title: Folding and rescue of a cystic fibrosis transmembrane conductance regulator trafficking mutant identified using human-murine chimeric proteins.
abstract: 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 Lys(584) interacts with residue Leu(581) in human CFTR Glu(584) interacts with Phe(581) in mouse CFTR. Introduction of the murine residue (Phe(581)) 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.
authors.affiliation: University of Lisboa, Faculty of Sciences, BioFIG-Centre for Biodiversity, Functional and Integrative Genomics, 1749-016 Lisboa, Portugal; Department of Genetics, National Institute of Health, 1649-016 Lisboa, Portugal.
authors.forename: Ana Carina
authors.initials: AC
authors.lastname: Da Paula
authors.affiliation: University of Lisboa, Faculty of Sciences, BioFIG-Centre for Biodiversity, Functional and Integrative Genomics, 1749-016 Lisboa, Portugal; Department of Genetics, National Institute of Health, 1649-016 Lisboa, Portugal.
authors.forename: Marisa
authors.initials: M
authors.lastname: Sousa
authors.affiliation: Department of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Xu
authors.affiliation: Medical Genetics Section, Molecular Medicine Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, United Kingdom.
authors.forename: Elizabeth S
authors.initials: ES
authors.lastname: Dawson
authors.affiliation: Medical Genetics Section, Molecular Medicine Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, United Kingdom.
authors.forename: A Christopher
authors.initials: AC
authors.lastname: Boyd
authors.affiliation: Department of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom.
authors.forename: David N
authors.initials: DN
authors.lastname: Sheppard
authors.affiliation: University of Lisboa, Faculty of Sciences, BioFIG-Centre for Biodiversity, Functional and Integrative Genomics, 1749-016 Lisboa, Portugal; Department of Genetics, National Institute of Health, 1649-016 Lisboa, Portugal. Electronic address: mdamaral@fc.ul.pt.
authors.forename: Margarida D
authors.initials: MD
authors.lastname: Amaral
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D020125:Mutation, Missense; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C505032:CFTR protein, human; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator