Rescue of Folding Defects in ABC Transporters Using Pharmacological Chaperones

The ATP-binding cassette (ABC) family of membrane transport proteins is the largest class of transporters in humans (48 members). The majority of ABC transporters function at the cell surface. Therefore, defective folding and trafficking of the protein to the cell surface can lead to serious health problems. The classic example is cystic fibrosis (CF). In most CF patients, there is a deletion of Phe508 in the CFTR protein (ΔF508 CFTR) that results in defective folding and intracellular retention of the protein (processing mutant). A potential treatment for most patients with CF would be to use a ligand(s) of CFTR that acts a pharmacological chaperone to correct the folding defect. The feasibility of such an approach was first demonstrated with the multidrug transporter P-glycoprotein (P-gp), an ABC transporter, and a sister protein of CFTR. It was found that P-gps with mutations at sites equivalent to those found in CFTR processing mutants were rescued when they were expressed in the presence of drug substrates or modulators of P-gp. These compounds acted as pharmacological chaperones and functioned by promoting interactions among the various domains in the protein during the folding process. Several groups have attempted to identify compounds that could rescue the folding defect in ΔF508 CFTR. The best compound identified through high-throughout screening is a quinazoline derivative (CFcor-325). Expression of ΔF508 CFTR as well as other CFTR processing mutants in the presence of 1 μM CFcor-325 promoted folding and trafficking of the mutant proteins to the cell surface in an active conformation. Therefore, CFcor-325 and other quinazoline derivates could be important therapeutic compounds for the treatment of CF.     

Organic solutes rescue the functional defect in delta F508 cystic fibrosis transmembrane conductance regulator

The most common defect in cystic fibrosis, deletion of phenylalanine from position 508 of the cystic fibrosis transmembrane conductance regulator (Delta F508 CFTR), decreases the trafficking of this protein to the cell surface membrane. Previous studies have shown that low temperature and high concentrations of glycerol or trimethylamine N-oxide can partially counteract the processing defect of Delta F508 CFTR. The present study investigates whether physiologically relevant concentrations of organic solutes, accumulated by cotransporter proteins, can rescue the misprocessing of Delta F508 CFTR. Myoinositol alone or myoinositol, betaine, and taurine given sequentially increased the processing of core-glycosylated, endoplasmic reticulum-arrested Delta F508 CFTR into the fully glycosylated form of CFTR in IB3 cells or NIH 3T3 cells stably expressing Delta F508 CFTR. Pulse-chase experiments using transiently transfected COS7 cells demonstrated that organic solutes also increased the processing of the core-glycosylated form of green fluorescent protein-Delta F508 CFTR. Moreover, the prolonged half-life of the complex-glycosylated form of GFP-Delta F508 CFTR suggests that this treatment stabilized the mature form of the protein. In vitro studies of purified NBD1 stability and aggregation showed that myoinositol stabilized both the Delta F508 and wild type CFTR and inhibited Delta F508 misfolding. Most significantly, treatment of CF bronchial airway cells with these transportable organic solutes restores cAMP-stimulated single channel activity of both CFTR and outwardly rectifying chloride channel in the cell surface membrane and also restores a forskolin-stimulated macroscopic 36Cl- efflux. We conclude that organic solutes can repair CFTR functions by enhancing the processing of Delta F508 CFTR to the plasma membrane by stabilizing the complex-glycosylated form of Delta F508 CFTR.     

Processing mutations located throughout the human multidrug resistance P-glycoprotein disrupt interactions between the nucleotide binding domains

