title: Rescue of F508del-CFTR by RXR motif inactivation triggers proteome modulation associated with the unfolded protein response.
abstract: F508del-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 Ca(2+)-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.
authors.affiliation: Laboratório de Proteómica, Departamento de Genética, Instituto Nacional de Saúde Dr Ricardo Jorge (INSA, I.P.), 1649-016 Lisboa, Portugal.
authors.forename: Patrícia
authors.initials: P
authors.lastname: Gomes-Alves
authors.forename: Francisco
authors.initials: F
authors.lastname: Couto
authors.forename: Cátia
authors.initials: C
authors.lastname: Pesquita
authors.forename: Ana V
authors.initials: AV
authors.lastname: Coelho
authors.forename: Deborah
authors.initials: D
authors.lastname: Penque
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D008954:Models, Biological; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040081:Protein Array Analysis; D020543:Proteome / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013312:Stress, Physiological; D053719:Tandem Mass Spectrometry; D056811:Unfolded Protein Response
chemical_list: D048948:14-3-3 Proteins; C505032:CFTR protein, human; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D050505:Mutant Proteins; D020543:Proteome; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator