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1.
Cell Biochem Biophys ; 78(1): 15-22, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31893350

RESUMO

Anions enter from the cytoplasm into the channel pore of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel not via a central pathway but via a single lateral portal or fenestration. High Cl- conductance is dependent on electrostatic attraction of cytoplasmic Cl- ions by four positively charged amino acid side-chains located within this portal. Here we use a mutagenic approach to investigate the functional effects of transplanting or supplementing these positive charges at nearby portal-lining sites. Using patch clamp recording, we find that the functionally important positive charges at K190 and R303 can be transplanted to four nearby sites (N186, L197, W356, and A367) with little loss of Cl- conductance. Introduction of additional positive charge at these sites had almost no effect on Cl- conductance, but did increase the sensitivity to channel block by intracellular suramin and Pt(NO2)42- anions. We suggest that it is the number of positive charges within the portal, rather than their exact location, that is the most important factor influencing Cl- conductance. The portal appears well optimized in terms of charge distribution to maximize Cl- conductance.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Citoplasma/metabolismo , Animais , Ânions/química , Ânions/metabolismo , Linhagem Celular , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo , Cricetinae , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Platina/química , Eletricidade Estática , Suramina/química , Suramina/metabolismo
2.
Nat Med ; 26(2): 244-251, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31959991

RESUMO

Mucociliary clearance, the physiological process by which mammalian conducting airways expel pathogens and unwanted surface materials from the respiratory tract, depends on the coordinated function of multiple specialized cell types, including basal stem cells, mucus-secreting goblet cells, motile ciliated cells, cystic fibrosis transmembrane conductance regulator (CFTR)-rich ionocytes, and immune cells1,2. Bronchiectasis, a syndrome of pathological airway dilation associated with impaired mucociliary clearance, may occur sporadically or as a consequence of Mendelian inheritance, for example in cystic fibrosis, primary ciliary dyskinesia (PCD), and select immunodeficiencies3. Previous studies have identified mutations that affect ciliary structure and nucleation in PCD4, but the regulation of mucociliary transport remains incompletely understood, and therapeutic targets for its modulation are lacking. Here we identify a bronchiectasis syndrome caused by mutations that inactivate NIMA-related kinase 10 (NEK10), a protein kinase with previously unknown in vivo functions in mammals. Genetically modified primary human airway cultures establish NEK10 as a ciliated-cell-specific kinase whose activity regulates the motile ciliary proteome to promote ciliary length and mucociliary transport but which is dispensable for normal ciliary number, radial structure, and beat frequency. Together, these data identify a novel and likely targetable signaling axis that controls motile ciliary function in humans and has potential implications for other respiratory disorders that are characterized by impaired mucociliary clearance.


Assuntos
Ciliopatias/imunologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Depuração Mucociliar , Quinases Relacionadas a NIMA/metabolismo , Adolescente , Adulto , Separação Celular , Criança , Ciliopatias/metabolismo , Células Epiteliais/metabolismo , Exoma , Feminino , Citometria de Fluxo , Células HEK293 , Homozigoto , Humanos , Microscopia de Contraste de Fase , Microscopia de Vídeo , Mutação , Fenótipo , Proteoma , Sistema Respiratório , Tomografia Computadorizada por Raios X , Microtomografia por Raio-X , Adulto Jovem
3.
PLoS One ; 15(1): e0227668, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31978131

