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Nonsense mutations account for 12 % of cystic fibrosis (CF) cases. The presence of a premature termination codon (PTC) leads to gene inactivation, which can be countered by the use of drugs stimulating PTC readthrough, restoring production of the full-length protein. We recently identified a new readthrough inducer, TLN468, more efficient than gentamicin. We measured the readthrough induced by these two drugs with different cystic fibrosis transmembrane conductance regulator (CFTR) PTCs. We then determined the amino acids inserted at the S1196X, G542X, W846X and E1417X PTCs of CFTR during readthrough induced by gentamicin or TLN468. TLN468 significantly promoted the incorporation of one specific amino acid, whereas gentamicin did not greatly modify the proportions of the various amino acids incorporated relative to basal conditions. The function of the engineered missense CFTR channels corresponding to these four PTCs was assessed with and without potentiator. For the recoded CFTR, except for E1417Q and G542W, the PTC readthrough induced by TLN468 allowed the expression of CFTR variants that were correctly processed and had significant activity that was enhanced by CFTR modulators. These results suggest that it would be relevant to assess the therapeutic benefit of TLN468 PTC suppression in combination with CFTR modulators in preclinical assays.
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Over time, cystic fibrosis has become a model of synergy between research in pathophysiology and cell biology, and clinical advances. Therapies targeting the CFTR protein, in particular CFTR modulators, have transformed the prognosis of patients, bringing the hope of a normal life with the possibility of starting a family and growing old, challenging established statistics. However, patients are not yet cured, and side effects remain insufficiently documented. Epidemiological changes create new challenges for the management of cystic fibrosis. Approximately 10 % of patients still lack a therapeutic option. The community of researchers, pharmaceutical industries, patient associations, and health authorities remains committed to monitor the long-term effects of these still poorly characterised treatments, and to explore new pharmacological approaches, such as gene therapies.
Title: Traitements de la mucoviscidose - Révolution clinique et nouveaux défis. Abstract: Avec le temps, la mucoviscidose est devenue un exemple de synergie entre la recherche en biologie cellulaire et les progrès cliniques. Les thérapies protéiques ont enfin apporté l'espoir d'une vie normale aux patients, bouleversant ainsi les statistiques épidémiologiques établies. Néanmoins, les patients ne guérissent pas, et l'évolution épidémiologique de la maladie ouvre de nouveaux défis pour la prise en charge des malades. Par ailleurs, environ 10 % des patients demeurent sans solution thérapeutique. De nouvelles stratégies sont ainsi envisagées et la communauté des chercheurs, industriels, patients et autorités de santé reste mobilisée pour suivre les effets à long terme de ces nouveaux traitements et explorer de nouvelles approches pharmacologiques.
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Fibrosis Quística , Humanos , Fibrosis Quística/genética , Fibrosis Quística/terapia , Mutación , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Terapia GenéticaRESUMEN
Podocin is a key membrane scaffolding protein of the kidney podocyte essential for intact glomerular filtration. Mutations in NPHS2, the podocin-encoding gene, represent the commonest form of inherited nephrotic syndrome (NS), with early, intractable kidney failure. The most frequent podocin gene mutation in European children is R138Q, causing retention of the misfolded protein in the endoplasmic reticulum. Here, we provide evidence that podocin R138Q (but not wild-type podocin) complexes with the intermediate filament protein keratin 8 (K8) thereby preventing its correct trafficking to the plasma membrane. We have also identified a small molecule (c407), a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator protein defect, that interrupts this complex and rescues mutant protein mistrafficking. This results in both the correct localization of podocin at the plasma membrane and functional rescue in both human patient R138Q mutant podocyte cell lines, and in a mouse inducible knock-in model of the R138Q mutation. Importantly, complete rescue of proteinuria and histological changes was seen when c407 was administered both via osmotic minipumps or delivered orally prior to induction of disease or crucially via osmotic minipump two weeks after disease induction. Thus, our data constitute a therapeutic option for patients with NS bearing a podocin mutation, with implications for other misfolding protein disorders. Further studies are necessary to confirm our findings.
