Full Rescue of F508del-CFTR Processing and Function by CFTR Modulators can be Achieved by Removal of Two Unique Regulatory Regions

Background and Purpose: Cystic Fibrosis (CF) is caused by mutations in the CF Transmembrane conductance Regulator (CFTR), the only ABC transporter functioning as a channel. Unique to CFTR are two highly conformationally dynamic regions: the regulatory extension (RE) and regulatory insertion (RI). Removal of the latter rescues the trafficking defect of CFTR with F508del, the most common CF-causing mutation. We aimed here to assess the impact of RE removal (alone or with RI or genetic revertants) on F508del-CFTR traffic and how CFTR modulator drugs corrector VX-809/lumacaftor and potentiator VX-770/ivacaftor rescue these combined variants so as to gain insight into the mechanism of action (MoA) of these drugs. Experimental Approach. We generated ∆RE and ∆RI CFTR variants (with and without genetic revertants) by site-directed mutagenesis and used them to stably transfect BHK cell lines. We studied CFTR expression and stability by Western blotting and pulse-chase respectively, plasma membrane levels by cell surface biotinylation and channel activity by the iodide efflux technique. Key Results. Our data demonstrate that ∆RI significantly enhanced rescue of F508del-CFTR by VX-809. Thus, while the presence of the regulatory insertion seems to be precluding full rescue of F508del-CFTR processing by VX-809, this region appears essential to rescue its function by VX-770, thus suggesting some contradictory role in rescue of F508del-CFTR by these two modulators. Nevertheless, this negative impact of RI removal on VX-770-stimulated currents on F508del-CFTR can be compensated by deletion of the regulatory extension which also leads to the stabilization of this mutant. We thus propose that, despite both these regions being conformationally active, RI precludes F508del-CFTR processing while RE affects mostly its stability and channel opening. Supporting Information: Additional figures with supplementary data


Introduction
Cystic Fibrosis (CF), a life-threatening recessive disorder affecting ~80,000 individuals worldwide, is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein present at the apical membrane of epithelial cells. This is the only member of the ATPbinding cassette (ABC) transporter family, functioning as a channel, actually a cAMP-dependent chloride (Cl -)/ bicarbonate (HCO 3 -) channel. It consists of two membrane-spanning domains (MSD1/2), two nucleotide binding domains (NBD1/2) and a cytoplasmic regulatory domain (RD), which is unique to CFTR 1 . The MSDs are linked via intra-and extra-cellular loops (ICLs, ECLs, respectively). ATP binding promoting NBD1:NBD2 dimerization and subsequent phosphorylation of RD at multiple sites lead to channel gating 2 . The NBD1:NBD2 and ICL4:NBD1 interfaces were shown to represent critical folding conformational sites [3][4][5] important for the gating and maturation of the CFTR protein 5,6 .
Although >2,000 CFTR gene mutations were reported (http://www.genet.sickkids.on.ca/cftr/app), one mutation F508del occurs in 85% of CF patients. This NBD1 mutant fails to traffic to the plasma membrane (PM) due to protein misfolding and retention by the endoplasmic reticulum quality control (ERQC) that targets it to premature proteasomal degradation. F508del-CFTR folding is a complex and inefficient process but it can be rescued, at least partially, by several treatments. These include low temperature incubation 7 , genetic revertants 4,5,[8][9][10][11][12][13] or pharmacological agents, like "corrector" VX-809 (lumacaftor) 14 , one of the first CFTR modulator drugs to receive FDA-approved in combination with potentiator VX-770/ivacaftor 1 . Determining the additive/ synergistic rescue of F508del-CFTR by small molecules correctors together with other rescuing agents/revertants is very valuable to determine the mechanism of action (MoA) of these CFTR modulators 5 .
Comparative studies of CFTR with other ABC transporters are very powerful to understand the uniqueness of some of its regions, their influence on CFTR maturation and function as well as how they affect distinctive binding of CFTR to modulator drugs. Two such unique regions are present in NBD1 of CFTR, which are absent in NBDs of other ABC transporters (Fig.S1) -the regulatory extension (RE) and regulatory insertion (RI). Both regions were described as highly conformationally dynamic 3,16 following PKA phosphorylation at certain ( 660 Ser, 670 Ser; 422 Ser) 18,19 . Importantly, removal the 32-amino acid RI (ΔRI) was reported to rescue traffic of F508del-CFTR 13 .
Our first goal here was to assess the impact of removing the 30-amino acid RE (ΔRE) alone or jointly with ΔRI on the traffic of F508del-CFTR. Secondly, we aimed to evaluate how RE and or RI removal from F508del-CFTR influences the rescue of this mutant by genetic revertants. Our third and final 1 www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm453565.htm Role of regulatory extension on CFTR stability.
4 goal was to determine how the traffic and function of these combined variants of F508del-CFTR (ΔRE, ΔRI plus genetic revertants) are rescued by CFTR modulator drugs VX-809 (corrector) and VX-770 (potentiator) to gain further insight into their MoA.
Our data show that although F508del-CFTR without RE did not traffic to the PM, it showed a dramatic stabilization of its immature form (evidencing a turnover rate ~3 x lower than that of wt-CFTR, being the turnover of F508del-CFTR itself is 2x faster vs. wt-CFTR). Results also show that, while ΔRI further increased processing of F508del-CFTR with revertants to almost wt-CFTR levels, RE removal completely abolished their processing, thus highlighting the different impact of the two dynamic regions on revertant-rescued F508del-CFTR. Most strikingly, although VX-809 rescued ΔRI-F508del-CFTR and ΔRE-ΔRI-F508del-CFTR processing to wt-CFTR levels, nevertheless to achieve maximal function of F508del-CFTR, removal of just RI was insufficient, as both RI and RE had to be absent from F508del-CFTR. These data indicate that removal of these two regions has a positive effect on the rescuing efficacy of F508del-CFTR by CFTR modulators.
Role of regulatory extension on CFTR stability.