The most common cause of cystic fibrosis is misfolding of the cystic fibrosis transmembrane conductance regulator (CFTR) protein because of deletion of residue Phe-508 (DeltaF508). P-glycoprotein (P-gp) is an ideal model protein for studying how mutations disrupt folding of ATP-binding cassette proteins such as CFTR because specific chemical chaperones can be used to correct folding defects. Interactions between the nucleotide binding domains (NBDs) are critical because ATP binds at the interface between the NBDs. Here, we used disulfide cross-linking between cysteines in the Walker A sites and the LSGGQ signature sequences to test whether processing mutations located throughout P-gp disrupted interactions between the NBDs. We found that mutations present in the cytoplasmic loops, transmembrane segments, and linker regions or deletion of Tyr-490 (equivalent to Phe-508 in CFTR) inhibited cross-linking between the NBDs. Deletion of Phe-508 in the P-gp/CFTR chimera also inhibited cross-linking between the NBDs. Cross-linking was restored, however, when the mutants were expressed in the presence of the chemical chaperone cyclosporin A. The "rescued" mutants exhibited drug-stimulated ATPase activity, and cross-linking between the NBDs was inhibited by vanadate trapping of nucleotide. These results together with our previous findings (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2002) J. Biol. Chem. 277, 27585-27588) indicate that processing mutations disrupt interactions among all four domains. It appears that cross-talk between the cytoplasmic and the transmembrane domains is required for establishment of proper domain-domain interactions that occur during folding of ATP-binding cassette protein transporters.     

A Highlights from MBoC Selection: Small heat shock proteins target mutant cystic fibrosis transmembrane conductance regulator for degradation via a small ubiquitin-like modifier–dependent pathway

Small heat shock proteins (sHsps) bind destabilized proteins during cell stress and disease, but their physiological functions are less clear. We evaluated the impact of Hsp27, an sHsp expressed in airway epithelial cells, on the common protein misfolding mutant that is responsible for most cystic fibrosis. F508del cystic fibrosis transmembrane conductance regulator (CFTR), a well-studied protein that is subject to cytosolic quality control, selectively associated with Hsp27, whose overexpression preferentially targeted mutant CFTR to proteasomal degradation. Hsp27 interacted physically with Ubc9, the small ubiquitin-like modifier (SUMO) E2 conjugating enzyme, implying that F508del SUMOylation leads to its sHsp-mediated degradation. Enhancing or disabling the SUMO pathway increased or blocked Hsp27’s ability to degrade mutant CFTR. Hsp27 promoted selective SUMOylation of F508del NBD1 in vitro and of full-length F508del CFTR in vivo, which preferred endogenous SUMO-2/3 paralogues that form poly-chains. The SUMO-targeted ubiquitin ligase (STUbL) RNF4 recognizes poly-SUMO chains to facilitate nuclear protein degradation. RNF4 overexpression elicited F508del degradation, whereas Hsp27 knockdown blocked RNF4’s impact on mutant CFTR. Similarly, the ability of Hsp27 to degrade F508del CFTR was lost during overexpression of dominant-negative RNF4. These findings link sHsp-mediated F508del CFTR degradation to its SUMOylation and to STUbL-mediated targeting to the ubiquitin–proteasome system and thereby implicate this pathway in the disposal of an integral membrane protein.     

Can correcting the ΔF508-CFTR proteostasis-defect rescue CF lung disease?

Protein homeostasis (proteostasis) generates and maintains individual proteins in their folded and functional-competent states. The components of the cellular proteostasis machinery also dictate the functional lifetime of a protein by constantly regulating its conformation, concentration and subcellular location. The autosomal recessive disease cystic fibrosis (CF) is caused by a proteostasis-defect in CF transmembrane conductance regulator (CFTR). The most common CF mutation leading to this proteostasis-defect is the deletion of a phenylalanine residue at position 508 (ΔF508) of the CFTR protein. This ΔF508-CFTR protein is prone to aberrant folding, increased ER-associated degradation, atypical intracellular trafficking and reduced stability at the apical membrane. This ΔF508-CF proteostasis-defect leads to an obstructive lung disease characterized by impaired ion transport in airway epithelial cells, mucus buildup in air space and chronic airway inflammation. We assess here whether correcting the underlying defect in ΔF508-CFTR protein processing using therapeutic proteostasis regulators can treat chronic CF lung disease. As a proof of concept, recent studies support that the selective modulation of mutant-CFTR proteostasis may offer promising therapies to reverse chronic CF lung disease.     

