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1.
Molecules ; 26(9)2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-33946490

RESUMO

The cystic fibrosis transmembrane conductance regulator (CFTR) gene is influenced by the fundamental cellular processes like epithelial differentiation/polarization, regeneration and epithelial-mesenchymal transition. Defects in CFTR protein levels and/or function lead to decreased airway surface liquid layer facilitating microbial colonization and inflammation. The SERPINA1 gene, encoding alpha1-antitrypsin (AAT) protein, is one of the genes implicated in CF, however it remains unknown whether AAT has any influence on CFTR levels. In this study we assessed CFTR protein levels in primary human lung epithelial cells grown at the air-liquid-interface (ALI) alone or pre-incubated with AAT by Western blots and immunohistochemistry. Histological analysis of ALI inserts revealed CFTR- and AAT-positive cells but no AAT-CFTR co-localization. When 0.5 mg/mL of AAT was added to apical or basolateral compartments of pro-inflammatory activated ALI cultures, CFTR levels increased relative to activated ALIs. This finding suggests that AAT is CFTR-modulating protein, albeit its effects may depend on the concentration and the route of administration. Human lung epithelial ALI cultures provide a useful tool for studies in detail how AAT or other pharmaceuticals affect the levels and activity of CFTR.


Assuntos
Barreira Alveolocapilar/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Mucosa Respiratória/metabolismo , alfa 1-Antitripsina/metabolismo , Biomarcadores , Linhagem Celular , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/metabolismo , Imunofluorescência , Humanos , Imuno-Histoquímica , alfa 1-Antitripsina/genética
2.
Medicine (Baltimore) ; 100(16): e25246, 2021 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-33879658

RESUMO

ABSTRACT: Lung adenocarcinoma (LUAD) is a lethal malignancy worldwide and a major public health concern. We explored the potential clinical significance for LUAD of ATP-binding cassette (ABC), sub-family C, consisting of ABCC1-6, 8-12, and cystic fibrosis transmembrane conductance regulator (CFTR).Five hundred LUAD patients from The Cancer Genome Atlas database were used for analysis, including differential expression and diagnostic and prognostic significance. Oncomine and MERAV databases were used to validate differential expression and diagnostic significance. A risk score model was constructed using prognosis-related ABCC members. Prognosis-related genes were further explored to correlate their expression with tumor stage progression. Interaction networks, including biological processes and metabolic pathways, were constructed using Cytoscape software and STRING website.ABCC1-3 consistently showed high expression in tumor tissues (all P ≤ 0.05). Most datasets indicated that ABCC5, 10, and 11 were highly expressed in tumor tissues whereas ABCC6, 9, and CFTR were highly expressed in nontumor tissues (all P ≤ 0.05). Diagnostic significance of ABCC3 and ABCC5 was consistently assessed and validated in three datasets (all area under the curve > 0.700) whereas ABCC6, 8, 10, 11, and CFTR were assessed in The Cancer Genome Atlas dataset and validated in one dataset (all area under the curve > 0.700). Prognostic analysis indicated that ABCC2, 6, and 8 mRNA expression was associated with survival of LUAD (all adjusted P ≤ .037). The risk score model constructed using ABCC2, 6, and 8 suggested prognostic significance for survival predictions. ABCC2 expression was associated with tumor stage, whereas ABCC6 and 8 were not. Interaction networks indicated that they were involved in establishment of localization, ion transport, plasma membrane, apical plasma membrane, adenylyl nucleotide binding, ABC transporters, ABC transporter disorders, ABC-family-protein-mediated transport, and bile secretion.Differentially expressed ABCC2 and ABCC5 might be diagnostic whereas ABCC2, 6, and 8 may be prognostic biomarkers for LUAD, possibly through ABC-family-mediated transporter disorders.