RESUMO

Cystic Fibrosis (CF) is an inherited disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. Mutations in CFTR cause impaired chloride ion transport in the epithelial tissues of patients leading to cardiopulmonary decline and pancreatic insufficiency in the most severely affected patients. CFTR is composed of twelve membrane-spanning domains, two nucleotide-binding domains (NBDs), and a regulatory domain. The most common mutation in CFTR is a deletion of phenylalanine at position 508 (ΔF508) in NBD1. Previous research has primarily concentrated on the structure and dynamics of the NBD1 domain; However numerous pathological mutations have also been found in the lesser-studied NBD2 domain. We have investigated the amino acid co-evolved network of interactions in NBD2, and the changes that occur in that network upon the introduction of CF and CF-related mutations (S1251N(T), S1235R, D1270N, N1303K(T)). Extensive coupling between the α- and ß-subdomains were identified with residues in, or near Walker A, Walker B, H-loop and C-loop motifs. Alterations in the predicted residue network varied from moderate for the S1251T perturbation to more severe for N1303T. The S1235R and D1270N networks varied greatly compared to the wildtype, but these CF mutations only affect ion transport preference and do not severely disrupt CFTR function, suggesting dynamic flexibility in the network of interactions in NBD2. Our results also suggest that inappropriate interactions between the ß-subdomain and Q-loop could be detrimental. We also identified mutations predicted to stabilize the NBD2 residue network upon introduction of the CF and CF-related mutations, and these predicted mutations are scored as benign by the MUTPRED2 algorithm. Our results suggest the level of disruption of the co-evolution predictions of the amino acid networks in NBD2 does not have a straightforward correlation with the severity of the CF phenotypes observed.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Fibrose Cística/metabolismo , Mutação , Algoritmos , Substituição de Aminoácidos , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Evolução Molecular , Estudos de Associação Genética , Humanos , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas/genética , Mapas de Interação de Proteínas/genética , Estabilidade Proteica , Alinhamento de Sequência , Deleção de Sequência
6.
Int J Mol Sci ; 20(19)2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31590401

RESUMO

Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene lead to cystic fibrosis (CF). The most common mutation F508del inhibits folding and processing of CFTR protein. FDA-approved correctors rescue the biosynthetic processing of F508del-CFTR protein, while potentiators improve the rescued CFTR channel function. Transforming growth factor (TGF-ß1), overexpressed in many CF patients, blocks corrector/potentiator rescue by inhibiting CFTR mRNA in vitro. Increased TGF-ß1 signaling and acquired CFTR dysfunction are present in other lung diseases. To study the mechanism of TGF-ß1 repression of CFTR, we used molecular, biochemical, and functional approaches in primary human bronchial epithelial cells from over 50 donors. TGF-ß1 destabilized CFTR mRNA in cells from lungs with chronic disease, including CF, and impaired F508del-CFTR rescue by new-generation correctors. TGF-ß1 increased the active pool of selected micro(mi)RNAs validated as CFTR inhibitors, recruiting them to the RNA-induced silencing complex (RISC). Expression of F508del-CFTR globally modulated TGF-ß1-induced changes in the miRNA landscape, creating a permissive environment required for degradation of F508del-CFTR mRNA. In conclusion, TGF-ß1 may impede the full benefit of corrector/potentiator therapy in CF patients. Studying miRNA recruitment to RISC under disease-specific conditions may help to better characterize the miRNAs utilized by TGF-ß1 to destabilize CFTR mRNA.


Assuntos
Brônquios/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , MicroRNAs/metabolismo , Estabilidade de RNA , Mucosa Respiratória/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Brônquios/citologia , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Inativação Gênica , Humanos , MicroRNAs/genética , Mucosa Respiratória/efeitos dos fármacos
7.
Medicina (B Aires) ; 79(4): 303-314, 2019.
Artigo em Espanhol | MEDLINE | ID: mdl-31487254

RESUMO

The chloride channels, sodium and bicarbonate channels, and aquaporin water channels are coordinated to maintain the airway surface liquid that is necessary for mucociliary clearance. The general mechanism for the transport of electrolytes and fluids depends mainly on the differential expression and distribution of ion transporters and pumps. Ions and water move through the paracellular or transcellular pathways. The transcellular route of electrolyte transport requires an active transport (dependent on ATP) or passive (following electrochemical gradients) of ions. The paracellular pathway is a passive process that is ultimately controlled by the predominant transepithelial electrochemical gradients. Cystic fibrosis is a hereditary disease that is produced by mutations in the gene that encode cystic fibrosis transmembrane conductance regulatory protein (CFTR) that acts as a chloride channel and performs functions of hydration of periciliary fluid and maintenance of luminal pH. The dysfunction of the chlorine channel in the respiratory epithelium determines an alteration in the bronchial secretions, with an increase in its viscosity and alteration of the mucociliary clearance and that associated with infectious processes can lead to irreversible lung damage. CFTR dysfunction has also been implicated in the pathogenesis of acute pancreatitis, chronic obstructive pulmonary disease, and bronchial hyperreactivity in asthma. There are drugs that exploit physiological mechanisms in the transport of ions with a therapeutic objective.