Western blot
To study the effect of removal of regulatory extension (RE) and or regulatory insertion (RI) in combination with genetic revertants and VX-809, cells were incubated for 48h at 37ºC with 3µM VX-809. After incubation, cells were lysed and extracts analysed by Western blot (WB) using the anti-CFTR antibody (Ab) 596 or anti-calnexin Ab as a loading control. Score corresponds to the percentage of band C to total CFTR (bands B+C) as normalized to the same ratio in samples from wt-CFTR expressing cells. Blot images were acquired using BioRad ChemiDoc XRC+ imaging system and band intensities were measured using Image Lab analysis software.

Pulse-chase and immunoprecipitation
BHK cells lines stably expressing CFTR variants were starved for 30 min in methionine-free α- sodium salicylate for 60 min. After drying at 80°C for 2h, gels were exposed to X-ray films and further analysed and quantified by densitometry.

Biochemical determination of the plasma membrane levels of CFTR
To determine plasma membrane levels of CFTR protein, we performed cell surface biotinylation in BHK cells cultured on permeable growth supports or tissue culture plates using cell membrane impermeable EZ-Link™ Sulfo-NHS-SS-Biotin, followed by cell lysis in buffer containing 25 mM HEPES, pH 8.0, 1% (v/v) Triton, 10% glycerol (v/v), and Complete Protease Inhibitor Mixture, as described previously 23,24 . Biotinylated proteins were isolated by streptavidin-agarose beads, eluted into SDSsample buffer, and separated by 7.5% (w/v) SDS-PAGE.

Multiple sequence alignment
Sequences for NBD domains of ABC transporters were obtained from Uniprot 25 (human and mouse CFTR) or the PDB 26 (1B0U, 1L2T, 1G29, 1G6H, 1JJ7). Alignments were performed with Jalview 27 using the T-Coffee 28 algorithm using the default parameters.

