
==== Front
ACS Chem Biol
ACS Chem Biol
cb
acbcct
ACS Chemical Biology
1554-8929
1554-8937
American Chemical Society

37730207
10.1021/acschembio.3c00310
Reviews
CFTR Folding: From Structure and Proteostasis to Cystic Fibrosis Personalized Medicine
McDonald Eli Fritz †‡
Meiler Jens *†‡§∥
https://orcid.org/0000-0003-4363-6116
Plate Lars *†⊥#
† Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235, United States
‡ Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37240, United States
§ Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37240, United States
∥ Institute for Drug Discovery, Leipzig University, Leipzig, SAC 04103, Germany
⊥ Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235, United States
# Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States
* Email: jens@meilerlab.org.
* Email: lars.plate@vanderbilt.edu.
20 09 2023
20 10 2023
20 09 2024
18 10 21282143
25 05 2023
02 08 2023
© 2023 The Authors. Published by American Chemical Society
2023
The Authors
https://creativecommons.org/licenses/by-nc-nd/4.0/ Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Cystic fibrosis (CF) is a lethal genetic disease caused by mutations in the chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR). Class-II mutants of CFTR lack intermolecular interactions important for CFTR structural stability and lead to misfolding. Misfolded CFTR is detected by a diverse suite of proteostasis factors that preferentially bind and route mutant CFTR toward premature degradation, resulting in reduced plasma membrane CFTR levels and impaired chloride ion conductance associated with CF. CF treatment has been vastly improved over the past decade by the availability of small molecules called correctors. Correctors directly bind CFTR, stabilize its structure by conferring thermodynamically favorable interactions that compensate for mutations, and thereby lead to downstream folding fidelity. However, each of over 100 Class-II CF causing mutations causes unique structural defects and shows a unique response to drug treatment, described as theratype. Understanding CFTR structural defects, the proteostasis factors evaluating those defects, and the stabilizing effects of CFTR correctors will illuminate a path toward personalized medicine for CF. Here, we review recent advances in our understanding of CFTR folding, focusing on structure, corrector binding sites, the mechanisms of proteostasis factors that evaluate CFTR, and the implications for CF personalized medicine.

National Heart, Lung, and Blood Institute 10.13039/100000050 F31 HL162483 Deutsche Forschungsgemeinschaft 10.13039/501100001659 SFB1423 National Institute of General Medical Sciences 10.13039/100000057 T32 GM065086 National Institute of General Medical Sciences 10.13039/100000057 R35 GM133552 National Institute of General Medical Sciences 10.13039/100000057 R01 GM129261 National Institute of General Medical Sciences 10.13039/100000057 R01 GM080403 National Heart, Lung, and Blood Institute 10.13039/100000050 R01 HL122010 document-id-old-9cb3c00310
document-id-new-14cb3c00310
ccc-price
==== Body
pmcIntroduction

Cystic fibrosis (CF) is a lethal genetic disease afflicting 85,000 people worldwide and is caused by ∼700 variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.1 CFTR is an ATP-gated anion channel expressed in the apical membrane of epithelial cells. Diverse defects in CFTR result in lack of chloride conduction in the epithelia, which is the cause of CF disease.2 Class-II CF causing mutations derail thermostability, leading to misfolding and premature degradation of the channel. This defect is illustrated by the most common mutation found in persons with CF: deletion of F508 (F508del). F508del CFTR is thermodynamically unstable and consequently recognized by the proteostasis network (PN).3,4 The PN evaluates F508del CFTR as misfolded and routes the mutant prematurely toward degradation.5 The only clinically approved, etiologically targeted treatment for CF entails small molecules called correctors which stabilize mutant CFTR, tipping the balance toward a properly folded, trafficking competent protein6 (Figure 1A). Potentiator small molecules are required for CFTR function in addition to correctors.7 However, current correctors fail to stabilize all CFTR missense mutations.8−11

Figure 1 A. CFTR folding is balanced by intrinsic and extrinsic factors. Mutations, heat, and unfoldase proteostasis factors tip the balance toward the unfolded state. By contrast pro-folding chaperones, corrector compounds, stabilizing mutations, and reduced temperature tip the balance toward to the folded state. B. CFTR protein homeostasis (proteostasis) network begins with translation of the CFTR polypeptide chain and translocation into the ER membrane. Molecular chaperones and cochaperones aid in CFTR folding while targeting misfolded CFTR to proteasomal degradation and autophagy. Properly folded CFTR is trafficked via the Golgi to the PM where it is further subjected to peripheral quality control (PQC) and endocytosis for lysosomal degradation.

The CFTR protein structure comprises two transmembrane domains, two cytosolic nucleotide binding domains, and a regulatory domain. This topology subjects CFTR to complex folding and evaluation from both membrane bound and cytosolic proteostasis factors. CFTR is translated and translocated into the endoplasmic reticulum (ER) membrane, where it folds co- and post-translationally. Folding is evaluated at multiple steps by heat shock proteins, cochaperones, and lectin binding chaperones. The chaperone network connects to the ubiquitin E3 ligase network that marks incorrectly folded CFTR for degradation. Degradation occurs via proteasomal, autophagosomal, or plasma membrane (PM) associated lysosomal recycling (Figure 1B). Once folding and core-glycosylation of CFTR in the ER are successful, CFTR traffics via the Golgi, where it is further glycosylated, to the PM (Figure 1B). At the PM, CFTR proteostasis continues and involves several overlapping cytosolic proteostasis factors.12

Different CF causing mutants experience divergent proteostasis states influencing how chaperone evaluation and subsequent degradation pathways are coordinated.9 Differences in mutant proteostasis likely stem from distinct structural defects. Whereas the pathways for the most common mutation F508del are well characterized, the proteostasis pathways of rarer CF causing mutations remain understudied. Despite the classification of many of these CF causing mutation as class-II, abnormal protein folding/trafficking, this classification scheme provides little basis for predicting CFTR therapeutic response, otherwise known as theratype.13 Given these divergent theratypes for many of the CF causing mutations, a better understanding of the differential structural defects of CFTR mutants and the subsequent interplay between proteostasis and theratype should lead to therapeutically relevant insight into improving precision medicine for CF.

Here, we review the current knowledge of CFTR biogenesis, focusing on CFTR structure and interactions with the proteostasis network. We begin with CFTR folding and recent structural biologic insights into the mutant CFTR conformation and the binding sites of correctors. We then discuss comprehensive CFTR mass-spectrometry-based interactomics studies, which reveal broad trends in CFTR proteostasis dependencies. Recent studies have shed new light on the mechanisms by which correctors stabilize CFTR structure and alter PN interactions with implications to overcome barriers for CF personalized medicine.

I CFTR Structure

CFTR is a member of the ABCC type transporter family composed of two transmembrane domains (TMD1 and TMD2), two nucleotide binding domains (NBD1 and NBD2), and an unstructured regulatory domain (RD) (Figure 2A). However, CFTR functions uniquely among ABC transporters as a passive anion channel. Unique unstructured regions such as the regulatory insertion (RI) region and RD are found in no other ABC transporters and likely give CFTR its passive anion channel properties.14 The active conformation resembles an outward facing ABC transporter with dimerized NBDs,15 while the inactive conformation resembles the inward facing ABC transporter with diminished NBD flexibility compared to other ABC transporters.16 Below, we review CFTR structural biology with an emphasis on folding kinetics, thermodynamics, effects of CF causing mutations, and corrector binding sites.

Figure 2 A. The transmembrane helix topology of CFTR colored from N to C terminus as ROYGBIV rainbow, NBDs and RD depicted in gray, and residue numbers listed throughout the protein. TH2/3 forms ICL1, TH4/5 forms ICL2, TH8/9 forms ICL3, and TH10/11 form ICL4. TH2/3 and TH10/11 crisscross to interact primarily with opposing TMD and NBD. B. In the folded state, ICL2 associates with NBD2 toward the C-terminus. Likewise, ICL4 associates with NBD1 at the N-terminus. C. Topology of CFTR helices is shown in published full-length model 5UAK.

Transmembrane Domains

CFTR TMDs consist of 12 transmembrane helices (TH) (Figure 2A), which are inserted via the Sec61 translocon and folded cooperatively in the ER membrane. Every two helices insert as hairpins, TH1/2, TH3/4, and TH5/6. In TMD2, TH7/8 act similarly to TH1/2—with a glycosylation site in between, TH9/10 act similarly to TH3/4, and so on.17,18 The TH intersection at TH2/3, TH4/5, TH9/8, and TH10/11 form intercellular loops (ICL) 1, ICL2, ICL3, and ICL4 respectively (Figure 2B,C).19,20 Thus, in the folded structure, the ICLs crisscross to interact with both NBDs: ICL1 and ICL4 interact with NBD1 whereas ICL2 and ICL3 interact with NBD2. F508 in NBD1 fits into an aromatic pocket formed by F1068, Y1073, and F1074 in ICL4. The stabilizing mutation R1070W, which suppresses the F508del defect, works by filling this aromatic pocket and recovering intermolecular interactions.21 TMD1/2 association may undergo the greatest post-translational conformational change22 alongside the post-translational folding of NBD2.23 A recent study suggests CFTR folds in two stages: first TMD1, TMD2, and NBD1 and second NBD2 and the association of TMDs with NBDs.24

TMD1 begins with an N-terminal region called the lasso motif that contains a membrane bound helix wrapped around TMD2 at TH10 and TH11 and two aliphatic helices half submerged on the cytosolic side of the membrane25 (Figure 2A). The lasso motif is unique to the ABCC subfamily of transporters and is present in CFTR and Multidrug resistant transporter (MR1–9).25 A well conserved charged region with acidic side chains in the first aliphatic helix (residues 46–61), interacts with the RD N-terminus, and is essential for CFTR gating.26 Like TMD1, TMD2 begins with an “elbow helix”, but its role remains unknown. F508del impairs TMD1/TMD2 assembly, presumably allosterically through the NBD1/TMD2 folding defect.27

Corrector Binding Sites

The TMDs form the binding sites of several small molecule, FDA-approved CF therapies (Figure 3A). The most effective CF combination therapy on the market, Trikafta, consists of three small molecules: two correctors VX-661 and VX-445 and one potentiator VX-770. CFTR TH8 is broken and consequentially displaces TM7 from its usual position compared to other ABCC subfamily members. The potentiator VX-770 binds here, presumably stabilizing the open gate conformation (Figure 3B).28 Type-I CFTR correctors such as VX-809 and VX-661 bind to a site in TMD1 (Figure 3C) as recently shown in cryo-EM structures29 and computational docking confirmed by mutagenesis binding assays.30 The compounds functionally stabilize the TMD1/NBD1 interface. Evidence supports putative alternative binding locations for VX-809/661, including at the TMD/NBD1 interface and NBD1.31−34 The binding site in TMD1 suggests that VX-809/661 stabilizes F508del by increasing the lifetime of TMD1 during cotranslational folding. Type-II correctors stabilize NBD2. Although no type-II correctors are FDA approved yet, several research compounds have been identified.11 Finally, the type-III CFTR corrector VX-445 binds at the interface of TH10/11 and the lasso motif (Figure 3D), and allosterically stabilizes NBD1.35,36 This binding site suggests that VX-445 stabilizes ICL4 by anchoring TH10/11 to the lasso motif and counteracting lost intermolecular interactions between F508 and the aromatic pocket in ICL4.

Figure 3 A. Chemical structures of the FDA approved CFTR drugs. VX-809 and VX-661 represent Type I correctors that stabilize the TMD1/NBD1 interface. VX-770 is a potentiator, and VX-445 is a Type III corrector that stabilizes NBD1. B. Cryo-EM structure of F508del CFTR bound to VX-661, VX-445, and VX-77036 (PDBID 8EIQ). C. Close up of the binding site for VX-661 in TMD1 interacting with TH1, TH2, TH3, and TH6. A salt bridge with R74 forms at the very beginning of TH1 near the lasso motif. D. VX-445 binds at the TMD2/lasso interface and interacts with TH10 and TH11 as well as N terminal residues in the lasso motif and TH2.