Assuntos
Transporte Biológico Ativo/fisiologia , Canais de Cloreto/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Transporte de Íons/fisiologia , Depuração Mucociliar/fisiologia , Canais de Cloreto/fisiologia , Fibrose Cística/fisiopatologia , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Humanos
8.
J Immunol Res ; 2019: 2180409, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31396541

RESUMO

The primary purpose of pulmonary ventilation is to supply oxygen (O2) for sustained aerobic respiration in multicellular organisms. However, a plethora of abiotic insults and airborne pathogens present in the environment are occasionally introduced into the airspaces during inhalation, which could be detrimental to the structural integrity and functioning of the respiratory system. Multiple layers of host defense act in concert to eliminate unwanted constituents from the airspaces. In particular, the mucociliary escalator provides an effective mechanism for the continuous removal of inhaled insults including pathogens. Defects in the functioning of the mucociliary escalator compromise the mucociliary clearance (MCC) of inhaled pathogens, which favors microbial lung infection. Defective MCC is often associated with airway mucoobstruction, increased occurrence of respiratory infections, and progressive decrease in lung function in mucoobstructive lung diseases including cystic fibrosis (CF). In this disease, a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene results in dehydration of the airway surface liquid (ASL) layer. Several mice models of Cftr mutation have been developed; however, none of these models recapitulate human CF-like mucoobstructive lung disease. As an alternative, the Scnn1b transgenic (Scnn1b-Tg+) mouse model overexpressing a transgene encoding sodium channel nonvoltage-gated 1, beta subunit (Scnn1b) in airway club cells is available. The Scnn1b-Tg+ mouse model exhibits airway surface liquid (ASL) dehydration, impaired MCC, increased mucus production, and early spontaneous pulmonary bacterial infections. High morbidity and mortality among mucoobstructive disease patients, high economic and health burden, and lack of scientific understanding of the progression of mucoobstruction warrants in-depth investigation of the cause of mucoobstruction in mucoobstructive disease models. In this review, we will summarize published literature on the Scnn1b-Tg+ mouse and analyze various unanswered questions on the initiation and progression of mucobstruction and bacterial infections.


Assuntos
Obstrução das Vias Respiratórias/imunologia , Obstrução das Vias Respiratórias/fisiopatologia , Fibrose Cística/imunologia , Fibrose Cística/fisiopatologia , Modelos Animais de Doenças , Canais Epiteliais de Sódio/genética , Obstrução das Vias Respiratórias/metabolismo , Obstrução das Vias Respiratórias/microbiologia , Animais , Fibrose Cística/genética , Fibrose Cística/microbiologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Desidratação/metabolismo , Desidratação/fisiopatologia , Canais Iônicos/deficiência , Canais Iônicos/genética , Leucócitos/imunologia , Pulmão/imunologia , Pulmão/fisiopatologia , Macrófagos/imunologia , Camundongos , Camundongos Transgênicos , Depuração Mucociliar/genética , Depuração Mucociliar/imunologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/fisiopatologia
9.
Cell Physiol Biochem ; 53(2): 400-412, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31403270