Data and Statistical analyses
The data and statistical analyses used in this study comply with the recommendations on Role of regulatory extension on CFTR stability. 8

Removal of short regulatory extension (RE S ) alone or with regulatory insertion (RI) has no impact on
F508del-CFTR processing The impact of deleting RE S -short RE (Δ 654 Ser-Gly 673 , Figure S1) was first assessed, either alone or together with RI in its short and long variants (ΔRI S , ΔRI L ), on the in vivo processing of wt-and  Removal of RE S together with RI L -long RI (Δ 404 Gly-Leu 435 , Figure S1) led to a maturation increase of F508del-CFTR from 3±2% to 71±3% (vs wt-CFTR levels), similarly to what had been previously reported by Alexandrov et al for ΔRI L alone 13 . In contrast, removal of RI S -short RI (Δ 412 Ala-Leu 428 , Figure S1) had no impact on ΔRE S -F508del-CFTR processing (Fig.1A, lanes 6,7; Fig.1E; Table1). So in summary, removal of RE S jointly with RI S or RI L had no effect on F508del-CFTR processing.
Interestingly, when RE S and RI S , were jointly removed from wt-CFTR, its processing was significantly reduced to 54±9%, while ΔRE S jointly with ΔRI L caused no impact (Fig.1A, lanes11,12, respectively; Fig.1D; Table1). Again, these data were equivalent to removal of ΔRI S or ΔRI L alone on wt-CFTR processing ( Fig.1A

R1070W
Role of regulatory extension on CFTR stability.

9
The differential effect caused by removal of RI S vs RI L on F508del-and wt-CFTR emphasize the importance of those 8 N-term ( 404 Gly-Ala 412 ) and 7 C-term (Leu 428 -Leu 435 ) amino acid residues that differ between those 2 RI regions for the folding and processing of CFTR.
ΔRI L synergises with VX-809, but not with revertants to rescue ΔRE S -F508del-CFTR processing To further test how the stabilized immature form of ΔRE S -F508del-CFTR could be pharmacologically rescued, we then assessed the impact of corrector VX-809 (which per se promotes maturation of the F508del-CFTR) on the processing of this and of the other variants, to obtain structural insight on the effects of this novel drug. Although ΔRE S -F508del-CFTR could not be rescued by VX-809, data show that this small molecule was able to rescue ΔRI S -ΔRE S -F508del-CFTR (from 7±2% to 20±4%) and further increased processing of ΔRI L -ΔRE S -F508del-CFTR to wt-CFTR levels (from 71±3% to 96±2%)  Table 1). Interestingly, the impaired processing of ΔRE S -R1070W-wt-CFTR (71±6%) was also reverted by VX-809 to 90±3% (Fig.3B, lane 6; Table 1).
ΔRI L -ΔRE S -F508del-CFTR levels at the plasma membrane are equivalent to those of wt-CFTR Role of regulatory extension on CFTR stability.

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To determine the fraction of the above CFTR variants that localize to the PM, we used quantitative cell surface biotinylation. These data showed that PM levels of ΔRI L -ΔRE S -F508del-CFTR were equivalent to those of wt-CFTR, while those of ΔRI L -F508del-CFTR were significantly lower (Fig.4A, lanes 5, 4; Fig.4B). Data also confirmed that ΔRE S did not induce appearance of F508del-CFTR at the cell surface (data not shown). Corrector VX-809 further increased the PM expression of ΔRI L -ΔRE S -F508del-CFTR to levels that are significantly higher than those of wt-CFTR (Fig.4A, lanes 2, 9, Fig.4B).
Given the very significant stabilization of immature ΔRE S -F508del-CFTR (Fig.1A), next, we determined how removal of ΔRE S affected the processing efficiency and the turnover of the F508del-and ΔRI L -F508del-CFTR variants. To this end, we performed pulse-chase experiments (Fig.4C, D) and indeed our results revealed that ΔRE S very significantly stabilized immature F508del-CFTR not just relatively to F508del-CFTR but to levels even significantly higher than those of wt-CFTR (Fig.4C, D).
Interestingly, this stabilizing effect was no longer significant for ΔRI L -ΔRE S -F508del-CFTR nor for ΔRI L -F508del-CFTR, the latter being equivalent to wt-CFTR (Fig.4C, D). Removal of RI S from F508del-CFTR did not stabilize its turnover (data not shown). As to removal of either RE S or RI S from wt-CFTR, it did not affect the processing efficiency or the turnover vs wt-CFTR ( Figure S3).