Nucleotide Binding Domains

The CFTR NBDs are conserved among ABC transporters37 and consist of three subdomains: 1) the beta subdomain (NBD1 residues 390–400 and 440–450), 2) the alpha helical subdomain (NBD1 residues 500–565), and 3) the ATPase subdomain (NBD1 residues 450–500 and 566–650) (Figure 4A). NBD1 is unique among ABC transporters in containing a disordered RI region (residues 400–439), that is post-translationally modified for regulatory purposes.38 The RI may exist in alternative beta-sheet conformations implicated in CFTR stability, where shortening of the RI loop accelerates F508del unfolding.39 Furthermore, F508del is stabilized by removing the RI40 or rescue mutations in NBD1.41 NBD1 folding kinetics follows an ordered compaction from the N to C terminus starting with the beta subdomain, followed by the alpha subdomain and ATPase subdomain (Figure 4B). Since part of the ATPase subdomain is translated first, ATPase subdomain folding is delayed by alpha subdomain folding and accelerated by ATP binding (Figure 4B).42−44

Figure 4 A. Ribbon diagram of the NBD1 structure50 depicting subdomains, helix and β sheet organization of NBD1, as well as highlighting important loops and unstructured regions. B. Cartoon depicting a current model of NBD1 folding kinetics with subdomains colored according to A. NBD1 folding begins with compaction of the N terminal residues 389–491 followed by the synthesis of alpha subdomain.42 Alpha subdomain collapse is delayed until the subsequent β sheet core (residues 568–603) is synthesized and folded.43 Binding of ATP at the degenerate ATPase site44 accelerates NBD1 N terminal folding.42 Finally, the C terminus/RE region (residues 605–670) is synthesized and folded. The alpha and beta subdomains of CFTR fold cooperatively as premature alpha subdomain collapse can hinder downstream core formation. S3, S6, S7, and S8 beta strands form the earliest folding intermediate of NBD1, but S6 cannot form a beta strand until the alpha subdomain followed by S7 emerges from the ribosome. Consequently, S6–S7 β sheet formation depends on the timing of the alpha subdomain collapse. Alpha subdomain collapse is delayed allowing for better folding of S5, S7, and S8. Rescue mutation D529F, fails to change CFTR thermodynamic stability, but instead rescues NBD1 through a kinetic mechanism that delays alpha subdomain collapse.43

F508 lies in the alpha subdomain of NBD1 where it interacts with the ICL4 of TMD2 and is allosterically connected to other regions of NBD1. Deletion of F508 destabilizes both the kinetic and thermodynamic folding of NBD1,45 which fails to fold spontaneously at physiological temperatures.45 Consistent with this, Cryo-EM structures of full length F508del CFTR revealed amorphous NBD1 electron density but definite NBD2 density.36 Rescue mutations in the RI (F429S, S434P), Q loop (F494N, S495P), and structurally diverse region (A534P, I539T) introduce salt bridges or prolines into intrinsically disordered loops and correct NBD1 as well as other rescue mutations that affect gating and folding intermediates.41,46 Correcting both NBD1 and the NBD1/ICL4 interface can fully restore F508del.45,47

The ICL4 binding pocket in NBD1 is allosterically coupled to the RI and NBD1 C-terminus, particularly S9/S10 and H9/H10.48 The F508 loop couples allosterically with the ATP binding core but the coupling is lost after F508 deletion, thereby destabilizing the ATP binding core subdomain including H5, H7, and H9.41 Interestingly, allosteric communication is restored with RI deletion.41 F508 interacts (through W496) with the Q loop in the ATP binding motif, and its deletion likely leads to an altered Q loop conformation.49 These lines of evidence suggest F508del impairs alpha subdomain collapse, which allosterically effects other regions of NBD1 such as loops, the beta core, and the C-terminus. Thus, therapeutics targeting F508del NBD1 should focus on stabilizing these regions.

Regulatory Domain

The RD is highly flexible and unique to CFTR among ABC transporter type proteins and may have evolved from an expressed intron.51 Stretching ∼200 residues from the RE region at the NBD1 C-terminus to the TM7 elbow helix at the TMD2 N-terminus, the RD is roughly defined as residues 645–845. RD post-translational modifications regulate CFTR gating. The space between the NBDs is occupied by the RD in the inactive conformation,52 although this region is poorly resolved in WT Cryo-EM structures and missing in F508del Cryo-EM structures36 and therefore exact structural details are lacking. Spontaneous disengagement from the NBD1/NBD2 interface likely precedes rapid phosphorylation.16 RD impacts the affinity of several proteostasis factors for CFTR in a phosphorylation dependent manner. This suggests proteostasis factors may evaluate the RD itself or unstructured RD/structured domain interactions.

The RE region (residue 645–675), which contains two phosphorylation sites at 660 and 670,53 constitutes the junction of NBD1 and RD. RE phosphorylation effects the NBD interface contacts and phosphorylated RE interacts with F508del NBD1 more than it interacts with WT NBD154 (Figure 5A). This observation implies unfolded F508del NBD1 has higher affinity for the RE regardless of phosphorylation state despite F508del increased phosphorylation compared to WT38 (Figure 5A). Removing both the RE and RI regions allows F508del CFTR to experience WT levels of rescue with VX-809.55 The RD C-terminus contains two clusters of negatively charged residues 725–733 (Neg1) and 817–823 (Neg2), which regulate phosphorylation dependent CFTR gating (Figure 5B).56 Residues 825–843 likely form the helix in the inner vestibule of the inactive state16 and act as a “master switch” in regulating gating.56 NMR structural and electrophysiology studies show that RD interacts with the lasso motif, NBD1, and NBD2 as transient alpha helices (Figure 5B).26,56,57

Figure 5 A. The RE region preferentially interacts with WT NBD1 in the dephosphorylated state; however, phosphorylation fails to affect the RE interaction with F508del NBD1 implying the unfolded mutant may have exposed regions providing enthalpically favorable interactions absent in the WT. B. Phosphorylation sites, organized by WT or F508del phosphorylation, respectively, projected onto R domain sequence. RD interacts with NBD1 as transient alpha helices at residues 661–681, 702–718, 748–778, 803–813, and 829–83457 in a phosphorylation dependent manner. Amino acids 702–718 and 661–681 disengage CFTR when phosphorylated.57 RE phosphorylation may release RD from CFTR by decreasing interactions between H8/H9 and S9/S10 strands of NBD1, which consists of H620, Y625, F626, and Y627–a sequence conserved among CFTR homologues, but not among ABCC transporters, implying a role in CFTR gating.59 The longest transient helix 748–778 interacts most strongly with NBD1.57 Also, RD interacts with the lasso motif and with the NBD2 C terminus26 – the last 40 residues of which are unique and conserved to CFTR.57 NBDs are more likely to interact with nonphosphorylated RD, whereas the C-terminus is more likely to interact with phosphorylated RD.57 Regions that form transient helices and interact with the structured domains of CFTR are colored in red. Negatively charged regions are colored in gold. NBD2 interacts with the RD at residues 661–673, 752–778, and 819–836 in the nonphosphorylated state and at residues 802–824 upon phosphorylation,57 not shown for clarity.

The RD engagement and disengagement with the structured regions of CFTR is interesting because expression of the RD changes the folding efficiency and affinity of proteostasis factors to CFTR27 (Figure 6A). On the other hand, when expressing the two structured halves of CFTR individually, CFTR may fold and assemble without the RD.58 Thus, it remains unclear what role the RD plays, if any, in CFTR folding and proteostasis.

Figure 6 A. The CFTR N-terminus (TMD1/NBD1) has a higher affinity for DNAJA1 and folds better than the TMD1/NBD1/RD constructs. By contrast, including the RD increases affinity for Hsp70 as measured by coimmunoprecipitation. B. Mapping chaperone and cochaperones binding sites onto NBD1 reveals beta core S8 as a hotspot of overlapping recognition. Mutations impeding proper folding of these regions may cause prolonged chaperone binding leading to detection and degradation. Interestingly, S8 folding depends on alpha helical subdomain collapse (see NBD1 section/Figure 4B) in a cooperative manner. Proteostasis factors binding to S8 may stabilize NBD1 temporarily during this cooperative folding. A peptide binding array study shows JA1 binds to NBD1 residues 469–478 (H1/S4), 593–602 (H6/S8) and 618–627 (S10), with JA2 recognizing one distinct peptide, 589–598 in S8. Additionally, Hsc70 binds TMD2 including 1092–1101 and 1100–1109, as well as NBD2 including 1295–1304 and 1394–1415.85 BAG2 binds NBD1 at 478–493 (S4/S6), 511–526 (H4), 553–565 (H5), and 586–604 (H6/S8).86 Hsp90 recognizes WT and F508del CFTR H8–H9 region in the NBD1 C terminus (residues 624–643).87 Additionally, Hsp90 recognizes WT CFTR at 422–443 (RI region), 453–464 (S3/Walker A motif/NBD dimer interface), 480–482 (S4), and 526–532 (H4).87

II CFTR Proteostasis

Numerous CFTR interactomics studies using proteomics methods (affinity-purification mass spectrometry3,9 or proximity ligation (Bio-ID)60), as well as two-hybrid high-throughput screening61 have revealed the proteostasis factors that interact with CFTR throughout its folding process.12,62 Translation, translocation, and co- and posttranslational folding occur at the ER membrane where CFTR is evaluated at multiple steps by membrane bound and cytosolic proteostasis factor. Translational dynamics at the ribosome impacts CFTR, with slower speeds increasing F508del folding63,64 and ribosomal frameshifting sites modulating F508del function.63 The Sec61 translocon, the ER membrane complex (EMC),60,65 Hsp70 and Hsp90 chaperones play an integral role, with a myriad of cochaperones such as Hsp40/J proteins, nucleotide exchange factors (NEFs), and lectin binding chaperones also involved. F508del experiences a greater number and prolonged interactions with proteostasis factors compared to WT.3,62 Similar proteins interact with either F508del or WT regardless of location in the ER or plasma membrane, however interactors diverge between F508del and WT - indicating folding competency dictates interactors more than cellular location.66 Kinases also play a role in CFTR proteostasis, for example kinase family member C1 (KIFC1) evaluates the Arginine framed tripeptide motifs of CFTR.67 Rescue conditions such as low-temperature and siRNA knockdown of key interactors shifts the F508del interactomes toward WT.3 Rarer class-II CF causing mutations such as P67L, G85E, and L206W experience aberrant interactomes like F508del.9,68 Since P67L and L206W respond well to VX-809, these mutants shift far closer toward the WT CFTR interactome under drug treatment than F508del.9 Different mutations likely fail to be evaluated at different steps, leading to their distinct proteostasis states. Yet, how these distinct proteostasis states are connected to different mutation-induced structural defects remains unclear.

Hsp40/J Proteins

Hsp40/J proteins are cochaperones that bind client proteins independent of ATP and likely determine the specificity of Hsp70 for clients.69 This role in client specificity underscores the importance of understanding J proteins recognition of WT CFTR vs mutant CFTR.

DNAJA1 (JA1) and Hsp70 together engage during NBD1 cotranslation folding, evident by decreased affinity for TMD1/NBD1/RD compared to TMD1/NBD170 (Figure 6A). Peptide binding array studies show JA1 binds NBD1 peptides segments (Figure 6B).71 JA1 knockdown decreases both B band (immature ER-localized) and C band (fully mature) WT CFTR, indicating a pro-folding role.72 In contrast, JA1 overexpression does not affect WT C band72 and stimulates CHIP ubiquitination of F508del,73 indicating a pro-degradation role. Furthermore, JA1 and Hsc70 coexpression prevents CFTR aggregation—demonstrating a possible holdase role.73

Despite being closely related to JA1, DNAJA2 (JA2) uniquely demonstrates independent folding capabilities.74 JA2 knockdown increases WT and F508del CFTR C band indicating a pro-degradation role.72,75 JA2 overexpression increases Hsc70 association—enhancing CHIP mediated proteasomal degradation of immature CFTR.72 JA1, JA2, and Hsc70 share overlapping binding sites on CFTR (Figure 6B).71

DNAJB12 (JB12) is an ER resident J protein with a single pass transmembrane helix in the ER and its J domain facing the cytosol.76 JB12 knockdown increases trafficking of F508del CFTR to the PM.76 Knockdown folds WT more efficiently than F508del, suggesting JB12 fails to rate limit F508del folding. JB12 overexpression increases degradation through a complex consisting of Derlin-1, an E2 ligase Ub26e, and an E3 ligase RMA1, which routes CFTR to the proteasome,77,78 indicating JB12 plays a pro-degradation role.76,79

Several other J proteins interact with CFTR. DNAJC5 (JC5) promotes degradation by increasing association with Hsc70 and CHIP80 and increases WT CFTR cell surface expression upon knockdown.81 JC5 competes with JA1 implying overlapping binding sites on the RD or NBD1/RD interface.80,81,70 DNAJB1 (JB1) overexpression with Hsp70 decreases WT degradation rates, but fails to effect F508del.82 DNAJB2 (JB2), decreases WT CFTR levels, indicating a pro-degradation role via the proteasome.83 DNAJB9 (JB9), an ER luminal J protein cochaperone, plays a pro-degradation role and likely binds extracellular loop 4 (ECL4).84

In conclusion, J proteins provide specificity toward downstream chaperoning of CFTR by binding specific regions and domains. These regions hint at which structural defects may be evaluated by chaperone networks and subsequently routed to diverse degradation pathways, depending on the structural defect.