RESUMO

BACKGROUND/AIMS: Mutations in ABCA4 cause Stargardt macular degeneration, which invariably ends in legal blindness. We studied two common mutants, A1038V (in NBD1) and G1961E (in NBD2), with the purpose of exploring how they interact with the cell's quality control mechanism. The study was designed to determine how these mutants can be rescued. METHODS: We expressed wt and mutant ABCA4 in HEK293 cells and studied the effect of the mutations on trafficking and processing and the ability of correctors to rescue them. We used a combination of western blotting, confocal microscopy and surface biotinylation coupled with pulldown of plasma membrane proteins. RESULTS: G1961E is sensitive to inhibitors of the aggresome, tubacin and the lysosome, bafilomycin A. Both mutants cause a reduction in heat shock protein, Hsp27. Incubation of HEK293 cells expressing the mutants with VX-809, an FDA approved drug for the treatment of cystic fibrosis, increased the levels of A1038V and G1961E by 2- to 3-fold. Importantly, VX-809 increased the levels of both mutants at the plasma membrane suggesting that trafficking had been restored. Transfecting additional Hsp27 to the cells also increased the steady state levels of both mutants. However, in combination with VX-809 the addition of Hsp27 caused a dramatic increase in the protein expression particularly in the G1961 mutant which increased approximately 5-fold. CONCLUSION: Our results provide a new mechanism for the rescue of ABCA4 trafficking mutants based on the restoration of Hsp27. Our results provide a pathway for the treatment of Stargardt disease.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Transportadores de Cassetes de Ligação de ATP/genética , Aminopiridinas/uso terapêutico , Anilidas/farmacologia , Benzodioxóis/uso terapêutico , Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Leupeptinas/farmacologia , Lisossomos/metabolismo , Degeneração Macular/congênito , Degeneração Macular/tratamento farmacológico , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Mutação , Transporte Proteico/efeitos dos fármacos
10.
Res Microbiol ; 170(8): 374-380, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31376483

RESUMO

FtsEX is a member of a small subclass of ABC transporters that uses mechano-transmission to perform work in the periplasm. FtsEX controls periplasmic peptidoglycan (PG) hydrolase activities in many Gram negative and positive organisms to ensure the safe separation of daughter cells during division. In these organisms FtsEX localizes to the Z ring and uses its ATPase activity to regulate its periplasmic effectors. In Escherichia coli, FtsEX also participates in building the divisome and coordinates PG synthesis with PG hydrolysis. This review discusses studies that are beginning to elucidate the mechanisms of FtsEX's various roles in cell division.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/crescimento & desenvolvimento , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Fenômenos Biomecânicos/fisiologia , Parede Celular/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Peptidoglicano/metabolismo
11.
Drug Des Devel Ther ; 13: 2405-2412, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31409974

RESUMO

Lumacaftor-ivacaftor is a combination of two small molecule therapies targeting the basic defect in cystic fibrosis (CF) at a cellular level. It is a precision medicine and its effects are specific to individuals with two copies of the p.Phe508del gene mutation. The drug combination works by restoring functioning CF transmembrane conductance regulator (CFTR) protein in cell surface membranes and was the first CFTR modulator licensed for the homozygous p.Phe508del genotype. The drug is a combination of a CFTR corrector and potentiator. Lumacaftor, the corrector, works by increasing the trafficking of CFTR proteins to the outer cell membrane. Ivacaftor, the potentiator, works by enabling the opening of what would otherwise be a dysfunctional chloride channel. In vivo lumacaftor-ivacaftor improves Phe508del-CFTR activity in airways, sweat ducts and intestine to approximately 10-20% of normal CFTR function with greater reductions in sweat chloride levels in children versus adults. Its use results in a modest improvement in lung function and a decreased rate of subsequent decline. Perhaps more importantly, those treated report increased levels of well-being and their rate of respiratory exacerbations is significantly improved. This review traces the development and use of this combination of CFTR modulators, the first licensed drug for treating the homozygous p.Phe508del CF genotype at the intracellular level by correcting the protein defect.


