Browsing by Author "Matos, Ana Margarida"
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- A molecular switch in the scaffold NHERF1 enables misfolded CFTR to evade the peripheral quality control checkpointPublication . Loureiro, Cláudia; Matos, Ana Margarida; Dias-Alves, Ãngela; Pereira, Joana Filipa; Uliyakina, Irina; Barros, Patrícia; Amaral, Margarida; Matos, PauloThe peripheral protein quality control (PPQC) checkpoint removes improperly folded proteins from the plasma membrane through a mechanism involving the E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70 interacting protein). PPQC limits the efficacy of some cystic fibrosis (CF) drugs, such as VX-809, that improve trafficking to the plasma membrane of misfolded mutants of the CF transmembrane conductance regulator (CFTR), including F508del-CFTR, which retains partial functionality. We investigated the PPQC checkpoint in lung epithelial cells with F508del-CFTR that were exposed to VX-809. The conformation of the scaffold protein NHERF1 (Na(+)/H(+) exchange regulatory factor 1) determined whether the PPQC recognized "rescued" F508del-CFTR (the portion that reached the cell surface in VX-809-treated cells). Activation of the cytoskeletal regulator Rac1 promoted an interaction between the actin-binding adaptor protein ezrin and NHERF1, triggering exposure of the second PDZ domain of NHERF1, which interacted with rescued F508del-CFTR. Because binding of F508del-CFTR to the second PDZ of NHERF1 precluded the recruitment of CHIP, the coexposure of airway cells to Rac1 activator nearly tripled the efficacy of VX-809. Interference with the NHERF1-ezrin interaction prevented the increase of efficacy of VX-809 by Rac1 activation, but the actin-binding domain of ezrin was not required for the increase in efficacy. Thus, rather than mainly directing anchoring of F508del-CFTR to the actin cytoskeleton, induction of ezrin activation by Rac1 signaling triggered a conformational change in NHERF1, which was then able to bind and stabilize misfolded CFTR at the plasma membrane. These insights into the cell surface stabilization of CFTR provide new targets to improve treatment of CF.
- Network Biology Identifies Novel Regulators of CFTR Trafficking and Membrane StabilityPublication . Loureiro, Cláudia Almeida; Santos, João D.; Matos, Ana Margarida; Jordan, Peter; Matos, Paulo; Farinha, Carlos M.; Pinto, Francisco R.In cystic fibrosis, the most common disease-causing mutation is F508del, which causes not only intracellular retention and degradation of CFTR, but also defective channel gating and decreased membrane stability of the small amount that reaches the plasma membrane (PM). Thus, pharmacological correction of mutant CFTR requires targeting of multiple cellular defects in order to achieve clinical benefit. Although small-molecule compounds have been identified and commercialized that can correct its folding or gating, an efficient retention of F508del CFTR at the PM has not yet been explored pharmacologically despite being recognized as a crucial factor for improving functional rescue of chloride transport. In ongoing efforts to determine the CFTR interactome at the PM, we used three complementary approaches: targeting proteins binding to tyrosine-phosphorylated CFTR, protein complexes involved in cAMP-mediated CFTR stabilization at the PM, and proteins selectively interacting at the PM with rescued F508del-CFTR but not wt-CFTR. Using co-immunoprecipitation or peptide-pull down strategies, we identified around 400 candidate proteins through sequencing of complex protein mixtures using the nano-LC Triple TOF MS technique. Key candidate proteins were validated for their robust interaction with CFTR-containing protein complexes and for their ability to modulate the amount of CFTR expressed at the cell surface of bronchial epithelial cells. Here, we describe how we explored the abovementioned experimental datasets to build a protein interaction network with the aim of identifying novel pharmacological targets to rescue CFTR function in cystic fibrosis (CF) patients. We identified and validated novel candidate proteins that were essential components of the network but not detected in previous proteomic analyses.
- Plasma membrane-specific interactome analysis reveals calpain 1 as a druggable modulator of rescued Phe508del-CFTR cell surface stabilityPublication . Matos, Ana Margarida; Pinto, Francisco R.; Barros, Patrícia; Amaral, Margarida D.; Pepperkok, Rainer; Matos, PauloCystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a chloride channel normally expressed at the surface of epithelial cells. The most frequent mutation, resulting in Phe-508 deletion, causes CFTR misfolding and its premature degradation. Low temperature or pharmacological correctors can partly rescue the Phe508del-CFTR processing defect and enhance trafficking of this channel variant to the plasma membrane (PM). Nevertheless, the rescued channels have an increased endocytosis rate, being quickly removed from the PM by the peripheral protein quality-control pathway. We previously reported that rescued Phe508del-CFTR (rPhe508del) can be retained at the cell surface by stimulating signaling pathways that coax the adaptor molecule ezrin (EZR) to tether rPhe508del–Na+/H+-exchange regulatory factor-1 (NHERF1) complexes to the actin cytoskeleton, thereby averting the rapid internalization of this channel variant. However, the molecular basis for why rPhe508del fails to recruit active EZR to the PM remains elusive. Here, using a proteomics approach, we characterized and compared the core components of wt-CFTR– or rPhe508del–containing macromolecular complexes at the surface of human bronchial epithelial cells. We identified calpain 1 (CAPN1) as an exclusive rPhe508del interactor that prevents active EZR recruitment, impairs rPhe508del anchoring to actin, and reduces its stability in the PM. We show that either CAPN1 downregulation or its chemical inhibition dramatically improves the functional rescue of Phe508del-CFTR in airway cells. These observations suggest that CAPN1 constitutes an attractive target for pharmacological intervention, as part of CF combination therapies restoring Phe508del-CFTR function.
- Search for new modulators of Phe508del-CFTR retention at the plasma membranePublication . Matos, Ana Margarida; Matos, Paulo; Pepperkok, RainerCystic 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.