Hsp70 and Cochaperones

Hsp70 family proteins are constitutively expressed (Hsc70/HSPA8) or heat shock induced (Hsp70/HSPA1) chaperones. Hsc70 stabilizes WT and F508del CFTR NBD1 in vitro by preventing and reversing aggregation.88 Aggregation increases following addition of ATP,88 implying the Hsc70 ATP driven cycling increases aggregation.89 Increasing ATP concentration also reduces Hsc70 binding to F508del mouse NBD1.90 Inhibition of Hsp70 with the allosteric effector MKT077, which locks the chaperone to the substrate,91,92 increases the F508del CFTR maturation.72 These studies suggest that both isoforms Hsc70 and Hsp70 stabilize CFTR when substrate bound in a “holdase” manner.

Hsc70 and Hsp70 have a higher affinity for F508del CFTR than WT.62,90,3 However, one study shows Hsp70 affinity for WT and F508del are the same.93 Hsp70 co-IPs with truncated 837-CFTR more compared to shorter 642-CFTR implying a high affinity for the RD, or RD driven NBD1 unfolding27 (Figure 6A). Following the RD, TMD2 translation decreases CFTR affinity for Hsp70 but affinity increases again with NBD2.27 These varying affinities for CFTR domains may be attributed to the co-operative nature of the domain–domain assembly.94 A peptide binding array study shows Hsc70 binds peptides segments in NBD1 (Figure 6B).71

Altering Hsp70 family protein levels has variable outcomes for CFTR. Hsc70 and Hsp70 knockdown increases trafficking of WT CFTR implying a pro-degradation role.72,75 In another study, Hsc70 knockdown fails to effect F508del C band.12 In yet another study, Hsp70 overexpression decreases C band WT CFTR through CHIP mediated lysosomal degradation at the PM.72 On the other hand, in a cell free reconstituted ERAD system, addition of Hsc70 or Hsp70 increases CFTR degradation rate via the proteasome.89 In the cell free system, Hsc70 shows a pro-folding role cotranslationally but a pro-degradation role post-translationally.89

Nucleotide exchange factors (NEFs) HspBP1, Hsp105/110, and BAG proteins regulate Hsp70 by exchanging ADP for ATP. HspBP1 overexpression increases the folding efficiency of WT and F508del CFTR via a CHIP/Hsc70/HspBP1 complex that inhibits the CHIP ubiquitination through an unknown mechanism, indicating a pro-folding role.95 As follows, HspBP1 knockdown increases degradation.95 Hsp105/110 stabilizes F508del and has a higher affinity for F508del than WT.96 Hsp105/110 knockdown increases WT and F508del synthesis, suggesting a pro-degradation role early in biogensis.96

BAG (Bcl2-associated athanogene) proteins are NEFs characterized by a BAG domain and a localization domain that allow them to route clients to specific degradation pathways. BAG1 (truncated construct residues 96–208) increases CFTR proteasomal degradation independent of CHIP, indicating a pro-degradation role.89 BAG-1 stabilizes F508del but not WT CFTR, likely by binding CFTR directly, in a ubiquitin like domain dependent manner that competes directly with ubiquitin.97 In contrast, BAG2 inhibits CHIP ubiquitination of Hsc70 substrates and BAG2 overexpression rescues CFTR, indicating a pro-folding role.86,98 BAG2 shows holdase effects, preventing F508del NBD1 from aggregating in vitro by binding CFTR NBD186 (Figure 6B). BAG3 knockdown increases F508del CFTR cell surface expression by decreasing lysosomal degradation in the ER compartment through autophagy, indicating a pro-degradation role.99 PI3K/Akt/mTOR inhibition rescues F508del by restoring/stimulating autophagy likely by increasing expression of BAG3.100

To summarize, Hsp70 family proteins likely function as both a holdase and pro-degradation conjugating chaperone depending on the region of CFTR bound and the cochaperones involved. Hsc70 centrality in proteostasis makes it a decisive check point that balances folded versus degraded CFTR. Current evidence shows Hsc70 plays different roles in folding and degradation depending on CFTR translation, cellular localization, and cochaperones.

Hsp90 and Cochaperones

CFTR is passed to Hsp90 from Hsp70 via the Hsp organizing protein (Hop) (Figure 7A). Hop competes with CHIP, which uniquely functions as both an E3 ubiquitin ligase and a cochaperone and thus recasts Hsp70 from a pro-folding to a pro-degradation role.101 Although neither of these cochaperones likely engage CFTR directly, CHIP and Hop play a central role in CFTR folding.

Figure 7 A. Model depicting how chaperone mediated folding fails for F508del CFTR compared to WT due to the lack of proper interdomain hydrophobic collapse, leading to continual cycling through the chaperone cycle. Both Hsp organizing protein (HOP) and the C-terminus of Hsp interaction protein (CHIP) bind to the EEVD motif of Hsp70 and Hsp90 and either pass CFTR from Hsp70 to Hsp90 or stochastically ubiquitinate CFTR as it passes through this cycle. B. CHIP knockdown fails to rescue CFTR unless NBD2 is expressed, suggesting that CHIP degradation may act during post-translation folding of NBD2. Thus, CHIP likely evaluates F508del and NBD2 mutants. C. The activator of 90 kDa heat shock protein ATPase homologue 1 (Aha1) regulates the dwell time of CFTR in the Hsp90 chaperone cycle. The RD region plays a poorly understood role in the recognition of mutant CFTR by Aha1/Hsp90. Expression of excess truncated NBD1/RD F508del CFTR competes with Aha1 away from full-length F508del CFTR and is sufficient to rescue trafficking. However, when the RD is not included, the competition is mitigated.93

Counterintuitively, Hop knockdown increases F508del CFTR cell surface expression in contrast with Hop’s presumed pro-folding role.102 CHIP knockdown increases mature CFTR expression indicating a pro-degradation role.103 Unsurprisingly, CHIP overexpression decreases CFTR PM expression and increases ER retention.101 In the ER, CHIP mediates degradation through the proteasome101 but may mediate lysosomal degradation at the PM.72 Interestingly, F508del CFTR is only sensitive to CHIP overexpression following NBD2 translation78 indicating CHIP evaluates CFTR folding after NBD2 is translated (Figure 7B).

WT CFTR passes efficiently through the Hsp90/cochaperone complex.87 The Hsp90 inhibitor Geldanamycin disrupts Hsp90-client interactions and consequently decreases levels of mature and ER WT and F508del by promoting proteasomal degradation.104,75 This highlights the overall pro-folding role of Hsp90.105 Cross-linking mass spectrometry shows Hsp90 recognizes WT and F508del CFTR H8–H9 region.87 Hsp90 preferentially recognizes WT CFTR at three additional NBD1 sites (Figure 6B), suggesting F508del may impair NBD1 assembly such that Hsp90 fails to engage F508del NBD1 properly.87

Cochaperone Aha1 regulates the dwell time CFTR spends on Hsp90 by modulating Hsp90 ATPase activity. Aha1 knockdown increases WT and F508del CFTR PM levels indicating a pro-degradation role.62 Likewise, Aha1 overexpression decreases WT and F508del CFTR PM expression.62,106 Whether Aha1 engages in CFTR directly remains unclear. Some studies found Aha1 affinity independent of Hsp90,93 whereas others observed a direct relationship between Aha1 and Hsp90 affinity.62 Strangely, F508del NBD1/RD competes with full-length F508del CFTR for Aha1/Hsp90 binding (Figure 7C)93 in an RD dependent manner - suggesting RD is required for recognition. This competition is sufficient to rescue full-length F508del CFTR trafficking and implies that Aha1/Hsp90 preferentially routes F508del NBD1/RD to degradation over the full-length protein. Finally, in vitro electrophysiology measurements show that both Hsp90 and Aha1 are required to decrease the thermal instability of F508del CFTR indicating Hsp90 ATPase rate modulated by Aha1 is required for Hsp90 folding of CFTR.75

Thus, Hsp90 plays a pro-folding role but may trap F508del CFTR in prolonged refolding cycles leading to eventual degradation. Differential Hp90 binding between WT and F508del suggests Hsp90 fails to engage F508del NBD1 properly. How Hsp90 engages and triages other CFTR mutants beyond F508del remains unknown.

Lectin Chaperones and Small Heat Shock Proteins

During glycosylation, lectin binding proteins play important roles in evaluating CFTR. Calnexin (CXN) has an N terminal lectin binding domain in the ER lumen and a C terminal single pass transmembrane helix in the ER membrane.107 While the lectin domain engages CFTR glycans on ECL4 following TM7 and TM8 insertion,108 CXN’s transmembrane helix may serve as a place holder helix stabilizing TMD2 during translation.107 CXN knockdown or mutating the N-linked glycosylation sites increases WT and F508del CFTR degradation through the proteasome, whereas CXN overexpression increases WT CFTR PM expression, both suggesting a pro-folding role.109,110 Treatment with castanospermine (which dissociates CXN from clients) accumulates CFTR in the ER but fails to rescue F508del CFTR.110

Calreticulin (CRT) is a lectin binding, soluble homologue of CXN localized in the ER lumen.107 CRT knockdown increases the cell surface expression of WT CFTR and hence plays a pro-degradation role.111 Neither knockdown nor overexpression of CRT has an effect on F508del CFTR,111 indicating the mutant fails to reach CRT evaluation. In conclusion, CXN and CRT modulation only effects WT, implying that glycan evaluation occurs after the rate limiting steps in F508del folding/degradation.

The small heat shock protein Hsp27 recognizes NBD1112 and has a higher affinity for F508del CFTR than WT CFTR.113 Hsp27 knockdown increases F508del steady states levels and, albeit to a lesser extent, steady state WT levels, indicating a pro-degradation role.113 Hsp27 overexpression increases F508del SUMOylation, ubiquitination, and consequently degradation via the proteasome but does not affect WT CFTR.113 Different SUMO paralogues have distinct effects of CFTR. SUMO2/3 promotes degradation through the Hsp27 and the RING-finger ubiquitin E3 ligase RNF4 pathway.114 In contrast, SUMO1 promotes biogenesis through a PIAS4 (protein inhibitor of activated STAT 4) mediated pathway.115 PIAS4 overexpression increases ER CFTR susceptibility to C18 correction (a VX-809 analogue), but only for certain CF causing mutants: P67L, R117H, R334W, S564R, D614G, L1065P, L1077P and N1303K.116 This data suggest that the chaperone axis is mutant specific.

III Implications for Cystic Fibrosis Personalized Medicine

Rare CF causing mutations diverge from the most common mutation F508del in their structural defects, proteostasis, and variable response to CF correctors (theratype). Mutations derail CFTR folding by either 1) affecting the stability of the final folded structure or 2) affecting intermediate states along the folding pathway. The former leads to local unfolding at thermodynamic equilibrium and recognition by proteostasis factors binding to unfolded regions. The later may result in failed collapse of a specific folding intermediate, kinetic stalling, and continued cycling through a proteostasis check point.117,118 Corrector compound binding reduces the folding free energy sufficiently to push the equilibrium toward folded CFTR. Below, we review what recent studies have revealed about the complex interplay between structure, proteostasis, and theratype for diverse CFTR mutations.