Assuntos
Aminofenóis/farmacologia , Aminofenóis/uso terapêutico , Aminopiridinas/farmacologia , Aminopiridinas/uso terapêutico , Benzodioxóis/farmacologia , Benzodioxóis/uso terapêutico , Fibrose Cística/tratamento farmacológico , Desenho de Fármacos , Quinolonas/farmacologia , Quinolonas/uso terapêutico , Aminofenóis/síntese química , Aminofenóis/química , Aminopiridinas/síntese química , Aminopiridinas/química , Benzodioxóis/síntese química , Benzodioxóis/química , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Combinação de Medicamentos , Quimioterapia Combinada , Humanos , Quinolonas/síntese química , Quinolonas/química
12.
Eur J Med Chem ; 180: 430-448, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31326599

RESUMO

Deletion of phenylalanine at position 508 (F508del) in the CFTR protein, is the most common mutation causing cystic fibrosis (CF). F508del causes misfolding and rapid degradation of CFTR protein a defect that can be targeted with pharmacological agents termed "correctors". Correctors belong to various chemical classes but are generally small molecules based on nitrogen sulfur or oxygen heterocycles. The mechanism of action of correctors is generally unknown but there is experimental evidence that some of them can directly act on mutant CFTR improving folding and stability. Here we overview the characteristics of the various F508del correctors described so far to obtain indications on key chemical structures and modifications that are required for mutant protein rescue.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Pirimidinonas/farmacologia , Tiazóis/farmacologia , Animais , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Mutação , Dobramento de Proteína/efeitos dos fármacos , Pirimidinonas/química , Tiazóis/química
13.
Nat Commun ; 10(1): 3124, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31311920

RESUMO

Cystic fibrosis (CF) is a genetic disorder caused by defective CF Transmembrane Conductance Regulator (CFTR) function. Insulin producing pancreatic islets are located in close proximity to the pancreatic duct and there is a possibility of impaired cell-cell signaling between pancreatic ductal epithelial cells (PDECs) and islet cells as causative in CF. To study this possibility, we present an in vitro co-culturing system, pancreas-on-a-chip. Furthermore, we present an efficient method to micro dissect patient-derived human pancreatic ducts from pancreatic remnant cell pellets, followed by the isolation of PDECs. Here we show that defective CFTR function in PDECs directly reduced insulin secretion in islet cells significantly. This uniquely developed pancreatic function monitoring tool will help to study CF-related disorders in vitro, as a system to monitor cell-cell functional interaction of PDECs and pancreatic islets, characterize appropriate therapeutic measures and further our understanding of pancreatic function.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/fisiopatologia , Células Epiteliais/patologia , Ilhotas Pancreáticas/fisiopatologia , Dispositivos Lab-On-A-Chip , Adolescente , Criança , Pré-Escolar , Técnicas de Cocultura/métodos , Fibrose Cística/patologia , Fibrose Cística/terapia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/metabolismo , Feminino , Humanos , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Microdissecção , Organoides , Ductos Pancreáticos/citologia , Ductos Pancreáticos/patologia , Cultura Primária de Células/métodos
14.
Gene Ther ; 26(9): 354-362, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31300729

RESUMO

Cystic fibrosis (CF) is a life-limiting disease caused by defective or deficient cystic fibrosis transmembrane conductance regulator (CFTR) activity. The recent advent of the FDA-approved CFTR modulator drug ivacaftor, alone or in combination with lumacaftor or tezacaftor, has enabled treatment of the majority of patients suffering from CF. Even before the identification of the CFTR gene, gene therapy was put forward as a viable treatment option for this genetic condition. However, initial enthusiasm has been hampered as CFTR gene delivery to the lungs has proven to be more challenging than expected. This review covers the contemporary clinical and scientific knowledge base for small molecule CFTR modulator drug therapy, gene delivery vectors and CRISPR/Cas9 gene editing and highlights the prospect of these technologies for future treatment options.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/terapia , Terapia Genética , Aminofenóis/uso terapêutico , Animais , Sistemas CRISPR-Cas , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Quinolonas/uso terapêutico
15.
Phytomedicine ; 63: 153004, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31301536

