metabolismo , Vibrio cholerae/metabolismo , Antiporters/química , Antiporters/genética , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Transporte Biológico , Western Blotting , Membrana Celular/metabolismo , DNA/metabolismo , Etídio/metabolismo , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Proteolipídeos/metabolismo , Força Próton-Motriz , Vibrio cholerae/genéticaRESUMO
Pdr5 is a plasma membrane-bound ABC transporter from Saccharomyces cerevisiae and is involved in the phenomenon of resistance against xenobiotics, which are clinically relevant in bacteria, fungi, and humans. Many fungal ABC transporters such as Pdr5 display an inherent asymmetry in their nucleotide-binding sites (NBS) unlike most of their human counterparts. This degeneracy of the NBSs is very intriguing and needs explanation in terms of structural and functional relevance. In this study, we mutated nonconsensus amino acid residues in the NBSs to its consensus counterpart and studied its effect on the function of the protein and effect on yeast cells. The completely "regenerated" Pdr5 protein was severely impaired in its function of ATP hydrolysis and of rhodamine 6G transport. Moreover, we observe alternative compensatory mechanisms to counteract drug toxicity in some of the mutants. In essence, we describe here the first attempts to restore complete symmetry in an asymmetric ABC transporter and to study its effects, which might be relevant to the entire class of asymmetric ABC transporters.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Membrana Celular/metabolismo , Resistência a Múltiplos Medicamentos/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/metabolismo , Sítios de Ligação/fisiologia , Transporte Biológico/fisiologia , Humanos , Cinética , Mutagênese , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Relação Estrutura-AtividadeRESUMO
The soil bacterium Streptomyces peucetius produces two widely used anticancer antibiotics, doxorubicin and daunorubicin. Present within the biosynthesis gene cluster in S. peucetius is the drrAB operon, which codes for a dedicated ABC (ATP binding cassette)-type transporter for the export of these two closely related antibiotics. Because of its dedicated nature, the DrrAB system is believed to belong to the category of single-drug transporters. However, whether it also contains specificity for other known substrates of multidrug transporters has never been tested. In this study we demonstrate under both in vivo and in vitro conditions that the DrrAB system can transport not only doxorubicin but is also able to export two most commonly studied MDR substrates, Hoechst 33342 and ethidium bromide. Moreover, we demonstrate that many other substrates (including verapamil, vinblastine, and rifampicin) of the well studied multidrug transporters inhibit DrrAB-mediated Dox transport with high efficiency, indicating that they are also substrates of the DrrAB pump. Kinetic studies show that inhibition of doxorubicin transport by Hoechst 33342 and rifampicin occurs by a competitive mechanism, whereas verapamil inhibits transport by a non-competitive mechanism, thus suggesting the possibility of more than one drug binding site in the DrrAB system. This is the first in-depth study of a drug resistance system from a producer organism, and it shows that a dedicated efflux system like DrrAB contains specificity for multiple drugs. The significance of these findings in evolution of poly-specificity in drug resistance systems is discussed.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Streptomyces/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Daunorrubicina/metabolismo , Doxorrubicina/metabolismo , Cinética , Dados de Sequência Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Mutação , Rifampina/metabolismo , Rifampina/farmacologia , Homologia de Sequência de Aminoácidos , Streptomyces/genética , Vanadatos/farmacologia , Verapamil/metabolismo , Verapamil/farmacologia , Vimblastina/metabolismo , Vimblastina/farmacologiaRESUMO
Pdr5 is the founding member of a large subfamily of evolutionarily distinct, clinically important fungal ABC transporters containing a characteristic, deviant ATP-binding site with altered Walker A, Walker B, Signature (C-loop), and Q-loop residues. In contrast to these motifs, the D-loops of the two ATP-binding sites have similar sequences, including a completely conserved aspartate residue. Alanine substitution mutants in the deviant Walker A and Signature motifs retain significant, albeit reduced, ATPase activity and drug resistance. The D-loop residue mutants D340A and D1042A showed a striking reduction in plasma membrane transporter levels. The D1042N mutation localized properly had nearly WT ATPase activity but was defective in transport and was profoundly hypersensitive to Pdr5 substrates. Therefore, there was a strong uncoupling of ATPase activity and drug efflux. Taken together, the properties of the mutants suggest an additional, critical intradomain signaling role for deviant ATP-binding sites.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Farmacorresistência Fúngica Múltipla/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Substituição de Aminoácidos , Sítios de Ligação , Transporte Biológico Ativo/fisiologia , Mutação de Sentido Incorreto , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
