Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect.

Guido Veit,Pirjo M Apaja,Sheng-Ting Lin,Gergely L Lukacs,Kathryn Oliver,Mert Icyuz,Aurélien Bidaud-Meynard,Jingyu Guo,Doranda Perdomo,John L Hartman,Eric J Sorscher,David Ron

doi:10.1371/journal.pbio.1002462

Guido Veit, Pirjo M Apaja + Show 10 more

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https://doi.org/10.1371/journal.pbio.1002462

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Abstract

The most common cystic fibrosis (CF) causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del), results in functional expression defect of the CF transmembrane conductance regulator (CFTR) at the apical plasma membrane (PM) of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER). Deletion of phenylalanine 670 (ΔF670) in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter) of Saccharomyces cerevisiae phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic) analysis of the Yor1-ΔF670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ΔF508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ΔF508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor) restored the mutant function to ~50% of the wild-type channel in primary CFTRΔF508/ΔF508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect.

Highlights

Cystic fibrosis (CF), caused by mutations in cystic fibrosis transmembrane conductance regulator (CFTR), is characterized by multiorgan pathology, mainly affecting the upper and lower airways, gastrointestinal tract, and endocrine system [1,2]
Our data suggest that reducing the translational elongation rate via RPL12 silencing can partially reverse the ΔF508-CFTR folding defect
RPL12 silencing in combination with the corrector drug VX-809, increased the mutant function to 50% of the wild-type CFTR channel, suggesting that the ribosomal stalk perturbation may represent a therapeutic target for rescuing the ΔF508-CFTR biogenesis defect

Summary

Introduction

Cystic fibrosis (CF), caused by mutations in cystic fibrosis transmembrane conductance regulator (CFTR), is characterized by multiorgan pathology, mainly affecting the upper and lower airways, gastrointestinal tract, and endocrine system [1,2]. Modifier genes may facilitate the ΔF508-CFTR functional rescue by enhancing the mRNA or protein expression, folding, stability, or by inhibiting its degradation at the ER and post-ER compartments [11,12, 23,24,25]. Strategies focusing on reverting the maladaptive stress response in CF have been proposed [32]. None of these approaches, appear to attain sufficient functional correction in preclinical studies to be therapeutically robust in patients with the most common CF mutation, in individuals carrying only one copy of ΔF508-CFTR, representing 40% of US CF patients [3]

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