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CFTR folding in the cell. The schematic shows the time scale of CFTR synthesis, folding and domain assembly in cells. The sub-domains of NBD1 (N-terminus (N), K377 -S492; α-helical ( α), Q493 -D565; α/ β-core ( β), A566 -Q637) and the position of the F508del mutation are indicated. Abbreviations: NBD, nucleotide-binding domain;
Source publication
The treatment of cystic fibrosis (CF) has been transformed by orally-bioavailable small molecule modulators of the cystic fibrosis transmembrane conductance regulator (CFTR), which restore function to CF mutants. However, CFTR modulators are not available to all people with CF and better modulators are required to prevent disease progression. Here,...
Contexts in source publication
Context 1
... process is influenced by the adjacent ribosome [ 10 , 11 ], the translation rate [ 12 , 13 ], cellular chaperones [ 14 , 15 ], and the cytosolic folding environment [16] . For CFTR, cotranslational folding requires the precise insertion of 12 transmembrane α-helices into the lipid bilayer, tertiary folding and packing of three cytosolic domains and two membrane-spanning domains (MSDs), and assembly of these domains into a mature tertiary structure ( Fig. 1 ). Many of these steps occur during the 7-10 minutes required to complete CFTR peptide synthesis and thus are truly cotranslational events [17] . ...
Context 2
... this FRET assay, Khushoo et al. [21] and Kim et al. [11] showed that NBD1 folds cotranslationally during synthesis on the ribosome via the sequential compaction of N-terminal, α-helical, and α/ β-core subdomains ( Fig. 1 ). The timing of these folding events is finely tuned by properties of the ribosome that delay collapse of the α-subdomain until synthesis of a 4-stranded parallel β-sheet core is completed. ...
Citations
... For example, the most prevalent CF mutation ΔF508 by introducing intrinsic structural flaws (Chen et al., 2019) blocks CFTR protein expression at the cell membrane (Cheng et al., 1990), whereas ΔF508, G551D and G1349D mutations greatly decrease channel activity (Dalemans et al., 1991;Cai and Sheppard, 2002;Cai et al., 2006;Bompadre et al., 2007;Chen et al., 2009;Chen et al., 2017) with further alterations in channel responses to gating potentiators (Hwang et al., 1997;Cai and Sheppard, 2002;Cai et al., 2006;Bompadre et al., 2007) and intracellular pH (Chen et al., 2009;Chen et al., 2017). Therefore, the current drug therapy for CF aims to elevate the protein expression and channel activity of mutant CFTRs by correctors and potentiators, respectively (Bose et al., 2020). ...
Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel by genetic mutations causes the inherited disease cystic fibrosis (CF). CF lung disease that involves multiple disorders of epithelial function likely results from loss of CFTR function as an anion channel conducting chloride and bicarbonate ions and its function as a cellular regulator modulating the activity of membrane and cytosol proteins. In the absence of CFTR activity, abundant mucus accumulation, bacterial infection and inflammation characterize CF airways, in which inflammation-associated tissue remodeling and damage gradually destroys the lung. Deciphering the link between CFTR dysfunction and bacterial infection in CF airways may reveal the pathogenesis of CF lung disease and guide the development of new treatments. Research efforts towards this goal, including high salt, low volume, airway surface liquid acidosis and abnormal mucus hypotheses are critically reviewed.
... smFRET experiments are increasingly employed to study the molecular mechanisms of small-molecule binding to target proteins in a variety of applications, ranging from enzyme-ligand interactions to probing the reversal effect of small molecule corrector compounds on protein misfolding [57][58][59][60][61] . However, larger-scale screenings of molecular compounds by smFRET, as used in pharmacological research and drug discovery, have been limited because tools to conduct such time-and labor-intensive measurements are lacking. ...