Much work has focused on mutations that exhibit selective responsiveness to correctors, such as a CF causing mutation in the lasso motif bend, P67L, which responds strongly to corrector VX-809.119 Interestingly, P67L VX-809 response requires the CFTR C-terminus (TMD2/NBD2)118 suggesting this mutation impairs lasso-TMD2 interactions, which are restored by VX-809. A computational structural modeling study suggests VX-809 stabilizes TMD1.120 A TMD1 mutation L188W rescues P67L by stabilizing the TMD1/2 interface, but fails to rescue F508del.121 Together these lines of evidence suggest that lasso-TMD2 interactions require TMD1 stability. Strangely, the Type III corrector VX-445 fails to rescue P67L despite binding at the lasso-TMD2 interface.10 Consequently, VX-445 TMD2 stabilization fails, whereas VX-809 TMD1 stabilization succeeds in rescuing P67L, highlighting that the order of TMD assembly is critical for mutant specific drug response.

Different proteostasis states are experienced by divergent mutations. Calnexin (CNX) stabilizes TMD2 during NBD2 translation, after which CXN no longer associates with CFTR.27 Interestingly, the corrector VX-809 lowers CNX interactions with P67L, but increases CXN binding to F508del–likely because F508del is triaged before CXN evaluation.9 The BAG family of proteins uses their localization domain to triage CFTR into specific pathways. Thus, different CFTR mutations respond differently to BAG protein knockdown. F508del is rescued by knocking down BAG1, 3, 4, and 5. N1303K, the second most frequent class II CF causing mutations, which also fails to respond to correctors, is rescued by knocking down BAG1, 3, and 4, but not BAG5 or 6.99 These results further highlight the divergent roles that proteostasis factors play in evaluating mutants with distinct structural defects.

A recent deep mutational scanning (DMS) study screened the changes in CFTR cell surface expression for >100 CF causing mutations under basal, VX-661, VX-445, and the combination therapy conditions.8 Interestingly, several rare CFTR mutants without corrector FDA approval (Class II mutations: L138ins, L927P, and nonclass II mutations: G970R, I1234V) responded well in vitro to the combination treatment compared to controls, warranting further studies of these mutants in CF bronchial epithelial cell and organoid models. The DMS data revealed that theratype correlates loosely with mutation location. TMD1 mutants P67L and Q98R respond selectively to VX-661, which has been corroborated in CFBE cell lines for several additional TMD CF-causing mutations.122 In contrast, TMD2 mutants Y1032C, T1036N, H1054D, and R1066H respond selectively to VX-445. This notion is reinforced in CFBEs for H1079P and Q1100P which show a VX-445 dependent drug response.123 However, R1066H responds well to VX-445 while R1066C fails to respond to VX-445 suggesting that proximity to the binding site alone is not sufficient to predict theratype. Still, the additional free energy of corrector binding has a stronger effect near the binding site, suggesting a targeted approach for future computational drug design efforts.

The DMS study also revealed two hotspots of unapproved mutations that respond poorly to current therapy in NBD1 and TMD2. In NBD1, the hotspot comprises the alpha helical and ATPase subdomain mutants I507del, V520F, L558S, A559T, R560K, R560T, A561E, and Y569D (Figure 8). Kinetic folding studies suggests L558S attenuates the collapse of the NBD1 beta core by disrupting folding as the H5 helix and S7 beta strand emerge from the ribosome.124 A recent study using spatial covariance unsupervised learning on 64 class-II CF causing mutations demonstrated robust temperature correction for mutations I507del, L558S, R560T, and R560 K.125 By contrast, mutations V520F, A559T, and Y569D fail to respond to temperature correction suggesting kinetically and thermodynamically impaired substitutions occur in this region.125 The authors suggested mutations in residues 512–536 may impair assembly of the diacidic exit motif (YKDAD) from 563 to 567 and consequently impede trafficking.125 Together, evidence suggests these nonresponsive, unapproved NBD1 mutations likely attenuate NBD1 folding kinetics or thermodynamics and may be treated with therapeutics targeting and stabilizing NBD1 specifically.

Figure 8 Hotspots of CFTR mutants that are nonresponsive to corrector drugs. NBD1 nonresponsive mutants I507del and V520F occur in the alpha helical subdomain (gold) along with F508del. Nonresponsive mutants L558S, A559T, R560K/T, A561E, and Y569D occur in the ATPase subdomain (red) and cluster near the NBD1/ICL4 interface. TMD2 nonresponsive mutants L1065P, R1066C, and W1098R (blue) cluster in the TH10-TH11loop/ICL4 in TMD2.

The second hotspot in TMD2 is composed of TH10-TH11 mutants L1065P, R1066C, and W1098R (Figure 8). These likely effect the TMD2/NBD interfaces which are evaluated by CXN and DNAJB12. For example, JB12 routes F508del and N1303K to distinct degradation pathways, proteasomal and ER-associated autophagy, respectively.126,127 We speculate that these TMD2 mutations may be nonresponsive to VX-445 because they experience aberrant proteostasis compared to responsive mutants in the same region. Interestingly, the spatial covariance study showed temperature correction of mutations L1065P and L1077P in same region in TMD2.125 Aberrant proteostasis could include preferential routing to autophagy such as NBD2 mutants S1235R and N1303K.128,129 Thus, future Type I and Type III correctors that stabilize the TMD/NBD interfaces will be needed to treat these CF causing mutations.

Finally, the RD plays an unclear role in the CFTR structure and proteostasis. No evidence suggests proteostasis factors bind to the RD, yet RD expression attracts Hsc70 and Hsp90. We speculate the disordered RD can slightly destabilize the neighboring NBD1–sufficient to unfold F508del NBD1, leading to the increased chaperone recognition. Since, the RD interacts with the lasso motif, NBD1, and NBD2, mutations in these domains may alter RD dynamics sufficiently to derail CFTR folding. RD dynamics may also hinder NBD1 folding kinetics, as NBD1 folding occurs during RD translation. Thus, the structure and proteostasis interplay between the RD and nonapproved mutants (Figure 8) highlight an important area for future CFTR theratype research.

Conclusion

Much research over the last decades has elucidated the complex folding of CFTR. Recently determined cryo-EM structures reveal the unique topology of CFTR including the lasso motif, which is important for the final assembly of the channel. Proper folding of the CFTR requires many key proteostasis factors. Mutant structural defects derail folding and drive premature degradation of CFTR leading to CF. Correctors stabilize CFTR structural defects by directly binding to CFTR. Recent literature has revealed how proteostasis factors evaluate specific CFTR structural defects and how current corrector molecules bind and stabilize CFTR. The insights reviewed here could guide computational corrector drug design for untreatable mutations. Finding effective treatments for persons with CF harboring rare CFTR missense mutations, which are nonresponsive to current correctors and not FDA-approved for treatment, remains an important focus of CF research in the future. Enumerating which CF causing mutants encounter which proteostasis factors and the role of interactions in CFTR folding will be a valuable focus for additional studies. Furthermore, the role of the disordered regulatory domain in the structure and proteostasis remains unclear. Understanding mutation drug response to emerging therapeutics de novo from structural and interactomics data has the exciting potential to guide future CF personalized medicine.

Author Contributions

Conceptualization, E.F.M., L.P. Funding acquisition, E.F.M., L.P., J.M.; Writing—original draft, E.F.M.; Writing—review and editing, E.F.M. and L.P. All authors have read and agreed to the published version of the manuscript.

The authors declare no competing financial interest.

Acknowledgments

This work was supported by NIH grants R35 GM133552, R01 GM080403, R01 HL122010, and R01 GM129261. The authors further acknowledge funding by the Deutsche Forschungsgemeinschaft (DFG) through SFB1423, project number 421152132. J.M. is supported by a Humboldt Professorship of the Alexander von Humboldt Foundation. E.F.M. was supported by a predoctoral fellowship from the National Heart, Lung, and Blood Institute (F31 HL162483-01A1). We would like to thank M. Kim for help and feedback on figures and manuscript.

Keywords

CFTR Cystic fibrosis transmembrane conductance regulator, an anion channel protein that malfunctions due to genetic mutations. CFTR is the causative protein implicated in persons with cystic fibrosis, a lethal genetic disease.

Molecular Chaperone A protein that binds to mostly hydrophobic regions of nascent polypeptide chains, unfolded proteins, or partially folded proteins to prevent aggregation. Chaperones also bind to sugar moieties (lectin chaperones) and to degradation factors to connect the folding and protein degradation process for misfolded proteins.

Interactomics Characterization of the ensemble of protein–protein interactions experienced throughout a protein lifecycle. Interactomics studies are typically carried out by mass spectrometry, yeast-two hybrid, or other high-throughput methods.

Proteostasis Protein homeostasis, the process by which functional protein levels are maintained through regulation of translation rates, degradation rates, subcellular localization, activation, and inhibition. Proteostasis is maintained by the coordinated activity of the proteostasis network, which evaluates the folding and functional fidelity of proteins.

Class-II Mutations CF causing mutations that result in misfolding and mis-trafficking defects that subsequently lead to dysfunction due to lack of sufficient functional CFTR levels in the apical plasma membrane of epithelial cells.

Correctors A class of small molecule drugs that promote the folding, trafficking, and thus cell surface expression of CFTR. Many correctors have been shown to bind CFTR directly and thermodynamically promote the folding process, others may work through alternate mechanisms.

Theratype A basis for predicting CFTR therapeutic response to a particular corrector drug.

Personalized Medicine An approach for treating patients based on the specific underlying genetic mutations or environmental experience of the individual. Here, personalized medicine refers specifically to selecting a CF drug treatment plan based on the genotype and subsequent theratype of a person with CF.
==== Refs
References

