Search for new modulators of Phe508del-CFTR retention at the plasma membrane of epithelial cells

Tese de doutoramento, Biologia (Biologia de Sistemas), Universidade de Lisboa, Faculdade de Ciências, 2018Cystic fibrosis (CF) is a complex inherited disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Around 2000 disease causing mutations are known for this gene, which encodes a Chloride (Cl−) channel expressed at the plasma membrane (PM) of epithelial cells. The most frequent CFTR mutation, the deletion of phenylalanine 508 (Phe508del), causes the protein to misfold and be prematurely degraded. Low temperature or pharmacological “correctors” can partly rescue Phe508del-CFTR processing defect and enhance the channel traffic to the cell surface. Nevertheless, the rescued channels show partial channel function and a highly decreased PM half-life, due to accelerated endocytosis and fast turnover. Given this accelerated endocytic rate, new strategies aiming to retain rescued Phe508del-CFTR at the cell surface could be relevant as to enhance the efficacy of currently available pharmacological correctors. For that reason, the major objective of this dissertation is to identify novel cellular pathways or key interactors for the modulation of CFTR surface retention. Previous results from the host laboratory had showed that stimulation of endogenous RAC1 by Hepatocyte Growth Factor (HGF) signaling potentiated the retention of rescued Phe508del-CFTR at the PM by promoting an interaction between the actin-binding adaptor ezrin (EZR) and the Na+/H+ exchange regulatory factor-1 (NHERF1), enhancing CFTR anchoring to the actin cytoskeleton. In chapter 2 we showed that the mechanism behind this stabilization lies on a conformational change in NHERF1, triggered by EZR activation upon RAC1 signaling, which is then able to bind and stabilize misfolded CFTR at the PM. However, HGF/RAC1 signaling pathway is known to have proliferative and pleiotropic biological functions, which limit its application for therapeutic intervention. Therefore, in chapter 3, we investigated the effect of HGF treatment in epithelium-like cellular models, in combination with the most common administrated drugs. Contrary to what would be commonly assumed, we found that prolonged co-administration of HGF actually prevented previously unrecognized epithelial dedifferentiation effects of prolonged exposure to the FDA-approved Phe508del-CFTR corrector VX-809. It also significantly increased the Phe508del-CFTR functional rescue by the FDA- and EMA-approved VX-809/VX-770 drug combination, preventing the destabilization of the PM rescued channels by prolonged exposure to the VX-770 potentiator drug. These results suggest that HGF co-administration could indeed be beneficial for CF patients and should be further clinically explored. Lastly, since we showed that the type of protein interactions that wt- and rescued Phe508del-CFTR establish at the cell surface can be major determinants of their different PM stabilities, in chapter 4 we identified, for the first time, the core components of the macromolecular complexes assembled around wt- and rescued Phe508del-CFTR proteins at the PM. By identifying exclusive PM interactions between rescued Phe508del-CFTR, NHERF1 and EZR, we were able to recognize Calpain 1 as a key contributor for the decreased surface stability of pharmacologically rescued Phe508del-CFTR, probably acting through the disruption of the EZR-actin cytoskeleton binding.Fundação para a Ciência e a Tecnologia (PD/BD/52490/2014