VX-809 jointly with RE and RI removal completely restored F508del-CFTR function as chloride channel
To investigate the channel function of the ΔRE S -and ΔRI L -F508del-CFTR variants, we used the iodide efflux technique. Removal of RE S alone had no impact on F508del-CFTR function ( Fig.5A; black dash line in Figure S4B) as expected from the lack of processed form for this variant (Fig.1A). However, when RE S and RI L were removed jointly from F508del-CFTR, functional levels reached 78±7% of wt-CFTR (Fig.5A, C; black dotted line in Figure S4B). As to removal of RI L alone from F508del-CFTR, it restored function to 92±7% of wt-CFTR (Fig.5A, C; black line in Figure S4B), as described 13 . Also, the delay in peak response observed for F508del-CFTR (min=4) vs wt-CFTR (min=2), was partially corrected (to min=3) both by removal of RI L alone from F508del-CFTR or together with RE S (Fig.5C; Figure S4B, black line and dotted line).
Role of regulatory extension on CFTR stability.
The effect of VX-809 (3µM, 48h at 37ºC) was also examined at functional level on the ΔRE S -, ΔRI L -ΔRE S -and ΔRI L -F508del-CFTR variants. Strikingly VX-809 caused an increase in ΔRE S -ΔRI L -F508del-CFTR function (84±8% of wt-CFTR) which was parallel to the observed increase in PM expression ( Fig.4; Fig.5A, C; black dotted line in Figure S4F). Also, ΔRE S -ΔRI L -F508del-CFTR fully corrected (to min=2) the delay in peak response of F508del-CFTR which was still partially present in ΔRI L -and ΔRI L - In contrast to the observed increase in processing, VX-809 caused no significant increase in the function of ΔRI L -F508del-CFTR and actually a slight, but not significant decrease was observed and there was still the same delay in peak response (Fig.5A, C; black line in Figure S4F).
A similar slight decrease in function was observed for VX-809 on the ΔRI L -R1070W-F508del-CFTR variant (Fig.5A, C; black line in Figure S4H), but the most striking result was observed for the significant decrease caused by VX-809 on function of ΔRI L -G550E-F508del from 76±8 to 41±3% (Fig.5A, C; black line in Figure S4G). Nevertheless both revertant variants of ΔRI L -F508del-CFTR showed no change in peak response which was still the same as wt-CFTR (min=2).

VX-770-stimulated currents of CFTR variants are dramatically decreased by ΔRI L but not by ΔRI L -ΔRE S
Given the interesting and diverse results observed for the ΔRE S and ΔRI L under VX-809, next we tested the effects of potentiator VX-770 (Ivacaftor), an approved drug for CF patients with gating mutations (Fig.5B) on these variants and in combination with VX-809, for F508del/F508del patents.
Most strikingly, our data show that VX-770/Fsk significantly decreased the function of ΔRI L -F508del-CFTR vs potentiation by Gen/Fsk (Fig.5B, C) from 92±7% to 58±7% of wt-CFTR (Fig.5B, C; black line in Figure S4J). As ΔRE S -F508del-CFTR was not processed, we did not test the effect of VX-770 on this variant, but when the ΔRE S -ΔRI L -F508del-CFTR variant was assessed for its function after acute application of VX-770 with Fsk, an increase in function (to 87±3% of wt-CFTR) was observed vs its function under Gen/Fsk (78±7% of wt-CFTR) (Fig.5B, C; black dotted line in Figure S4J).
Role of regulatory extension on CFTR stability. 14

Discussion
The main goal of this study was to understand how the removal of the regulatory extension (ΔRE) alone or with the regulatory insertion (ΔRI)-two highly conformationally dynamic regions -impacted on the rescue of F508del-CFTR processing and function by two compounds -VX-809 and VX-770which in combination were recently approved o clinically treat F508del-homozygous patients.
Indeed, CFTR is the sole ABC that functions as a channel and thus, these highly dynamic RE and RI regions which are absent in other ABC transporters, may be of high relevance to understand how CFTR differs from other ABCs, namely in its function as a channel.
These regions RE and RI were originally suggested to be positioned to impede formation of the NBD1-NBD2 dimer required for channel gating 16,17 (Fig.S1). Later, however, these limits were proposed to be 404 Gly-Leu 435 13,21 or 405 Phe-Leu 436 22 .