Comparative processing and function of human and ferret cystic fibrosis transmembrane conductance regulator

The most common cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation is ΔF508, and this causes cystic fibrosis (CF). New CF models in the pig and ferret have been generated that develop lung, pancreatic, liver, and intestinal pathologies that reflect disease in CF patients. Species-specific biology in the processing of CFTR has demonstrated that pig and mouse ΔF508-CFTR proteins are more effectively processed to the apical membrane of airway epithelia than human ΔF508-CFTR. The processing behavior of ferret WT- and ΔF508-CFTR proteins remains unknown, and such information is important to predicting the utility of a ΔF508-CFTR ferret. To this end, we sought to compare processing, membrane stability, and function of human and ferret WT- and ΔF508-CFTR proteins in a heterologous expression system using HT1080, HEK293T, BHK21, and Cos7 cells as well as human and ferret CF polarized airway epithelia. Analysis of the protein processing and stability by metabolic pulse-chase and surface On-Cell Western blots revealed that WT-fCFTR half-life and membrane stability were increased relative to WT-hCFTR. Furthermore, in BHK21, Cos7, and CuFi cells, human and ferret ΔF508-CFTR processing was negligible, whereas low levels of processing of ΔF508-fCFTR could be seen in HT1080 and HEK293T cells. Only the WT-fCFTR, but not ΔF508-fCFTR, produced functional cAMP-inducible chloride currents in both CF human and ferret airway epithelia. Further elucidation of the mechanism responsible for elevated fCFTR protein stability may lead to new therapeutic approaches to augment CFTR function. These findings also suggest that generation of a ferret CFTR(ΔF508/ΔF508) animal model may be useful.     

Reduced PDZ Interactions of Rescued ΔF508CFTR Increases Its Cell Surface Mobility

Deletion of phenylalanine 508 (ΔF508) in the cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane chloride channel is the most common cause of cystic fibrosis (CF). Though several maneuvers can rescue endoplasmic reticulum-retained ΔF508CFTR and promote its trafficking to the plasma membrane, rescued ΔF508CFTR remains susceptible to quality control mechanisms that lead to accelerated endocytosis, ubiquitination, and lysosomal degradation. To investigate the role of scaffold protein interactions in rescued ΔF508CFTR surface instability, the plasma membrane mobility of ΔF508CFTR was measured in live cells by quantum dot single particle tracking. Following rescue by low temperature, chemical correctors, thapsigargin, or overexpression of GRASP55, ΔF508CFTR diffusion was more rapid than that of wild-type CFTR because of reduced interactions with PDZ domain-containing scaffold proteins. Knock-down of the plasma membrane quality control proteins CHIP and Hsc70 partially restored ΔF508CFTR-scaffold association. Quantitative comparisons of CFTR cell surface diffusion and endocytosis kinetics suggested an association between reduced scaffold binding and CFTR internalization. Our surface diffusion measurements in live cells indicate defective scaffold interactions of rescued ΔF508CFTR at the cell surface, which may contribute to its defective peripheral processing.     

Rescue of F508del-CFTR by RXR motif inactivation triggers proteome modulation associated with the unfolded protein response

