Tese de doutoramento, Biologia (Biologia de Sistemas), Universidade de Lisboa, Faculdade de Ciências, 2018Cystic fibrosis (CF), the most common life-shortening autosomal recessive disorder in Caucasians, is caused by >2,000 different mutations in the gene encoding the CF Transmembrane Conductance Regulator (CFTR). CFTR is a Cl- and HCO3- permeant channel expressed in the epithelial apical membrane. Clinically, the majority of CF patients suffer mostly from severe lung disease. A possible approach to treat CF is through the stimulation of alternative channels, such as anoctamins, that could compensate for absence of CFTR mediated Cl- secretion, thus being called “bypass” approach. The prime objective of this PhD project focusses on identifying novel anoctamin regulators as possible drug targets for CF. The first chapter describes the generation of CFBE-3HA-ANO1-eGFP cells and their use in a siRNA traffic screen. As a result, nine hits were selected for functional validation: Extended Synaptotagmin Member 1 (ESYT1) and Casein Kinase 2 (CSNK2) as negative modulators and Peptidoglycan Recognition Protein 3 (PGLYRP3) and Coat Protein Complex I subunit β1 (COPβ1) as positive hits. These screen data also showed a positive regulation of ANO1 by GPCRs, which were further characterized in chapter two. Our data illustrate an overlap between Ca2+- and cAMP- induced currents, which prevent a clear distinction between their respective Cl- currents. Our data in the third chapter describes the relationship between ANO1 and mucus and show that IL4 treatment (an inducer of ANO1 expression) does not affect MUC5AC levels, in contrast to other studies which proposed a causative role for ANO1 in mucus production. In the fourth chapter, the relationship between ANO1, ANO6 and CFTR was further investigated and our data suggest that CFTR activation requires at least one anoctamin member trafficking to the PM, to probably modulate cellular Ca2+ signalling and activate CFTR by SOcAMPs and Ca2+-sensitive adenylyl cyclases.Fundação para a Ciência e a Tecnologia (FCT), SFRH/BD/52489/201
