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

PLoS Biol. 2016 May 11;14(5):e1002462. doi: 10.1371/journal.pbio.1002462. eCollection 2016 May.

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.

MeSH terms

  • ATP-Binding Cassette Transporters / metabolism
  • Aminopyridines / pharmacology
  • Benzodioxoles / pharmacology
  • Bronchi / drug effects
  • Bronchi / metabolism
  • Bronchi / pathology
  • Cells, Cultured
  • Cystic Fibrosis / drug therapy
  • Cystic Fibrosis / genetics
  • Cystic Fibrosis / pathology
  • Cystic Fibrosis Transmembrane Conductance Regulator / chemistry
  • Cystic Fibrosis Transmembrane Conductance Regulator / genetics*
  • Cystic Fibrosis Transmembrane Conductance Regulator / metabolism*
  • Epithelial Cells / drug effects
  • Gene Knockdown Techniques
  • Gene Silencing
  • High-Throughput Screening Assays
  • Humans
  • Peptide Elongation Factor 2 / genetics
  • Peptide Elongation Factor 2 / metabolism
  • Protein Folding
  • Protein Stability
  • RNA, Small Interfering
  • Ribosomal Proteins / genetics*
  • Ribosomal Proteins / metabolism
  • Saccharomyces cerevisiae Proteins / metabolism
  • Yeasts / genetics

Substances

  • ATP-Binding Cassette Transporters
  • Aminopyridines
  • Benzodioxoles
  • CFTR protein, human
  • Peptide Elongation Factor 2
  • RNA, Small Interfering
  • RPL12 protein, human
  • Ribosomal Proteins
  • Saccharomyces cerevisiae Proteins
  • YOR1 protein, S cerevisiae
  • Cystic Fibrosis Transmembrane Conductance Regulator
  • lumacaftor