ABCMdb

ABCC7 p.Ile539Thr

CF databases:	c.1616T>C , p.Ile539Thr (CFTR1) ? , The above mutation was detected by DGGE with chemical clamps and characterized by direct sequencing.
Predicted by SNAP2:	A: D (66%), C: N (66%), D: D (85%), E: D (80%), F: D (66%), G: D (80%), H: D (75%), K: D (85%), L: D (53%), M: D (63%), N: D (75%), P: D (85%), Q: D (75%), R: D (80%), S: D (75%), T: N (87%), V: N (57%), W: D (75%), Y: D (75%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N,

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[hide] Mutations in the nucleotide binding domain 1 signa... J Biol Chem. 2002 Sep 27;277(39):35896-905. Epub 2002 Jul 10. DeCarvalho AC, Gansheroff LJ, Teem JL
Mutations in the nucleotide binding domain 1 signature motif region rescue processing and functional defects of cystic fibrosis transmembrane conductance regulator delta f508.
J Biol Chem. 2002 Sep 27;277(39):35896-905. Epub 2002 Jul 10., 2002-09-27 [PMID:12110684]

Abstract [show]
The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP binding cassette (ABC) transporter that functions as a phosphorylation- and nucleotide-regulated chloride channel, is mutated in cystic fibrosis (CF) patients. Deletion of a phenylalanine at amino acid position 508 (DeltaF508) in the first nucleotide binding domain (NBD1) is the most prevalent CF-causing mutation and results in defective protein processing and reduced CFTR function, leading to chloride impermeability in CF epithelia and heterologous systems. Using a STE6/CFTRDeltaF508 chimera system in yeast, we isolated two novel DeltaF508 revertant mutations, I539T and G550E, proximal to and within the conserved ABC signature motif of NBD1, respectively. Western blot and functional analysis in mammalian cells indicate that mutations I539T and G550E each partially rescue the CFTRDeltaF508 defect. Furthermore, a combination of both revertant mutations resulted in a 38-fold increase in CFTRDeltaF508-mediated chloride current, representing 29% of wild type channel activity. The G550E mutation increased the sensitivity of CFTRDeltaF508 and wild type CFTR to activation by cAMP agonists and blocked the enhancement of CFTRDeltaF508 channel activity by 2 mm 3-isobutyl-1-methylxanthine. The data show that the DeltaF508 defect can be significantly rescued by second-site mutations in the nucleotide binding domain 1 region, that includes the LSGGQ consensus motif.