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Síndrome Nefrótico , Animales , Niño , Humanos , Ratones , Péptidos y Proteínas de Señalización Intracelular/genética , Queratina-8/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/genética , Mutación , Síndrome Nefrótico/tratamiento farmacológico , Síndrome Nefrótico/genética , Síndrome Nefrótico/patologíaRESUMEN
Introduction: Cystic fibrosis (CF) is caused by defective Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) proteins. CFTR controls chloride (Cl-) and bicarbonate (HCO3 -) transport into the Airway Surface Liquid (ASL). We investigated the impact of F508del-CFTR correction on HCO3 - secretion by studying transepithelial HCO3 - fluxes. Methods: HCO3 - secretion was measured by pH-stat technique in primary human respiratory epithelial cells from healthy subjects (WT) and people with CF (pwCF) carrying at least one F508del variant. Its changes after CFTR modulation by the triple combination VX445/661/770 and in the context of TNF-α+IL-17 induced inflammation were correlated to ASL pH and transcriptional levels of CFTR and other HCO3 - transporters of airway epithelia such as SLC26A4 (Pendrin), SLC26A9 and NBCe1. Results: CFTR-mediated HCO3 - secretion was not detected in F508del primary human respiratory epithelial cells. It was rescued up to â¼ 80% of the WT level by VX-445/661/770. In contrast, TNF-α+IL-17 normalized transepithelial HCO3 - transport and increased ASL pH. This was related to an increase in SLC26A4 and CFTR transcript levels. VX-445/661/770 induced an increase in pH only in the context of inflammation. Effects on HCO3 - transport were not different between F508del homozygous and F508del compound heterozygous CF airway epithelia. Conclusion: Our studies show that correction of F508del-CFTR HCO3 - is not sufficient to buffer acidic ASL and inflammation is a key regulator of HCO3 - secretion in CF airways. Prediction of the response to CFTR modulators by theratyping should take into account airway inflammation.
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BACKGROUND: Around 20% of people with cystic fibrosis (pwCF) do not have access to the triple combination elexacaftor/tezacaftor/ivacaftor (ETI) in Europe because they do not carry the F508del allele on the CF transmembrane conductance regulator (CFTR) gene. Considering that pwCF carrying rare variants may benefit from ETI, including variants already validated by the US Food and Drug Administration (FDA), a compassionate use programme was launched in France. PwCF were invited to undergo a nasal brushing to investigate whether the pharmacological rescue of CFTR activity by ETI in human nasal epithelial cell (HNEC) cultures was predictive of the clinical response. METHODS: CFTR activity correction was studied by short-circuit current in HNEC cultures at basal state (dimethyl sulfoxide (DMSO)) and after ETI incubation and expressed as percentage of normal (wild-type (WT)) CFTR activity after sequential addition of forskolin and Inh-172 (ΔI ETI/DMSO%WT). RESULTS: 11 pwCF carried variants eligible for ETI according to the FDA label and 28 carried variants not listed by the FDA. ETI significantly increased CFTR activity of FDA-approved CFTR variants (I601F, G85E, S492F, M1101K, R347P, R74W;V201M;D1270N and H1085R). We point out ETI correction of non-FDA-approved variants, including N1303K, R334W, R1066C, Q552P and terminal splicing variants (4374+1G>A and 4096-3C>G). ΔI ETI/DMSO%WT was significantly correlated to change in percentage predicted forced expiratory volume in 1â s and sweat chloride concentration (p<0.0001 for both). G85E, R74W;V201M;D1270N, Q552P and M1101K were rescued more efficiently by other CFTR modulator combinations than ETI. CONCLUSIONS: Primary nasal epithelial cells hold promise for expanding the prescription of CFTR modulators in pwCF carrying rare mutants. Additional variants should be discussed for ETI indication.
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Fibrosis Quística , Humanos , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Dimetilsulfóxido , MutaciónRESUMEN
RATIONALE: Limited information is available on the clinical status of people with Cystic Fibrosis (pwCF) carrying 2 nonsense mutations (PTC/PTC). The main objective of this study was to compare disease severity between pwCF PTC/PTC, compound heterozygous for F508del and PTC (F508del/PTC) and homozygous for F508del (F508del+/+). METHODS: Based on the European CF Society Patient Registry clinical data of pwCF living in high and middle income European and neighboring countries, PTC/PTC (n = 657) were compared with F508del+/+ (n = 21,317) and F508del/PTC(n = 4254).CFTR mRNA and protein activity levels were assessed in primary human nasal epithelial (HNE) cells sampled from 22 PTC/PTC pwCF. MAIN RESULTS: As compared to F508del+/+ pwCF; both PTC/PTC and F508del/PTC pwCF exhibited a significantly faster rate of decline in Forced Expiratory Volume in 1 s (FEV1) from 7 years (-1.33 for F508del +/+, -1.59 for F508del/PTC; -1.65 for PTC/PTC, p < 0.001) until respectively 30 years (-1.05 for F508del +/+, -1.23 for PTC/PTC, p = 0.048) and 27 years (-1.12 for F508del +/+, -1.26 for F508del/PTC, p = 0.034). This resulted in lower FEV1 values in adulthood. Mortality of pediatric pwCF with one or two PTC alleles was significantly higher than their F508del homozygous pairs. Infection with Pseudomonas aeruginosa was more frequent in PTC/PTC versus F508del+/+ and F508del/PTC pwCF. CFTR activity in PTC/PTC pwCF's HNE cells ranged between 0% to 3% of the wild-type level. CONCLUSIONS: Nonsense mutations decrease the survival and accelerate the course of respiratory disease in children and adolescents with Cystic Fibrosis.