F508del-CFTR, the most common mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, disrupts intracellular trafficking leading to cystic fibrosis (CF). The trafficking defect of F508del-CFTR can be rescued by simultaneous inactivation of its four RXR motifs (4RK). Proteins involved in the F508del-CFTR trafficking defect and/or rescue are therefore potential CF therapeutic targets. We sought to identify these proteins by investigating differential proteome modulation in BHK cells over-expressing wt-CFTR, F508del-CFTR or the revertant F508del/4RK-CFTR. By 2-dimensional electrophoresis-based proteomics and western blot approaches we demonstrated that over-expression of F508del/4RK-CFTR modulates the expression of a large number of proteins, many of which are reported interactors of CFTR and/or 14-3-3 with potential roles in CFTR trafficking. GRP78/BiP, a marker of ER stress and unfolded protein response (UPR), is up-regulated in cells over-expressing either F508del-CFTR or F598del/4RK-CFTR. However, over-expression of F508del/4RK-CFTR induces the up-regulation of many other UPR-associated proteins (e.g. GRP94, PDI, GRP75/mortalin) and, interestingly, the down-regulation of proteasome components associated with CFTR degradation, such as the proteasome activator PA28 (PSME2) and COP9 signalosome (COPS5/CSN5). Moreover, the F508del-CFTR-induced proteostasis imbalance, which involves some heat shock chaperones (e.g. HSP72/Hpa2), ER-EF-hand Ca²⁺-binding proteins (calumenin) and the proteasome activator PA28 (PSME2), tends to be ‘restored’, i.e., in BHK cells over-expressing F508del/4RK-CFTR those proteins tend to have expression levels similar to the wild-type ones. These findings indicate that a particular cellular environment orchestrated by the UPR contributes to and/or is compatible with F508del/4RK-CFTR rescue.     

Strategies for Correcting the AF508 CFTR Protein-Folding Defect

Many human diseases arise as a result of mutations within genes encoding essential proteins. In many cases, the mutations are not so severe as to render the protein biologically inactive. Rather, the mutations oftentimes result in only subtle protein-folding abnormalities. In the case of the CFTR protein, a mutation leading to the loss of a single amino acid is responsible for the diseased state in the majority of individuals with cystic fibrosis. Here the newly synthesized mutant CFTR protein, missing a phenylalanine residue at position 508 (F508 CFTR), is unable to transit from the endoplasmic reticulum to the plasma membrane, where it functions as a regulator of chloride transport. All of the available evidence indicate that the newly synthesized F508 CFTR protein adopts a slightly altered conformation and therefore is retained at the level of the endoplasmic reticulum, ostensibly by the actions of the cellular quality control system. Because the mutant protein is capable of functioning as a chloride channel, developing ways to elicit its release out of the ER and to the plasma membrane has important clinical implications. Herein, we discuss our recent studies showing that the protein folding defect associated with the F508 CFTR mutation, as well as a number of other temperature-sensitive mutations, can be overcome by strategies designed to influence protein folding inside the cell. Specifically we show that a number of low-molecular-weight compounds, all of which are known to stabilize proteins in their native conformation, are effective in rescuing the folding and/or processing defects associated with different mutations that oftentimes lead to human disease.     

Protein kinase CK2, cystic fibrosis transmembrane conductance regulator, and the deltaF508 mutation: F508 deletion disrupts a kinase-binding site

Deletion of phenylalanine 508 (DeltaF508) from the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common mutation in cystic fibrosis. The F508 region lies within a surface-exposed loop that has not been assigned any interaction with associated proteins. Here we demonstrate that the pleiotropic protein kinase CK2 that controls protein trafficking, cell proliferation, and development binds wild-type CFTR near F508 and phosphorylates NBD1 at Ser-511 in vivo and that mutation of Ser-511 disrupts CFTR channel gating. Importantly, the interaction of CK2 with NBD1 is selectively abrogated by the DeltaF508 mutation without disrupting four established CFTR-associated kinases and two phosphatases. Loss of CK2 association is functionally corroborated by the insensitivity of DeltaF508-CFTR to CK2 inhibition, the absence of CK2 activity in DeltaF508 CFTR-expressing cell membranes, and inhibition of CFTR channel activity by a peptide that mimics the F508 region of CFTR (but not the equivalent DeltaF508 peptide). Disruption of this CK2-CFTR association is the first described DeltaF508-dependent protein-protein interaction that provides a new molecular paradigm in the most frequent form of cystic fibrosis.     

