Chaperone displacement from mutant cystic fibrosis transmembrane conductance regulator restores its function in human airway epithelia

Abstract

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). The most common mutation, DeltaF508, omits the phenylalanine residue at position 508 in the first nucleotide binding domain (NBD1) of CFTR. The mutant protein is retained in the endoplasmic reticulum and degraded by the ubiquitin-proteasome system. We demonstrate that expression of NBD1 plus the regulatory domain (RD) of DeltaF508 CFTR (DeltaFRD) restores the biogenesis of mature DeltaF508 CFTR protein. In addition, DeltaFRD elicited a cAMP-stimulated anion conductance response in primary human bronchial epithelial (HBE) cells isolated from homozygous DeltaF508 CF patients. A protein transduction domain (PTD) could efficiently transduce (approximately 90%) airway epithelial cells. When fused to a PTD, direct addition of the DeltaFRD peptide conferred a dose-dependent, cAMP-stimulated anion efflux to DeltaF508 HBE cells. Hsp70 and Hsp90 associated equally with WT and DeltaF508 CFTR, whereas nearly twice as much of the Hsp90 cochaperone, Aha1, associated with DeltaF508 CFTR. Expression of DeltaFRD produced a dose-dependent removal of Aha1 from DeltaF508 CFTR that correlated with its functional rescue. These findings indicate that disruption of the excessive association of the cochaperone, Aha1, with DeltaF508 CFTR is associated with the correction of its maturation, trafficking and regulated anion channel activity in human airway epithelial cells. Thus, PTD-mediated DeltaFRD fragment delivery may provide a therapy for CF.

Transcomplementation by a Truncation Mutant of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Enhances ΔF508 Processing through a Biomolecular Interaction

“…Our earlier work and that of Sun et al (35) concluded that the truncation mutants were competing for and displacing ⌬F508 CFTR from key elements in the quality control mechanism, in our case, VCP (19) and HDAC6 and in theirs Aha1 (35). Low temperature rescue of ⌬F508 CFTR allows the processing of immature band B to mature band C but does not correct channel activity (see Fig.…”

“…This would contain the complete TMD1 and NBD1. Sun et al utilized a smaller fragment that included only NBD1 and the R domain (35). Given the results from these reports and the data here, the one common element of all these truncated CFTRs is the presence of the NBD1 domain.…”

Emergent Properties of Proteostasis in Managing Cystic Fibrosis

“…PN components facilitating folding of the cytosolic NBD1 and NBD2 are now recognized to include the Hsp40/Hsc-Hsp70 (Meacham et al 1999;Meacham et al 2001;Younger et al 2004;Younger et al 2006;Morito et al 2008;Rosser et al 2008;Grove et al 2009) and Hsp90 chaperone systems (Loo et al 1998;Fuller and Cuthbert 2000;Wang et al 2006;Sun et al 2008;Koulov et al 2010). Calnexin, a membrane-anchored ER-associated chaperone, has been shown to interact with the immature forms of both WT and DF508 vCFTR (Pind et al 1994), although its exact role in folding remains controversial Okiyoneda et al 2002;Amaral 2004;Okiyoneda et al 2004;Norez et al 2006;Okiyoneda et al 2008).…”

Improving Cell Surface Functional Expression of F508 CFTR: A Quest for Therapeutic Targets

“…Such transcomplementation does not result in changes in Hsc70 association but is believed to improve F508 CFTR forward folding through intra-and/or inter-molecular domaindomain interactions. A related but distinct approach to promote F508 CFTR maturation is to co-express a fragment of F508 CFTR containing NBD1 and R domains (Sun et al, 2008). This mutant fragment of CFTR can actually sequester key chaperone components from the endogenous F508 CFTR and lead to its rescue.…”

“…2). Due to misfolding, F508 CFTR has more extensive association with molecular chaperones (Yang et al, 1993;Jiang et al, 1998;Meacham et al, 1999;Wang et al, 2006;Sun et al, 2008;Roy et al, 2010). Therefore, the impact of chaperone machinery on F508 CFTR is greater than on wild-type CFTR.…”