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Fibrosis Quística , Adolescente , Humanos , Niño , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Codón sin Sentido , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Volumen Espiratorio Forzado , ARN Mensajero , MutaciónRESUMEN
Accumulation of senescent dermal fibroblasts drives skin aging. The reactivation of proliferation is one strategy to modulate cell senescence. Recently, we reported the exact chemical composition of the hydrophilic extract of Oenothera biennis cell cultures (ObHEx) and we showed its skin anti-aging properties. The aim of this work is to assess its biological effect specifically on cell senescence. ObHEx action has been evaluated on normal human dermal fibroblasts subjected to stress-induced premature senescence (SIPS) through an ultra-deep proteomic analysis, leading to the most global senescence-associated proteome so far. Mass spectrometry data show that the treatment with ObHEx re-establishes levels of crucial mitotic proteins, strongly downregulated in senescent cells. To validate our proteomics findings, we proved that ObHEx can, in part, restore the activity of 'senescence-associated-ß-galactosidase', the most common hallmark of senescent cells. Furthermore, to assess if the upregulation of mitotic protein levels translates into a cell cycle re-entry, FACS experiments have been carried out, demonstrating a small but significative reactivation of senescent cell proliferation by ObHEx. In conclusion, the deep senescence-associated global proteome profiling published here provides a panel of hundreds of proteins deregulated by SIPS that can be used by the community to further understand senescence and the effect of new potential modulators. Moreover, proteomics analysis pointed to a specific promitotic effect of ObHEx on senescent cells. Thus, we suggest ObHEx as a powerful adjuvant against senescence associated with skin aging.
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Oenothera biennis , Proteómica , Humanos , Fibroblastos/metabolismo , Senescencia Celular , Piel , Células CultivadasRESUMEN
ABC transporters are large membrane proteins sharing a complex architecture, which comprises two nucleotide-binding domains (NBDs) and two membrane-spanning domains (MSDs). These domains are susceptible to mutations affecting their folding and assembly. In the CFTR (ABCC7) protein, a groove has been highlighted in the MSD1 at the level of the membrane inner leaflet, containing both multiple mutations affecting folding and a binding site for pharmaco-chaperones that stabilize this region. This groove is also present in ABCB proteins, however it is covered by a short elbow helix, while in ABCC proteins it remains unprotected, due to a lower position of the elbow helix in the presence of the ABCC-specific lasso motif. Here, we identified a MSD1 second-site mutation located in the vicinity of the CFTR MSD1 groove that partially rescued the folding defect of cystic fibrosis causing mutations located within MSD1, while having no effect on the most frequent mutation, F508del, located within NBD1. A model of the mutated protein 3D structure suggests additional interaction between MSD1 and MSD2, strengthening the assembly at the level of the MSD intracellular loops. Altogether, these results provide insightful information in understanding key features of the folding and function of the CFTR protein in particular, and more generally, of type IV ABC transporters.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Humanos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Estructura Terciaria de Proteína , Fibrosis Quística/genética , Mutación , Membranas/metabolismoRESUMEN
The endoplasmic reticulum exit of some polytopic plasma membrane proteins (PMPs) is controlled by arginin-based retention motifs. PRAF2, a gatekeeper which recognizes these motifs, was shown to retain the GABAB-receptor GB1 subunit in the ER. We report that PRAF2 can interact on a stoichiometric basis with both wild type and mutant F508del Cystic Fibrosis (CF) Transmembrane Conductance Regulator (CFTR), preventing the access of newly synthesized cargo to ER exit sites. Because of its lower abundance, compared to wild-type CFTR, CFTR-F508del recruitment into COPII vesicles is suppressed by the ER-resident PRAF2. We also demonstrate that some pharmacological chaperones that efficiently rescue CFTR-F508del loss of function in CF patients target CFTR-F508del retention by PRAF2 operating with various mechanisms. Our findings open new therapeutic perspectives for diseases caused by the impaired cell surface trafficking of mutant PMPs, which contain RXR-based retention motifs that might be recognized by PRAF2.