Examining the Cystic Fibrosis Transmembrane Conductance Regulator Protein Interactome and its Modulations Upon Mutation and Exposure to Frontline Therapies

Cystic fibrosis (CF) is a fatal inherited disease caused by mutations in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) that lead to varying clinical manifestations and severity. CF affects approximately 85,000 people and presents heterogeneous distribution globally. The limited efficacy of therapies for CF could be partly explained by molecular defects caused by single mutations. This highlights the importance of understanding the dynamic network of interacting proteins that impact its synthesis, folding, stability, localization and function. Proximity-dependent biotin identification (BioID) is a powerful approach for comprehensive interactome mapping as it can reveal novel interactions and monitor alterations under different conditions. Affinity-purification mass spectrometry (AP-MS) combined with BioID are a novel and effective means of mapping stable and transient protein-protein interactions in living cells. The focus of my work has been on utilizing BioID to map the CFTR interactome and examine alterations based on different culturing conditions, mutations, and exposure to therapies. The CFTR protein interaction network, and how it may be modulated by treatment, is poorly understood. This knowledge may help to guide theratyping efforts and provide co-targeting strategies for improving precision drugs. We have generated interactomes for WT CFTR and two of its variants, ΔF508 and G551D, in different culturing and drug-exposed (Kalydeco, Orkambi, Trikafta) conditions. For the first time, we also performed BioID on bronchial epithelial lines grown in normal culturing conditions and at the air-liquid interface (ALI). The ALI protocol induces further differentiation which reconstitutes many aspects of the in vivo airway. It is likely that this model has specialized interactions vital for CFTR biology. Moreover, we curated a list of high-confidence interactors of CFTR from recent proteomic studies that attempted to map out the CFTR interactome. Here we propose the first-ever CFTR surfaceome – the CFTR interaction network in the context of the cell surface. This highlighted increasing evidence surrounding the importance that early subunits of clathrin-mediated endocytosis have on CFTR activity and stability at the plasma membrane, which is supported in further investigations performed in our studies. We identify several novel interactors which have been characterized to be of importance for trafficking and function of CFTR. The proteomic data collected throughout this research represent rich datasets that could act as resources for researchers in the CF field and could be further mined to reveal additional candidates for the functional rescue of CFTR.