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Stimulation of RAC1/PAK1 signalling upregulates DNA damage repair genes via STAT5 stimulation of BCL6 repressed loci

Publication . Barros, Patrícia; Amaral, Andreia; Abrantes, Leonor; Oliveira, Tiago; Louro, Henriqueta; Silva, Maria João; Jordan, Peter; Gama-Carvalho, Margarida; Matos, Paulo

Colorectal cancer is one of the most prevalent types of cancer worldwide. The GTPase RAC1 and its effector PAK1 have been found overexpressed or hyperactivated in colorectal cancers, particularly those with more aggressive and invasive features, leading to unfavourable clinical prognosis, often resulting from chemoresistance.

2017-05-08Conference object

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Activation of RAC1/PAK1 axis potentiates transcriptional upregulation of DNA damage response genes via the BCL6/STAT5 switch

Publication . Barros, Patrícia; Amaral, Andreia; Abrantes, Leonor; Oliveira, Tiago; Lourio, Henriqueta; Silva, Maria João; Jordan, Peter; Gama-Carvalho, Margarida; Matos, Paulo

2017-06-01Conference object

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Stimulation of RAC1/PAK1 signalling upregulates DNA damage repair genes via the BCL6/STAT5-switch

Publication . Barros, Patrícia; Amaral, Andreia; Abrantes, Leonor; Oliveira, Tiago; Louro, Henriqueta; Silva, Maria João; Jordan, Peter; Gama-Carvalho, Margarida; Matos, Paulo

2017-09-08Conference object

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Plasma membrane-specific interactome analysis reveals calpain 1 as a druggable modulator of rescued Phe508del-CFTR cell surface stability

Publication . Matos, Ana Margarida; Pinto, Francisco R.; Barros, Patrícia; Amaral, Margarida D.; Pepperkok, Rainer; Matos, Paulo

Cystic 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.

2019-10-18Conference object

Open access

Prolonged co-treatment with HGF sustains epithelial integrity and improves pharmacological rescue of Phe508del-CFTR

Publication . Matos, Ana M.; Gomes-Duarte, Andreia; Faria, Márcia; Barros, Patrícia; Jordan, Peter; Amaral, Margarida D.; Matos, Paulo

Cystic fibrosis (CF), the most common inherited disease in Caucasians, is caused by mutations in the CFTR chloride channel, the most frequent of which is Phe508del. Phe508del causes not only intracellular retention and premature degradation of the mutant CFTR protein, but also defective channel gating and decreased half-life when experimentally rescued to the plasma membrane (PM). Despite recent successes in the functional rescue of several CFTR mutations with small-molecule drugs, the folding-corrector/gating-potentiator drug combinations approved for Phe508del-CFTR homozygous patients have shown only modest benefit. Several factors have been shown to contribute to this outcome, including an unexpected intensification of corrector-rescued Phe508del-CFTR PM instability after persistent co-treatment with potentiator drugs. We have previously shown that acute co-treatment with hepatocyte growth factor (HGF) can significantly enhance the chemical correction of Phe508del-CFTR. HGF coaxes the anchoring of rescued channels to the actin cytoskeleton via induction of RAC1 GTPase signalling. Here, we demonstrate that a prolonged, 15-day HGF treatment also significantly improves the functional rescue of Phe508del-CFTR by the VX-809 corrector/VX-770 potentiator combination, in polarized bronchial epithelial monolayers. Importantly, we found that HGF treatment also prevented VX-770-mediated destabilization of rescued Phe508del-CFTR and enabled further potentiation of the rescued channels. Most strikingly, prolonged HGF treatment prevented previously unrecognized epithelial dedifferentiation effects of sustained exposure to VX-809. This was observed in epithelium-like monolayers from both lung and intestinal origin, representing the two systems most affected by adverse symptoms in patients treated with VX-809 or the VX-809/VX-770 combination. Taken together, our findings strongly suggest that co-administration of HGF with corrector/potentiator drugs could be beneficial for CF patients.

2018-08-29Journal article

Open access