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Mutación , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Proteins interacting with CFTR and its mutants have been intensively studied using different experimental approaches. These studies provided information on the cellular processes leading to proper protein folding, routing to the plasma membrane, recycling, activation and degradation. Recently, new approaches have been developed based on the proximity labeling of protein partners or proteins in close vicinity and their subsequent identification by mass spectrometry. In this study, we evaluated TurboID- and APEX2-based proximity labeling of WT CFTR and compared the obtained data to those reported in databases. The CFTR-WT interactome was then compared to that of two CFTR (G551D and W1282X) mutants and the structurally unrelated potassium channel KCNK3. The two proximity labeling approaches identified both known and additional CFTR protein partners, including multiple SLC transporters. Proximity labeling approaches provided a more comprehensive picture of the CFTR interactome and improved our knowledge of the CFTR environment.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística , Pliegue de Proteína , Membrana Celular/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Espectrometría de Masas , MutaciónRESUMEN
Early recognition and enhanced degradation of misfolded proteins by the endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD) cause defective protein secretion and membrane targeting, as exemplified for Z-alpha-1-antitrypsin (Z-A1AT), responsible for alpha-1-antitrypsin deficiency (A1ATD) and F508del-CFTR (cystic fibrosis transmembrane conductance regulator) responsible for cystic fibrosis (CF). Prompted by our previous observation that decreasing Keratin 8 (K8) expression increased trafficking of F508del-CFTR to the plasma membrane, we investigated whether K8 impacts trafficking of soluble misfolded Z-A1AT protein. The subsequent goal of this study was to elucidate the mechanism underlying the K8-dependent regulation of protein trafficking, focusing on the ERAD pathway. The results show that diminishing K8 concentration in HeLa cells enhances secretion of both Z-A1AT and wild-type (WT) A1AT with a 13-fold and fourfold increase, respectively. K8 down-regulation triggers ER failure and cellular apoptosis when ER stress is jointly elicited by conditional expression of the µs heavy chains, as previously shown for Hrd1 knock-out. Simultaneous K8 silencing and Hrd1 knock-out did not show any synergistic effect, consistent with K8 acting in the Hrd1-governed ERAD step. Fractionation and co-immunoprecipitation experiments reveal that K8 is recruited to ERAD complexes containing Derlin2, Sel1 and Hrd1 proteins upon expression of Z/WT-A1AT and F508del-CFTR. Treatment of the cells with c407, a small molecule inhibiting K8 interaction, decreases K8 and Derlin2 recruitment to high-order ERAD complexes. This was associated with increased Z-A1AT secretion in both HeLa and Z-homozygous A1ATD patients' respiratory cells. Overall, we provide evidence that K8 acts as an ERAD modulator. It may play a scaffolding protein role for early-stage ERAD complexes, regulating Hrd1-governed retrotranslocation initiation/ubiquitination processes. Targeting K8-containing ERAD complexes is an attractive strategy for the pharmacotherapy of A1ATD.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística , Degradación Asociada con el Retículo Endoplásmico , Queratina-8/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células HeLa , Humanos , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Here, we present a standardized protocol for isolation, maintenance, and polarization of the respiratory epithelial primary cells from patient samples acquired from nasal brushing, polyp specimens, or lung explants. This protocol generates a clearly defined polarized layer of epithelial cells on filters, with a good number of ciliated cells and a thin layer of mucus. We detail the steps for samples prepared from patients with cystic fibrosis as well as from subjects without cystic fibrosis.