Cutting G. R. Cystic Fibrosis Genetics: From Molecular Understanding to Clinical Application. Nat. Rev. Genet. 2015, 16 , 45–56. 10.1038/nrg3849.25404111
Veit G. ; Avramescu R. G. ; Chiang A. N. ; Houck S. A. ; Cai Z. ; Peters K. W. ; Hong J. S. ; Pollard H. B. ; Guggino W. B. ; Balch W. E. ; Skach W. R. ; Cutting G. R. ; Frizzell R. A. ; Sheppard D. N. ; Cyr D. M. ; Sorscher E. J. ; Brodsky J. L. ; Lukacs G. L. From CFTR Biology toward Combinatorial Pharmacotherapy: Expanded Classification of Cystic Fibrosis Mutations. Mol. Biol. Cell 2016, 27 (3 ), 424–433. 10.1091/mbc.e14-04-0935.26823392
Pankow S. ; Bamberger C. ; Calzolari D. ; Martínez-Bartolomé S. ; Lavallée-Adam M. ; Balch W. E. ; Yates J. R. Δf508 CFTR Interactome Remodelling Promotes Rescue of Cystic Fibrosis. Nature 2015, 528 (7583 ), 510–516. 10.1038/nature15729.26618866
Welsh M. J. ; Smith A. E. Molecular Mechanisms of CFTR Chloride Channel Dysfunction in Cystic Fibrosis. Cell. 1993, 73 , 1251–1254. 10.1016/0092-8674(93)90353-R.7686820
Cheng S. H. ; Gregory R. J. ; Marshall J. ; Paul S. ; Souza D. W. ; White G. A. ; O’Riordan C. R. ; Smith A. E. Defective Intracellular Transport and Processing of CFTR Is the Molecular Basis of Most Cystic Fibrosis. Cell 1990, 63 (4 ), 827–834. 10.1016/0092-8674(90)90148-8.1699669
Van Goor F. ; Hadida S. ; Grootenhuis P. D. J. ; Burton B. ; Stack J. H. ; Straley K. S. ; Decker C. J. ; Miller M. ; McCartney J. ; Olson E. R. ; Wine J. J. ; Frizzell R. A. ; Ashlock M. ; Negulescu P. A. Correction of the F508del-CFTR Protein Processing Defect in Vitro by the Investigational Drug VX-809. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (46 ), 18843–18848. 10.1073/pnas.1105787108.21976485
Van Goor F. Rescue of CF Airway Epithelial Cell Functino in Vitro by a CFTR Potentiator, VX-770. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (44 ), 18825–18830. 10.1073/pnas.0904709106.19846789
McKee A. G. ; McDonald E. F. ; Penn W. D. ; Kuntz C. P. ; Noguera K. ; Chamness L. M. ; Roushar F. J. ; Meiler J. ; Oliver K. E. ; Plate L. ; Schlebach J. P. General Trends in the Effects of VX-661 and VX-445 on the Plasma Membrane Expression of Clinical CFTR Variants. Cell Chem. Biol. 2023, 30 (6 ), 632–642.e5. 10.1016/j.chembiol.2023.05.001.37253358
McDonald E. F. ; Sabusap C. M. P. ; Kim M. ; Plate L. Distinct Proteostasis States Drive Pharmacologic Chaperone Susceptibility for Cystic Fibrosis Transmembrane Conductance Regulator Misfolding Mutants. Mol. Biol. Cell 2022, 33 , mbc.E21-11-0578 10.1091/mbc.E21-11-0578.
Kim M. ; Mcdonald E. F. ; Sabusap C. M. P. ; Timalsina B. ; Joshi D. ; Hong J. S. ; Rab A. ; Sorscher E. J. ; Plate L. Elexacaftor/VX-445-Mediated CFTR Interactome Remodeling Reveals Differential Correction Driven by Mutation-Specific Translational Dynamics. bioRxiv, Feb. 4, 2023.10.1101/2023.02.04.527134.
Veit G. ; Xu H. ; Dreano E. ; Avramescu R. G. ; Bagdany M. ; Beitel L. K. ; Roldan A. ; Hancock M. A. ; Lay C. ; Li W. ; Morin K. ; Gao S. ; Mak P. A. ; Ainscow E. ; Orth A. P. ; McNamara P. ; Edelman A. ; Frenkiel S. ; Matouk E. ; Sermet-Gaudelus I. ; Barnes W. G. ; Lukacs G. L. Structure-Guided Combination Therapy to Potently Improve the Function of Mutant CFTRs. Nat. Med. 2018, 24 (11 ), 1732–1742. 10.1038/s41591-018-0200-x.30297908
Okiyoneda T. ; Barrière H. ; Bagdány M. ; Rabeh W. M. ; Du K. ; Höhfeld J. ; Young J. C. ; Lukacs G. L. Peripheral Protein Quality Control Removes Unfolded CFTR from the Plasma Membrane. Science (80-.) 2010, 329 (5993 ), 805–810. 10.1126/science.1191542.
Clancy J. P. ; Cotton C. U. ; Donaldson S. H. ; Solomon G. M. ; VanDevanter D. R. ; Boyle M. P. ; Gentzsch M. ; Nick J. A. ; Illek B. ; Wallenburg J. C. ; Sorscher E. J. ; Amaral M. D. ; Beekman J. M. ; Naren A. P. ; Bridges R. J. ; Thomas P. J. ; Cutting G. ; Rowe S. ; Durmowicz A. G. ; Mense M. ; Boeck K. D. ; Skach W. ; Penland C. ; Joseloff E. ; Bihler H. ; Mahoney J. ; Borowitz D. ; Tuggle K. L. CFTR Modulator Theratyping: Current Status, Gaps and Future Directions. J. Cyst. Fibros. 2019, 18 (1 ), 22–34. 10.1016/j.jcf.2018.05.004.29934203
Hwang T.-C. ; Yeh J.-T. ; Zhang J. ; Yu Y.-C. ; Yeh H.-I. ; Destefano S. Structural Mechanisms of CFTR Function and Dysfunction. J. Gen. Physiol. 2018, 150 (4 ), 539–570. 10.1085/jgp.201711946.29581173
Zhang Z. ; Liu F. ; Chen J. Molecular Structure of the ATP-Bound, Phosphorylated Human CFTR. Proc. Natl. Acad. Sci. U. S. A. 2018, 115 (50 ), 12757–12762. 10.1073/pnas.1815287115.30459277
Liu F. ; Zhang Z. ; Csanády L. ; Gadsby D. C. ; Chen J. Molecular Structure of the Human CFTR Ion Channel. Cell 2017, 169 (1 ), 85–92. 10.1016/j.cell.2017.02.024.28340353
Pitonzo D. ; Yang Z. ; Matsumura Y. ; Johnson A. E. ; Skach W. R. Sequence-Specific Retention and Regulated Integration of a Nascent Membrane Protein by the Endoplasmic Reticulum Sec61 Translocon. Mol. Biol. Cell 2009, 20 (2 ), 685–698. 10.1091/mbc.e08-09-0902.19019984
Lu Y. ; Xiong X. ; Helm A. ; Kimani K. ; Bragin A. ; Skach W. R. Co- and Posttranslational Translocation Mechanisms Direct Cystic Fibrosis Transmembrane Conductance Regulator N Terminus Transmembrane Assembly *. J. Biol. Chem. 1998, 273 (1 ), 568–576. 10.1074/jbc.273.1.568.9417117
Carveth K. ; Buck T. ; Anthony V. ; Skach W. R. Cooperativity and Flexibility of Cystic Fibrosis Transmembrane Conductance Regulator Transmembrane Segments Participate in Membrane Localization of a Charged Residue *. J. Biol. Chem. 2002, 277 (42 ), 39507–39514. 10.1074/jbc.M205759200.12186867
Enquist K. ; Fransson M. ; Boekel C. ; Bengtsson I. ; Geiger K. ; Lang L. ; Pettersson A. ; Johansson S. ; von Heijne G. ; Nilsson I. Membrane-Integration Characteristics of Two ABC Transporters, CFTR and P-Glycoprotein. J. Mol. Biol. 2009, 387 (5 ), 1153–1164. 10.1016/j.jmb.2009.02.035.19236881
Thibodeau P. H. ; Richardson J. M. ; Wang W. ; Millen L. ; Watson J. ; Mendoza J. L. ; Du K. ; Fischman S. ; Senderowitz H. ; Lukacs G. L. ; Kirk K. ; Thomas P. J. The Cystic Fibrosis-Causing Mutation ΔF508 Affects Multiple Steps in Cystic Fibrosis Transmembrane Conductance Regulator Biogenesis. J. Biol. Chem. 2010, 285 (46 ), 35825–35835. 10.1074/jbc.M110.131623.20667826
Kleizen B. ; Van Vlijmen T. ; De Jonge H. R. ; Braakman I. Folding of CFTR Is Predominantly Cotranslational. Mol. Cell 2005, 20 (2 ), 277–287. 10.1016/j.molcel.2005.09.007.16246729
Du K. ; Sharma M. ; Lukacs G. L. The ΔF508 Cystic Fibrosis Mutation Impairs Domain-Domain Interactions and Arrests Post-Translational Folding of CFTR. Nat. Struct. Mol. Biol. 2005, 12 (1 ), 17–25. 10.1038/nsmb882.15619635
Im J. ; Hillenaar T. ; Yeoh H. Y. ; Sahasrabudhe P. ; Mijnders M. ; van Willigen M. ; Hagos A. ; de Mattos E. ; van der Sluijs P. ; Braakman I. ABC-Transporter CFTR Folds with High Fidelity through a Modular, Stepwise Pathway. Cell. Mol. Life Sci. 2023, 80 (1 ), 33 10.1007/s00018-022-04671-x.36609925
Zhang Z. ; Chen J. Atomic Structure of the Cystic Fibrosis Transmembrane Conductance Regulator. Cell 2016, 167 (6 ), 1586–1597.e9. 10.1016/j.cell.2016.11.014.27912062
Naren A. P. ; Cormet-Boyaka E. ; Fu J. ; Villain M. ; Blalock J. E. ; Quick M. W. ; Kirk K. L. CFTR Chloride Channel Regulation by an Interdomain Interaction. Science (80-.) 1999, 286 (5439 ), 544–548. 10.1126/science.286.5439.544.
Rosser M. F. N. ; Grove D. E. ; Chen L. ; Cyr D. M. Assembly and Misassembly of Cystic Fibrosis Transmembrane Conductance Regulator: Folding Defects Caused by Deletion of F508 Occur Before and After the Calnexin-Dependent Association of Membrane Spanning Domain (MSD) 1 and MSD2. Mol. Biol. Cell 2008, 19 (11 ), 4570–4579. 10.1091/mbc.e08-04-0357.18716059
Liu F. ; Zhang Z. ; Levit A. ; Levring J. ; Touhara K. K. ; Shoichet B. K. ; Chen J. Structural Identification of a Hotspot on CFTR for Potentiation. Science (80-.) 2019, 364 (6446 ), 1184–1188. 10.1126/science.aaw7611.
Fiedorczuk K. ; Chen J. Mechanism of CFTR Correction by Type I Folding Correctors. Cell 2022, 185 (1 ), 158–168.e11. 10.1016/j.cell.2021.12.009.34995514
Baatallah N. ; Elbahnsi A. ; Mornon J.-P. ; Chevalier B. ; Pranke I. ; Servel N. ; Zelli R. ; Décout J.-L. ; Edelman A. ; Sermet-Gaudelus I. ; Callebaut I. ; Hinzpeter A. Pharmacological Chaperones Improve Intra-Domain Stability and Inter-Domain Assembly via Distinct Binding Sites to Rescue Misfolded CFTR. Cell. Mol. Life Sci. 2021, 78 , 7813 10.1007/s00018-021-03994-5.34714360
Okiyoneda T. ; Veit G. ; Dekkers J. F. ; Bagdany M. ; Soya N. ; Xu H. ; Roldan A. ; Verkman A. S. ; Kurth M. ; Simon A. ; Hegedus T. ; Beekman J. M. ; Lukacs G. L. Mechanism-Based Corrector Combination Restores ΔF508-CFTR Folding and Function. Nat. Chem. Biol. 2013, 9 (7 ), 444–454. 10.1038/nchembio.1253.23666117
Laselva O. ; Molinski S. ; Casavola V. ; Bear C. E. Correctors of the Major Cystic Fibrosis Mutant Interact through Membrane-Spanning Domains. Mol. Pharmacol. 2018, 93 (6 ), 612–618. 10.1124/mol.118.111799.29618585
He L. ; Kota P. ; Aleksandrov A. A. ; Cui L. ; Jensen T. ; Dokholyan N. V. ; Riordan J. R. Correctors of ??F508 CFTR Restore Global Conformational Maturation without Thermally Stabilizing the Mutant Protein. FASEB J. 2013, 27 (2 ), 536–545. 10.1096/fj.12-216119.23104983