Impact of RE and RI on CFTR processing and function
Our data shown here on CFTR variants depleted of different versions of the RE dynamic region demonstrate that unlike RI deletion, removal of short RE -ΔRE S (Δ 654 Ser-Gly 673 ) did not per se rescue F508del-CFTR processing. Nevertheless, ΔRE S dramatically stabilized the immature form of F508del-CFTR (see Fig.6). In fact, our pulse-chase experiments show that the immature form of ΔRE S -F508del-CFTR exhibits a turnover rate which is ~2.7 fold lower than that of wt-CFTR. Of note, when Aleksandrov et al removed the dynamic region RI from F508del-CFTR they found a dramatic increase Role of regulatory extension on CFTR stability.

15
in the channel thermostability, even augmented for higher temperatures 13 . Interestingly however, removal of the RE S from ΔRI L -F508del-CFTR (ΔRI L : Δ 404 Gly-Leu 435 ) while not affect processing, it further reduced its function (from 92% to 78%).
The latter findings on function of ΔRE S -wt-CFTR and ΔRE S -ΔRI L -F508del-CFTR were somewhat surprising since the RE was previously described to impede putative NBD1:NBD2 dimerization that is required for channel gating 21 . Accordingly, an increase in CFTR activity would be expected upon  19,20 . Although the RD contains more than ten PKA phospho-sites and no individual one is essential, phosphorylation of increasing numbers of sites enables progressively greater channel activity 31 .
Removal of a longer RE version (ΔRE L : Δ 647 Cys-Ser 678 ) was without effect on F508del-CFTR processing and significantly reduced that of wt-CFTR to 88%. Such differential impact on wt-and F508del-CFTR is consistent with the conformational heterogeneity between these two proteins lacking both RI and RE 32 .
Removal of RI short version, RI s (Δ 412 Ala-Leu 428 ) significantly reduced wt-CFTR processing to less than half of its normal levels, while not rescuing F508del-CFTR processing, thus being essential for CFTR proper folding. This is in contrast to removal of long RI (ΔRI L ) which led to 78% processing F508del-CFTR, as reported 13 but without impact on wt-CFTR. These data indicate that those 8/ 7 amino acid residues at the N-term/ C-term of RI S (and absent in RI L ) impair the folding efficiency and processing of both wt-and F508del-CFTR.
The recently published cryo-EM structure of human CFTR 33 indicates that these regions are structurally disordered. Consistently, the RI loop in NBD1 is described in the of zebrafish CFTR cryostructure to contribute to the amorphous density that is observed between NBD1 and the elbow helix of TMD2 34

Effect of VX-809 on F508del-CFTR variants lacking RE and RI
As for rescue of CFTR variants lacking RE and RI by CFTR modulators, VX-809 restored the processing of both ΔRE S -ΔRI L -and ΔRI L -F508del-CFTR variants equally well and to wt-CFTR levels (from 71-78% to 92-96%. These data suggest a strong synergistic effect between VX-809 and ΔRI L to rescue ΔRE S -F508del-CFTR processing and thus some possible interference of the regulatory insertion with VX-809 binding to F508del-CFTR (see Fig.6). Indeed, although previous studies 5,36,37 suggested putative binding of VX-809 to NBD1:MSD2 (ICL4) interface (see Fig.6), they also suggested scope for further F508del-CFTR correction at distinct conformational sites, so data shown here suggest that VX-809 may also bind to the regulatory insertion.
Interestingly, the processing defect of ΔRI S -wt-CFTR (but not of ΔRI S -F508del-CFTR) was rescuable by VX-809 to 90%, indicating that the amino acid stretches of the RI L that remain present in RI S do not affect the rescue of ΔRI S -wt-CFTR but preclude rescue of F508del-CFTR by VX-809.
We also tested here the impact of removing helix 9 (H9) which precedes the RE ( 635 Gln-Gly 646 ), just after H8 ( 630 Phe-Leu 634 ), both helices proposed to interact with the NBD1:NBD2 heterodimer interface by folding onto the NBD1 ß-subdomain 20,32 as well as to bind ICL4 near Phe508. When H9 is present, ΔRE L -wt-CFTR processing appears to be favoured by VX-809, suggesting some synergy of this small molecule with H9 helix to correct the conformational defect(s) caused by ΔRE L on wt-CFTR.
These data are in contrast to ΔH9-F508del-CFTR which exhibits 0% processing with or without ΔRE L and no rescue by VX-809.
Very strikingly, and in contrast to its effect on processing, was the effect of ΔRI L on the VX-770 stimulated currents which were decreased by almost half vs those stimulated by Gen/Fsk (92% to 58%). These data seem to indicate that the absence of the regulatory insertion could impair (and its presence favour) binding of VX-770 to CFTR (see Fig.6). Surprisingly, the removal of RE s from ΔRI L -F508del-CFTR could correct this defect and restore the maximal function by VX-770 (see Fig.6).

