Browsing by Author "Gomes-Duarte, Andreia"
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- eIF3: a factor for human health and diseasePublication . Gomes-Duarte, Andreia; Lacerda, Rafaela; Menezes, Juliane; Romão, LuísaThe eukaryotic initiation factor 3 (eIF3) is one of the most complex translation initiation factors in mammalian cells, consisting of several subunits (eIF3a to eIF3m). It is crucial in translation initiation and termination, and in ribosomal recycling. Accordingly, deregulated eIF3 expression is associated with different pathological conditions, including cancer. In this manuscript, we discuss the interactome and function of each subunit of the human eIF3 complex. Furthermore, we review how altered levels of eIF3 subunits correlate with neurodegenerative disorders and cancer onset and development; in addition, we evaluate how such misregulation may also trigger infection cascades. A deep understanding of the molecular mechanisms underlying eIF3 role in human disease is essential to develop new eIF3-targeted therapeutic approaches and thus, overcome such conditions.
- G418 as a suppression therapy for beta-thalassemia diseasePublication . Dias, Patrícia; Gomes-Duarte, Andreia; Romão, LuísaPremature translation-termination codons (PTCs or nonsense codons) can arise from mutations in germ or somatic cells. The introduction of a PTC into an mRNA can trigger nonsense-mediated decay (NMD), an important mRNA surveillance mechanism that typically recognizes and degrades mRNAs containing PTCs to prevent the synthesis of C-terminally truncated proteins potentially toxic for the cell. The physiological relevance of NMD is manifested by the fact that about one third of genetic disease-associated mutations generate PTCs, including -thalassemia. In recent years, a novel therapeutic approach entitled suppression therapy has been developed based on low molecular weight compounds to induce the translation machinery to recode a PTC into a sense codon, the so called “readthrough” (or suppression). Here, by using a model of constructs containing the 5’ part of the normal, or nonsense-mutated, human -globin gene fused to the firefly luciferase gene as a reporter, we intend to prove the principle that the suppression therapy can restore enough -globin protein to outweight the manifestations of -thalassemia. Our results from bioluminescence assays and Western blot analyses have shown that the aminoglycoside G418 is able to suppress a nonsense mutation at codon 15 or 39 of the human -globin mRNA, in cultured HEK293 cells. We are now interested in stablishing how NMD inhibition can increase the efficiency of suppression therapy. A depper study on the suppression therapy is crucial, as it offers a major approach to treat a wide range of inherited pathologies.
- Prolonged co-treatment with HGF sustains epithelial integrity and improves pharmacological rescue of Phe508del-CFTRPublication . Matos, Ana M.; Gomes-Duarte, Andreia; Faria, Márcia; Barros, Patrícia; Jordan, Peter; Amaral, Margarida D.; Matos, PauloCystic 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.
- Suppression therapy as novel approach for genetic diseases and cancerPublication . Gomes-Duarte, Andreia; Dias, Parícia; Romão, LuísaPremature translation-termination codons (PTCs or nonsense codons) can arise from mutations in germ or somatic cells. The introduction of a PTC into an mRNA can trigger nonsense-mediated decay (NMD), an important mRNA surveillance mechanism that typically recognizes and degrades mRNAs containing PTCs to prevent the synthesis of C-terminally truncated proteins potentially toxic for the cell. The physiological importance of NMD is manifested by the fact that about one third of genetic disease-associated mutations generate PTCs, including beta-thalassemia. In recent years, a novel therapeutic approach entitled suppression therapy has been developed based on low molecular weight compounds to induce the translation machinery to recode a PTC into a sense codon, the so called “readthrough”. Here, by using a model of constructs containing the firefly luciferase gene as a reporter gene for beta-globin transcripts that result from PTCs, we intend to prove the principle that the suppression therapy can restore enough beta-globin protein to outweight the disease manifestations of beta-thalassemia. Our preliminary results show that both the aminoglycoside G418 and non-aminoglycoside PTC124 do not seem to be able to suppress the nonsense mutation at codon 26 or 39 of the human beta-globin mRNA in cultured HeLa cells, as reflected on the firefly luciferase activity and protein levels assessed by bioluminescence assays and Western blot, respectively. Regarding future directions, a deeper study on the use of G418 and PTC124 as efficient suppression agents for the treatment of PTC-associated diseases will be performed as it offers a major potential to treat a wide range of inherited pathologies.