RESUMO

BACKGROUND: PM2.5 is closely related to the incidence and mortality of respiratory diseases. Diesel particulate matter (DPM) is the main component of particulate air pollution and an important source of PM2.5. HYPOTHESIS/PURPOSE: This study mainly explored the effect of DPM on airway surface liquid (ASL) secretion and the regulation of naringin in this process, to evaluate therapeutic potentials of naringin for the treatment of abnormal secretion of the respiratory tract caused by PM2.5. METHODS: The concentration of lysozyme was measured by Lysozyme Assay Kit. Total protein content was determined by the BCA Protein Assay Kit. The concentration of cAMP and MUC5AC, expressions of CFTR, AQP1, and AQP5 proteins were measured by ELISA. Expressions of CFTR, AQP1 and AQP5 mRNA were determined by qPCR. Amount of CFTR on the cell membrane was determined by immunofluorescence. RESULTS: The in vitro and in vivo studies had indicated that DPM could inhibit ASL secretion and increased the viscosity of the liquid. Naringin had the functions to attenuate DPM-induced injury, reduce liquid viscosity by reducing MUC5AC and total protein secretion, increase DPM-induced CFTR, AQP1, and AQP5 mRNA and protein expression, positively regulate apical CFTR insertion and promote CFTR activation by increasing intracellular cAMP. CONCLUSION: These results demonstrated that naringin had regulating effects on the DPM-induced abnormal secretion of the respiratory tract.


Assuntos
Poluentes Atmosféricos/toxicidade , Flavanonas/farmacologia , Pulmão/efeitos dos fármacos , Material Particulado/toxicidade , Emissões de Veículos , Animais , Aquaporina 1/genética , Aquaporina 1/metabolismo , Aquaporina 5/genética , Aquaporina 5/metabolismo , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos Endogâmicos BALB C , Mucina-5AC/metabolismo
16.
Elife ; 82019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31205003

RESUMO

The devastating inherited disease cystic fibrosis (CF) is caused by mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel. The recent approval of the CFTR potentiator drug ivacaftor (Vx-770) for the treatment of CF patients has marked the advent of causative CF therapy. Currently, thousands of patients are being treated with the drug, and its molecular mechanism of action is under intensive investigation. Here we determine the solubility profile and true stimulatory potency of Vx-770 towards wild-type (WT) and mutant human CFTR channels in cell-free patches of membrane. We find that its aqueous solubility is ~200 fold lower (~60 nanomolar), whereas the potency of its stimulatory effect is >100 fold higher, than reported, and is unexpectedly fully reversible. Strong, but greatly delayed, channel activation by picomolar Vx-770 identifies multiple sequential slow steps in the activation pathway. These findings provide solid guidelines for the design of in vitro studies using Vx-770.


Assuntos
Aminofenóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Fibrose Cística/tratamento farmacológico , Ativação do Canal Iônico/efeitos dos fármacos , Quinolonas/farmacologia , Aminofenóis/química , Aminofenóis/uso terapêutico , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Feminino , Humanos , Ativação do Canal Iônico/genética , Transporte de Íons/efeitos dos fármacos , Mutação , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/fisiologia , Quinolonas/química , Quinolonas/uso terapêutico , Solubilidade , Solventes/química , Xenopus laevis
17.
Nat Commun ; 10(1): 2636, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201318

RESUMO

The leading cause of cystic fibrosis (CF) is the deletion of phenylalanine 508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR). The mutation affects the thermodynamic stability of the domain and the integrity of the interface between NBD1 and the transmembrane domain leading to its clearance by the quality control system. Here, we develop nanobodies targeting NBD1 of human CFTR and demonstrate their ability to stabilize both isolated NBD1 and full-length protein. Crystal structures of NBD1-nanobody complexes provide an atomic description of the epitopes and reveal the molecular basis for stabilization. Furthermore, our data uncover a conformation of CFTR, involving detachment of NBD1 from the transmembrane domain, which contrast with the compact assembly observed in cryo-EM structures. This unexpected interface rearrangement is likely to have major relevance for CF pathogenesis but also for the normal function of CFTR and other ABC proteins.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Modelos Moleculares , Cristalografia por Raios X , Regulador de Condutância Transmembrana em Fibrose Cística/isolamento & purificação , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas/genética , Estabilidade Proteica , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Anticorpos de Domínio Único/metabolismo
18.
FEBS Open Bio ; 9(6): 1119-1127, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30985981