Functional analysis of F508del CFTR in native human colon

The major cystic fibrosis mutation F508del has been classified by experiments in animal and cell culture models as a temperature-sensitive mutant defective in protein folding, processing and trafficking, but literature data on F508del CFTR maturation and function in human tissue are inconsistent. In the present study the molecular pathology of F508del CFTR was characterized in freshly excised rectal mucosa by bioelectric measurement of the basic defect and CFTR protein analysis by metabolic labelling or immunoblot. The majority of investigated F508del homozygous subjects expressed low amounts of complex-glycosylated mature F508del CFTR and low residual F508del CFTR-mediated chloride secretory activity in the rectal mucosa. The finding that some F508del CFTR escapes the ER quality control in vivo substantiates the hope that the defective processing and trafficking of F508del CFTR can be corrected by pharmacological agents.     

Analysis of the localization of STE6/CFTR chimeras in a Saccharomyces cerevisiae model for the cystic fibrosis defect CFTRΔF508

The use of yeast as a model system to study mammalian proteins is attractive, because yeast genetic tools can be utilized if a suitable phenotype is created. STE6, the Saccharomyces cerevisiae a-factor mating pheromone transporter, and CFTR, the mammalian cystic fibrosis transmembrane conductance regulator, are both members of the ATP binding cassette (ABC) superfamily. Teem et al . (1993) described a yeast model for studying a mutant form of the cystic fibrosis protein, CFTRΔF508. The model involved expression of a chimeric molecule in which a portion of yeast STE6 was replaced with the corresponding region from mammalian CFTR. The STE6/CFTR chimera complemented a ste6 mutant strain for mating, indicating that it could export a-factor. However, mating efficiency was dramatically reduced upon introduction of ΔF508, providing a yeast phenotype for this mutation. In human cells, the ΔF508 mutation results in retention of CFTR in the endoplasmic reticulum (ER), and possibly in reduction of its chloride-channel activity. Here we examine the basis for the differences in STE6 activity promoted by the wild-type and mutant STE6/CFTR chimeras. By analysis of protein stability and subcellular localization, we find that the mutant chimera is not ER-retained in yeast. We conclude that the molecular basis for the reduced mating of the STE6/CFTRΔF508 chimera must reflect a reduction in its capacity to transport a-factor, rather than mistrafficking. Thus, STE6/CFTRΔF508 in yeast appears to be a good genetic model to probe certain aspects of protein function, but not to study protein localization.     

Correctors promote folding of the CFTR in the endoplasmic reticulum

Cystic fibrosis (CF) is most commonly caused by deletion of a residue (DeltaF508) in the CFTR (cystic fibrosis transmembrane conductance regulator) protein. The misfolded mutant protein is retained in the ER (endoplasmic reticulum) and is not trafficked to the cell surface (misprocessed mutant). Corrector molecules such as corr-2b or corr-4a are small molecules that increase the amount of functional CFTR at the cell surface. Correctors may function by stabilizing CFTR at the cell surface or by promoting folding in the ER. To test whether correctors promoted folding of CFTR in the ER, we constructed double-cysteine CFTR mutants that would be retained in the ER and only undergo cross-linking when the protein folds into a native structure. The mature form, but not the immature forms, of M348C(TM6)/T1142C(TM12) (where TM is transmembrane segment), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutants were efficiently cross-linked. Mutations to the COPII (coatamer protein II) exit motif (Y(563)KDAD(567)) were then made in the cross-linkable cysteine mutants to prevent the mutant proteins from leaving the ER. Membranes were prepared from the mutants expressed in the absence or presence of correctors and subjected to disulfide cross-linking analysis. The presence of correctors promoted folding of the mutants as the efficiency of cross-linking increased from approx. 2-5% to 22-35%. The results suggest that correctors interact with CFTR in the ER to promote folding of the protein into a native structure.     

