SYVN1, NEDD8, and FBXO2 Proteins Regulate ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Ubiquitin-mediated Proteasomal Degradation

J Biol Chem. 2016 Dec 2;291(49):25489-25504. doi: 10.1074/jbc.M116.754283. Epub 2016 Oct 18.

Abstract

We previously reported that delivery of a microRNA-138 mimic or siRNA against SIN3A to cultured cystic fibrosis (ΔF508/ΔF508) airway epithelia partially restored ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR)-mediated cAMP-stimulated Cl- conductance. We hypothesized that dissecting this microRNA-138/SIN3A-regulated gene network would identify individual proteins contributing to the rescue of ΔF508-CFTR function. Among the genes in the network, we rigorously validated candidates using functional CFTR maturation and electrolyte transport assays in polarized airway epithelia. We found that depletion of the ubiquitin ligase SYVN1, the ubiquitin/proteasome system regulator NEDD8, or the F-box protein FBXO2 partially restored ΔF508-CFTR-mediated Cl- transport in primary cultures of human cystic fibrosis airway epithelia. Moreover, knockdown of SYVN1, NEDD8, or FBXO2 in combination with corrector compound 18 further potentiated rescue of ΔF508-CFTR-mediated Cl- conductance. This study provides new knowledge of the CFTR biosynthetic pathway. It suggests that SYVN1 and FBXO2 represent two distinct multiprotein complexes that may degrade ΔF508-CFTR in airway epithelia and identifies a new role for NEDD8 in regulating ΔF508-CFTR ubiquitination.

Keywords: E3 ubiquitin ligase; airway epithelia; cystic fibrosis; endoplasmic-reticulum-associated protein degradation (ERAD); neddylation; proteostasis; ubiquitylation (ubiquitination).

MeSH terms

  • Amino Acid Sequence*
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Cystic Fibrosis Transmembrane Conductance Regulator / genetics
  • Cystic Fibrosis Transmembrane Conductance Regulator / metabolism*
  • Epithelial Cells / metabolism*
  • Epithelial Cells / pathology
  • F-Box Proteins / genetics
  • F-Box Proteins / metabolism*
  • Gene Knockdown Techniques
  • HeLa Cells
  • Humans
  • Ion Transport / genetics
  • NEDD8 Protein
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism*
  • Proteasome Endopeptidase Complex / genetics
  • Proteasome Endopeptidase Complex / metabolism*
  • Proteolysis*
  • Respiratory Mucosa / metabolism*
  • Respiratory Mucosa / physiology
  • Sequence Deletion*
  • Ubiquitin / genetics
  • Ubiquitin / metabolism
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism*
  • Ubiquitination*
  • Ubiquitins / metabolism*

Substances

  • CFTR protein, human
  • Cell Cycle Proteins
  • F-Box Proteins
  • FBXO2 protein, human
  • NEDD8 Protein
  • NEDD8 protein, human
  • Nerve Tissue Proteins
  • Ubiquitin
  • Ubiquitins
  • Cystic Fibrosis Transmembrane Conductance Regulator
  • SYVN1 protein, human
  • Ubiquitin-Protein Ligases
  • Proteasome Endopeptidase Complex