Percorrer por autor "Gomes-Alves, Patrícia"
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- Proteomics uncovering possible key players in F508del-CFTR processing and traffickingPublication . Gomes-Alves, Patrícia; Penque, DeborahThe achievement and maintenance of a protein native conformation is a very complex cellular process involving a multitude of key factors whose contribution to a successful folding remains to be elucidated. On top of this, it is known that correct folding is crucial for proteins to play their normal role and, consequently, for the maintenance of cellular homeostasis or proteostasis. If the folding process is affected, the protein is unable to achieve its native conformation, compromising its life and function, and a pathological condition may arise. Protein-misfolding diseases are characterized by either formation of protein aggregates that are toxic to the cell (gain-of-toxic-function diseases) or by an incorrect processing of proteins, which leads to a deficiency in protein activity (loss-of-function diseases). In this article we have focused on proteomics advances in the molecular knowledge of protein-misfolding diseases with direct impact on possible key players in F508del-CFTR processing and trafficking.
- Rescue of F508del-CFTR by RXR motif inactivation triggers proteome modulation associated with the unfolded protein responsePublication . Gomes-Alves, Patrícia; Couto, Francisco; Pesquita, Cátia; Coelho, Ana V.; Penque, DeborahF508del-CFTR, the most common mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, disrupts intracellular trafficking leading to cystic fibrosis (CF). The trafficking defect of F508del-CFTR can be rescued by simultaneous inactivation of its four RXR motifs (4RK). Proteins involved in the F508del-CFTR trafficking defect and/or rescue are therefore potential CF therapeutic targets. We sought to identify these proteins by investigating differential proteome modulation in BHK cells over-expressing wt-CFTR, F508del-CFTR or the revertant F508del/4RK-CFTR. By 2-dimensional electrophoresis-based proteomics and western blot approaches we demonstrated that over-expression of F508del/4RK-CFTR modulates the expression of a large number of proteins, many of which are reported interactors of CFTR and/ or 14-3-3 with potential roles in CFTR trafficking. GRP78/BiP, a marker of ER stress and unfolded protein response (UPR), is up-regulated in cells over-expressing either F508del-CFTR or F598del/4RK-CFTR. However, over-expression of F508del/4RK-CFTR induces the up-regulation of many other UPR-associated proteins (e.g. GRP94, PDI, GRP75/mortalin) and, interestingly, the down-regulation of proteasome components associated with CFTR degradation, such as the proteasome activator PA28 (PSME2) and COP9 signalosome (COPS5/CSN5). Moreover, the F508del-CFTR-induced proteostasis imbalance, which involves some heat shock chaperones (e.g. HSP72/Hpa2), ER-EF-hand Ca2+-binding proteins (calumenin) and the proteasome activator PA28 (PSME2), tends to be ‘restored’, i.e., in BHK cells over-expressing F508del/4RK-CFTR those proteins tend to have expression levels similar to the wild-type ones. These findings indicate that a particular cellular environment orchestrated by the UPR contributes to and/or is compatible with F508del/4RK-CFTR rescue.
- SELDI-TOF biomarker signatures for cystic fibrosis, asthma and chronic obstructive pulmonary diseasePublication . Gomes-Alves, Patrícia; Imrie, Margaret; Gray, Robert D.; Nogueira, Paulo; Ciordia, Sergio; Pacheco, Paula; Azevedo, Pilar; Lopes, Carlos; De Almeida, António Bugalho; Guardiano, Micaela; Porteous, David J.; Albard, Juan P.; Boyd, A. Christopher; Penque, DeborahOBJECTIVES: The aim of this work was to establish protein profiles in serum and nasal epithelial cells of cystic fibrosis individuals in comparison with controls, asthma and chronic obstructive pulmonary disease patients for specific biomarker signatures identification. DESIGN AND METHODS: Protein extracts were analyzed by Surface Enhanced Laser Desorption/Ionization Time-Of-Flight Mass-Spectrometry (SELDI-TOF-MS). RESULTS: The mass spectra revealed a set of peaks with differential expression in serum and nasal cells among the different groups studied, resulting into peak signatures representative/specific of each pathology. Logistic regressions were applied to those peaks; sensitivity, specificity, Youden's indexes and area under the curve (AUC) of the respective receiver operating characteristic (ROC) curves were compared. DISCUSSION: Multivariate analysis demonstrated that combination of peaks has a better predictive value than the individual ones. These protein signatures may serve as diagnostic/prognostic markers for the studied diseases with common clinical features, or as follow-up assessment markers of therapeutic interventions.