The endoplasmic reticulum-associated Hsp40 DNAJB12 and Hsc70 cooperate to facilitate RMA1 E3-dependent degradation of nascent CFTRDeltaF508

Relative contributions of folding kinetics versus protein quality control (QC) activity in the partitioning of non-native proteins between life and death are not clear. Cystic fibrosis transmembrane conductance regulator (CFTR) biogenesis serves as an excellent model to study this question because folding of nascent CFTR is inefficient and deletion of F508 causes accumulation of CFTRΔF508 in a kinetically trapped, but foldable state. Herein, a novel endoplasmic reticulum (ER)-associated Hsp40, DNAJB12 (JB12) is demonstrated to play a role in control of CFTR folding efficiency. JB12 cooperates with cytosolic Hsc70 and the ubiquitin ligase RMA1 to target CFTR and CFTRΔF508 for degradation. Modest elevation of JB12 decreased nascent CFTR and CFTRΔF508 accumulation while increasing association of Hsc70 with ER forms of CFTR and the RMA1 E3 complex. Depletion of JB12 increased CFTR folding efficiency up to threefold and permitted a pool of CFTRΔF508 to fold and escape the ER. Introduction of the V510D misfolding suppressor mutation into CFTRΔF508 modestly increased folding efficiency, whereas combined inactivation of JB12 and suppression of intrinsic folding defects permitted CFTRΔF508 to fold at 50% of wild-type efficiency. Therapeutic correction of CFTRΔF508 misfolding in cystic fibrosis patients may require repair of defective folding kinetics and suppression of ER QC factors, such as JB12.     

Structure and Dynamics of NBD1 from CFTR Characterized Using Crystallography and Hydrogen/Deuterium Exchange Mass Spectrometry

The ΔF508 mutation in nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the predominant cause of cystic fibrosis. Previous biophysical studies on human F508 and ΔF508 domains showed only local structural changes restricted to residues 509-511 and only minor differences in folding rate and stability. These results were remarkable because ΔF508 was widely assumed to perturb domain folding based on the fact that it prevents trafficking of CFTR out of the endoplasmic reticulum. However, the previously reported crystal structures did not come from matched F508 and ΔF508 constructs, and the ΔF508 structure contained additional mutations that were required to obtain sufficient protein solubility. In this article, we present additional biophysical studies of NBD1 designed to address these ambiguities. Mass spectral measurements of backbone amide ¹H/²H exchange rates in matched F508 and ΔF508 constructs reveal that ΔF508 increases backbone dynamics at residues 509-511 and the adjacent protein segments but not elsewhere in NBD1. These measurements also confirm a high level of flexibility in the protein segments exhibiting variable conformations in the crystal structures. We additionally present crystal structures of a broader set of human NBD1 constructs, including one harboring the native F508 residue and others harboring the ΔF508 mutation in the presence of fewer and different solubilizing mutations. The only consistent conformational difference is observed at residues 509-511. The side chain of residue V510 in this loop is mostly buried in all non-ΔF508 structures but completely solvent exposed in all ΔF508 structures. These results reinforce the importance of the perturbation ΔF508 causes in the surface topography of NBD1 in a region likely to mediate contact with the transmembrane domains of CFTR. However, they also suggest that increased exposure of the 509-511 loop and increased dynamics in its vicinity could promote aggregation in vitro and aberrant intermolecular interactions that impede trafficking in vivo.     

Cytoskeleton and CFTR

Cystic Fibrosis Transmembrane conductance Regulator, CFTR, is a membrane protein expressed in epithelia. A protein kinase A (PKA)-regulated Cl⁻ channel, it is a rate-limiting factor in fluid transport. Mutations in CFTR are responsible for cystic fibrosis, CF, an autosomal recessive disease. The most frequent mutation is deletion of phenylalanine at position 508, ΔF508. The regulation of trafficking and degradation of CFTR/ΔF508CFTR as well as its function(s) is a complex process which involves a number of proteins including chaperones and adaptors. It is now known that cytoskeletal proteins, previously considered only as structural proteins, are also important factors in the regulation of cellular processes and functions. The aim of the present review is to focus on how microfilaments, microtubules and intermediary filaments form a dynamic interactome with CFTR to participate in the regulation of CFTR-dependent transepithelial ion transport, CFTR trafficking and degradation.This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances.     