Assuntos
Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/mortalidade , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Idoso , Biomarcadores Tumorais/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Bases de Dados Genéticas , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Prognóstico , Medição de Risco , Fatores de Risco , Análise de Sobrevida
3.
Int J Mol Sci ; 22(9)2021 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-33923202

RESUMO

The emergence of highly effective CFTR modulator therapy has led to significant improvements in health care for most patients with cystic fibrosis (CF). For some, however, these therapies remain inaccessible due to the rarity of their individual CFTR variants, or due to a lack of biologic activity of the available therapies for certain variants. One proposed method of addressing this gap is the use of primary human cell-based models, which allow preclinical therapeutic testing and physiologic assessment of relevant tissue at the individual level. Nasal cells represent one such tissue source and have emerged as a powerful model for individual disease study. The ex vivo culture of nasal cells has evolved over time, and modern nasal cell models are beginning to be utilized to predict patient outcomes. This review will discuss both historical and current state-of-the art use of nasal cells for study in CF, with a particular focus on the use of such models to inform personalized patient care.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/patologia , Células Epiteliais/patologia , Modelos Biológicos , Mucosa Nasal/patologia , Medicina de Precisão , Animais , Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Humanos , Mucosa Nasal/metabolismo
4.
Int J Mol Sci ; 22(7)2021 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-33806154

RESUMO

Knowledge on the mechanisms of acid and base secretion in airways has progressed recently. The aim of this review is to summarize the known mechanisms of airway surface liquid (ASL) pH regulation and their implication in lung diseases. Normal ASL is slightly acidic relative to the interstitium, and defects in ASL pH regulation are associated with various respiratory diseases, such as cystic fibrosis. Basolateral bicarbonate (HCO3-) entry occurs via the electrogenic, coupled transport of sodium (Na+) and HCO3-, and, together with carbonic anhydrase enzymatic activity, provides HCO3- for apical secretion. The latter mainly involves CFTR, the apical chloride/bicarbonate exchanger pendrin and paracellular transport. Proton (H+) secretion into ASL is crucial to maintain its relative acidity compared to the blood. This is enabled by H+ apical secretion, mainly involving H+/K+ ATPase and vacuolar H+-ATPase that carry H+ against the electrochemical potential gradient. Paracellular HCO3- transport, the direction of which depends on the ASL pH value, acts as an ASL protective buffering mechanism. How the transepithelial transport of H+ and HCO3- is coordinated to tightly regulate ASL pH remains poorly understood, and should be the focus of new studies.


Assuntos
Bicarbonatos/química , Anidrases Carbônicas/metabolismo , Epitélio/metabolismo , Mucosa Respiratória/metabolismo , Animais , Antiporters/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Coelhos , Transportadores de Sulfato/metabolismo , Traqueia/metabolismo
5.
Nutrients ; 13(5)2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33919435

RESUMO

The purpose of this study was to compare the nutritional status between deltaF508 CFTR hetero- and homozygous paediatric patients with cystic fibrosis. We assessed the percentage profiles of fatty acids measured in erythrocyte membranes and the serum levels of vitamins A, D3, E and K1 in the studied groups. We also measured the weights and heights and calculated the body mass indexes (BMIs). The studied groups consisted of 34 heterozygous and 30 homozygous patients. No statistically significant differences were found in the serum vitamins or erythrocyte membrane fatty acid profiles between the hetero- and homozygous patient groups, except for heptadecanoic acid (p = 0.038). The mean percentiles of height, weight and BMI did not differ significantly between the two groups. The homozygous and heterozygous paediatric patients with cystic fibrosis were similar in terms of their nutritional statuses.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Estado Nutricional , Adolescente , Índice de Massa Corporal , Estudos de Casos e Controles , Criança , Pré-Escolar , Fibrose Cística/sangue , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Membrana Eritrocítica/metabolismo , Ácidos Graxos/sangue , Heterozigoto , Homozigoto , Humanos , Lactente , Mutação/genética , Vitaminas/sangue
6.
Nutrients ; 13(4)2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33917155

