Tese de doutoramento, Bioquímica (Genética Molecular), Universidade de Lisboa, Faculdade de Ciências, 2015Cystic fibrosis (CF), the most common life-shortening genetic disorder among Caucasians, is caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, an anion (chloride/ bicarbonate) channel that is expressed at the apical membrane of epithelial cells to control salt and water transport. Clinically, CF is characterized by multiple manifestations in different organs, but the disease is dominated by the respiratory symptoms, the main cause of morbidity and mortality. The very thick mucus, a hallmark of CF lead to inefficient mucociliary clearance (MCC) and recurrent chronic bacterial infections, mostly by Pseudomonas aeruginosa, and to chronic exacerbated inflammation contributing to progressive loss of respiratory function and ultimately to death. Other CF classical symptoms include elevated sweat electrolytes, exocrine pancreatic insufficiency (85%), intestinal obstruction (meconium ileus in 15-20% of CF newborns and/or distal intestinal obstruction syndrome at a later age) and male infertility (95%). To date almost 2,000 CFTR gene mutations (most disease-causing) were reported, but one single mutation - F508del that impairs CFTR plasma membrane (PM) traffic – occurs in ~85% of CF patients. Although there is some degree of correlation between the CFTR genotype and disease severity, there is also a huge variation in the severity of clinical phenotypes, namely in lung disease. CFTR protein is an atypical ABC transporter (ABCC7) that functions as an apical membrane channel in epithelial cells and causes CF when defective. Unique to CFTR is its regulatory domain (RD) which includes a highly conformationally dynamic region - the regulatory extension (RE). The first nucleotide-binding domain (NBD1), where the most common CF-causing mutation (F508del) is located, contains the regulatory insertion (RI) also equally dynamic. Because CFTR is a negative regulator of the epithelial Na+ channel (ENaC), besides impaired cAMP-dependent chloride (Cl-)/ bicarbonate secretion, enhanced sodium (Na+) absorption by ENaC also occurs in CF airways. Novel therapies modulating the basic defect caused by F508del and other CFTR mutations are emerging. Indeed, high-throughput screens (HTS) have identified several novel small molecules with potential to treat the basic defect in CF and some have approved for clinical use. These include the potentiator VX-770/ivacaftor for patients with G551D and other mutations affecting channel gating and the VX-809/lumacaftor corrector (that partially rescues the trafficking defect of F508del-CFTR) in combination with VX-770 for F508del-homozygous patients However, the latter combination drug only showed 3% improvement in lung function and the respective mechanism of action (MoA) is not established, a step that is considered essential to identify more efficacious drugs. Despite these advances, not all CFTR mutations can be pharmacologically rescued (e.g., frameshift mutations or large deletions). For these, major hopes are on the ‘bypass’ therapeutic strategies which aim to correct the ionic imbalance in CF by downregulating ENaC hyperactivity and/or stimulating non-CFTR Cl- channels, such as the calcium (Ca2+)-, activated Cl- channels (CaCCs), to compensate for the absence of functional CFTR. The first objective (Chapter I) of this doctoral work was to assess the expression and function of the CFTR mutants in epithelial cells and tissues from the CF patients to determine its respective molecular/cellular defect and to evaluate the effect of potential therapeutic compounds in correcting the CF basic defect. One of these compounds is INO-4995 which is shown here to activate the endogenous CaCCs-TMEM16A in both CF and non-CF primary human bronchial epithelial cells. Within this objective, we also established a new methodology to culture human nasal epithelial cells from the nasal brushings in order to perform electrophysiological measurements in this novel cellular model, namely to assess patients’ responses to therapies in their own cells in a personalized pharmacotherapeutic approach. The second objective (Chapter II) of the present work, aimed to study the mechanisms that regulate processing and function of CFTR protein (both wt- and F508del) upon removal of two dynamic regions – the regulatory extension (RE) and the regulatory insertion (RI), which are absent in the other ABC transporters. Our results show that the deletion of the RE region leads to a dramatic stabilization of the immature form of the mutant protein. Moreover, our data demonstrate that VX-809 significantly rescued ΔRI-F508del-CFTR processing. More strikingly, its activity was only restored to normal CFTR levels when RE was also absent. Altogether, these data suggest that the two dynamic regions negatively impact the rescuing of F508del-CFTR by VX-809, but while RI deletion is essential to achieve full processing, only by RE removal is maximal channel function reached. The third and final objective (Chapter III) of this work, aimed to functionally characterize 270 genes, localized at two loci identified in a genomewide association and linkage study (GWAS) as significantly associated with severity of lung disease in CF, by finding their possible role in CFTR traffic or ENaC activity. To this end, we used cell-based microscopy assays and a loss-of function approach, (siRNA reverse transfection) to assess the respective effects by high-content automatic microscopy. We thus identified 66 genes regulating the traffic of wt-CFTR, 49 regulators of F508del-CFTR traffic and 41 regulators of ENaC function. Further, in order to clarify the MoA by which the hit siRNAs rescue F508del-CFTR, the 127 most significant genes were selected for the secondary screen using similar double-tagged constructs of F508del-CFTR but containing revertant mutations, i.e. second-site mutations which revert the F508del-CFTR traffic defect. Some of the hits identified within this study are potential drug targets that enhance CFTR traffic or decrease ENaC function in the context of CF lung disease. Further hit characterization is expected to generate a valuable resource for the community regarding both insight into those pathways and the establishment of novel non-CFTR therapeutic targets for CF. Altogether, results included in the present thesis bring new insights into our understanding of expression, traffic, function and regulation of the normal and mutant CFTR protein both in heterologous cellular systems and in primary human airway cell cultures. The relevance of developing the “by-pass therapies” aiming at overcoming the ionic defects caused in epithelia by the loss of CFTR via ENaC and CaCCs is also a major point of the current work, such as understanding the mechanism of action of the novel therapeutical compounds approved for a use in CF patients, such as VX-770 and VX-809. The characterization of genetically identified modifier genes by functional approaches (siRNA screens), constitutes an original approach linking genetics to functional genomics towards a global understanding of disease pathways.Fundação para a Ciência e a Tecnologia (FCT), SFRH/BD/69180/201