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2 Using a STE6/CFTR⌬F508 chimera system in yeast, we isolated two novel ⌬F508 revertant mutations, I539T and G550E, proximal to and within the conserved ABC signature motif of NBD1, respectively. Login to comment
3 Western blot and functional analysis in mammalian cells indicate that mutations I539T and G550E each partially rescue the CFTR⌬F508 defect. Login to comment
25 Here we used the STE/CFTR⌬F508 chimera system to identify novel amino acid substitutions just upstream (I539T) and within (G550E) the ABC signature motif of CFTR * This work was supported by National Institutes of Health Grant HL61234 and a Program Enhancement Grant from Florida State University Research Foundation (to J. L. T.). Login to comment
101 Two novel point mutations were isolated in the CFTR sequence that substantially rescued the H5-⌬F508 mating defect (Table I), resulting in the change of Ile-539 of the CFTR sequence to a Thr residue (I539T) and of Gly to Glu at the 550 position (G550E). Login to comment
102 Mutations I539T and G550E Partially Rescue CFTR⌬F508- The two novel ⌬F508 revertant mutations isolated in yeast were located either just upstream (I539T) or within (G550E) the CFTR NBD1 signature motif (Fig. 1). Login to comment
104 To evaluate the effect of the novel revertant mutations on CFTR⌬F508 processing, I539T and G550E mutations were introduced into the full-length CFTR⌬F508 cDNA (⌬F/I539T and ⌬F/G550E) for expression in mammalian cells. Login to comment
105 To test whether the combination of the I539T and G550E mutations would result in an additive or synergistic effect in correcting the ⌬F508 phenotype, we also constructed a CFTR⌬F508 allele containing both revertant mutations (⌬F/DB). Login to comment
110 We observed that I539T and, to a lesser extent, G550E partially rescued the CFTR⌬F508-processing defect. Login to comment
122 STE6/CFTR (H5) variant Mating efficiency % of H5 ⌬F508 0.28 Ϯ 0.04 ⌬F508/I539T 42.70 Ϯ 0.40 ⌬F508/G550E 79.90 Ϯ 4.50 Mutations in the ABC Signature Motif Region Rescue CFTR⌬F50835898 fected with CFTR wt respond to cAMP agonists with a rapid increase in Isc, reflecting increased chloride permeability (48). Login to comment
124 The ⌬F508 revertants CFTR⌬F/I539T and CFTR⌬F/G550E exhibited 6- and 12-fold increases in chloride current relative to CFTR⌬F508, respectively (Fig. 2B). Login to comment
126 To assess the effect of the ⌬F508 revertant mutations on CFTR wt chloride channel function, CFTRG550E, CFTRI539T, and a CFTR allele containing both I539T and G550E mutations (CFTRDB) were transiently expressed in FRT cells, and chloride current was measured after activation with 10 ␮M forskolin and 100 ␮M IBMX. Login to comment
129 The G550E Mutation Increases the Sensitivity of CFTR and CFTR⌬F508 to Activation by cAMP Agonists in FRT Cells Transiently Expressing CFTR-As shown in Fig. 2B, the G550E mutation was more effective than I539T in restoring the chloride channel function of CFTR⌬F508 (12-fold versus 6-fold increase), yet CFTR⌬F/G550E cell lysates contained lower levels of mature protein relative to CFTR⌬F/I539T (Fig. 2A). Login to comment
131 The I539T and G550E mutations partially rescue CFTR⌬F508-processing and functional defects. Login to comment
150 Characterization of FRT Cell Lines Stably Expressing the CFTR⌬F508 Revertants-We obtained FRT cell lines stably expressing CFTR⌬F/I539T, CFTR⌬F/G550E, and CFTR⌬F/DB to further characterize the effect of the revertant mutations on CFTR⌬F508 processing and function. Login to comment
152 Results from CFTR immunoblotting analysis (Fig. 4A) and functional studies (Fig. 4B) confirmed the suppression of the CFTR ⌬F508 processing and chloride impermeability defects by I539T and G550E mutations, as observed for the transient expression experiments (Fig. 2, A and B). Login to comment
153 Functional assays showed a 13-fold increase in transepithelial chloride current for FRT-CFTR⌬F/I539T in relation to FRT-CFTR⌬F508, which is in agreement with the substantial amount of mature protein observed for this cell line (Fig. 4, A and B). Login to comment
154 Whereas the Isc was not significantly different between FRT-CFTR⌬F/DB and FRT-CFTR⌬F/I539T, we observed decreased levels of both mature (band C) and immature (band B) protein for FRT-CFTR⌬F/DB. Login to comment
170 We also investigated the effect of the revertant mutations I539T and G550E on the temperature sensitivity of CFTR ⌬F508. Login to comment
174 Effect of I539T and G550E mutations on CFTR ⌬F508 temperature sensitivity and dose response to forskolin activation in FRT stable cell lines. Login to comment
175 A, Western blot analysis of the steady-state level of CFTR⌬F508, CFTR⌬F/I539T, CFTR⌬F/G550E, and CFTR⌬F/DB stably expressed in FRT cells. Login to comment
187 The I539T mutation rendered CFTR⌬F508 and CFTR⌬F/G550E insensitive to incubation at 30 °C (Fig. 4B). Login to comment