Impact of F508del-revertants on CFTR variants lacking RE and RI
Another goal of the present study was to assess how presence of the F508del-CFTR revertants G550E and R1070W 5,10,11 influence variants without RE and RI. Remarkably, our data show that the presence of either of these revertants did not affect ΔRE S -F508del-CFTR processing, but both of them further increased processing (but not function) of ΔRI L -F508del-CFTR to almost levels of wt-CFTR: 92-96% (G550E) and 71-76% (R1070W).
In contrast, removal of RI S from either of these F508del-CFTR revertants completely abolished their processing, emphasizing how important the different residues between RI S and RI L are for F508del-CFTR conformers partially rescued by the revertants.
Regarding wt-CFTR processing, G550E (at the NBD1:NBD2 dimer interface) also partially recovered the negative effect caused by RI S removal. In contrast, R1070W (at the NBD1:ICL4 interface), negatively affected processing of wt-CFTR (to 69%) and of ΔRI S -wt-CFTR (from 44% to 11%), while not affecting ΔRI L -wt-CFTR. R1070W rescues F508del-CFTR because Trp1070 fills the gap created by deletion of residue Phe508 38 . It is not surprising that it perturbs CFTR folding due to clashing of 508 Phe and 1070 Trp residues. It is, nevertheless curious that R1070W affect more the processing ΔRI Swt-CFTR than wt-CFTR, to levels of F508del-CFTR, suggesting that both changes affect the same region of the molecule. Rescue of both R1070W-wt-CFTR and R1070W-ΔRI S -wt-CFTR by VX-809 further supports this concept.
The most striking effect of ΔRI L however, was the almost complete abolition of VX-770-stimulated current of G550E-F508del-CFTR to levels even lower than those observed for ΔRI L -F508del-CFTR (see above). It was suggested that VX-770 binds directly to CFTR to the MSDs (although it is not defined the exact binding site), in phosphorylation-dependent but ATP-independent manner and away from the canonical catalytic site 39,40 . Both RD and RI were suggested by Eckford and colleagues as putative binding regions for VX-770 39 . Nevertheless, the pharmacological effect of VX-770 remains robust in the absence of the RD 40 and here we demonstrate that it does indeed require RI since VX-770 is unable to stimulate either ΔRI L -F508del-CFTR or ΔRI L -G550E-F508del-CFTR (see Fig.6).
Overall, our data show that while the presence of the regulatory insertion seems to be precluding full rescue of F508del-CFTR processing by VX-809, this region appears essential to rescue its function Role of regulatory extension on CFTR stability.
18 by VX-770, thus suggesting some contradictory role in rescue of F508del-CFTR by these two modulators. Nevertheless, this negative impact of RI L removal on VX-770-stimulated currents on F508del-CFTR can be compensated by deletion of the regulatory extension which also leads to the stabilization of this mutant. We thus propose that, despite both these regions being conformationally active, RI precludes F508del-CFTR processing while RE affects mostly its stability and channel opening. (5) (3)