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.

activity. Three select representative traces for interactors whose expression did not affect channel activity are also shown for each.
Appendix Figure S8 -Hit siRNAs in the high-content CFTR trafficking assay. Representative images of CFBE cells expressing the wt-or F508del-variants of the mCherry-Flag-CFTR traffic reporter. Hoechst stains nuclei, mCherry labels all CFTR molecules and immunostained Flag in unpermeabilized cells labels CFTR molecules located at the PM. Cells treated with the non-targeting Neg1 siRNA or F508del-CFTR correctors (VX-809, VX-661) were taken as negative or positive controls, respectively. Significant loss of CFTR expression in cells treated with a CFTR-targeting siRNA indicates high transfection efficiency. The remaining conditions represent cells treated with siRNAs which rescue F508del-CFTR PM localization (Z-score > 1), as seen by increased fluorescence intensity in the Flag channel versus the negative control. One of the siRNAs targeting FGL2, which did not meet the Z-score hit threshold in the traffic screen, is also shown. Corresponding channels are shown in the same lookup table, except for wt-CFTR expressing cells, where contrast required adjustment due to the overall larger fluorescence intensity. Scale bar: 50 µm.
Appendix Figure S9 -Validation of selected interactions via co-immunoprecipitation (Co-IP) in HEK293T cells. A) Co-IP experiments showing 17 successfully validated interactions (out of 30 tested). Co-IPs were performed using anti-FLAG antibody directed against overexpressed FLAG-tagged protein corresponding to identified interactors, followed by Western blotting using anti-FLAG antibody (top panels) and anti-V5 antibody directed against the V5-tagged wildtype CFTR (lower panels). Red asterisks indicate bands of the size corresponding to the FLAG-tagged interactor protein being tested. Images are representative of n=2 biological replicates. B) Co-IPs performed to confirm the FGL2/CFTR interaction. Pull-downs were performed using anti-V5 antibody, followed by Western blotting with anti-V5 and anti-FLAG antibody (top panel) or using anti-FLAG antibody, followed by Western blotting with anti-FLAG and anti-V5 antibody (bottom panel). C) Control co-IPs for all tested samples, performed using unrelated anti-GAPDH antibody, showing that over-expressed FLAG-tagged interactors do not non-specifically pull-down unrelated protein.
Appendix Figure S10 -Overview of the validation strategy used in this work. Created with BioRender.com.
Appendix Figure S1-Overview of the MaMTH system. Integral membrane 'bait', fused to the Cterminus of ubiquitin (Cub) and an artificial transcription factor (TF), is expressed alongside cytosolic or membrane-bound 'prey' fused to the N-terminus of ubiquitin (Nub) in cells carrying a reporter expression system. Interaction of bait and prey leads to ubiquitin reconstitution, proteolytic cleavage by deubiquitinating enzymes (DUBs) and subsequent release of the TF, which then enters the nucleus of the cell and activates reporter (luciferase in the traditional MaMTH system or eGFP in MaMTH-HTS).

C) D)
Appendix Figure S7 -FLIPR membrane potential traces for interactors whose transient expression in HEK293 cells affects CFTR channel activity. A) Interactors affecting F508del-CFTR channel activity. B) Interactors affecting wt-CFTR channel activity. Three select representative traces for interactors whose expression did not affect channel activity are also shown for each.  Figure S8 -Hit siRNAs in the high-content CFTR trafficking assay. Representative images of CFBE cells expressing the wt-or F508del-variants of the mCherry-Flag-CFTR traffic reporter. Hoechst stains nuclei, mCherry labels all CFTR molecules and immunostained Flag in unpermeabilized cells labels CFTR molecules located at the PM. Cells treated with the non-targeting Neg1 siRNA or F508del-CFTR correctors (VX-809, VX-661) were taken as negative or positive controls, respectively. Significant loss of CFTR expression in cells treated with a CFTR-targeting siRNA indicates high transfection efficiency. The remaining conditions represent cells treated with siRNAs which rescue F508del-CFTR PM localization (Z-score > 1), as seen by increased fluorescence intensity in the Flag channel versus the negative control. One of the siRNAs targeting FGL2, which did not meet the Z-score hit threshold in the traffic screen, is also shown. Corresponding channels are shown in the same lookup table, except for wt-CFTR expressing cells, where contrast required adjustment due to the overall larger fluorescence intensity. Scale bar: 50 µm.