191 To assess the effect of G550E and I539T on the modulation of sensitivity of CFTR⌬F508 to activation while minimizing the potential effect of different channel levels at the plasma membrane, we compared the sensitivity to forskolin activation of CFTR ⌬F508 rescued by incubation for 48 h at 30 °C, with CFTR⌬F/G550E, CFTR⌬F/I539T, and CFTR⌬F/DB incubated at 37 °C. FRT monolayers stably expressing each CFTR variant were mounted in Ussing chambers, and the cAMP-activated chloride current was measured in response to decreasing concentrations of forskolin. Login to comment
195 FRT-CFTR⌬F/DB (containing both I539T and G550E) also exhibited a significant increase in sensitivity to activation relative to FRT-CFTR⌬F508 over the entire range of suboptimal forskolin concentrations tested. Login to comment
196 However, in contrast to the variants containing G550E, increased sensitivity to activation by suboptimal concentrations of cAMP agonist was not observed for the variant containing I539T alone. Login to comment
221 Mutations in the ABC Signature Motif Region Rescue CFTR⌬F50835902 IBMX and 50 ␮M genistein on the PKA-dependent activity of CFTR⌬F508, CFTR⌬F/I539T, CFTR⌬F/G550E, CFTR⌬F/DB, and CFTR wt. Login to comment
227 Genistein significantly increased the PKA-activated Isc for all the cell lines containing the ⌬F508 mutation, although it produced a smaller increase for cell lines containing the G550E mutation; compare 58 and 70% increase for CFTR⌬F508 and CFTR⌬F/I539T, respectively, with 45 and 25% for CFTR⌬F/G550E and CFTR⌬F/DB (Fig. 6). Login to comment
231 Two millimolar IBMX resulted in ϳ80% increase in Isc for FRT-CFTR⌬F508 and FRT-CFTR⌬F/ I539T (Fig. 6). Login to comment
233 DISCUSSION Two novel ⌬F508 revertant mutations were identified just upstream (I539T) and within (G550E) the core consensus ABC signature motif LSGGQ in the NBD1 of CFTR. Login to comment
234 Each mutation partially restored processing of mutant CFTR⌬F508 expressed in HeLa cells, with I539T being the most effective. Login to comment
235 Increased cAMP-activated chloride permeability was also observed in FRT monolayers expressing CFTR⌬F/I539T and CFTR⌬F/ G550E to levels 6- and 12-fold higher than CFTR⌬F508, respectively. Login to comment
236 The larger fraction of processed CFTR⌬F/I539T and CFTR⌬F/G550E observed relative to CFTR⌬F508, thus, represents functional channels localized at the plasma membrane. Login to comment
237 Furthermore, functional studies using a double revertant allele (CFTR⌬F/DB) showed that I539T and G550E mutations act synergistically to increase CFTR⌬F508 chloride currents to ϳ29% of CFTR wt, representing a 38-fold increase over the CF mutant. Login to comment
240 The I539T and G550E mutations were identified as revertants of the CF-causing mutation ⌬F508. Login to comment
244 In support of the latter possibility, it was observed that the combination of I539T and G550E within wild type CFTR increased functional activity. Login to comment
245 Further experiments will be necessary to determine the extent to which ⌬F508 revertant mutations I539T and G550E suppress other CF mutations within the NBD1 that are associated with defective protein processing. Login to comment
246 To further assess the effects of the revertant mutations on CFTR⌬F508, we compared the functional activity of CFTR⌬F508, CFTR⌬F/G550E, and CFTR⌬F/I539T under various experimental conditions. Login to comment
248 The I539T mutation, when introduced in either CFTR⌬F508 or CFTR⌬F/G550E, rendered these variants insensitive to low temperature treatment. Login to comment
249 It is possible that the I539T revertant mutation and the low temperature treatment stabilize the same step in the mutant protein folding pathway; thus, their effects are not additive. Login to comment
251 Effect of the I539T and G550E mutations on CFTR ⌬F508 activation by 2 mM IBMX and 50 ␮M genistein. Login to comment
256 In contrast to I539T, G550E had little effect on CFTR⌬F508 temperature sensitivity, suggesting that it affects the protein folding pathway differently. Login to comment
260 Our results show that the G550E mutation decreased the concentration of forskolin required for half-maximal stimulation of all the CFTR variants tested, CFTR wt, CFTR⌬F508, and CFTR⌬F/I539T. Login to comment
264 Because IBMX and genistein are known to enhance the functional activity of CFTR⌬F508 (23-25), we assessed the effect of these molecules on CFTR⌬F/G550E, CFTR⌬F/I539T, and CFTR⌬F/DB. Login to comment
268 The effect of 2 mM IBMX on the activation of CFTR⌬F/I539T was similar to the effect observed for CFTR⌬F508. However, 2 mM IBMX did not increase the PKA-dependent currents of FRT-CFTR⌬F/G550E or FRT-CFTR⌬F/DB. Login to comment
270 Genistein significantly enhanced PKA-activated chloride currents of CFTR⌬F508, CFTR⌬F/G550E, CFTR⌬F/I539T, and CFTR⌬F/DB, although the currents mediated by CFTR variants containing the G550E mutation were stimulated to a lesser extent. Login to comment
273 The revertant mutations I539T and G550E did not preclude genistein enhancement of the PKA-dependent activity of CFTR⌬F508 implying that, similarly to the CF mutant, the revertants could be underphosphorylated at maximal PKA activity. Login to comment
274 I539T significantly enhanced the processing of CFTR⌬F508. Login to comment
294 We speculate that G550E and I539T mutations could restore the LSGGQ-mediated interactions disrupted by the ⌬F508 mutation. Login to comment