The primary folding defect and rescue of ΔF508 CFTR emerge during translation of the mutant domain

In the vast majority of cystic fibrosis (CF) patients, deletion of residue F508 from CFTR is the cause of disease. F508 resides in the first nucleotide binding domain (NBD1) and its absence leads to CFTR misfolding and degradation. We show here that the primary folding defect arises during synthesis, as soon as NBD1 is translated. Introduction of either the I539T or G550E suppressor mutation in NBD1 partially rescues ΔF508 CFTR to the cell surface, but only I539T repaired ΔF508 NBD1. We demonstrated rescue of folding and stability of NBD1 from full-length ΔF508 CFTR expressed in cells to isolated purified domain. The co-translational rescue of ΔF508 NBD1 misfolding in CFTR by I539T advocates this domain as the most important drug target for cystic fibrosis.     

Human heat shock protein 105/110 kDa (Hsp105/110) regulates biogenesis and quality control of misfolded cystic fibrosis transmembrane conductance regulator at multiple levels

Heat shock protein 105/110-kDa (Hsp105/110), a member of the Hsp70 super family of molecular chaperones, serves as a nucleotide exchange factor for Hsc70, independently prevents the aggregation of misfolded proteins, and functionally relates to Hsp90. We investigated the roles of human Hsp105α, the constitutively expressed isoform, in the biogenesis and quality control of the cystic fibrosis transmembrane conductance regulator (CFTR). In the endoplasmic reticulum (ER), Hsp105 facilitates CFTR quality control at an early stage in its biosynthesis but promotes CFTR post-translational folding. Deletion of Phe-508 (ΔF508), the most prevalent mutation causing cystic fibrosis, interferes with de novo folding of CFTR, impairing its export from the ER and accelerating its clearance in the ER and post-Golgi compartments. We show that Hsp105 preferentially associates with and stabilizes ΔF508 CFTR at both levels. Introduction of the Hsp105 substrate binding domain potently increases the steady state level of ΔF508 CFTR by reducing its early-stage degradation. This in turn dramatically enhances ΔF508 CFTR cell surface functional expression in cystic fibrosis airway epithelial cells. Although other Hsc70 nucleotide exchange factors such as HspBP1 and BAG-2 inhibit CFTR post-translational degradation in the ER through cochaperone CHIP, Hsp105 has a primary role promoting CFTR quality control at an earlier stage. The Hsp105-mediated multilevel regulation of ΔF508 CFTR folding and quality control provides new opportunities to understand how chaperone machinery regulates the homeostasis and functional expression of misfolded proteins in the cell. Future studies in this direction will inform therapeutics development for cystic fibrosis and other protein misfolding diseases.     

Introduction of the most common cystic fibrosis mutation (Delta F508) into human P-glycoprotein disrupts packing of the transmembrane segments

The most common mutation in cystic fibrosis (deletion of phenylalanine 508 (DeltaF508) in the cystic fibrosis conductance transmembrane regulator (CFTR) gene) causes defective synthesis of CFTR protein. To understand how this deletion interferes with protein folding, we made the equivalent deletion (DeltaY490) in P-glycoprotein (P-gp). A Cys-less P-gp with cysteines in transmembrane (TM) 4 or TM5 can be cross-linked with a cysteine in TM12. Deleting Tyr(490) in P-gp resulted in an inactive and defectively processed mutant in which no cross-linking between TM4 or TM5 and TM12 was detected. Expression of the DeltaY490 mutant in the presence of a chemical chaperone corrected the processing defect and yielded active P-gp mutants that could be cross-linked between TM4 or TM5 and TM12. Cross-linking between TM4 or TM5 and TM12 was also detected when residues (483)TIAENIRYG(491) in P-gp were replaced with residues (501)TIKENIIFG(509) from CFTR (P-gp/CFTR). Deleting Phe(508) in the P-gp/CFTR chimera, however, caused defective processing of the mutant protein and no detectable cross-linking between TM4 or TM5 and TM12. The processing defect was corrected with a chemical chaperone and yielded active P-gp/CFTR mutant proteins that could be cross-linked. These results show that deletion at residue 490 disrupts packing of the TM segments possibly by affecting interaction between the first nucleotide-binding domain (Tyr(490)) and the first cytoplasmic loop (Glu(184)).