RESUMO

Exposure to gluten, a protein present in wheat rye and barley, is the major inducer for human Celiac Disease (CD), a chronic autoimmune enteropathy. CD occurs in about 1% worldwide population, in genetically predisposed individuals bearing human leukocyte antigen (HLA) DQ2/DQ8. Gut epithelial cell stress and the innate immune activation are responsible for the breaking oral tolerance to gliadin, a gluten component. To date, the only treatment available for CD is a long-term gluten-free diet. Several studies have shown that an altered composition of the intestinal microbiota (dysbiosis) could play a key role in the pathogenesis of CD through the modulation of intestinal permeability and the regulation of the immune system. Here, we show that gliadin induces a chronic endoplasmic reticulum (ER) stress condition in the small intestine of a gluten-sensitive mouse model and that the coadministration of probiotics efficiently attenuates both the unfolded protein response (UPR) and gut inflammation. Moreover, the composition of probiotics formulations might differ in their activity at molecular level, especially toward the three axes of the UPR. Therefore, probiotics administration might potentially represent a new valuable strategy to treat gluten-sensitive patients, such as those affected by CD.


Assuntos
Suplementos Nutricionais , Estresse do Retículo Endoplasmático , Intolerância Alimentar/terapia , Trato Gastrointestinal/patologia , Gliadina/efeitos adversos , Glutens/efeitos adversos , Inflamação/patologia , Probióticos/uso terapêutico , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Células CACO-2 , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Trato Gastrointestinal/efeitos dos fármacos , Humanos , Camundongos Endogâmicos BALB C , Permeabilidade , Probióticos/administração & dosagem , Transglutaminases/metabolismo , Regulação para Cima
7.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33802742

RESUMO

New anti-inflammatory treatments are needed for CF airway disease. Studies have implicated the endoplasmic reticulum stress transducer inositol requiring enzyme 1α (IRE1α) in CF airway inflammation. The activation of IRE1α promotes activation of its cytoplasmic kinase and RNase, resulting in mRNA splicing of X-box binding protein-1 (XBP-1s), a transcription factor required for cytokine production. We tested whether IRE1α kinase and RNase inhibition decreases cytokine production induced by the exposure of primary cultures of homozygous F508del CF human bronchial epithelia (HBE) to supernatant of mucopurulent material (SMM) from CF airways. We evaluated whether IRE1α expression is increased in freshly isolated and native CF HBE, and couples with increased XBP-1s levels. A FRET assay confirmed binding of the IRE1α kinase and RNase inhibitor, KIRA6, to the IRE1α kinase. F508del HBE cultures were exposed to SMM with or without KIRA6, and we evaluated the mRNA levels of XBP-1s, IL-6, and IL-8, and the secretion of IL-6 and IL-8. IRE1α mRNA levels were up-regulated in freshly isolated CF vs. normal HBE and coupled to increased XBP-1s mRNA levels. SMM increased XBP-1s, IL-6, and IL-8 mRNA levels and up-regulated IL-6 and IL-8 secretion, and KIRA6 blunted these responses in a dose-dependent manner. Moreover, a triple combination of CFTR modulators currently used in the clinic had no effect on SMM-increased XBP-1s levels coupled with increased cytokine production in presence or absence of KIRA6. These findings indicate that IRE1α mediates cytokine production in CF airways. Small molecule IRE1α kinase inhibitors that allosterically reduce RNase-dependent XBP-1s may represent a new therapeutic strategy for CF airway inflammation.


Assuntos
Fibrose Cística/tratamento farmacológico , Fibrose Cística/patologia , Endorribonucleases/metabolismo , Inflamação/tratamento farmacológico , Inflamação/patologia , Pulmão/patologia , Terapia de Alvo Molecular , Proteínas Serina-Treonina Quinases/metabolismo , Células Cultivadas , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Citocinas/biossíntese , Endorribonucleases/genética , Epitélio/efeitos dos fármacos , Epitélio/patologia , Humanos , Imidazóis/química , Imidazóis/farmacologia , Inflamação/genética , Modelos Biológicos , Naftalenos/química , Naftalenos/farmacologia , Proteínas Serina-Treonina Quinases/genética , Pirazinas/química , Pirazinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Proteína 1 de Ligação a X-Box/metabolismo
8.
Int J Mol Sci ; 22(5)2021 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-33800499