[hide] Processing of CFTR: traversing the cellular maze--... Pediatr Pulmonol. 2005 Jun;39(6):479-91. Amaral MD
Processing of CFTR: traversing the cellular maze--how much CFTR needs to go through to avoid cystic fibrosis?
Pediatr Pulmonol. 2005 Jun;39(6):479-91., [PMID:15765539]

Abstract [show]
Biosynthesis of the cystic fibrosis transmembrane conductance regulator (CFTR), like other proteins aimed at the cell surface, involves transport through a series of membranous compartments, the first of which is the endoplasmic reticulum (ER), where CFTR encounters the appropriate environment for folding, oligomerization, maturation, and export from the ER. After exiting the ER, CFTR has to traffic through complex pathways until it reaches the cell surface. Although not yet fully understood, the fine details of these pathways are starting to emerge, partially through identification of an increasing number of CFTR-interacting proteins (CIPs) and the clarification of their roles in CFTR trafficking and function. These aspects of CFTR biogenesis/degradation and by membrane traffic and CIPs are discussed in this review. Following this description of complex pathways and multiple checkpoints to which CFTR is subjected in the cell, the basic question remains of how much CFTR has to overcome these barriers and be functionally expressed at the plasma membrane to avoid CF. This question is also discussed here.

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No. Sentence Comment
49 Indeed, by replacing either glycineat position550 by aglutamic acid residue (G550E) or isoleucine 539 by threonine (I539T), in cis with F508del it is possible to rescue both its membrane localization and channel activity.28 Another study showed that removal from F508del-CFTR of sequence signals, called arginine-framed tripeptide (AFT)-sequences (responsible for the ER retention or retrieval of other ion channels),29 permits nascent F508del-CFTR to mature and generate functional ClÀ channels at the cell surface.30 There are four of these arginine (R) sequences in CFTR: one in the N-terminal cytoplasmic domain (R29), two in NBD1 (R516 and R555), and one in the R domain (R766). Login to comment
51 Whether these AFT motifs are just ER-retention signals or, like G550E and I539T, also act as intrinsic folding determinants remains to be fully clarified. Login to comment

[hide] Restoration of domain folding and interdomain asse... FASEB J. 2010 Aug;24(8):3103-12. Epub 2010 Mar 16. He L, Aleksandrov LA, Cui L, Jensen TJ, Nesbitt KL, Riordan JR
Restoration of domain folding and interdomain assembly by second-site suppressors of the DeltaF508 mutation in CFTR.
FASEB J. 2010 Aug;24(8):3103-12. Epub 2010 Mar 16., [PMID:20233947]

Abstract [show]
Deletion of PHE508 (DeltaF508) from the first nucleotide-binding domain (NBD1) of CFTR, which causes most cystic fibrosis, disrupts the folding and assembly of the protein. Although the folding pathways and yield of isolated NBD1 are altered, its global structure is not, and details of the changes in the rest of the protein remain unclear. To gain further insight into how the whole mutant protein is altered, we have determined the influence of known second-site suppressor mutations in NBD1 on the conformation of this domain and key interfaces between domains. We found that the suppressors restored maturation of only those processing mutations located in NBD1, but not in other domains, including those in the C-terminal cytoplasmic loop of the second membrane-spanning domain, which forms an interface with the NBD1 surface. Nevertheless, the suppressors promoted the formation of this interface and others in the absence of F508. The suppressors restored maturation in a DeltaF508 construct from which NBD2 was absent but to a lesser extent than in the full-length, indicating that DeltaF508 disrupts interactions involving NBD2, as well as other domains. Rescue of DeltaF508-CFTR by suppressors required the biosynthesis of the entire full-length protein in continuity, as it did not occur when N- and C-terminal "halves" were coexpressed. Simultaneous with these interdomain perturbations, DeltaF508 resulted in suppressor reversed alterations in accessibility of residues both in the F508-containing NBD1 surface loop and in the Q loop within the domain core. Thus, in the context of the full-length protein, DeltaF508 mutation causes detectable changes in NBD1 conformation, as well as interdomain interactions.