RESUMO

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF cells and tissues exhibit various mitochondrial abnormalities. However, the underlying molecular mechanisms remain elusive. Here, we examined the mechanisms through which CFTR regulates Bcl-2 family proteins, which in turn regulate permeabilization of the mitochondrial outer membrane. Notably, inhibition of CFTR activated Bax and Bad, but inhibited Bcl-2. Moreover, degradation of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT increased significantly in CFTR-knockdown cells. Dysfunction of CFTR decreased heat-shock protein 90 (Hsp90) mRNA levels, and CFTR was found to interact with Hsp90. Inhibition of Hsp90 by SNX-2112 induced the degradation of phosphorylated AKT and ERK1/2 in Caco2 and HRT18 cells. These findings may help provide insights into the physiological role of CFTR in CF-related diseases.


Assuntos
Neoplasias Colorretais/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células CACO-2 , Neoplasias Colorretais/patologia , Fibrose Cística/genética , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Mitocôndrias/metabolismo , Fosforilação , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução Genética , Regulação para Cima/genética , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismo
19.
Gene Ther ; 26(6): 240-249, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30962536

RESUMO

Adeno-associated virus (AAV) has been investigated to transfer the cystic fibrosis transmembrane conductance regulator (CFTR) to airways. Inhaled AAV2-CFTR in people with cystic fibrosis (CF) is safe, but inefficient. In vitro, AAV2 transduction of human airway epithelia on the apical (luminal) side is inefficient, but efficient basolaterally. We previously selected AAV2.5T, a novel capsid that apically transduces CF human airway epithelia and efficiently restores CFTR function. We hypothesize the AAV receptor (AAVR) is basolaterally localized, and that AAV2.5T utilizes an alternative apical receptor. We found AAVR in human airway epithelia by western blot and RNA-Seq analyses. Using immunocytochemistry we did not find endogenous AAVR at membranes but overexpression localized AAVR to the basolateral membrane, where it preferentially increased transduction. Anti-AAVR antibodies blocked transduction by AAV2 from the basolateral side but not AAV2.5T from the apical side, suggesting a unique apical receptor. Finally, we found infection by AAV2 but not AAV2.5T was blocked by CRISPR knockout of AAVR in cell lines. Our data suggest the absence of apical AAVR is rate limiting for AAV2, and efficient transduction by AAV2.5T is accomplished using an AAVR independent pathway. Our findings inform the development of gene therapy for CF, and AAV vectors in general.


Assuntos
Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Receptores de Superfície Celular/genética , Transfecção/métodos , Linhagem Celular , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Receptores de Superfície Celular/metabolismo , Mucosa Respiratória/metabolismo
20.
Nat Commun ; 10(1): 1763, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30992452

RESUMO

Personalized approaches for systematically assessing ciliary beat dynamics and for drug testing would improve the challenging task of diagnosing and treating respiratory disorders. In this pilot study, we show how multiscale differential dynamic microscopy (multi-DDM) can be used to characterize collective ciliary beating in a non-biased automated manner. We use multi-DDM to assess the efficacy of different CFTR-modulating drugs in human airway epithelial cells derived from subjects with cystic fibrosis (ΔF508/ΔF508 and ∆F508/-) based on ciliary beat frequency and coordination. Similar to clinical observations, drug efficacy is variable across donors, even within the same genotype. We show how our assay can quantitatively identify the most efficient drugs for restoring ciliary beating for each individual donor. Multi-DDM provides insight into ciliary beating responses following treatment with drugs, and has application in the broader context of respiratory disease and for drug screening.


Assuntos
Brônquios/metabolismo , Cílios/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/metabolismo , Algoritmos , Aminofenóis/química , Aminopiridinas/química , Benzodioxóis/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Progressão da Doença , Células Epiteliais/metabolismo , Genótipo , Humanos , Microscopia , Oscilometria , Fenótipo , Quinolonas/química , Gravação em Vídeo
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