Farinha C. M. ; King-Underwood J. ; Sousa M. ; Correia A. R. ; Henriques B. J. ; Roxo-Rosa M. ; Da Paula A. C. ; Williams J. ; Hirst S. ; Gomes C. M. ; Amaral M. D. Revertants, Low Temperature, and Correctors Reveal the Mechanism of F508del-CFTR Rescue by VX-809 and Suggest Multiple Agents for Full Correction. Chem. Biol. 2013, 20 (7 ), 943–955. 10.1016/j.chembiol.2013.06.004.23890012
Veit G. ; Roldan A. ; Hancock M. A. ; Da Fonte D. F. ; Xu H. ; Hussein M. ; Frenkiel S. ; Matouk E. ; Velkov T. ; Lukacs G. L. Allosteric Folding Correction of F508del and Rare CFTR Mutants by Elexacaftor-Tezacaftor-Ivacaftor (Trikafta) Combination. JCI insight 2020, 5 (18 ), e139983 10.1172/jci.insight.139983.32853178
Fiedorczuk K. ; Chen J. Molecular Structures Reveal Synergistic Rescue of Δ508 CFTR by Trikafta Modulators. Science 2022, 378 (6617 ), 284–290. 10.1126/science.ade2216.36264792
Mendoza J. L. ; Schmidt A. ; Li Q. ; Nuvaga E. ; Barrett T. ; Bridges R. J. ; Feranchak A. P. ; Brautigam C. A. ; Thomas P. J. Requirements for Efficient Correction of Δf508 CFTR Revealed by Analyses of Evolved Sequences. Cell 2012, 148 , 164 10.1016/j.cell.2011.11.023.22265409
Pankow S. ; Bamberger C. ; Yates J. R. A Posttranslational Modification Code for CFTR Maturation Is Altered in Cystic Fibrosis. Sci. Signal. 2019, 12 (562 ), 1–14. 10.1126/scisignal.aan7984.
Scholl D. ; Sigoillot M. ; Overtus M. ; Martinez R. C. ; Martens C. ; Wang Y. ; Pardon E. ; Laeremans T. ; Garcia-Pino A. ; Steyaert J. ; Sheppard D. N. ; Hendrix J. ; Govaerts C. A Topological Switch in CFTR Modulates Channel Activity and Sensitivity to Unfolding. Nat. Chem. Biol. 2021, 17 (9 ), 989–997. 10.1038/s41589-021-00844-0.34341587
Aleksandrov A. A. ; Kota P. ; Aleksandrov L. A. ; He L. ; Jensen T. ; Cui L. ; Gentzsch M. ; Dokholyan N. V. ; Riordan J. R. Regulatory Insertion Removal Restores Maturation, Stability and Function of ΔF508 CFTR. J. Mol. Biol. 2010, 401 (2 ), 194–210. 10.1016/j.jmb.2010.06.019.20561529
Aleksandrov A. A. ; Kota P. ; Cui L. ; Jensen T. ; Alekseev A. E. ; Reyes S. ; He L. ; Gentzsch M. ; Aleksandrov L. A. ; Dokholyan N. V. ; Riordan J. R. Allosteric Modulation Balances Thermodynamic Stability and Restores Function of Δf508 CFTR. J. Mol. Biol. 2012, 419 (1–2 ), 41–60. 10.1016/j.jmb.2012.03.001.22406676
Khushoo A. ; Yang Z. ; Johnson A. E. ; Skach W. R. Ligand-Driven Vectorial Folding of Ribosome-Bound Human CFTR NBD1. Mol. Cell 2011, 41 (6 ), 682–692. 10.1016/j.molcel.2011.02.027.21419343
Kim S. J. ; Yoon J. S. ; Shishido H. ; Yang Z. ; Rooney L. A. A. ; Barral J. M. ; Skach W. R. Translational Tuning Optimizes Nascent Protein Folding in Cells. Science (80-.) 2015, 348 (6233 ), 444–448. 10.1126/science.aaa3974.
Vergani P. ; Lockless S. W. ; Nairn A. C. ; Gadsby D. C. CFTR Channel Opening by ATP-Driven Tight Dimerization of Its Nucleotide-Binding Domains. Nature 2005, 433 (7028 ), 876–880. 10.1038/nature03313.15729345
Rabeh W. M. ; Bossard F. ; Xu H. ; Okiyoneda T. ; Bagdany M. ; Mulvihill C. M. ; Du K. ; Di Bernardo S. ; Liu Y. ; Konermann L. ; Roldan A. ; Lukacs G. L. Correction of Both NBD1 Energetics and Domain Interface Is Required to Restore Δf508 CFTR Folding and Function. Cell 2012, 148 (1–2 ), 150–163. 10.1016/j.cell.2011.11.024.22265408
Protasevich I. ; Yang Z. ; Wang C. ; Atwell S. ; Zhao X. ; Emtage S. ; Wetmore D. ; Hunt J. F. ; Brouillette C. G. Thermal Unfolding Studies Show the Disease Causing F508del Mutation in CFTR Thermodynamically Destabilizes Nucleotide-Binding Domain 1. Protein Sci. 2010, 19 (10 ), 1917–1931. 10.1002/pro.479.20687133
Mendoza J. L. ; Schmidt A. ; Li Q. ; Nuvaga E. ; Barrett T. ; Bridges R. J. ; Feranchak A. P. ; Brautigam C. A. ; Thomas P. J. Requirements for Efficient Correction of Δf508 CFTR Revealed by Analyses of Evolved Sequences. Cell 2012, 148 (1–2 ), 164–174. 10.1016/j.cell.2011.11.023.22265409
Dawson J. E. ; Farber P. J. ; Forman-Kay J. D. Allosteric Coupling between the Intracellular Coupling Helix 4 and Regulatory Sites of the First Nucleotide-Binding Domain of CFTR. PLoS One 2013, 8 (9 ), e74347 10.1371/journal.pone.0074347.24058550
Chong P. A. ; Farber P. J. ; Vernon R. M. ; Hudson R. P. ; Mittermaier A. K. ; Forman-Kay J. D. Deletion of Phenylalanine 508 in the First Nucleotide-Binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator Increases Conformational Exchange and Inhibits Dimerization. J. Biol. Chem. 2015, 290 (38 ), 22862–22878. 10.1074/jbc.M115.641134.26149808
Lewis H. A. ; Buchanan S. G. ; Burley S. K. ; Conners K. ; Dickey M. ; Dorwart M. ; Fowler R. ; Gao X. ; Guggino W. B. ; Hendrickson W. A. ; Hunt J. F. ; Kearins M. C. ; Lorimer D. ; Maloney P. C. ; Post K. W. ; Rajashankar K. R. ; Rutter M. E. ; Sauder J. M. ; Shriver S. ; Thibodeau P. H. ; Thomas P. J. ; Zhang M. ; Zhao X. ; Emtage S. Structure of Nucleotide-Binding Domain 1 of the Cystic Fibrosis Transmembrane Conductance Regulator. EMBO J. 2004, 23 (2 ), 282–293. 10.1038/sj.emboj.7600040.14685259
Sebastian A. ; Rishishwar L. ; Wang J. ; Bernard K. F. ; Conley A. B. ; McCarty N. A. ; Jordan I. K. Origin and Evolution of the Cystic Fibrosis Transmembrane Regulator Protein R Domain. Gene 2013, 523 (2 ), 137–146. 10.1016/j.gene.2013.02.050.23578801
Liu F. ; Zhang Z. ; Csanády L. ; Gadsby D. C. ; Chen J. Molecular Structure of the Human CFTR Ion Channel. Cell 2017, 169 (1 ), 85–95.e8. 10.1016/j.cell.2017.02.024.28340353
Csanády L. ; Chan K. W. ; Nairn A. C. ; Gadsby D. C. Functional Roles of Nonconserved Structural Segments in CFTR’s NH 2-Terminal Nucleotide Binding Domain. J. Gen. Physiol. 2005, 125 (1 ), 43–55. 10.1085/jgp.200409174.15596536
Kanelis V. ; Hudson R. P. ; Thibodeau P. H. ; Thomas P. J. ; Forman-Kay J. D. NMR Evidence for Differential Phosphorylation-Dependent Interactions in WT and DF508 CFTR. EMBO J. 2010, 29 (1 ), 263–277. 10.1038/emboj.2009.329.19927121
Uliyakina I. ; Botelho H. M. ; da Paula A. C. ; Afonso S. ; Lobo M. J. ; Felício V. ; Farinha C. M. ; Amaral M. D. Full Rescue of F508DEL-CFTR Processing and Function by CFTR Modulators Can Be Achieved by Removal of Two Regulatory Regions. Int. J. Mol. Sci. 2020, 21 (12 ), 4524 10.3390/ijms21124524.32630527
Xie J. ; Adams L. M. ; Zhao J. ; Gerken T. A. ; Davis P. B. ; Ma J. A Short Segment of the R Domain of Cystic Fibrosis Transmembrane Conductance Regulator Contains Channel Stimulatory and Inhibitory Activities That Are Separable by Sequence Modification. J. Biol. Chem. 2002, 277 (25 ), 23019–23027. 10.1074/jbc.M201661200.11950844
Bozoky Z. ; Krzeminski M. ; Muhandiram R. ; Birtley J. R. ; Al-Zahrani A. ; Thomas P. J. ; Frizzell R. A. ; Ford R. C. ; Forman-Kay J. D. Regulatory R Region of the CFTR Chloride Channel Is a Dynamic Integrator of Phospho-Dependent Intra- and Intermolecular Interactions. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (47 ), E4427 10.1073/pnas.1315104110.24191035
Csanády L. ; Chan K. W. ; Seto-Young D. ; Kopsco D. C. ; Nairn A. C. ; Gadsby D. C. Severed Channels Probe Regulation of Gating of Cystic Fibrosis Transmembrane Conductance Regulator by Its Cytoplasmic Domains. J. Gen. Physiol. 2000, 116 (3 ), 477–500. 10.1085/jgp.116.3.477.10962022
Hudson R. P. ; Chong P. A. ; Protasevich I. I. ; Vernon R. ; Noy E. ; Bihler H. ; An J. L. ; Kalid O. ; Sela-Culang I. ; Mense M. ; Senderowitz H. ; Brouillette C. G. ; Forman-Kay J. D. Conformational Changes Relevant to Channel Activity and Folding within the First Nucleotide Binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator. J. Biol. Chem. 2012, 287 (34 ), 28480–28494. 10.1074/jbc.M112.371138.22722932
Iazzi M. ; Astori A. ; St-Germain J. ; Raught B. ; Gupta G. D. Proximity Profiling of the CFTR Interaction Landscape in Response to Orkambi. International Journal of Molecular Sciences 2022, 23 , 2442 10.3390/ijms23052442.35269585
Lim S. H. ; Snider J. ; Birimberg-Schwartz L. ; Ip W. ; Serralha J. C. ; Botelho H. M. ; Lopes-Pacheco M. ; Pinto M. C. ; Moutaoufik M. T. ; Zilocchi M. ; Laselva O. ; Esmaeili M. ; Kotlyar M. ; Lyakisheva A. ; Tang P. ; López Vázquez L. ; Akula I. ; Aboualizadeh F. ; Wong V. ; Grozavu I. ; Opacak-Bernardi T. ; Yao Z. ; Mendoza M. ; Babu M. ; Jurisica I. ; Gonska T. ; Bear C. E. ; Amaral M. D. ; Stagljar I. CFTR Interactome Mapping Using the Mammalian Membrane Two-Hybrid High-Throughput Screening System. Mol. Syst. Biol. 2022, 18 (2 ), e10629 10.15252/msb.202110629.35156780
Wang X. ; Venable J. ; LaPointe P. ; Hutt D. M. ; Koulov A. V. ; Coppinger J. ; Gurkan C. ; Kellner W. ; Matteson J. ; Plutner H. ; Riordan J. R. ; Kelly J. W. ; Yates J. R. ; Balch W. E. Hsp90 Cochaperone Aha1 Downregulation Rescues Misfolding of CFTR in Cystic Fibrosis. Cell 2006, 127 (4 ), 803–815. 10.1016/j.cell.2006.09.043.17110338
Carmody P. ; Roushar F. J. ; Tedman A. ; Herwig M. ; Kim M. ; Eli F. ; Wong-roushar J. ; Poirier J. ; Zelt N. B. ; Pockrass B. T. ; Andrew G. ; Kuntz C. P. ; Plate L. ; Penn W. D. ; Schlebach J. P. Ribosomal Frameshifting Selectively Modulates the Biosynthesis, Assembly, and Function of a Misfolded CFTR Variant. bioRxiv, May 3, 2023.10.1101/2023.05.02.539166.