RESUMO

While approximately 2000 mutations have been discovered in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), only a small amount (about 10%) is associated with clinical cystic fibrosis (CF) disease. The discovery of the association between CFTR and the hyperactive epithelial sodium channel (ENaC) has raised the question of the influence of ENaC on the clinical CF phenotype. ENaC disturbance contributes to the pathological secretion, and overexpression of one ENaC subunit, the ß-unit, can give a CF-like phenotype in mice with normal acting CFTR. The development of ENaC channel modulators is now in progress. Both CFTR and ENaC are located in the cell membrane and are influenced by its lipid configuration. Recent studies have emphasized the importance of the interaction of lipids and these proteins in the membranes. Linoleic acid deficiency is the most prevailing lipid abnormality in CF, and linoleic acid is an important constituent of membranes. The influence on sodium excretion by linoleic acid supplementation indicates that lipid-protein interaction is of importance for the clinical pathophysiology in CF. Further studies of this association can imply a simple clinical adjuvant in CF therapy.


Assuntos
Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Canais Epiteliais de Sódio/metabolismo , Ácido Linoleico/deficiência , Animais , Membrana Celular/genética , Membrana Celular/patologia , Fibrose Cística/genética , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Canais Epiteliais de Sódio/genética , Humanos , Ácido Linoleico/metabolismo , Camundongos
9.
Am J Physiol Lung Cell Mol Physiol ; 320(5): L845-L879, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33655758

RESUMO

Airway submucosal gland serous cells are important sites of fluid secretion in conducting airways. Serous cells also express the cystic fibrosis (CF) transmembrane conductance regulator (CFTR). Protease-activated receptor 2 (PAR-2) is a G protein-coupled receptor that activates secretion from intact airway glands. We tested if and how human nasal serous cells secrete fluid in response to PAR-2 stimulation using Ca2+ imaging and simultaneous differential interference contrast imaging to track isosmotic cell shrinking and swelling reflecting activation of solute efflux and influx pathways, respectively. During stimulation of PAR-2, serous cells exhibited dose-dependent increases in intracellular Ca2+. At stimulation levels >EC50 for Ca2+, serous cells simultaneously shrank ∼20% over ∼90 s due to KCl efflux reflecting Ca2+-activated Cl- channel (CaCC, likely TMEM16A)-dependent secretion. At lower levels of PAR-2 stimulation (

Assuntos
Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Glândulas Exócrinas/patologia , Infecções por Pseudomonas/microbiologia , Receptor PAR-2/metabolismo , Mucosa Respiratória/patologia , Membrana Serosa/patologia , Cálcio/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Glândulas Exócrinas/metabolismo , Glândulas Exócrinas/microbiologia , Humanos , Pseudomonas aeruginosa/isolamento & purificação , Mucosa Respiratória/metabolismo , Mucosa Respiratória/microbiologia , Membrana Serosa/metabolismo , Membrana Serosa/microbiologia
10.
Int J Mol Sci ; 22(4)2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33669352

RESUMO

Cystic fibrosis (CF) is an inherited disorder caused by mutations in the gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ATP-gated chloride channel expressed on the apical surface of airway epithelial cells. CFTR absence/dysfunction results in defective ion transport and subsequent airway surface liquid dehydration that severely compromise the airway microenvironment. Noxious agents and pathogens are entrapped inside the abnormally thick mucus layer and establish a highly inflammatory environment, ultimately leading to lung damage. Since chronic airway inflammation plays a crucial role in CF pathophysiology, several studies have investigated the mechanisms responsible for the altered inflammatory/immune response that, in turn, exacerbates the epithelial dysfunction and infection susceptibility in CF patients. In this review, we address the evidence for a critical role of dysfunctional inflammation in lung damage in CF and discuss current therapeutic approaches targeting this condition, as well as potential new treatments that have been developed recently. Traditional therapeutic strategies have shown several limitations and limited clinical benefits. Therefore, many efforts have been made to develop alternative treatments and novel therapeutic approaches, and recent findings have identified new molecules as potential anti-inflammatory agents that may exert beneficial effects in CF patients. Furthermore, the potential anti-inflammatory properties of CFTR modulators, a class of drugs that directly target the molecular defect of CF, also will be critically reviewed. Finally, we also will discuss the possible impact of SARS-CoV-2 infection on CF patients, with a major focus on the consequences that the viral infection could have on the persistent inflammation in these patients.


