The endoplasmic reticulum quality control : dissecting protein networks in cystic fibrosis

Tese de doutoramento, Biologia (Biologia de Sistemas), Universidade de Lisboa, Faculdade de Ciências, 2018Cystic Fibrosis (CF) is the most common lethal monogenic autosomal recessive disease among Caucasian population and it is caused by dysfunction of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein which is located at the apical membrane of epithelial cells. The most common CF-causing mutation is a deletion of phenylalanine 508 - F508del, present in approximately 85% of CF patients - and leads to CFTR misfolding which is recognized by the endoplasmic reticulum (ER) quality control (ERQC) resulting in ER retention and degradation. CFTR traffic from the ER is mediated by specific sorting motifs that include retention motifs, such as the arginineframed tripeptides (AFTs) and of the diacidic (DAD) exit code that controls the interaction with the COPII machinery. Here, we aimed at identifying traffic factors that regulate CFTR exit from the ER at these specific QC checkpoints. Using protein interaction profiling and global bioinformatic analysis we identified factors that interact specifically with F508del-CFTR (with/without mutated AFT motifs) and wt-CFTR (with/without abrogation of the DAD code). Proteins showing differential interactions were selected/validated. A high number of potential interacting proteins (> 800) was identified. In those with stronger interaction with F508del-CFTR (198), there is an enrichment in proteins involved in RNA processing and complex organization and a decrease in those related to epithelial integrity when compared to the interactome of F508del-CFTR with abrogated AFT motifs. Among these 198 proteins a subset of proteins was selected and validated. We found 4 proteins involved in the regulation of F508del-CFTR retention and degradation. For one of the hits, KIFC1, we identified that it is involved in CFTR targeting to degradation and also suggested, through the use of an available inhibitor, that the approach used here is able to identify novel putative therapeutic targets that can be ultimately used to the benefit of CF patients.BioSys - Sistemas Biológicos, Genómica Funcional & Integrativa, progama doutoral, FCT/PD/00065/2012; Fundação para a Ciência e a Tecnologia (FCT), SFRH/BD/52491/201