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Fibrosis Quística , Pólipos , Fibrosis Quística/patología , Células Epiteliales/patología , Humanos , Pulmón , Moco , Mucosa Nasal/patología , Pólipos/patologíaRESUMEN
Human nasal epithelial (HNE) cells are easy to collect by simple, non-invasive nasal brushing. Patient-derived primary HNE cells can be amplified and differentiated into a pseudo-stratified epithelium in air-liquid interface conditions to quantify cyclic AMP-mediated Chloride (Cl-) transport as an index of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) function. If critical steps such as quality of nasal brushing and cell density upon cryopreservation are performed efficiently, HNE cells can be successfully biobanked. Moreover, short-circuit current studies demonstrate that freeze-thawing does not significantly modify HNE cells' electrophysiological properties and response to CFTR modulators. In the culture conditions used in this study, when less than 2 x 106 cells are frozen per cryovial, the failure rate is very high. We recommend freezing at least 3 x 106 cells per cryovial. We show that dual therapies combining a CFTR corrector with a CFTR potentiator have a comparable correction efficacy for CFTR activity in F508del-homozygous HNE cells. Triple therapy VX-445 + VX-661 + VX-770 significantly increased correction of CFTR activity compared to dual therapy VX-809 + VX-770. The measure of CFTR activity in HNE cells is a promising pre-clinical biomarker useful to guide CFTR modulator therapy.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Bancos de Muestras Biológicas , Recuento de Células , Cloruros , Fibrosis Quística/tratamiento farmacológico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/metabolismo , Humanos , Mutación , Medicina de PrecisiónRESUMEN
Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) are responsible for Cystic Fibrosis (CF). The most common CF-causing mutation is the deletion of the 508th amino-acid of CFTR (F508del), leading to dysregulation of the epithelial fluid transport in the airway's epithelium and the production of a thickened mucus favoring chronic bacterial colonization, sustained inflammation and ultimately respiratory failure. c407 is a bis-phosphinic acid derivative which corrects CFTR dysfunction in epithelial cells carrying the F508del mutation. This study aimed to investigate c407 in vivo activity in the F508del Cftrtm1Eur murine model of CF. Using nasal potential difference measurement, we showed that in vivo administration of c407 by topical, short-term intraperitoneal and long-term subcutaneous route significantly increased the CFTR dependent chloride (Cl-) conductance in F508del Cftrtm1Eur mice. This functional improvement was correlated with a relocalization of F508del-cftr to the apical membrane in nasal epithelial cells. Importantly, c407 long-term administration was well tolerated and in vitro ADME toxicologic studies did not evidence any obvious issue. Our data provide the first in vivo preclinical evidence of c407 efficacy and absence of toxicity after systemic administration for the treatment of Cystic Fibrosis.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Animales , Cloruros , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Transporte Iónico , Ratones , Mutación , Ácidos FosfínicosRESUMEN
Dysfunction of the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR) causes a wide spectrum of disease, including cystic fibrosis (CF) and CFTR-related diseases (CFTR-RDs). Here, we investigate genotype-phenotype-CFTR function relationships using human nasal epithelial (hNE) cells from a small cohort of non-CF subjects and individuals with CF and CFTR-RDs and genotypes associated with either residual or minimal CFTR function using electrophysiological techniques. Collected hNE cells were either studied directly with the whole-cell patch-clamp technique or grown as primary cultures at an air-liquid interface after conditional reprogramming. The properties of cAMP-activated whole-cell Cl- currents in freshly isolated hNE cells identified them as CFTR-mediated. Their magnitude varied between hNE cells from individuals within the same genotype and decreased in the rank order: non-CF > CFTR residual function > CFTR minimal function. CFTR-mediated whole-cell Cl- currents in hNE cells isolated from fully differentiated primary cultures were identical to those in freshly isolated hNE cells in both magnitude and behaviour, demonstrating that conditional reprogramming culture is without effect on CFTR expression and function. For the cohort of subjects studied, CFTR-mediated whole-cell Cl- currents in hNE cells correlated well with CFTR-mediated transepithelial Cl- currents measured in vitro with the Ussing chamber technique, but not with those determined in vivo with the nasal potential difference assay. Nevertheless, they did correlate with the sweat Cl- concentration of study subjects. Thus, this study highlights the complexity of genotype-phenotype-CFTR function relationships, but emphasises the value of conditionally reprogrammed hNE cells in CFTR research and therapeutic testing. KEY POINTS: The genetic disease cystic fibrosis is caused by pathogenic variants in the cystic fibrosis transmembrane conductance regulator (CFTR), an ion channel, which controls anion flow across epithelia lining ducts and tubes in the body. This study investigated CFTR function in nasal epithelial cells from people with cystic fibrosis and CFTR variants with a range of disease severity. CFTR function varied widely in nasal epithelial cells depending on the identity of CFTR variants, but was unaffected by conditional reprogramming culture, a cell culture technique used to grow large numbers of patient-derived cells. Assessment of CFTR function in vitro in nasal epithelial cells and epithelia, and in vivo in the nasal epithelium and sweat gland highlights the complexity of genotype-phenotype-CFTR function relationships.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Cloruros/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Genotipo , Humanos , Mucosa Nasal/metabolismo , Mucosa Nasal/patología , FenotipoRESUMEN