ATP-binding cassette (ABC) transporters belong to one of the largest protein superfamilies that expands from prokaryotes to man. Recent x-ray crystal structures of bacterial and mammalian ABC exporters suggest a common alternating access mechanism of substrate transport, which has also been biochemically substantiated. However, the current model does not yet explain the coupling between substrate binding and ATP hydrolysis that underlies ATP-dependent substrate transport. In our studies on the homodimeric multidrug/lipid A ABC exporter MsbA from Escherichia coli, we performed cysteine cross-linking, fluorescence energy transfer, and cysteine accessibility studies on two reporter positions, near the nucleotide-binding domains and in the membrane domains, for transporter embedded in a biological membrane. Our results suggest for the first time that substrate binding by MsbA stimulates the maximum rate of ATP hydrolysis by facilitating the dimerization of nucleotide-binding domains in a state, which is markedly distinct from the previously described nucleotide-free, inward-facing and nucleotide-bound, outward-facing conformations of ABC exporters and which binds ATP.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Benzimidazóis/metabolismo , Sítios de Ligação/genética , Transporte Biológico , Membrana Celular/metabolismo , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Etídio/metabolismo , Transferência Ressonante de Energia de Fluorescência , Hidrólise , Mutação , Ligação Proteica , Multimerização Proteica , Especificidade por Substrato , Verapamil/metabolismoRESUMO
The TolC-like protein HgdD of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 is part of multiple three-component "AB-D" systems spanning the inner and outer membranes and is involved in secretion of various compounds, including lipids, metabolites, antibiotics, and proteins. Several components of HgdD-dependent tripartite transport systems have been identified, but the diversity of inner membrane energizing systems is still unknown. Here we identified six putative resistance-nodulation-cell division (RND) type factors. Four of them are expressed during late exponential and stationary growth phase under normal growth conditions, whereas the other two are induced upon incubation with erythromycin or ethidium bromide. The constitutively expressed RND component Alr4267 has an atypical predicted topology, and a mutant strain (I-alr4267) shows a reduction in the content of monogalactosyldiacylglycerol as well as an altered filament shape. An insertion mutant of the ethidium bromide-induced all7631 did not show any significant phenotypic alteration under the conditions tested. Mutants of the constitutively expressed all3143 and alr1656 exhibited a Fox(-) phenotype. The phenotype of the insertion mutant I-all3143 parallels that of the I-hgdD mutant with respect to antibiotic sensitivity, lipid profile, and ethidium efflux. In addition, expression of the RND genes all3143 and all3144 partially complements the capability of Escherichia coli ΔacrAB to transport ethidium. We postulate that the RND transporter All3143 and the predicted membrane fusion protein All3144, as homologs of E. coli AcrB and AcrA, respectively, are major players for antibiotic resistance in Anabaena sp. PCC 7120.
Assuntos
Anabaena/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/metabolismo , Farmacorresistência Bacteriana Múltipla/fisiologia , Anabaena/genética , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Transporte/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Eritromicina/farmacologia , Etídio/farmacologia , Regulação Bacteriana da Expressão Gênica , Mutagênese InsercionalRESUMO
The ABC transporter ABCC4 is recognized as an ATP-dependent exporter of endogenous substances as well as an increasing variety of anionic chemotherapeutics. A loss-of-function variant of zebrafish Abcc4 was identified with a single amino acid substitution in the cytoplasmic loop T804M. Because this substituted amino acid is highly conserved among ABCC4 orthologs and is located in cytoplasmic loop 3 (CL3), we investigated the impact of this mutation on human and zebrafish Abcc4 expression. We demonstrate that zebrafish Abcc4 T804M or human ABCC4 T796M exhibit substantially reduced expression, coupled with impaired plasma membrane localization. To understand the molecular basis for the localization defect, we developed a homology model of zebrafish Abcc4. The homology model suggested that the bulky methionine substitution disrupted side-chain contacts. Molecular dynamic simulations of a fragment of human or zebrafish CL3 containing a methionine substitution indicated altered helicity coupled with reduced thermal stability. Trifluoroethanol challenge coupled with circular dichroism revealed that the methionine substitution disrupted the ability of this fragment of CL3 to readily form an α-helix. Furthermore, expression and plasma membrane localization of these mutant ABCC4/Abcc4 proteins are mostly rescued by growing cells at subphysiological temperatures. Because the cystic fibrosis transmembrane conductance regulator (ABCC7) is closely related to ABCC4, we extended this by engineering certain pathogenic CFTR-CL3 mutations, and we showed they destabilized human and zebrafish ABCC4. Altogether, our studies provide the first evidence for a conserved domain in CL3 of ABCC4 that is crucial in ensuring its proper plasma membrane localization.