Assuntos
Anti-Inflamatórios/uso terapêutico , Fibrose Cística/tratamento farmacológico , Inflamação/tratamento farmacológico , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Anti-Inflamatórios/farmacologia , Azitromicina/farmacologia , Azitromicina/uso terapêutico , COVID-19/complicações , COVID-19/tratamento farmacológico , COVID-19/metabolismo , Canabinoides/farmacologia , Canabinoides/uso terapêutico , Fibrose Cística/complicações , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Eicosanoides/metabolismo , Humanos , Inflamação/complicações , Inflamação/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Roscovitina/farmacologia , Roscovitina/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Timalfasina/farmacologia , Timalfasina/uso terapêutico
11.
Eur J Med Chem ; 213: 113195, 2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33524685

RESUMO

Cystic fibrosis (CF) is the most frequent life-limiting autosomal recessive disorder in the Caucasian population. It is due to mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Current symptomatic CF therapies, which treat the downstream consequences of CFTR mutations, have increased survival. Better knowledge of the CFTR protein has enabled pharmacologic therapy aiming to restore mutated CFTR expression and function. These CFTR "modulators" have revolutionised the CF therapeutic landscape, with the potential to transform prognosis for a considerable number of patients. This review provides a brief summary of their mechanism of action and presents a thorough review of the results obtained from clinical trials of CFTR modulators.


Assuntos
Aminofenóis/farmacologia , Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/tratamento farmacológico , Desenvolvimento de Medicamentos , Indóis/farmacologia , Quinolonas/farmacologia , Aminofenóis/síntese química , Aminofenóis/química , Aminopiridinas/síntese química , Benzodioxóis/síntese química , Ensaios Clínicos como Assunto , Fibrose Cística/diagnóstico , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Indóis/síntese química , Quinolonas/síntese química , Quinolonas/química
12.
Eur J Med Chem ; 213: 113186, 2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33472120

RESUMO

Computational drug repositioning is of growing interest to academia and industry, for its ability to rapidly screen a huge number of candidates in silico (exploiting comprehensive drug datasets) together with reduced development cost and time. The potential of drug repositioning has not been fully evaluated yet for cystic fibrosis (CF), a disease mainly caused by deletion of Phe 508 (F508del) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. F508del-CFTR is thus withheld in the endoplasmic reticulum and rapidly degraded by the ubiquitin/proteasome system. CF is still a fatal disease. Nowadays, it is treatable by some CFTR-rescuing drugs, but new-generation drugs with stronger therapeutic benefits and fewer side effects are still awaited. In this manuscript we report about the results of a pilot computational drug repositioning screening in search of F508del-CFTR-targeted drugs performed on AIFA library by means of a dedicated computational pipeline and surface plasmon resonance binding assay to experimentally validate the computational findings.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Fenilalanina/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Conjuntos de Dados como Assunto , Relação Dose-Resposta a Droga , Reposicionamento de Medicamentos , Humanos , Estrutura Molecular , Fenilalanina/metabolismo , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
13.
Gene ; 765: 145045, 2021 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-32777524