Single-molecule FRET (smFRET) has become a versatile tool for probing the structure and functional dynamics of biomolecular systems, and is extensively used to address questions ranging from biomolecular folding to drug discovery. Confocal smFRET measurements are amongst the widely used smFRET assays and are typically performed in a single-well format. Thus, sampling of many experimental parameters is laborious and time consuming. To address this challenge, we extend here the capabilities of confocal smFRET beyond single-well measurements by integrating a multiwell plate functionality to allow for continuous and automated smFRET measurements. We demonstrate the broad applicability of the multiwell plate assay towards DNA hairpin dynamics, protein folding, competitive and cooperative protein–DNA interactions, and drug-discovery, revealing insights that would be very difficult to achieve with conventional single-well format measurements. For the adaptation into existing instrumentations, we provide a detailed guide and open-source acquisition and analysis software.
... Though there are no known cures for CF, several medications known to alleviate symptoms of the disease have been clinically approved [14,15]. These medications, termed modulators, are divided in two categories; as examples: Ivacaftor (VX-770) is a CFTR potentiator that works by binding to CFTR at a protein-lipid interface to open chloride channels, while Lumacaftor (VX-809) is a corrector of CFTR that inhibits misfolding of mutant CFTR during biosynthesis [15,16]. ...
Cystic fibrosis (CF) is caused by mutations in the gene that codes for the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). Recent advances in CF treatment have included use of small-molecule drugs known as modulators, such as Lumacaftor (VX-809), but their detailed mechanism of action and interplay with the surrounding lipid membranes, including cholesterol, remain largely unknown. To examine these phenomena and guide future modulator development, we prepared a set of wild type (WT) and mutant helical hairpin constructs consisting of CFTR transmembrane (TM) segments 3 and 4 and the intervening extracellular loop (termed TM3/4 hairpins) that represent minimal membrane protein tertiary folding units. These hairpin variants, including CF-phenotypic loop mutants E217G and Q220R, and membrane-buried mutant V232D, were reconstituted into large unilamellar phosphatidylcholine (POPC) vesicles, and into corresponding vesicles containing 70 mol% POPC +30 mol% cholesterol, and studied by single-molecule FRET and circular dichroism experiments. We found that the presence of 30 mol% cholesterol induced an increase in helicity of all TM3/4 hairpins, suggesting an increase in bilayer cross-section and hence an increase in the depth of membrane insertion compared to pure POPC vesicles. Importantly, when we added the corrector VX-809, regardless of the presence or absence of cholesterol, all mutants displayed folding and helicity largely indistinguishable from the WT hairpin. Fluorescence spectroscopy measurements suggest that the corrector alters lipid packing and water accessibility. We propose a model whereby VX-809 shields the protein from the lipid environment in a mutant-independent manner such that the WT scaffold prevails. Such ‘normalization’ to WT conformation is consistent with the action of VX-809 as a protein-folding chaperone.
... Understanding of the folding of this helical class is advancing and now ranges from in vitro studies on artificially denatured proteins and detergent mixtures, through lipid bilayers to co-translational work probing folding events as the protein is made on the ribosome. Diseases caused by misfolded membrane proteins include retinitis pigmentosa, in which misfolding of rhodopsin causes blindness [1], and cystic fibrosis which is associated with mistrafficking and misfolding of the chloride channel CFTR [2,3]. ...
How alpha-helical membrane proteins fold correctly in the highly hydrophobic membrane interior is not well understood. Their folding is known to be highly influenced by the lipids within the surrounding bilayer, but the majority of folding studies have focused on detergent-solubilized protein rather than protein in a lipid environment. There are different ways to study folding in lipid bilayers, and each method has its own advantages and disadvantages. This review will discuss folding methods which can be used to study alpha-helical membrane proteins in bicelles, liposomes, nanodiscs or native membranes. These folding methods include in vitro folding methods in liposomes such as denaturant unfolding studies, and single-molecule force spectroscopy studies in bicelles, liposomes and native membranes. This review will also discuss recent advances in co-translational folding studies, which use cell-free expression with liposomes or nanodiscs or are performed in vivo with native membranes.
... In CF patients, more changes are observed at the pulmonary level leading to chronic inflammatory diseases of the respiratory tract characterized by pulmonary infections, generally Sensors 2022, 22, 1902 2 of 13 due to the colonization of Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, or Stenotrophomonas maltophilia [6]. CF is multisystemic and life-threatening, although the advancement in treatment and care have increased life expectancy to 40-50 years [7,8]. ...