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No. Sentence Comment
27 These suppressor mutations (I539T, G550E, R553M/Q, and R555K) promote ⌬F508-CFTR maturation and trafficking to the cell surface, and also restore channel activity (16). Login to comment
72 RESULTS Suppressor mutations restore folding mutations in NBD1 but not elsewhere Four suppressor mutations (I539T, G550E, R553M, and R555K) were originally found to rescue ⌬F508-CFTR maturation in a yeast mating screen using STE6/CFTR chimeras (14-16). Login to comment
79 B, C) HEK293 cells were transiently transfected with CFTR variants with maturation-compromising mutations introduced in different domains, in the presence or absence of single or combined suppressor mutations (4S: I539T/G550E/R4553M/R555K; 3S: G550E/R553M/R555K). Login to comment
85 The addition of G550E and R553M to R555K (3S) further increased its maturation, but no additional effect was detected by the addition of the fourth mutation I539T (4S) (Fig. 1A). Login to comment
112 Stable BHK cells overexpressing WT-CFTR and ⌬F508-CFTR with and without 4 suppressor mutations (I539T/G550E/R553M/R555K, ⌬F/ 4S) were pulse labeled with 100 ␮Ci/ml [35 S] methionine for 20 min, followed by 0, 1, 2, and 4 h chase. Login to comment
124 To test whether these NBD/CL interfaces not formed in ⌬F508-CFTR could be restored by the suppressor mutations, the 4 combined suppressor mutations, I539T/G550E/R553M/R555K (4S) were introduced into the ⌬F508-CFTR constructs with the Cys pairs at the potential interfaces. Login to comment
128 The rescue of ⌬F508 by suppressor mutations is diminished in the absence of NBD2 According to a structural model of CFTR (18), some of the suppressor mutations (I539T and G550E) are located at the NBD1/NBD2 interface (Fig. 1A), and ⌬F508 is known to destabilize NBD2 (6, 7). Login to comment
139 HEK293 cells were transiently transfected with Cys-less ⌬F508-CFTR in the presence or absence of suppressor mutations I539T/G550E/R553M/R555K, with Cys pairs introduced at CL2/NBD2 (A) or CL4/NBD1 (B) interfaces. Login to comment
154 HEK293 cells were transiently transfected with 1172X-CFTR or ⌬F508-1172X-CFTR in the presence or absence of single or combined suppressor mutations (4S: I539T/G550E/R4553M/R555K; 3S: G550E/R553M/R555K). Login to comment

[hide] Folding and rescue of a cystic fibrosis transmembr... J Biol Chem. 2010 Aug 27;285(35):27033-44. Epub 2010 Jun 15. Da Paula AC, Sousa M, Xu Z, Dawson ES, Boyd AC, Sheppard DN, Amaral MD
Folding and rescue of a cystic fibrosis transmembrane conductance regulator trafficking mutant identified using human-murine chimeric proteins.
J Biol Chem. 2010 Aug 27;285(35):27033-44. Epub 2010 Jun 15., 2010-08-27 [PMID:20551307]

Abstract [show]
Impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel causes cystic fibrosis, a fatal genetic disease. Here, to gain insight into CFTR structure and function, we exploited interspecies differences between CFTR homologues using human (h)-murine (m) CFTR chimeras containing murine nucleotide-binding domains (NBDs) or regulatory domain on an hCFTR backbone. Among 15 hmCFTR chimeras analyzed, all but two were correctly processed, one containing part of mNBD1 and another containing part of mNBD2. Based on physicochemical distance analysis of divergent residues between human and murine CFTR in the two misprocessed hmCFTR chimeras, we generated point mutations for analysis of respective CFTR processing and functional properties. We identified one amino acid substitution (K584E-CFTR) that disrupts CFTR processing in NBD1. No single mutation was identified in NBD2 that disrupts protein processing. However, a number of NBD2 mutants altered channel function. Analysis of structural models of CFTR identified that although Lys(584) interacts with residue Leu(581) in human CFTR Glu(584) interacts with Phe(581) in mouse CFTR. Introduction of the murine residue (Phe(581)) in cis with K584E in human CFTR rescued the processing and trafficking defects of K584E-CFTR. Our data demonstrate that human-murine CFTR chimeras may be used to validate structural models of full-length CFTR. We also conclude that hmCFTR chimeras are a valuable tool to elucidate interactions between different domains of CFTR.

