CFTR interactome mapping using the mammalian membrane two‐hybrid high‐throughput screening system

Abstract

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride and bicarbonate channel in secretory epithelia with a critical role in maintaining fluid homeostasis. Mutations in CFTR are associated with Cystic Fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasians. While remarkable treatment advances have been made recently in the form of modulator drugs directly rescuing CFTR dysfunction, there is still considerable scope for improvement of therapeutic effectiveness. Here, we report the application of a high-throughput screening variant of the Mammalian Membrane Two-Hybrid (MaMTH-HTS) to map the protein-protein interactions of wild-type (wt) and mutant CFTR (F508del), in an effort to better understand CF cellular effects and identify new drug targets for patient-specific treatments. Combined with functional validation in multiple disease models, we have uncovered candidate proteins with potential roles in CFTR function/CF pathophysiology, including Fibrinogen Like 2 (FGL2), which we demonstrate in patient-derived intestinal organoids has a significant effect on CFTR functional expression.

Effects of Elexacaftor/Tezacaftor/Ivacaftor on Sputum Viscoelastic Properties, Airway Infection and Inflammation in Patients with Cystic Fibrosis

“…We speculate that pharmacologically corrected F508del-CFTR may have specific binding partners during processing and trafficking that are only part of the CFTR-interactome when the protein is folded and not prematurely degraded. This notion is supported by a recent study showing large differences in the wild-type and F508del-CFTR interactomes [49].…”

Differential CFTR-Interactome Proximity Labeling Procedures Identify Enrichment in Multiple SLC Transporters

“…Of note, around half of the identified proteins were unique to each method (547 out of 1095 for APEX2, 446 out of 941 for TurboID and 466 out of 729 for coIP). In the same line, the recently developed MaMTH-HTS method, which used full-length CFTR-WT as a bait to screen a library of around 10,000 ORF [ 31 ], only marginally overlapped with proximity labeling or co-IP. Another important issue is the cell type used.…”

“…These strategies have provided detailed CFTR interactome maps and are constantly improving. While, yeast two-hybrid screens remain challenging for transmembrane proteins leading to the use of CFTR fragments as baits, technological advances now enable to screen full-length CFTR in mammalian cells [ 31 ] Immunoprecipitation approaches require cell lysis with detergents that may modify the interactome compared to interactions taking place in a living cell [ 32 , 33 , 34 , 35 ]. Furthermore, the specificity of the obtained interactomes depends on the availability and specificity of antibodies as well as the experimental conditions.…”

In Utero Mapping and Development Role of CFTR in Lung and Gastrointestinal Tract of Cystic Fibrosis Patients

“…Lim et al used a high-throughput screening variant of mammalian membrane two-hybrid technology to screen the human open reading frame (ORF)eome V8.1. collection (a library designed to screen for integral- and membrane associated proteins, consisting of >10,000 ORFs) of wildtype and F508del-CFTR mammalian cells . Furthermore, the authors used computational and functional analysis of the CFTR interactomes to identify interactors for essential CFTR roles, e.g., trafficking and regulation.…”

“…collection (a library designed to screen for integral-and membrane associated proteins, consisting of >10,000 ORFs) of wildtype and F508del-CFTR mammalian cells. 40 Furthermore, the authors used computational and functional analysis of the CFTR interactomes to identify interactors for essential CFTR roles, e.g., trafficking and regulation. They discovered a number of candidate proteins, including fibrinogen like 2 (FGL2) with likely influential roles in CFTR function and hence CF pathophysiology.…”