Proximity Profiling of the CFTR Interaction Landscape in Response to Orkambi.

Melissa Iazzi,Audrey Astori,Jonathan St-Germain,Brian Raught,Gagan D Gupta

doi:10.3390/ijms23052442

Abstract

Deletion of phenylalanine 508 (∆F508) of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel protein is the leading cause of Cystic Fibrosis (CF). Here, we report the analysis of CFTR and ∆F508-CFTR interactomes using BioID (proximity-dependent biotin identification), a technique that can also detect transient associations. We identified 474 high-confidence CFTR proximity-interactors, 57 of which have been previously validated, with the remainder representing novel interaction space. The ∆F508 interactome, comprising 626 proximity-interactors was markedly different from its wild type counterpart, with numerous alterations in protein associations categorized in membrane trafficking and cellular stress functions. Furthermore, analysis of the ∆F508 interactome in cells treated with Orkambi identified several interactions that were altered as a result of this drug therapy. We examined two candidate CFTR proximity interactors, VAPB and NOS1AP, in functional assays designed to assess surface delivery and overall chloride efflux. VAPB depletion impacted both CFTR surface delivery and chloride efflux, whereas NOS1AP depletion only affected the latter. The wild type and ∆F508-CFTR interactomes represent rich datasets that could be further mined to reveal additional candidates for the functional rescue of ∆F508-CFTR.

Highlights

Cystic fibrosis (CF) is a fatal inherited disease caused by mutations in CFTR that lead to varying clinical manifestations and severity [1,2]
The spike in fluorescence observed after the addition of the cAMP agonist forskolin (Fsk) in cells expressing FLAG-BirA*-CFTR (Figure 1b) is similar to what has been observed with wild type (WT) CFTR [20]
The FLAG-BirA*-CFTR fusion is able to retain its ability to localize to the plasma membrane (PM) and perform chloride efflux, and was selected for biotin identification (BioID) implementation

Summary

Introduction

Cystic fibrosis (CF) is a fatal inherited disease caused by mutations in CFTR that lead to varying clinical manifestations and severity [1,2]. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein resides at the apical membrane of epithelial cells and functions as an ion channel that mediates the flux of chloride and bicarbonate ions [3,4]. In 2015, ivacaftor-lumacaftor (Orkambi) was approved for use in patients aged 12 years or higher and homozygous for the ∆F508 mutation and was recently extended to include those aged 6–11 years old [7,9]. Orkambi is a combination treatment consisting of a small molecule corrector compound (VX-809, lumacaftor) that promotes protein stability and forward trafficking [10], and a small molecule potentiator (VX-770, ivacaftor) that promotes the open state of the channel [10]. Orkambi is associated with a variable clinical response [11,12]

Methods

Results

Conclusion