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

Shyam Ramachandran,Samantha R Osterhaus,Kalpaj R Parekh,Ashley M Jacobi,Mark A Behlke,Paul B Mccray

doi:10.1074/jbc.m116.754283

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.

Highlights

A major focus of current CF research and therapeutics development is understanding factors regulating CFTR expression and biosynthesis and the cellular mechanisms underlying ⌬F508-CFTR degradation and exploiting this knowledge to identify drugs or small molecules that might restore ⌬F508CFTR function
MiR-138 and SIN3A Regulate Genes Influencing CFTR Biosynthesis—We previously reported that miR-138 overexpression or SIN3A knockdown improved ⌬F508-CFTR trafficking and function in airway epithelia and altered transcript levels of target genes [9]
These results suggest that FBXO2 and NEDD8 act via the same pathway, which is distinct from the effects of synovial apoptosis inhibitor 1 (SYVN1), and that the SCFFBXO2 complex is involved in ⌬F508-CFTR ubiquitination and degradation in airway epithelia

Summary

Edited by George DeMartino

We previously reported that delivery of a microRNA-138 mimic or siRNA against SIN3A to cultured cystic fibrosis (⌬F508/⌬F508) airway epithelia partially restored ⌬F508-cystic fibrosis transmembrane conductance regulator (CFTR)-mediated cAMP-stimulated Cl؊ conductance. We found that depletion of the ubiquitin ligase SYVN1, the ubiquitin/proteasome system regulator NEDD8, or the F-box protein FBXO2 partially restored ⌬F508-CFTR-mediated Cl؊ transport in primary cultures of human cystic fibrosis airway epithelia. Both interventions increased the abundance of fully glycosylated ⌬F508-CFTR and partially restored ⌬F508-CFTR-mediated ClϪ transport in primary CF epithelia from multiple human donors [9] This novel finding identified a previously unrecognized mechanism regulating CFTR expression and biosynthesis. We identified synovial apoptosis inhibitor 1 (SYVN1) (Hrd; E3 ubiquitin ligase), neural precursor cell expressed, developmentally down-regulated 8 (NEDD8; neddylation), and FBXO2 (Fbs; E3 ubiquitin ligase) as members of this gene network that mediate partial restoration of ⌬F508-CFTR trafficking and function. RNAi-mediated depletion of each of these factors increased ⌬F508-CFTR protein maturation and significantly improved ⌬F508-CFTR-mediated ClϪ transport These findings provide a new understanding of how CFTR expression is regulated in airway epithelia

Results

Discussion

Experimental Procedures