LSC Abstract – Identification of novel traffic factors involved in CFTR exit from the ER

Abstract

Cystic Fibrosis (CF) is an autosomal recessive disorder caused by dysfunction of the CF Transmembrane conductance Regulator (CFTR), a cAMP-regulated chloride channel expressed at the apical surface of epithelial cells. The most common disease-causing mutation, F508del, leads to CFTR misfolding, recognition by the endoplasmic reticulum (ER) quality control (ERQC), ER retention and early degradation. It is known that the retention of misfolded CFTR at ER is mediated by the exposure of arginine-framed tripeptides (AFTs). The recognition mechanism is not clear. Here, we aim to identify AFT interactor proteins involved in CFTR exit from the ER that may be used as novel therapeutic targets in the rescue of F508del-CFTR. Pull-down assays using CFBE cells were performed to isolate AFT interactors. Synthetic peptides designed to mimic mutated or non-mutated CFTR at the AFT regions were used. Samples were analyzed by LC-MS/MS and proteins showing differential interactions with the two sets of peptides were selected. A high number of AFT interactors was identified with the majority corresponding to proteins localized to the ER or involved in cytoskeleton regulation. Several of these novel interactors were not previously directly associated with CFTR regulation but some are associated with the activation of cAMP-dependent PKA related pathways, suggesting a connection with CFTR regulation. The identification of specific CFTR interactors/regulators and their validation which is in progress, is a promising approach in the identification of novel therapeutic targets that could ultimately benefit CF patients. Supported by FCT – Portugal: centre grant PEst-OE/BIA/UI4046/2011 (BioISI); PhD fellowship PD/BD/106084/2015 (JDS).