Combination of Correctors Rescue ΔF508-CFTR by Reducing Its Association with Hsp40 and Hsp27.
Correcting the processing of ΔF508-CFTR, the most common mutation in cystic fibrosis, is the major goal in the development of new therapies for this disease. Here, we determined whether ΔF508 could be rescued by a combination of small-molecule correctors, and identified the mechanism by which correctors rescue the trafficking mutant of cystic fibrosis transmembrane conductance regulator (CFTR). We transfected COS-7 cells with ΔF508, created HEK-293 stably expressing ΔF508, and utilized CFBE41o(-) cell lines stably transduced with ΔF508. As shown previously, ΔF508 expressed less protein, was unstable at physiological temperature, and rapidly degraded. When the cells were treated with the combination C18 + C4 the mature C-band was expressed at the cell surface. After treatment with C18 + C4, we saw a lower rate of protein disappearance after translation was stopped with cycloheximide. To understand how this rescue occurs, we evaluated the change in the binding of proteins involved in endoplasmic reticulum-associated degradation, such as Hsp27 (HspB1) and Hsp40 (DnaJ). We saw a dramatic reduction in binding to heat shock proteins 27 and 40 following combined corrector therapy. siRNA experiments confirmed that a reduction in Hsp27 or Hsp40 rescued CFTR in the ΔF508 mutant, but the rescue was not additive or synergistic with C4 + 18 treatment, indicating these correctors shared a common pathway for rescue involving a network of endoplasmic reticulum-associated degradation proteins.
From the Departments of Medicine and Physiology, Johns Hopkins University, Baltimore, Maryland 21205 and the Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
Miquéias
M
Lopes-Pacheco
From the Departments of Medicine and Physiology, Johns Hopkins University, Baltimore, Maryland 21205 and.
Clément
C
Boinot
From the Departments of Medicine and Physiology, Johns Hopkins University, Baltimore, Maryland 21205 and.
Inna
I
Sabirzhanova
the Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
Marcelo M
MM
Morales
From the Departments of Medicine and Physiology, Johns Hopkins University, Baltimore, Maryland 21205 and.
William B
WB
Guggino
From the Departments of Medicine and Physiology, Johns Hopkins University, Baltimore, Maryland 21205 and lcebotaru@jhmi.edu.
Liudmila
L
Cebotaru
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D055551:HSP27 Heat-Shock Proteins / Q000378:metabolism*; D050956:HSP40 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D013696:Temperature
C505032:CFTR protein, human; D055551:HSP27 Heat-Shock Proteins; D050956:HSP40 Heat-Shock Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
ABC transporter; chaperonin; electrophysiology; heat shock protein (HSP); lung; membrane; mutant; protein degradation; protein folding; protein processing