Protein misfolding diseases (PMDs) are associated with either aggregation of misfolded proteins leading to toxic gain-of-function phenotypes or with protein degradation leading to loss-of-function phenotypes. Cystic Fibrosis (CF) is an example of a loss of function resulting from genetic mutations within CFTR. F508del, by far the most frequent mutation, is associated with protein misfolding, reduced channel function and cell surface stability. Mutant channel maturation and function can be partially rescued with small molecules referred to as correctors, while channel function is efficiently enhanced by potentiators. Nonetheless, this treatment combination is still suboptimal in clinics and there are still no means to increase channel cell surface stability. Our working hypothesis is to increase channel rescue by targeting specific protein-protein interactions, which retain abnormally folded proteins. This novel strategy is supported by our previous work, which revealed that an interaction with intermediary filament, Keratin 8, retains F508del-CFTR in the endoplasmic reticulum (ER) and that disruption of this interaction restores CFTR-F508del functional expression, leading to the identification of a new class of CFTR correctors. Preliminary data indicate that the two classes of correctors, targeting either CFTR directly or protein-protein interactions, present an additive effect. Our new results identified PRAF2 as a novel key regulator of CFTR exit from the ER, suggesting that PRAF2/F508del-CFTR interaction could also be a target for pharmacotherapy. We have previously shown that PRAF2 controls the ER exit of cargo transmembrane proteins (i.e. receptors, transporters…) in a stoichiometric manner and that this regulation involves the presence of a molecular code-bar (RXR motifs) located in the intracellular domains of PRAF2-regulated proteins. The first aim of this project is therefore to understand the role of PRAF2 in the ER exit of CFTR by identifying both the specific binding motifs and the additional molecular actors implicated. This will set the basis for a molecular screen aimed at identifying chemical compounds targeting the PRAF2/CFTR interaction. In parallel, we will identify new (i) differential (WT or mutant CFTR-specific) interatomics and (ii) compartment-specific protein-protein interactions for F508del-CFTR using the newest proteomic approaches: proximity labeling with APEX2 and APEX2-complementation coupled to mass spectrometry. The importance of these “new” molecular partners will be evaluated both functionally and biochemically. The best targets will be combined with CFTR modulators available today to enhance treatment efficacy and tested on primary epithelial cells. The end point to these studies would be the identification of new drugs capable of enhancing the efficacy of current treatments. We believe, indeed, that the release of partially functional misfolded proteins from specific protein-protein interactions will restore to some extent functional activity. For cystic fibrosis, this could be further enhanced by combination with available CFTR modulators. The data obtained so far support our strategy of targeting protein-protein interactions in PMDs and provide the flowchart to study other loss of function PMDs such as alpha-1-antitrypsin deficiency, diabetes, nephritic syndrome, and also to some extend other diseases such Chronic obstructive pulmonary disease (COPD)…. Consequently, the methodology developed in this project could in fine lead to the identification of key protein-protein interactions in other diseases which could be targeted and represent potentially treatments, which could similarly be improved be improved with a new class of drugs.