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.

A specialized Hsp40 protein, DNAJB12, was found to function on the cytoplasmic face of the ER with the RMA1 E3 ligase to regulate the folding efficiency of CFTR.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic membrane protein that functions as a Cl ؊ channel and consists of two membrane spanning domains (MSDs), two cytosolic nucleotide binding domains (NBDs), and a cytosolic regulatory domain. Cytosolic 70-kDa heat shock protein (Hsp70), and endoplasmic reticulum-localized calnexin are chaperones that facilitate CFTR biogenesis. Hsp70 functions in both the cotranslational folding and posttranslational degradation of CFTR. Yet, the mechanism for calnexin action in folding and quality control of CFTR is not clear. Investigation of this question revealed that calnexin is not essential for CFTR or CFTR⌬F508 degradation. We identified a dependence on calnexin for proper assembly of CFTR's membrane spanning domains. Interestingly, efficient folding of NBD2 was also found to be dependent upon calnexin binding to CFTR. Furthermore, we identified folding defects caused by deletion of F508 that occurred before and after the calnexin-dependent association of MSD1 and MSD2. Early folding defects are evident upon translation of the NBD1 and R-domain and are sensed by the RMA-1 ubiquitin ligase complex. INTRODUCTIONCystic fibrosis transmembrane conductance regulator (CFTR) is a membrane glycoprotein that is localized to the apical surface of epithelial cells that line ducts of glands and airways. CFTR functions as an ATP-gated Cl Ϫ channel that is critical for proper hydration of the mucosal layer that lines lung airways (Welsh and Smith, 1993). Individuals who inherit two mutant forms of CFTR have exceedingly viscous mucous and, due to chronic lung infections, develop cystic fibrosis and often die from lung failure. CFTR is a member of the ATP-binding cassette (ABC) transporter superfamily (Hyde et al., 1990) and is a 1480-amino acid protein that contains two membrane spanning domains (MSDs), MSD1 and MSD2; two cytosolic nucleotide binding domains (NBDs), NBD1 and NBD2; and a regulatory (R) domain (Riordan et al., 1989). The proper folding and assembly of CFTR subdomains in the endoplasmic reticulum (ER) is required for CFTR to engage the COPII machinery and be packaged into vesicles for transport to the plasma membrane (Kopito, 1999;Wang et al., 2004). The folding pathway of this complex polytopic membrane protein has been a topic of great interest, because misfolding results in premature recognition of CFTR by the ER quality control system (ERQC) and degradation by the ubiquitin proteasome system (Skach, 2000). In fact, the most common disease-causing mutation of CFTR, ⌬F508CFTR, results in almost complete degradation of the protein by the ERQC system, which gives rise to a loss of function phenotype and lung disease (Ward and Kopito, 1994).The assembly of CFTR into an ion channel is complicated because it requires the coordinated folding and assembly of its membrane and cytoplasmic domains into a functional unit (Du et al., 2005;Riordan, 2005;Cui et al., 2007). CFTR is a modular protein, and its domains can collapse to a protease-resistant conformation i...

Premature degradation of CFTR⌬F508 causes cystic fibrosis (CF). CFTR⌬F508 folding defects are conditional and folding correctors are being developed as CF therapeutics. How the cellular environment impacts CFTR⌬F508 folding efficiency and the identity of CFTR⌬F508's correctable folding defects is unclear. We report that inactivation of the RMA1 or CHIP ubiquitin ligase permits a pool of CFTR⌬F508 to escape the endoplasmic reticulum. Combined RMA1 or CHIP inactivation and Corr-4a treatment enhanced CFTR⌬F508 folding to 3-7-fold greater levels than those elicited by Corr-4a. Some, but not all, folding defects in CFTR⌬F508 are correctable. CHIP and RMA1 recognize different regions of CFTR and a large pool of nascent CFTR⌬F508 is ubiquitinated by RMA1 before Corr-4a action. RMA1 recognizes defects in CFTR⌬F508 related to misassembly of a complex that contains MSD1, NBD1, and the R-domain. Corr-4a acts on CFTR⌬F508 after MSD2 synthesis and was ineffective at rescue of ⌬F508 dependent folding defects in amino-terminal regions. In contrast, misfolding caused by the rare CF-causing mutation V232D in MSD1 was highly correctable by Corr-4a. Overall, correction of folding defects recognized by RMA1 and/or global modulation of ER quality control has the potential to increase CFTR⌬F508 folding and provide a therapeutic approach for CF. INTRODUCTIONCystic fibrosis (CF) is a lethal inherited disorder that is caused by mutations in the gene coding for the CF transmembrane conductance regulator (CFTR; Riordan et al., 1989). The CFTR protein is a cAMP-regulated chloride channel that is localized to the apical surface of epithelial cells (Li et al., 1988;Anderson et al., 1991) where it functions to regulate water and salt homeostasis. Inheritance of the CFTR⌬F508 gene, in which phenylalanine 508 is deleted from the nucleotide binding domain 1 (NBD1), is the major cause of CF. This prevalent mutation is found in ϳ90% of patients afflicted with CF (Bobadilla et al., 2002). Loss of functional CFTR alters the hydration of airway epithelial and gives rise to microbial infections, which can ultimately lead to lung failure and death (Rowe et al., 2005).CFTR, an ATP-binding cassette (ABC) transporter superfamily member (Hyde et al., 1990), is a 1480-residue polytopic membrane glycoprotein that is predicted to contain two membrane-spanning domains (MSD), two nucleotide-binding domains (NBD), and a regulatory domain (R; Riordan et al., 1989). The folding of CFTR into a functional chloride channel is complex because it requires the coordinated folding and assembly of its membrane and cytoplasmic domains Cui et al., 2007). Consequently, CFTR folding is a relatively inefficient process with ϳ70% of the wild type and 99% of the ⌬F508 mutant being partitioned from a folding to a degradation pathway (Ward and Kopito, 1994). However, although early stage biogenic intermediates of CFTR and CFTR⌬F508 appear to have similar conformations (Zhang et al., 1998), the folding of CFTR⌬F508 becomes arrested at a poorly defined step. Interestingly, deletion ...

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.