Misfolding of cystic fibrosis transmembrane conductance regulator protein (CFTR) causes the fatal lung disease cystic fibrosis. VX-809 was developed to suppress disease-related folding defects in CFTR. VX-809 suppresses folding defects in CFTR by modulating the conformation of membrane-spanning domain 1. VX-808 is thereby able to partially restore function to F508del-CFTR and other disease-related mutants.

Cystic fibrosis (CF) is a lethal recessive genetic disease caused primarily by the F508del mutation in the CF transmembrane conductance regulator (CFTR). The potentiator VX-770 was the first CFTR modulator approved by the FDA for treatment of CF patients with the gating mutation G551D. Orkambi is a drug containing VX-770 and corrector VX809 and is approved for treatment of CF patients homozygous for F508del, which has folding and gating defects. At least 30% of CF patients are heterozygous for the F508del mutation with the other allele encoding for one of many different rare CFTR mutations. Treatment of heterozygous F508del patients with VX-809 and VX-770 has had limited success, so it is important to identify heterozygous patients that respond to CFTR modulator therapy. R117H is a more prevalent rare mutation found in over 2,000 CF patients. In this study we investigated the effectiveness of VX-809/VX-770 therapy on restoring CFTR function in human bronchial epithelial (HBE) cells from R117H/F508del CF patients. We found that VX-809 stimulated more CFTR activity in R117H/F508del HBEs than in F508del/F508del HBEs. R117H expressed exclusively in immortalized HBEs exhibited a folding defect, was retained in the ER, and degraded prematurely. VX-809 corrected the R117H folding defect and restored channel function. Because R117 is involved in ion conductance, VX-770 acted additively with VX-809 to restore CFTR function in chronically treated R117H/F508del cells. Although treatment of R117H patients with VX-770 has been approved, our studies indicate that Orkambi may be more beneficial for rescue of CFTR function in these patients.

DNAJB12 (JB12) is an endoplasmic reticulum (ER)-associated Hsp40 family protein that recruits Hsp70 to the ER surface to coordinate the function of ER-associated and cytosolic chaperone systems in protein quality control. Hsp70 is stress-inducible, but paradoxically, we report here that JB12 was degraded by the proteasome during severe ER stress. Destabilized JB12 was degraded by ER-associated degradation complexes that contained HERP, Sel1L, and gp78. JB12 was the only ER-associated chaperone that was destabilized by reductive stress. JB12 knockdown by siRNA led to the induction of caspase processing but not the unfolded protein response. ER stress-induced apoptosis is regulated by the highly labile and ER-associated BCL-2 family member BOK, which is controlled at the level of protein stability by ER-associated degradation components. We found that JB12 was required in human hepatoma cell line 7 (Huh-7) liver cancer cells to maintain BOK at low levels, and BOK was detected in complexes with JB12 and gp78. Depletion of JB12 during reductive stress or by shRNA from Huh-7 cells was associated with accumulation of BOK and activation of Caspase 3, 7, and 9. The absence of JB12 sensitized Huh-7 to death caused by proteotoxic agents and the proapoptotic chemotherapeutic LCL-161. In summary, JB12 is a stress-sensitive Hsp40 whose degradation during severe ER stress provides a mechanism to promote BOK accumulation and induction of apoptosis.

ABSTRACT. Misfolded proteins in the endoplasmic reticulum (ER) are dislocated out of the ER to the cytosol, polyubiquitinated, and degraded by the ubiquitin-proteasome system in a process collectively termed ERassociated degradation (ERAD). Recent studies have established that a mammalian ER-localized transmembrane J-protein, DNAJB12, cooperates with Hsc70, a cytosolic Hsp70 family member, to promote the ERAD of misfolded membrane proteins. Interestingly, mammalian genomes have another J-protein called DNAJB14 that shows a high sequence similarity to DNAJB12. Yet, very little was known about this protein. Here, we report the characterization of DNAJB14. Immunofluorescence study and protease protection assay showed that, like DNAJB12, DNAJB14 is an ER-localized, single membrane-spanning J-protein with its J-domain facing the cytosol. We used co-immunoprecipitation assay to find that DNAJB14 can also specifically bind Hsc70 via its J-domain to recruit this chaperone to ER membrane. Remarkably, the overexpression of DNAJB14 accelerated the degradation of misfolded membrane proteins including a mutant of cystic fibrosis transmembrane conductance regulator (CFTRΔF508), but not that of a misfolded luminal protein. Furthermore, the DNAJB14-dependent degradation of CFTRΔF508 was compromised by MG132, a proteasome inhibitor, indicating that DNAJB14 can enhance the degradation of a misfolded membrane protein using the ubiquitin-proteasome system. Thus, the mammalian ER possesses two analogous J-proteins (DNAJB14 and DNAJB12) that both can promote the ERAD of misfolded transmembrane proteins. Compared with DNAJB12 mRNA that was widely expressed in mouse tissues, DNAJB14 mRNA was expressed more weakly, being most abundant in testis, implying its specific role in this tissue.