Veit G. ; Oliver K. ; Apaja P. M. ; Perdomo D. ; Bidaud-Meynard A. ; Lin S.-T. ; Guo J. ; Icyuz M. ; Sorscher E. J. ; Hartman J. L. IV ; Lukacs G. L. Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect. PLOS Biol. 2016, 14 (5 ), e1002462 10.1371/journal.pbio.1002462.27168400
Louie R. J. ; Guo J. ; Rodgers J. W. ; White R. ; Shah N. A. ; Pagant S. ; Kim P. ; Livstone M. ; Dolinski K. ; McKinney B. A. ; Hong J. ; Sorscher E. J. ; Bryan J. ; Miller E. A. ; Hartman J. L. A Yeast Phenomic Model for the Gene Interaction Network Modulating CFTR-ΔF508 Protein Biogenesis. Genome Med. 2012, 4 (12 ), 103 10.1186/gm404.23270647
Santos J. D. ; Canato S. ; Carvalho A. S. ; Botelho H. M. ; Aloria K. ; Amaral M. D. ; Matthiesen R. ; Falcao A. O. ; Farinha C. M. Folding Status Is Determinant over Traffic-Competence in Defining CFTR Interactors in the Endoplasmic Reticulum. Cells 2019, 8 (4 ), 353 10.3390/cells8040353.31014000
Canato S. ; Santos J. D. ; Carvalho A. S. ; Aloria K. ; Amaral M. D. ; Matthiesen R. ; Falcao A. O. ; Farinha C. M. Proteomic Interaction Profiling Reveals KIFC1 as a Factor Involved in Early Targeting of F508del-CFTR to Degradation. Cell. Mol. Life Sci. 2018, 75 (24 ), 4495–4509. 10.1007/s00018-018-2896-7.30066085
Hutt D. M. ; Loguercio S. ; Campos A. R. ; Balch W. E. A Proteomic Variant Approach (ProVarA) for Personalized Medicine of Inherited and Somatic Disease. J. Mol. Biol. 2018, 430 (18 ), 2951–2973. 10.1016/j.jmb.2018.06.017.29924966
Kampinga H. H. ; Craig E. A. The HSP70 Chaperone Machinery: J Proteins as Drivers of Functional Specificity. Nat. Rev. Mol. Cell Biol. 2010, 11 (8 ), 579–592. 10.1038/nrm2941.20651708
Meacham G. C. The Hdj-2/Hsc70 Chaperone Pair Facilitates Early Steps in CFTR Biogenesis. EMBO J. 1999, 18 (6 ), 1492–1505. 10.1093/emboj/18.6.1492.10075921
Baaklini I. ; Gonçalves C. de C. ; Lukacs G. L. ; Young J. C. Selective Binding of HSC70 and Its Co-Chaperones to Structural Hotspots on CFTR 2020, 10 , 1–12. 10.1038/s41598-020-61107-x.
Kim Chiaw P. ; Hantouche C. ; Wong M. J. H. ; Matthes E. ; Robert R. ; Hanrahan J. W. ; Shrier A. ; Young J. C. Hsp70 and DNAJA2 Limit CFTR Levels through Degradation. PLoS One 2019, 14 (8 ), e0220984 10.1371/journal.pone.0220984.31408507
Younger J. M. ; Ren H. Y. ; Chen L. ; Fan C. Y. ; Fields A. ; Patterson C. ; Cyr D. M. A Foldable CFTRΔF508 Biogenic Intermediate Accumulates upon Inhibition of the Hsc70-CHIP E3 Ubiquitin Ligase. J. Cell Biol. 2004, 167 (6 ), 1075–1085. 10.1083/jcb.200410065.15611333
Baaklini I. ; Wong M. J. H. ; Hantouche C. ; Patel Y. ; Shrier A. ; Young J. C. The DNAJA2 Substrate Release Mechanism Is Essential for Chaperone-Mediated Folding. J. Biol. Chem. 2012, 287 (50 ), 41939–41954. 10.1074/jbc.M112.413278.23091061
Bagdany M. ; Veit G. ; Fukuda R. ; Avramescu R. G. ; Okiyoneda T. ; Baaklini I. ; Singh J. ; Sovak G. ; Xu H. ; Apaja P. M. ; Sattin S. ; Beitel L. K. ; Roldan A. ; Colombo G. ; Balch W. ; Young J. C. ; Lukacs G. L. Chaperones Rescue the Energetic Landscape of Mutant CFTR at Single Molecule and in Cell. Nat. Commun. 2017, 8 (1 ), 398 10.1038/s41467-017-00444-4.28855508
Yamamoto Y.-h. ; Kimura T. ; Momohara S. ; Takeuchi M. ; Tani T. ; Kimata Y. ; Kadokura H. ; Kohno K. A Novel ER J-Protein DNAJB12 Accelerates ER-Associated Degradation of Membrane Proteins Including CFTR. Cell Struct. Funct. 2010, 35 (2 ), 107–116. 10.1247/csf.10023.21150129
Sun F. ; Zhang R. ; Gong X. ; Geng X. ; Drain P. F. ; Frizzell R. A. Derlin-1 Promotes the Efficient Degradation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and CFTR Folding Mutants. J. Biol. Chem. 2006, 281 (48 ), 36856–36863. 10.1074/jbc.M607085200.16954204
Younger J. M. ; Chen L. ; Ren H. Y. ; Rosser M. F. N. ; Turnbull E. L. ; Fan C. Y. ; Patterson C. ; Cyr D. M. Sequential Quality-Control Checkpoints Triage Misfolded Cystic Fibrosis Transmembrane Conductance Regulator. Cell 2006, 126 (3 ), 571–582. 10.1016/j.cell.2006.06.041.16901789
Grove D. E. ; Fan C.-Y. ; Ren H. Y. ; Cyr D. M. The Endoplasmic Reticulum-Associated Hsp40 DNAJB12 and Hsc70 Cooperate to Facilitate RMA1 E3-Dependent Degradation of Nascent CFTR F508. Mol. Biol. Cell 2011, 22 (3 ), 301–314. 10.1091/mbc.e10-09-0760.21148293
Zhang H. ; Peters K. W. ; Sun F. ; Marino C. R. ; Lang J. ; Burgoyne R. D. ; Frizzell R. A. Cysteine String Protein Interacts with and Modulates the Maturation of the Cystic Fibrosis Transmembrane Conductance Regulator. J. Biol. Chem. 2002, 277 (32 ), 28948–28958. 10.1074/jbc.M111706200.12039948
Schmidt B. Z. ; Watts R. J. ; Aridor M. ; Frizzell R. A. Cysteine String Protein Promotes Proteasomal Degradation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Increasing Its Interaction with the C Terminus of Hsp70-Interacting Protein and Promoting CFTR Ubiquitylation. J. Biol. Chem. 2009, 284 (7 ), 4168–4178. 10.1074/jbc.M806485200.19098309
Farinha C. M. ; Nogueira P. ; Mendes F. ; Penque D. ; Amaral M. D. The Human DnaJ Homologue (Hdj)-1/Heat-Shock Protein (Hsp) 40 Co-Chaperone Is Required for the in Vivo Stabilization of the Cystic Fibrosis Transmembrane Conductance Regulator by Hsp70. Society 2002, 366 , 797–806. 10.1042/bj20011717.
Westhoff B. ; Chapple J. P. ; Van Der Spuy J. ; Höhfeld J. ; Cheetham M. E. HSJ1 Is a Neuronal Shuttling Factor for the Sorting of Chaperone Clients to the Proteasome. Curr. Biol. 2005, 15 (11 ), 1058–1064. 10.1016/j.cub.2005.04.058.15936278
Huang Y. ; Arora K. ; Mun K. S. ; Yang F. ; Moon C. S. ; Yarlagadda S. ; Jegga A. ; Weaver T. ; Naren A. P. Targeting DNAJB9, a Novel ER Luminal Co-Chaperone, to Rescue ΔF508-CFTR. Sci. Rep. 2019, 9 (1 ), 1–11. 10.1038/s41598-019-46161-4.30626917
Baaklini I. ; Gonçalves C. de C. ; Lukacs G. L. ; Young J. C. Selective Binding of HSC70 and Its Co-Chaperones to Structural Hotspots on CFTR. Sci. Rep. 2020, 10 (1 ), 4176 10.1038/s41598-020-61107-x.32144307
Arndt V. ; Daniel C. ; Nastainczyk W. ; Alberti S. ; Höhfeld J. BAG-2 Acts as an Inhibitor of the Chaperone-Associated Ubiquitin Ligase CHIP. Mol. Biol. Cell 2005, 16 (November ), 5891–5900. 10.1091/mbc.e05-07-0660.16207813
Coppinger J. A. ; Hutt D. M. ; Razvi A. ; Koulov A. V. ; Pankow S. ; Yates J. R. ; Balch W. E. A Chaperone Trap Contributes to the Onset of Cystic Fibrosis. PLoS One 2012, 7 (5 ), e37682 10.1371/journal.pone.0037682.22701530
Strickland E. ; Qu B. H. ; Millen L. ; Thomas P. J. The Molecular Chaperone Hsc70 Assists the in Vitro Folding of the N- Terminal Nucleotide-Binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator. J. Biol. Chem. 1997, 272 (41 ), 25421–25424. 10.1074/jbc.272.41.25421.9325249
Matsumura Y. ; David L. L. ; Skach W. R. Role of Hsc70 Binding Cycle in CFTR Folding and Endoplasmic Reticulum-Associated Degradation. Mol. Biol. Cell 2011, 22 (16 ), 2797–2809. 10.1091/mbc.e11-02-0137.21697503
Scott-Ward T. S. ; Amaral M. D. Deletion of Phe508 in the First Nucleotide-Binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator Increases Its Affinity for the Heat Shock Cognate 70 Chaperone. FEBS J. 2009, 276 (23 ), 7097–7109. 10.1111/j.1742-4658.2009.07421.x.19878303
Leu J. I.-J. ; Zhang P. ; Murphy M. E. ; Marmorstein R. ; George D. L. Structural Basis for the Inhibition of HSP70 and DnaK Chaperones by Small-Molecule Targeting of a C-Terminal Allosteric Pocket. ACS Chem. Biol. 2014, 9 (11 ), 2508–2516. 10.1021/cb500236y.25148104
Wadhwa R. ; Sugihara T. ; Yoshida A. ; Nomura H. ; Reddel R. R. ; Simpson R. ; Maruta H. ; Kaul S. C. Selective Toxicity of MKT-077 to Cancer Cells Is Mediated by Its Binding to the Hsp70 Family Protein Mot-2 and Reactivation of P53 Function. Cancer Res. 2000, 60 (24 ), 6818–6821.11156371
Sun F. ; Mi Z. ; Condliffe S. B. ; Bertrand C. A. ; Gong X. ; Lu X. ; Zhang R. ; Latoche J. D. ; Pilewski J. M. ; Robbins P. D. ; Frizzell R. A. Chaperone Displacement from Mutant Cystic Fibrosis Transmembrane Conductance Regulator Restores Its Function in Human Airway Epithelia. FASEB J. 2008, 22 (9 ), 3255–3263. 10.1096/fj.07-105338.18556464
Du K. ; Lukacs G. L. Cooperative Assembly and Misfolding of CFTR Domains In Vivo. Mol. Biol. Cell 2009, 20 , 1903–1915. 10.1091/mbc.e08-09-0950.19176754
Alberti S. ; Böhse K. ; Arndt E. ; Schmitz A. ; Höhfeld J. The Cochaperone HspBP1 Inhibits the CHIP Ubiquitin Ligase and Stimulates the Maturation of the Cystic Fibrosis Transmembrane Conductance Regulator. Mol. Biol. Cell 2004, 15 (December ), 4003–4010. 10.1091/mbc.e04-04-0293.15215316
Saxena A. ; Banasavadi-Siddegowda Y. K. ; Fan Y. ; Bhattacharya S. ; Roy G. ; Giovannucci D. R. ; Frizzell R. A. ; Wang X. Human Heat Shock Protein 105/110 KDa (Hsp105/110) Regulates Biogenesis and Quality Control of Misfolded Cystic Fibrosis Transmembrane Conductance Regulator at Multiple Levels. J. Biol. Chem. 2012, 287 (23 ), 19158–19170. 10.1074/jbc.M111.297580.22505710
Mendes F. ; Farinha C. M. ; Felício V. ; Alves P. C. ; Vieira I. ; Amaral M. D. BAG-1 Stabilizes Mutant F508del-CFTR in a Ubiquitin-like-Domain-Dependent Manner. Cell. Physiol. Biochem. 2012, 30 (5 ), 1120–1133. 10.1159/000343303.23178238