RESUMO

To find the variant spectrum of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and evaluate its frequent variants in Chinese congenital absence of vas deferens (CAVD) patients. A total of 276 patients with azoospermia and CAVD (aged from 21 to 44 years old) were investigated from May 2013 to September 2019 in the Third Affiliated Hospital of Sun Yat-sen University. Additionally, 50 healthy, unrelated volunteers were recruited as controls (aged from 21 to 46 years old). The 5'-UTR, exons and their flanking side of the CFTR gene were sequenced by high-throughput sequencing technology. The results were compared with those retrieved from the Ensembl Genome Browser. In addition, all 13 novel variants were further confirmed independently by Sanger sequencing and evaluated in the bioinformatics web servers. A schematic of the variant spectrum of the CFTR gene, including 13 novel variants (12 in CAVD patients, one in the control group), is shown, and the frequent variants in Chinese CAVD patients were 5 T (27.54%), c.-8G > C (7.25%), p.Q1352H (5.98%), and p.I556V (3.08%). 5 T was found to be the most frequent variant. p.Q1352H had a significantly high allelic frequency in CAVD patients (P < 0.05). c.-8G > C and p.I556V had high allelic frequencies but showed no difference between patients and controls (P > 0.05). p.Q1352H is the most common and important missense variant in Chinese patients with CAVD, while the pathological effects of C.-8G > C and p.I556V may be weak after evaluation.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Doenças Urogenitais Masculinas/genética , Ducto Deferente/anormalidades , Adulto , Alelos , Grupo com Ancestrais do Continente Asiático/genética , Azoospermia/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Análise Mutacional de DNA/métodos , Éxons/genética , Frequência do Gene/genética , Humanos , Infertilidade Masculina/genética , Masculino , Doenças Urogenitais Masculinas/metabolismo , Mutação/genética , Ducto Deferente/metabolismo
14.
J Med Chem ; 63(24): 15773-15784, 2020 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-33314931

RESUMO

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, encoding for a chloride ion channel. Membrane expression of CFTR is negatively regulated by CFTR-associated ligand (CAL). We previously showed that inhibition of the CFTR/CAL interaction with a cell-permeable peptide improves the function of rescued F508del-CFTR. In this study, optimization of the peptidyl inhibitor yielded PGD97, which exhibits a KD value of 6 nM for the CAL PDZ domain, ≥ 130-fold selectivity over closely related PDZ domains, and a serum t1/2 of >24 h. In patient-derived F508del homozygous cells, PGD97 (100 nM) increased short-circuit currents by ∼3-fold and further potentiated the therapeutic effects of small-molecule correctors (e.g., VX-661) by ∼2-fold (with an EC50 of ∼10 nM). Our results suggest that PGD97 may be used as a novel treatment for CF, either as a single agent or in combination with small-molecule correctors/potentiators.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fibrose Cística/tratamento farmacológico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Estabilidade de Medicamentos , Humanos , Cinética , Ligantes , Simulação de Acoplamento Molecular , Mutação , Domínios PDZ , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacologia , Permeabilidade/efeitos dos fármacos , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/metabolismo
15.
Artigo em Inglês | MEDLINE | ID: mdl-32750662

RESUMO

Cystic fibrosis (CF) is a recessively inherited fatal disease that is the subject of extensive research and ongoing development of therapeutics targeting the defective protein, cystic fibrosis transmembrane conductance regulator (CFTR). Despite progress, the link between CFTR and clinical symptoms is incomplete. The severe CF phenotypes are associated with a deficiency of linoleic acid, which is the precursor of arachidonic acid. The release of arachidonic acid from membranes via phospholipase A2 is the rate-limiting step for eicosanoid synthesis and is increased in CF, which contributes to the observed inflammation. A potential deficiency of docosahexaenoic acid may lead to decreased levels of specialized pro-resolving mediators. This pathophysiology may contribute to an early and sterile inflammation, mucus production, and to bacterial colonization, which further increases inflammation and potentiates the clinical symptoms. Advances in lipid technology will assist in elucidating the role of lipid metabolism in CF, and stimulate therapeutic modulations of inflammation.


