Proteomic interaction profiling reveals KIFC1 as a factor involved in early targeting of F508del-CFTR to degradation

Abstract

Misfolded F508del-CFTR, the main molecular cause of the recessive disorder cystic fibrosis, is recognized by the endoplasmic reticulum (ER) quality control (ERQC) resulting in its retention and early degradation. The ERQC mechanisms rely mainly on molecular chaperones and on sorting motifs, whose presence and exposure determine CFTR retention or exit through the secretory pathway. Arginine-framed tripeptides (AFTs) are ER retention motifs shown to modulate CFTR retention. However, the interactions and regulatory pathways involved in this process are still largely unknown. Here, we used proteomic interaction profiling and global bioinformatic analysis to identify factors that interact differentially with F508del-CFTR and F508del-CFTR without AFTs (F508del-4RK-CFTR) as putative regulators of this specific ERQC checkpoint. Using LC-MS/MS, we identified kinesin family member C1 (KIFC1) as a stronger interactor with F508del-CFTR versus F508del-4RK-CFTR. We further validated this interaction showing that decreasing KIFC1 levels or activity stabilizes the immature form of F508del-CFTR by reducing its degradation. We conclude that the current approach is able to identify novel putative therapeutic targets that can be ultimately used to the benefit of CF patients.

KLF4 Acts as a wt-CFTR Suppressor through an AKT-Mediated Pathway

“…It is possible that the intrinsic instability of F508del-CFTR protein [ 30 ] could mask the enhancement of immature F508del–CFTR protein by KLF4. In parallel, since it is established that F508del- and wt-CFTR have different interactomes [ 31 , 32 , 33 , 34 , 35 ], F508del–CFTR as an immature/unstable protein does not establish the required interactions for KLF4 to exert its signaling as an F508del–CFTR modulator. Moreover, alterations in intracellular pH caused by defective CFTR may also have an impact on the signaling exerted by KLF4 [ 36 ] In fact, KLF4 KD/KO and overexpression experiments showed no major impact on F508del–CFTR expression.…”

Folding Status Is Determinant over Traffic-Competence in Defining CFTR Interactors in the Endoplasmic Reticulum

“…To further characterize the ERQC, we identified proteins involved in recognizing the AFTs (i.e., at the third ERQC checkpoint). Previously, we used differential interactomics to identify such regulators [18]. Now, we used a pull-down approach in non-CFTR expressing CFBE cells with peptides mimicking such CFTR regions: 10 amino acid-long peptides corresponding to the CFTR sequence around each of the AFTs (see Materials and Methods).…”

“…CFBE41o− (cystic fibrosis bronchial epithelial) cells stably expressing DD/AA-CFTR were generated as previously described [18]. Briefly, CFTR cDNA bearing D565A and D567A (DD/AA) was introduced, cloned into the lentiviral expression vector pLVX-Puro by homologous recombination using the In-Fusion HD Cloning Kit (Clontech-Takara, #121416, Saint-Germain-en-Laye, France).…”

“…For lentiviral particles’ production, plasmids with the correct sequence were used to calcium phosphate-transfect HEK 293T cell. Lentiviral particles were collected 48 h after transfection and were used to transduce parental CFBE41o− cells [18].…”

“…Similar studies have confirmed the relevance of the protein biogenesis machinery in distinguishing these two CFTR variants, through identification of the eukaryotic translation initiation factor 3 [17] as a stronger interactor of F508del-CFTR whose modulation can rescue the mutant protein to the cell surface. Most of these studies however were focused on the wt- and F508del-CFTR interactomes, although recent work also compared the F508del-CFTR interactome with that of the F508del-4RK-CFTR variant, identifying critical differential interactions, which when modulated can lead to the rescue of F508del-CFTR to the PM [18] or others focused on the interactomes of other disease-occurring CFTR variants.…”