Dai Q. ; Qian S.-B. ; Li H.-H. ; McDonough H. ; Borchers C. ; Huang D. ; Takayama S. ; Younger J. M. ; Ren H. Y. ; Cyr D. M. ; Patterson C. Regulation of the Cytoplasmic Quality Control Protein Degradation Pathway by BAG2. J. Biol. Chem. 2005, 280 (46 ), 38673–38681. 10.1074/jbc.M507986200.16169850
Hutt D. M. ; Mishra S. K. ; Roth D. M. ; Larsen M. B. ; Angles F. ; Frizzell R. A. ; Balch W. E. Silencing of the Hsp70-Specific Nucleotide-Exchange Factor BAG3 Corrects the F508del-CFTR Variant by Restoring Autophagy. J. Biol. Chem. 2018, 293 (35 ), 13682–13695. 10.1074/jbc.RA118.002607.29986884
Reilly R. ; Mroz M. S. ; Dempsey E. ; Wynne K. ; Keely S. J. ; McKone E. F. ; Hiebel C. ; Behl C. ; Coppinger J. A. Targeting the PI3K/Akt/MTOR Signalling Pathway in Cystic Fibrosis. Sci. Rep. 2017, 7 (1 ), 7642 10.1038/s41598-017-06588-z.28794469
Meacham G. C. ; Patterson C. ; Zhang W. ; Younger J. M. ; Cyr D. M. The Hsc70 Co-Chaperone CHIP Targets Immature CFTR for Proteasomal Degradation. Nat. Cell Biol. 2001, 3 (1 ), 100–105. 10.1038/35050509.11146634
Marozkina N. V. ; Yemen S. ; Borowitz M. ; Liu L. ; Plapp M. ; Sun F. ; Islam R. ; Erdmann-Gilmore P. ; Townsend R. R. ; Lichti C. F. ; Mantri S. ; Clapp P. W. ; Randell S. H. ; Gaston B. ; Zaman K. Hsp 70/Hsp 90 Organizing Protein as a Nitrosylation Target in Cystic Fibrosis Therapy. Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (25 ), 11393–11398. 10.1073/pnas.0909128107.20534503
Grove D. E. ; Rosser M. F. N. ; Ren H. Y. ; Naren A. P. ; Cyr D. M. Mechanisms for Rescue of Correctable Folding Defects in CFTRΔF508. Mol. Biol. Cell 2009, 20 (18 ), 4059–4069. 10.1091/mbc.e08-09-0929.19625452
Loo M. A. ; Jensen T. J. ; Cui L. ; Hou Y. X. ; Chang X. B. ; Riordan J. R. Perturbation of Hsp90 Interaction with Nascent CFTR Prevents Its Maturation and Accelerates Its Degradation by the Proteasome. EMBO J. 1998, 17 (23 ), 6879–6887. 10.1093/emboj/17.23.6879.9843494
Morán Luengo T. ; Mayer M. P. ; Rüdiger S. G. D. The Hsp70–Hsp90 Chaperone Cascade in Protein Folding. Trends Cell Biol. 2019, 29 (2 ), 164–177. 10.1016/j.tcb.2018.10.004.30502916
Koulov A. V. ; Balch W. ; Yates J. R. III Biological and Structural Basis for Aha1 Regulation of Hsp90 ATPase ACtivity in Maintain Proteostatis in the Human Disease Cystic Fibrosis. Mol. Biol. Cell 2010, 21 (22 ), 4042–4056. 10.1091/mbc.E09.20861316
Marinko J. T. ; Huang H. ; Penn W. D. ; Capra J. A. ; Schlebach J. P. ; Sanders C. R. Folding and Misfolding of Human Membrane Proteins in Health and Disease: From Single Molecules to Cellular Proteostasis. Chem. Rev. 2019, 119 (9 ), 5537–5606. 10.1021/acs.chemrev.8b00532.30608666
Farinha C. M. ; Canato S. From the Endoplasmic Reticulum to the Plasma Membrane: Mechanisms of CFTR Folding and Trafficking. Cell. Mol. Life Sci. 2017, 74 (1 ), 39–55. 10.1007/s00018-016-2387-7.27699454
Okiyoneda T. ; Niibori A. ; Harada K. ; Kohno T. ; Michalak M. ; Duszyk M. ; Wada I. ; Ikawa M. ; Shuto T. ; Suico M. A. ; Kai H. Role of Calnexin in the ER Quality Control and Productive Folding of CFTR; Differential Effect of Calnexin Knockout on Wild-Type and ΔF508 CFTR. Biochim. Biophys. Acta - Mol. Cell Res. 2008, 1783 (9 ), 1585–1594. 10.1016/j.bbamcr.2008.04.002.
Farinha C. M. ; Amaral M. D. Most F508del-CFTR Is Targeted to Degradation at an Early Folding Checkpoint and Independently of Calnexin. Mol. Cell. Biol. 2005, 25 (12 ), 5242–5252. 10.1128/MCB.25.12.5242-5252.2005.15923638
Harada K. ; Okiyoneda T. ; Hashimoto Y. ; Ueno K. ; Nakamura K. ; Yamahira K. ; Sugahara T. ; Shuto T. ; Wada I. ; Suico M. A. ; Kai H. Calreticulin Negatively Regulates the Cell Surface Expression of Cystic Fibrosis Transmembrane Conductance Regulator. J. Biol. Chem. 2006, 281 (18 ), 12841–12848. 10.1074/jbc.M512975200.16527813
Gong X. ; Ahner A. ; Roldan A. ; Lukacs G. L. ; Thibodeau P. H. ; Frizzell R. A. Non-Native Conformers of Cystic Fibrosis Transmembrane Conductance Regulator NBD1 Are Recognized by Hsp27 and Conjugated to SUMO-2 for Degradation *. J. Biol. Chem. 2016, 291 (4 ), 2004–2017. 10.1074/jbc.M115.685628.26627832
Ahner A. ; Gong X. ; Schmidt B. Z. ; Peters K. W. ; Rabeh W. M. ; Thibodeau P. H. ; Lukacs G. L. ; Frizzell R. A. Small Heat Shock Proteins Target Mutant Cystic Fibrosis Transmembrane Conductance Regulator for Degradation via a Small Ubiquitin-like Modifier-Dependent Pathway. Mol. Biol. Cell 2013, 24 (2 ), 74–84. 10.1091/mbc.e12-09-0678.23155000
Tatham M. H. ; Jaffray E. ; Vaughan O. A. ; Desterro J. M. P. ; Botting C. H. ; Naismith J. H. ; Hay R. T. Polymeric Chains of SUMO-2 and SUMO-3 Are Conjugated to Protein Substrates by SAE1/SAE2 and Ubc9. J. Biol. Chem. 2001, 276 (38 ), 35368–35374. 10.1074/jbc.M104214200.11451954
Gong X. ; Liao Y. ; Ahner A. ; Larsen M. B. ; Wang X. ; Bertrand C. A. ; Frizzell R. A. Different SUMO Paralogues Determine the Fate of Wild-Type and Mutant CFTRs: Biogenesis versus Degradation. Mol. Biol. Cell 2019, 30 (1 ), 4–16. 10.1091/mbc.E18-04-0252.30403549
Peters K. W. ; Gong X. ; Frizzell R. A. Cystic Fibrosis Transmembrane Conductance Regulator Folding Mutations Reveal Differences in Corrector Efficacy Linked to Increases in Immature Cystic Fibrosis Transmembrane Conductance Regulator Expression. Frontiers in Physiology. 2021, 10.3389/fphys.2021.695767.
Kleizen B. ; van Willigen M. ; Mijnders M. ; Peters F. ; Grudniewska M. ; Hillenaar T. ; Thomas A. ; Kooijman L. ; Peters K. W. ; Frizzell R. ; van der Sluijs P. ; Braakman I. Co-Translational Folding of the First Transmembrane Domain of ABC-Transporter CFTR Is Supported by Assembly with the First Cytosolic Domain. J. Mol. Biol. 2021, 433 (13 ), 166955 10.1016/j.jmb.2021.166955.33771570
Sabusap C. M. ; Joshi D. ; Simhaev L. ; Oliver K. E. ; Senderowitz H. ; van Willigen M. ; Braakman I. ; Rab A. ; Sorscher E. J. ; Hong J. S. The CFTR P67L Variant Reveals a Key Role for N-Terminal Lasso Helices in Channel Folding, Maturation, and Pharmacologic Rescue. J. Biol. Chem. 2021, 296 , 100598 10.1016/j.jbc.2021.100598.33781744
Sabusap C. M. ; Wang W. ; McNicholas C. M. ; Chung W. J. ; Fu L. ; Wen H. ; Mazur M. ; Kirk K. L. ; Collawn J. F. ; Hong J. S. ; Sorscher E. J. Analysis of Cystic Fibrosis–Associated P67L CFTR Illustrates Barriers to Personalized Therapeutics for Orphan Diseases. JCI Insight 2016, 1 (14 ), 1–10. 10.1172/jci.insight.86581.
McDonald E. F. ; Woods H. ; Smith S. T. ; Kim M. ; Schoeder C. T. ; Plate L. ; Meiler J. Structural Comparative Modeling of Multi-Domain F508del CFTR. Biomolecules. 2022, 12 , 471 10.3390/biom12030471.35327663
Baatallah N. ; Elbahnsi A. ; Chevalier B. ; Castanier S. ; Mornon J.-P. ; Pranke I. ; Edelman A. ; Sermet-Gaudelus I. ; Callebaut I. ; Hinzpeter A. Acting on the CFTR Membrane-Spanning Domains Interface Rescues Some Misfolded Mutants. International Journal of Molecular Sciences 2022, 23 , 16225 10.3390/ijms232416225.36555865
Zacarias S. ; Batista M. S. P. ; Ramalho S. S. ; Victor B. L. ; Farinha C. M. Rescue of Rare CFTR Trafficking Mutants Highlights a Structural Location-Dependent Pattern for Correction. International Journal of Molecular Sciences 2023, 24 , 3211 10.3390/ijms24043211.36834620
Ramalho S. S. ; Silva I. A. L. ; Amaral M. D. ; Farinha C. M. Rare Trafficking CFTR Mutations Involve Distinct Cellular Retention Machineries and Require Different Rescuing Strategies. International Journal of Molecular Sciences 2022, 23 , 24 10.3390/ijms23010024.
Shishido H. ; Yoon J. S. ; Yang Z. ; Skach W. R. CFTR Trafficking Mutations Disrupt Cotranslational Protein Folding by Targeting Biosynthetic Intermediates. Nat. Commun. 2020, 11 (1 ), 4258 10.1038/s41467-020-18101-8.32848127
Anglès F. ; Wang C. ; Balch W. E. Spatial Covariance Analysis Reveals the Residue-by-Residue Thermodynamic Contribution of Variation to the CFTR Fold. Commun. Biol. 2022, 5 (1 ), 1–16. 10.1038/s42003-022-03302-2.34987157
He L. ; Kennedy A. S. ; Houck S. ; Aleksandrov A. ; Quinney N. L. ; Cyr-Scully A. ; Cholon D. M. ; Gentzsch M. ; Randell S. H. ; Ren H. Y. ; Cyr D. M. DNAJB12 and Hsp70 triage arrested intermediates of N1303K-CFTR for endoplasmic reticulum-associated autophagy. Mol. Biol. Cell 2021, 32 , 538–553. 10.1091/mbc.E20-11-0688.33534640
Grove D. E. ; Fan C.-Y. ; Ren H. Y. ; Cyr D. M. The Endoplasmic Reticulum–Associated Hsp40 DNAJB12 and Hsc70 Cooperate to Facilitate RMA1 E3–Dependent Degradation of Nascent CFTRΔF508. Mol. Biol. Cell 2011, 22 (3 ), 301–314. 10.1091/mbc.e10-09-0760.21148293
Liu Q. ; Sabirzhanova I. ; Yanda M. K. ; Bergbower E. A. S. ; Boinot C. ; Guggino W. B. ; Cebotaru L. Rescue of CFTR NBD2Mutants N1303K and S1235R Is Influenced by the Functioning of the Autophagosome. J. Cyst. Fibros. 2018, 17 (5 ), 582–594. 10.1016/j.jcf.2018.05.016.29936070
Rapino D. ; Sabirzhanova I. ; Lopes-Pacheco M. ; Grover R. ; Guggino W. B. ; Cebotaru L. Rescue of NBD2Mutants N1303K and S1235R of CFTR by Small-Molecule Correctors and Transcomplementation. PLoS One 2015, 10 (3 ), e0119796 10.1371/journal.pone.0119796.25799511