Physical training at home by making individuals play active video games is a new therapeutic strategy to improve the condition of patients with cystic fibrosis (CF). We reviewed studies on the use of video games and their benefits in the treatment of CF. We conducted a systematic review with data from six databases (PubMed, Medline, Scopus, Web of Science, PEDro, and Cochrane library plus) since 2010, according to PRISMA standards. The descriptors were: “Cystic Fibrosis”, “Video Game”, “Gaming Console”, “Pulmonary Rehabilitation”, “Physiotherapy”, and “Physical Therapy”. Nine articles with 320 participants met the inclusion criteria and the study objective. Patients who played active video games showed a high intensity of exercise and higher ventilatory and aerobic capacity compared to the values of these parameters in tests such as the cardiopulmonary stress test or the six-minute walk test. Adequate values of metabolic demand in these patients were recorded after playing certain video games. A high level of treatment adherence and satisfaction was observed in both children and adults. Although the quality of the included studies was moderate, the evidence to confirm these results was insufficient. More robust studies are needed, including those on evaluation and health economics, to determine the effectiveness of the treatment.
... Cystic fibrosis was an exemplar for the promise of gene therapies, and recent work has renewed the impetus [18]. In the meantime, however, major therapeutic advances have arisen through the development of compounds to rescue misfolding of the CFTR protein within the ER and stabilise its active conformation within the cell membrane [19][20][21]. ER stress is now known to mediate many chronic diseases and, in interstitial lung diseases, it correlates with severity of fibrosis rather than disease subtype [22,23]. ...
... which might bind at the NBD1:NBD2 dimer interface in a position distinct from the ATP-binding sites (Bose et al., 2020;Moran et al., 2005). Co-potentiation occurred when a class I potentiator was used with a class II potentiator, but not when two potentiators from the same class were employed (Phuan et al., 2019). ...
Background and Purpose
Cystic fibrosis transmembrane conductance regulator (CFTR) potentiators are small molecules developed to treat the genetic disease cystic fibrosis (CF). They interact directly with CFTR Cl⁻ channels at the plasma membrane to enhance channel gating. Here, we investigate the action of a new CFTR potentiator, CP‐628006 with a distinct chemical structure.
Experimental Approach
Using electrophysiological assays with CFTR‐expressing heterologous cells and CF patient‐derived human bronchial epithelial (hBE) cells, we compared the effects of CP‐628006 with the marketed CFTR potentiator ivacaftor.
Key Results
CP‐628006 efficaciously potentiated CFTR function in epithelia from cultured hBE cells. Its effects on the predominant CFTR variant F508del‐CFTR were larger than those with the gating variant G551D‐CFTR. In excised inside‐out membrane patches, CP‐628006 potentiated wild‐type, F508del‐CFTR, and G551D‐CFTR by increasing the frequency and duration of channel openings. CP‐628006 increased the affinity and efficacy of F508del‐CFTR gating by ATP. In these respects, CP‐628006 behaved like ivacaftor. CP‐628006 also demonstrated notable differences with ivacaftor. Its potency and efficacy were lower than those of ivacaftor. CP‐628006 conferred ATP‐dependent gating on G551D‐CFTR, whereas the action of ivacaftor was ATP‐independent. For G551D‐CFTR, but not F508del‐CFTR, the action of CP‐628006 plus ivacaftor was greater than ivacaftor alone. CP‐628006 delayed, but did not prevent, the deactivation of F508del‐CFTR at the plasma membrane, whereas ivacaftor accentuated F508del‐CFTR deactivation.
Conclusions and Implications
CP‐628006 has distinct effects compared to ivacaftor, suggesting a different mechanism of CFTR potentiation. The emergence of CFTR potentiators with diverse modes of action makes therapy with combinations of potentiators a possibility.
... Figure 3 represents the different functions of CFTR modulators. Ivacaftor was approved for G551D and 37 additional gating mutations [169][170][171][172]; lumacaftor-ivacaftor combination therapy for children (<12 years old) F508del homozygous [173] and tezacaftor-ivacaftor combination therapy for adults with homozygous F508del or subjects with F508del and a residual function allele [174,175]. The most recent combination approved in the United States and United Kingdom (not in Europe yet) was elexacaftor-tezacaftor-ivacaftor. ...