Protein misfolding is involved in a large number of diseases, among which cystic fibrosis. Complex intra- and inter-domain folding defects associated with mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, among which p.Phe508del (F508del), have recently become a therapeutical target. Clinically approved correctors such as VX-809, VX-661, and VX-445, rescue mutant protein. However, their binding sites and mechanisms of action are still incompletely understood. Blind docking onto the 3D structures of both the first membrane-spanning domain (MSD1) and the first nucleotide-binding domain (NBD1), followed by molecular dynamics simulations, revealed the presence of two potential VX-809 corrector binding sites which, when mutated, abrogated rescue. Network of amino acids in the lasso helix 2 and the intracellular loops ICL1 and ICL4 allosterically coupled MSD1 and NBD1. Corrector VX-445 also occupied two potential binding sites on MSD1 and NBD1, the latter being shared with VX-809. Binding of both correctors on MSD1 enhanced the allostery between MSD1 and NBD1, hence the increased efficacy of the corrector combination. These correctors improve both intra-domain folding by stabilizing fragile protein-lipid interfaces and inter-domain assembly via distant allosteric couplings. These results provide novel mechanistic insights into the rescue of misfolded proteins by small molecules.
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Aminopiridinas/farmacología , Benzodioxoles/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Fibrosis Quística/tratamiento farmacológico , Mutación , Pliegue de Proteína/efectos de los fármacos , Pirazoles/farmacología , Piridinas/farmacología , Pirrolidinas/farmacología , Sitios de Unión , Agonistas de los Canales de Cloruro/farmacología , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Quimioterapia Combinada , Células HEK293 , Humanos , Dominios Proteicos , Estructura Terciaria de ProteínaRESUMEN
With the advent of CFTR modulators, surrogate outcome parameters that accurately quantify the improvement in CFTR activity are needed. In vivo biomarkers that reflect CFTR ion transport and can serve as outcomes in the treatment of CFTR modulators are the sweat Cl- test (SCT), the nasal potential difference (NPD) measurement or the intestinal current measurement (ICM). This review focus on the SCT and NPD. The SCT displays a low intra-patient variability in contrast to the NPD. It has been used extensively as a biomarker of CFTR function in clinical trials of CFTR modulator therapies and provides evidence for change in the short term. The level of functional rescue in the NPD increases up to 40% of normal CFTR in patients with a Gly551Asp treated with ivacaftor monotherapy, while in F508del homozygous patients treated with ivacaftor-lumacaftor, activity increased on average up to ~20% of normal activity. While both tests provide evidence of the effect on CFTR activity, they cannot be used at an individual level to predict the response to any CFTR modulators. Nevertheless, their rapid modification, reflecting electrophysiological properties, highlight their potential use in proof-of-concept studies for CFTR modulators.
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C407 is a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein carrying the p.Phe508del (F508del) mutation. We investigated the corrector effect of c407 and its derivatives on F508del-CFTR protein. Molecular docking and dynamics simulations combined with site-directed mutagenesis suggested that c407 stabilizes the F508del-Nucleotide Binding Domain 1 (NBD1) during the co-translational folding process by occupying the position of the p.Phe1068 side chain located at the fourth intracellular loop (ICL4). After CFTR domains assembly, c407 occupies the position of the missing p.Phe508 side chain. C407 alone or in combination with the F508del-CFTR corrector VX-809, increased CFTR activity in cell lines but not in primary respiratory cells carrying the F508del mutation. A structure-based approach resulted in the synthesis of an extended c407 analog G1, designed to improve the interaction with ICL4. G1 significantly increased CFTR activity and response to VX-809 in primary nasal cells of F508del homozygous patients. Our data demonstrate that in-silico optimized c407 derivative G1 acts by a mechanism different from the reference VX-809 corrector and provide insights into its possible molecular mode of action. These results pave the way for novel strategies aiming to optimize the flawed ICL4-NBD1 interface.