Assuntos
Citoplasma/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Filogenia , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Células NIH 3T3 , Homologia de Sequência de Aminoácidos , Peixe-ZebraRESUMO
In ATP-binding cassette proteins, the two nucleotide-binding domains (NBDs) work as dimers to bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is controversial. It is still unresolved whether hydrolysis leads to dissociation of the ATP-induced dimers or partial opening of the dimers such that the NBDs remain in contact during the hydrolysis cycle. We studied the bacterial lipid flippase MsbA by luminescence resonance energy transfer (LRET). The LRET signal between optical probes reacted with single-cysteine mutants was employed to follow NBD association/dissociation in real time. The intermonomer distances calculated from LRET data indicate that the NBDs separate completely following ATP hydrolysis, even in the presence of mm MgATP, and that the dissociation occurs following each hydrolysis cycle. The results support association/dissociation, as opposed to constant contact models, for the mode of operation of ATP-binding cassette proteins.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Nucleotídeos/metabolismo , Salmonella typhimurium/metabolismo , Adenosina Trifosfatases/metabolismo , Biocatálise , Compostos de Boro/metabolismo , Transferência de Energia , Hidrólise , Cinética , Lipossomos/metabolismo , Luminescência , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Fatores de TempoRESUMO
Vesicular monoamine transporter 2 (VMAT2) transports monoamines into storage vesicles in a process that involves exchange of the charged monoamine with two protons. VMAT2 is a member of the DHA12 family of multidrug transporters that belongs to the major facilitator superfamily of secondary transporters. Tetrabenazine (TBZ) is a non-competitive inhibitor of VMAT2 that is used in the treatment of hyperkinetic disorders associated with Huntington disease and Tourette syndrome. Previous biochemical studies suggested that the recognition site for TBZ and monoamines is different. However, the precise mechanism of TBZ interaction with VMAT2 remains unknown. Here we used a random mutagenesis approach and selected TBZ-resistant mutants. The mutations clustered around the lumenal opening of the transporter and mapped to either conserved proline or glycine, or to residues immediately adjacent to conserved proline and glycine. Directed mutagenesis provides further support for the essential role of the latter residues. Our data strongly suggest that the conserved α-helix breaking residues identified in this work play an important role in conformational rearrangements required for TBZ binding and substrate transport. Our results provide a novel insight into the mechanism of transport and TBZ binding by VMAT2.
Assuntos
Inibidores da Captação Adrenérgica/farmacologia , Tetrabenazina/farmacologia , Proteínas Vesiculares de Transporte de Monoamina/antagonistas & inibidores , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Inibidores da Captação Adrenérgica/farmacocinética , Animais , Sítios de Ligação , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/fisiologia , Células HEK293 , Humanos , Doença de Huntington/tratamento farmacológico , Doença de Huntington/genética , Doença de Huntington/metabolismo , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Ratos , Saccharomyces cerevisiae , Tetrabenazina/farmacocinética , Síndrome de Tourette/tratamento farmacológico , Síndrome de Tourette/genética , Síndrome de Tourette/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/genéticaRESUMO
The functional unit of ATP-binding cassette (ABC) transporters consists of two transmembrane domains and two nucleotide-binding domains (NBDs). ATP binding elicits association of the two NBDs, forming a dimer in a head-to-tail arrangement, with two nucleotides "sandwiched" at the dimer interface. Each of the two nucleotide-binding sites is formed by residues from the two NBDs. We recently found that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii dimerizes in response to ATP binding and dissociates completely following ATP hydrolysis. However, it is still unknown whether dissociation of NBD dimers follows ATP hydrolysis at one or both nucleotide-binding sites. Here, we used luminescence resonance energy transfer to study heterodimers formed by one active (donor-labeled) and one catalytically defective (acceptor-labeled) NBD. Rapid mixing experiments in a stop-flow chamber showed that NBD heterodimers with one functional and one inactive site dissociated at a rate indistinguishable from that of dimers with two hydrolysis-competent sites. Comparison of the rates of NBD dimer dissociation and ATP hydrolysis indicated that dissociation followed hydrolysis of one ATP. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dimer dissociation.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas Arqueais/química , Methanocaldococcus/química , Multimerização Proteica/fisiologia , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Hidrólise , Methanocaldococcus/genética , Methanocaldococcus/metabolismo , Estrutura Terciária de ProteínaRESUMO