... However, 10% of CF people still do not have curative treatment [172]. Fortunately, there are clinical trials trying to find other modulator therapy and also amplifier and stabilizer CFTR treatment. ...
Cystic fibrosis (CF) is a genetic disease that causes absence or dysfunction of a protein named transmembrane conductance regulatory protein (CFTR) that works as an anion channel. As a result, the secretions of the organs where CFTR is expressed are very viscous, so their functionality is altered. The main cause of morbidity is due to the involvement of the respiratory system as a result of recurrent respiratory infections by different pathogens. In recent decades, survival has been increasing, rising by around age 50. This is due to the monitoring of patients in multidisciplinary units, early diagnosis with neonatal screening, and advances in treatments. In this chapter, we will approach the different therapies used in CF for the treatment of symptoms, obstruction, inflammation, and infection. Moreover, we will discuss specific and personalized treatments to correct the defective gene and repair the altered protein CFTR. The obstacle for personalized CF treatment is to predict the drug response of patients due to genetic complexity and heterogeneity of uncommon mutations.
... Figure 3 represents the different functions of CFTR modulators. Ivacaftor was approved for G551D and 37 additional gating mutations [169][170][171][172]; lumacaftor-ivacaftor combination therapy for children (<12 years old) F508del homozygous [173] and tezacaftor-ivacaftor combination therapy for adults with homozygous F508del or subjects with F508del and a residual function allele [174,175]. The most recent combination approved in the United States and United Kingdom (not in Europe yet) was elexacaftor-tezacaftor-ivacaftor. ...
... However, 10% of CF people still do not have curative treatment [172]. Fortunately, there are clinical trials trying to find other modulator therapy and also amplifier and stabilizer CFTR treatment. ...
Cystic fibrosis (CF) is a genetic disease that causes absence or dysfunction of a protein named transmembrane conductance regulatory protein (CFTR) that works as an anion channel. As a result, the secretions of the organs where CFTR is expressed, are very viscous so their functionality is altered. The main cause of morbidity is due to the involvement of the respiratory system as a result of recurrent respiratory infections by different pathogens. In recent decades, survival has been increasing rising by around age 50. This is due to the monitoring of patients in multi-disciplinary units, early diagnosis with neonatal screening and advances in treatments. In this chapter we will approach the different therapies used in CF for the treatment of symptoms, obstruction, inflammation and infection. Moreover, we will discuss specific and personalized treatments to correct the defective gene and repair the altered protein CFTR. The obstacle for CF treatment personalized is to predict the drug response of patients due to genetic complexity and heterogeneity uncommon mutations.
... [1][2][3] In addition, modulators must be administered daily, are costly, only partially restore function, and do not cover the full spectrum of CFTR mutations. 2,4,5 Thus, there is a continued need to develop a durable therapy that can treat 100% of CF patients. ...
Cystic fibrosis (CF) is a monogenic disease caused by impaired production and/or function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Although we have previously shown correction of the most common pathogenic mutation, there are many other pathogenic mutations throughout the CF gene. An autologous airway stem cell therapy in which the CFTR cDNA is precisely inserted into the CFTR locus may enable the development of a durable cure for almost all CF patients, irrespective of the causal mutation. Here, we use CRISPR/Cas9 and two adeno-associated viruses (AAV) carrying the two halves of the CFTR cDNA to sequentially insert the full CFTR cDNA along with a truncated CD19 (tCD19) enrichment tag in upper airway basal stem cells (UABCs) and human bronchial basal stem cells (HBECs). The modified cells were enriched to obtain 60-80% tCD19⁺ UABCs and HBECs from 11 different CF donors with a variety of mutations. Differentiated epithelial monolayers cultured at air-liquid interface showed restored CFTR function that was >70% of the CFTR function in non-CF controls. Thus, our study enables the development of a therapy for almost all CF patients, including patients who cannot be treated using recently approved modulator therapies.
