Disrupting abnormal protein-protein interactions to correct the functional defect of F508del-CFTR – CFTRgateway

The interactome of the wild and mutated CFTR channel was carried out by proximity labeling coupled with mass spectrometry. This new technology is well suited for the study of transmembrane proteins such as CFTR. We evaluated two methodologies by merging on CFTR either APEX2 or TurboID, which do not modify the channel characteristics. These different enzymes allow the addition to Lysines present on the partner protein of a phenol-biotin group (APEX2) or biotin group (TurboID) which enables to purify them with streptavidin beads and their subsequent identification by mass spectrometry. This methodology identified around 1000 CFTR partner proteins, with around 50% of proteins common to both methods. Comparison of the normal protein with CFTR mutants revealed an atypical mutant partially present at the surface, partners involved in the degradation of mutant CFTR, its stability on the cell surface and partners not restored by current treatments. The implication in CFTR biogenesis or activity of approximately 50 identified partners was evaluated on normal and mutated CFTR in combination with CFTR modulators.

This project implemented a new methodology to identify protein partners of CFTR involved in cystic fibrosis as well as KCNK3 involved in arterial hypertension. The results unmasks the CFTR-PRAF2 interaction as a target of the corrector VX-445 which relieves the retention of the mutated protein in the endoplasmic reticulum, identifies an atypical mutant present on the surface and partners involved in the degradation of CFTR or modulating its stability on the surface of cells which represent targets to increase the effectiveness of current treatments.

This project established a new methodology to identify protein partners of CFTR involved in cystic fibrosis.

This methodology identified an atypical defect induced by the N1303K mutation, improving the treatment of this mutation, better defined the mechanism of action of the VX-445 corrector which could be applied to other proteins having RXR motifs, identified CFTR partners involved in the ubiquitination of the mutated protein or modulating surface stability. The inhibition of certain partners could therefore increase the correction of CFTR with the correctors VX-661 and VX-445 in combination with the potentiator VX-770 (Trikafta treatment effective in patients), a treatment which does not correct all the defects associated with F508del. Among the partners identified, those involved in the degradation of CFTR (e.g. ubiquitination) or modulating its surface stability (e.g. CAL), represent therapeutic targets to increase the effectiveness of current treatments. This methodology also made it possible to establish the interactome of the KCNK3 channel involved in arterial hypertension.

The exploration of the partners involved in the ubiquitination of CFTR will be continued in order to increase the stability of corrected CFTR. Reducing CFTR ubiquitination at an early stage would increase the efficacy of the correctors, stabilize the protein at the surface and restore the interactome, particularly with SLC transporters.
The stabilization of CFTR at the surface is an important issue for increasing the effectiveness of current treatments. As part of the ANR STABTHER (ANR-23-CE18-0003-01) led by Magali Taulan (INSERM U1046, PhyMedExp, University of Montpellier) and in which I am a partner, the CFTR-CAL interaction will be targeted using of peptides.
Finally, in order to better understand the mechanism of action of apigenin on CFTR WT and N1303K, an IDEX project was submitted in collaboration with Maria Miteva (INSERM U1268, UMR 8038 CNRS).

These new analysis methods were compared and used to identify new CFTR partners. Among the partners, we demonstrate that PRAF2 is a key player in the retention of CFTR in the endoplasmic reticulum and that the CFTR-PRAF2 interaction is targeted by the corrector VX-445. This method identifies an atypical mutant present on the surface allowing to adapt the treatment and has been used successfully on another channel involved in arterial hypertension.

Publications acceptées
Saha K, Chevalier B, Doly S, Baatallah N, Guilbert T, Pranke I, Scott MGH, Enslen H, Guerrera C, Chuon C, Edelman A, Sermet-Gaudelus I, Hinzpeter A*, Marullo S*. Pharmacological chaperone-rescued cystic fibrosis CFTR-F508del mutant overcomes PRAF2-gated access to endoplasmic reticulum exit sites. Cell Mol Life Sci. 2022 Sep 27;79(10):530.

Chevalier B, Baatallah N, Najm M, Castanier S, Jung V, Pranke I, Golec A, Stoven V, Marullo S, Antigny F, Guerrera IC, Sermet-Gaudelus I, Edelman A, Hinzpeter A. Differential CFTR-Interactome Proximity Labeling Procedures Identify Enrichment in Multiple SLC Transporters. Int J Mol Sci. 2022 Aug 11;23(16):8937.

Le Ribeuz H, Saint-Martin Willer A, Chevalier B, Sancho M, Masson B, Eyries M, Jung V, Guerrera C, Dutheil M, El Jekmek K, Laubry L, Carpentier G, Perez-Vizcaino F, Tu L, Guignabert C, Chaumais MC, Péchoux C, Humbert M, Hinzpeter A, Mercier O, Capuano V, Montani D and Antigny F. Role of KCNK3 Dysfunction in Dasatinib-Associated PAH and Endothelial Cell Dysfunction, American Journal of Respiratory Cell and Molecular Biology (accepté le 04/03/2024)

Publication en reviewing
Pranke I*, Capurro V*, Chevalier B*, Pesce E*, Tomati V, Pastorino C, Hatton A, Urien S, Lena M, Dréano E, Bocciardi R, Zara F, Pantano S, Terlizzi V, Lucanto C, Costa S, Claut L, Daccò V, Poli P, Maschio M, Fabrizzi B, Caporelli N, Cipolli M, Volpi S, Jung V, Roger K, Chedevergne F, Cosson L, Macey J, LeBihan J, Weiss L, Grenet D, LeClainche Viala L, Douvry B, Ravoninjatovo B, Audousset C, Tatopoulos A, Richaud Thiriez B, Baravalle M, Thouvenin G, Labbé G, Mittaine M, Reix P, Durieu I, Mankikian J, Bui S, Kelly-Aubert M, Nguyen–Khoa T, Khoukh K, Martin C, Guerrera C, Da Silva J, di Carli P, Castellani C, Cresta F, Galietta L, Guillemaut A, Bouazza N, Girodon E, Remus N, Burgel PR, Sermet-Gaudelus I#, Hinzpeter A#, Pedemonte N (*: co-first and #: co-last)
Beyond Kaftrio: mechanistic insights to maximize N1303K-CFTR rescue

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.