Browsing by Author "Boyd, A. Christopher"
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- Folding and rescue of a cystic fibrosis transmembrane conductance regulator trafficking mutant identified using human-murine chimeric proteinsPublication . Da Paula, Ana Carina; Sousa, Marisa; Xu, Zhe; Dawson, Elizabeth S.; Boyd, A. Christopher; Sheppard, David N.; Amaral, Margarida D.Impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel causes cystic fibrosis, a fatal genetic disease. Here, to gain insight into CFTR structure and function, we exploited interspecies differences between CFTR homologues using human (h)-murine (m) CFTR chimeras containing murine nucleotide-binding domains (NBDs) or regulatory domain on an hCFTR backbone. Among 15 hmCFTR chimeras analyzed, all but two were correctly processed, one containing part of mNBD1 and another containing part of mNBD2. Based on physicochemical distance analysis of divergent residues between human and murine CFTR in the two misprocessed hmCFTR chimeras, we generated point mutations for analysis of respective CFTR processing and functional properties. We identified one amino acid substitution (K584E-CFTR) that disrupts CFTR processing in NBD1. No single mutation was identified in NBD2 that disrupts protein processing. However, a number of NBD2 mutants altered channel function. Analysis of structural models of CFTR identified that although Lys584 interacts with residue Leu581 in human CFTR Glu584 interacts with Phe581 in mouse CFTR. Introduction of the murine residue (Phe581) in cis with K584E in human CFTR rescued the processing and trafficking defects of K584E-CFTR. Our data demonstrate that human-murine CFTR chimeras may be used to validate structural models of full-length CFTR. We also conclude that hmCFTR chimeras are a valuable tool to elucidate interactions between different domains of CFTR.
- 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.