Assuntos
Ácido Araquidônico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Ácidos Docosa-Hexaenoicos/deficiência , Ácido Linoleico/deficiência , Ácido Araquidônico/deficiência , Fibrose Cística/fisiopatologia , Humanos , Inflamação/metabolismo , Ácido Linoleico/metabolismo , Metabolismo dos Lipídeos , Pulmão/metabolismo , Pulmão/microbiologia , Pulmão/fisiopatologia , Muco/metabolismo
16.
Phytochemistry ; 179: 112464, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32798746

RESUMO

A phytochemical study on the root extracts of Neorautanenia mitis, a Nigerian medicinal plant used in the management of diarrhea, led to the isolation of one new and 19 known natural products. These compounds and crude extracts were evaluated for Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl- channel and calcium-activated Cl- channel (TMEM16A) inhibitory activities in T84 and Calu-3 cells, respectively. Four compounds namely dolineon, neodulin, pachyrrhizine, and neotenone inhibited cAMP-induced Cl- secretion across T84 cell monolayers with IC50 values of ~0.81 µM, ~2.42 µM, ~2.87 µM, and ~4.66 µM, respectively. Dolineon having the highest inhibitory activity also inhibited a Ca + activated Cl- channel (TMEM16A) with an IC50 value of ~4.38 µM. The in vitro antidiarrheal activity of dolineon was evaluated on cholera toxin (CT) induced chloride secretion in T84 cells, where it inhibited CT-induced chloride secretion by >70% at 100 µM. Dolineon also inhibited CT-induced fluid secretion by ~70% in an in vivo mouse closed loop model at a dose of 16.9 µg/loop. The cytotoxicity of the extracts and compounds was evaluated on KB, Vero and BHK21 cells, dolineon showed low cytotoxicity of >29.6 µM and 57.30 + 6.77 µM against Vero and BHK21 cells, respectively. Our study revealed that several compounds isolated from N. mitis showed antidiarrheal activity. The most active compound dolineon can potentially serve as a lead compound towards the development of CFTR and TMEM16A inhibitors as future therapeutics for secretory diarrhea.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Chumbo , Animais , Transporte Biológico , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Diarreia/tratamento farmacológico , Camundongos
17.
Proc Natl Acad Sci U S A ; 117(35): 21740-21746, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817533

RESUMO

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel is essential for epithelial salt-water balance. CFTR mutations cause cystic fibrosis, a lethal incurable disease. In cells CFTR is activated through the cAMP signaling pathway, overstimulation of which during cholera leads to CFTR-mediated intestinal salt-water loss. Channel activation is achieved by phosphorylation of its regulatory (R) domain by cAMP-dependent protein kinase catalytic subunit (PKA). Here we show using two independent approaches--an ATP analog that can drive CFTR channel gating but is unsuitable for phosphotransfer by PKA, and CFTR mutants lacking phosphorylatable serines--that PKA efficiently opens CFTR channels through simple binding, under conditions that preclude phosphorylation. Unlike when phosphorylation happens, CFTR activation by PKA binding is completely reversible. Thus, PKA binding promotes release of the unphosphorylated R domain from its inhibitory position, causing full channel activation, whereas phosphorylation serves only to maintain channel activity beyond termination of the PKA signal. The results suggest two levels of CFTR regulation in cells: irreversible through phosphorylation, and reversible through R-domain binding to PKA--and possibly also to other members of a large network of proteins known to interact with the channel.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Ânions/metabolismo , Fenômenos Biofísicos , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Ativação do Canal Iônico/fisiologia , Mutagênese Sítio-Dirigida , Nucleotídeos/metabolismo , Oócitos/metabolismo , Técnicas de Patch-Clamp/métodos , Fosforilação , Ligação Proteica/fisiologia , Serina/metabolismo , Canais de Ânion Dependentes de Voltagem/fisiologia , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo
18.
Cells ; 9(9)2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32847034