P-glycoprotein (P-gp) is an ATP binding cassette transporter that effluxes a variety of structurally diverse compounds including anticancer drugs. Computational models of human P-gp in the apo- and nucleotide-bound conformation show that the adenine group of ATP forms hydrogen bonds with the conserved Asp-164 and Asp-805 in intracellular loops 1 and 3, respectively, which are located at the interface between the nucleotide binding domains and transmembrane domains. We investigated the role of Asp-164 and Asp-805 residues by substituting them with cysteine in a cysteine-less background. It was observed that the D164C/D805C mutant, when expressed in HeLa cells, led to misprocessing of P-gp, which thus failed to transport the drug substrates. The misfolded protein could be rescued to the cell surface by growing the cells at a lower temperature (27 °C) or by treatment with substrates (cyclosporine A, FK506), modulators (tariquidar), or small corrector molecules. We also show that short term (4-6 h) treatment with 15 µM cyclosporine A or FK506 rescues the pre-formed immature protein trapped in the endoplasmic reticulum in an immunophilin-independent pathway. The intracellularly trapped misprocessed protein associates more with chaperone Hsp70, and the treatment with cyclosporine A reduces the association of mutant P-gp, thus allowing it to be trafficked to the cell surface. The function of rescued cell surface mutant P-gp is similar to that of wild-type protein. These data demonstrate that the Asp-164 and Asp-805 residues are not important for ATP binding, as proposed earlier, but are critical for proper folding and maturation of a functional transporter.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Ciclosporina/farmacologia , Proteínas de Choque Térmico HSP70/metabolismo , Mutação , Dobramento de Proteína/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSP70/genética , Células HeLa , Humanos , Imunossupressores/farmacologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Tacrolimo/farmacologiaRESUMO
A representative of a new class of potent antimalarials with an unknown mode of action was recently described. To identify the molecular target of this class of antimalarials, we employed a photo-reactive affinity capture method to find parasite proteins specifically interacting with the capture compound in living parasitized cells. The capture reagent retained the antimalarial properties of the parent molecule (ACT-213615) and accumulated within parasites. We identified several proteins interacting with the capture compound and established a functional interaction between ACT-213615 and PfMDR1. We surmise that PfMDR1 may play a role in the antimalarial activity of the piperazine-containing compound ACT-213615.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Antimaláricos/farmacologia , Plasmodium falciparum/fisiologia , Raios Ultravioleta , AnimaisRESUMO
The fungal ATP-binding cassette (ABC) transporter Cdr1 protein (Cdr1p), responsible for clinically significant drug resistance, is composed of two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). We have probed the nature of the drug binding pocket by performing systematic mutagenesis of the primary sequences of the 12 transmembrane segments (TMSs) found in the TMDs. All mutated proteins were expressed equally well and localized properly at the plasma membrane in the heterologous host Saccharomyces cerevisiae, but some variants differed significantly in efflux activity, substrate specificity, and coupled ATPase activity. Replacement of the majority of the amino acid residues with alanine or glycine yielded neutral mutations, but about 42% of the variants lost resistance to drug efflux substrates completely or selectively. A predicted three-dimensional homology model shows that all the TMSs, apart from TMS4 and TMS10, interact directly with the drug-binding cavity in both the open and closed Cdr1p conformations. However, TMS4 and TMS10 mutations can also induce total or selective drug susceptibility. Functional data and homology modeling assisted identification of critical amino acids within a drug-binding cavity that, upon mutation, abolished resistance to all drugs tested singly or in combinations. The open and closed Cdr1p models enabled the identification of amino acid residues that bordered a drug-binding cavity dominated by hydrophobic residues. The disposition of TMD residues with differential effects on drug binding and transport are consistent with a large polyspecific drug binding pocket in this yeast multidrug transporter.