RESUMO

The preservation of cellular homeostasis requires the synthesis of new proteins (proteostasis) and organelles, and the effective removal of misfolded or impaired proteins and cellular debris. This cellular homeostasis involves two key proteostasis mechanisms, the ubiquitin proteasome system and the autophagy-lysosome pathway. These catabolic pathways have been known to be involved in respiratory exacerbations and the pathogenesis of various lung diseases, such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), idiopathic pulmonary fibrosis (IPF), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and coronavirus disease-2019 (COVID-19). Briefly, proteostasis and autophagy processes are known to decline over time with age, cigarette or biomass smoke exposure, and/or influenced by underlying genetic factors, resulting in the accumulation of misfolded proteins and cellular debris, elevating apoptosis and cellular senescence, and initiating the pathogenesis of acute or chronic lung disease. Moreover, autophagic dysfunction results in an impaired microbial clearance, post-bacterial and/or viral infection(s) which contribute to the initiation of acute and recurrent respiratory exacerbations as well as the progression of chronic obstructive and restrictive lung diseases. In addition, the autophagic dysfunction-mediated cystic fibrosis transmembrane conductance regulator (CFTR) immune response impairment further exacerbates the lung disease. Recent studies demonstrate the therapeutic potential of novel autophagy augmentation strategies, in alleviating the pathogenesis of chronic obstructive or restrictive lung diseases and exacerbations such as those commonly seen in COPD, CF, ALI/ARDS and COVID-19.


Assuntos
Autofagia/imunologia , Betacoronavirus , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/metabolismo , Progressão da Doença , Pneumonia Viral/imunologia , Pneumonia Viral/metabolismo , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/metabolismo , Infecções por Coronavirus/virologia , Fibrose Cística/imunologia , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Homeostase , Humanos , Fibrose Pulmonar Idiopática/imunologia , Fibrose Pulmonar Idiopática/metabolismo , Lisossomos/metabolismo , Pandemias , Pneumonia Viral/virologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , /metabolismo
19.
Nat Commun ; 11(1): 4258, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848127

RESUMO

Protein misfolding causes a wide spectrum of human disease, and therapies that target misfolding are transforming the clinical care of cystic fibrosis. Despite this success, however, very little is known about how disease-causing mutations affect the de novo folding landscape. Here we show that inherited, disease-causing mutations located within the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) have distinct effects on nascent polypeptides. Two of these mutations (A455E and L558S) delay compaction of the nascent NBD1 during a critical window of synthesis. The observed folding defect is highly dependent on nascent chain length as well as its attachment to the ribosome. Moreover, restoration of the NBD1 cotranslational folding defect by second site suppressor mutations also partially restores folding of full-length CFTR. These findings demonstrate that nascent folding intermediates can play an important role in disease pathogenesis and thus provide potential targets for pharmacological correction.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Mutação , Substituição de Aminoácidos , Sítios de Ligação/genética , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Células HEK293 , Humanos , Técnicas In Vitro , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Domínios Proteicos , Dobramento de Proteína , Modificação Traducional de Proteínas/genética , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribossomos/metabolismo , Supressão Genética , Temperatura
20.
Genes Immun ; 21(4): 260-262, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32606316

RESUMO

Cystic fibrosis (CF) is one of the most common autosomal recessive life-limiting conditions affecting Caucasians. The resulting defect in the cystic fibrosis transmembrane conductance regulator protein (CFTR) results in defective chloride and bicarbonate secretion, as well as dysregulation of epithelial sodium channels (ENaC). These changes bring about defective mucociliary clearance, reduced airway surface liquid and an exaggerated proinflammatory response driven, in part, by infection. In this short article we explore the overlap in the pathophysiology of CF and COVID-19 infection and discuss how understanding the interaction between both diseases may shed light on future treatments.


Assuntos
Infecções por Coronavirus/metabolismo , Fibrose Cística/metabolismo , Pneumonia Viral/metabolismo , Animais , Infecções por Coronavirus/complicações , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Fibrose Cística/complicações , Fibrose Cística/tratamento farmacológico , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Citocinas/metabolismo , Humanos , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/complicações , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia
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