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124 Thus, we identified six residues in 12b-NBD1 (E527Q, E528Q, S531T, K536Q, I539T, and K584E) and 12 residues in 114c-NBD2 (T1263I, P1290T, K1302Q, Y1307N, Q1309K, S1311K, R1325K, V1338T, C1344Y, L1367I, D1394G, and E1409D) (see supplemental Fig. 1 and supplemental Table 1, A and B). Login to comment
187 TABLE 1 Summary information of CFTR point mutants analyzed in present study CFTR variants Clinical dataa Band C/band Bb (؎S.E., n ‫؍‬ 5) Processingc Normalized processingd Normalized iodide efflux functione (؎S.E., n ‫؍‬ 6) Iodide efflux to processed proteinf % % % % peak intensity % WT-CFTR -g 83 Ϯ 3 77 100 100 Ϯ 8 - Murine - 86 Ϯ 5 66 86 74 Ϯ 4 86 Ϯ 4 E527Q Mild CF 64 Ϯ 5 49 63 46 Ϯ 4 73 Ϯ 4 E528Q - 86 Ϯ 5 79 102 135 Ϯ 16 132 Ϯ 10 S531T - 87 Ϯ 6 81 105 71 Ϯ 5 67 Ϯ 5 K536Q - 69 Ϯ 3 42 54 51 Ϯ 4 94 Ϯ 3 I539T Revertant 112 Ϯ 5 81 105 49 Ϯ 6 46 Ϯ 5 L581F - 118 Ϯ 3 83 107 72 Ϯ 5 67 Ϯ 3 L581F/K584E - 125 Ϯ 2 77 100 100 Ϯ 12 100 Ϯ 8 T1263I Mild CF 75 Ϯ 3 76 98 31 Ϯ 8 31 Ϯ 5 P1290T Asymptomatic 87 Ϯ 3 82 106 92 Ϯ 10 86 Ϯ 6 K1302Q - 72 Ϯ 3 77 100 37 Ϯ 2 37 Ϯ 2 Y1307N - 82 Ϯ 2 76 98 70 Ϯ 5 71 Ϯ 3 Q1309K - 79 Ϯ 4 77 100 26 Ϯ 2 26 Ϯ 3 S1311K - 73 Ϯ 4 72 93 33 Ϯ 7 35 Ϯ 5 R1325K - 64 Ϯ 6 78 101 47 Ϯ 2 46 Ϯ 4 V1338T - 88 Ϯ 2 77 100 37 Ϯ 11 37 Ϯ 6 C1344Y - 71 Ϯ 4 76 98 86 Ϯ 4 87 Ϯ 4 L1367I - 72 Ϯ 5 80 103 36 Ϯ 5 34 Ϯ 5 D1394G - 78 Ϯ 4 86 111 93 Ϯ 12 83 Ϯ 8 E1409D - 70 Ϯ 3 70 90 43 Ϯ 5 47 Ϯ 4 a Data from the CFTR mutation database. Login to comment
285 Among the 20 point mutants described in this study (the above 18 point mutants plus L581F and L581F/K584E), we found four that are listed in the CFTR mutation database (CFTR mutation database), namely E527Q, T1263I, and P1290T, which are described in association with mild CF, and I539T, which is described as an F508del-revertant mutation (Table 1). Login to comment
289 The most striking differences for the NBD1 mutants (Table 1 and Fig. 2, compare C with D) were (Iodide efflux to processed protein (%) far right column) E527Q (64/46), I539T (112/49), and L581F (118/72), whereas for the NBD2 mutants, they were T1263I (75/31), K1302Q (72/37), Q1309K (79/26), S1311K (73/33), V1338T (88/37), L1367I (72/36), and E1409D (70/43). Login to comment
290 Curiously, the point mutant with the highest discrepancy was I539T, which rescues the trafficking defect of F508del-CFTR (43). Login to comment

[hide] The V510D suppressor mutation stabilizes DeltaF508... Biochemistry. 2010 Aug 3;49(30):6352-7. Loo TW, Bartlett MC, Clarke DM
The V510D suppressor mutation stabilizes DeltaF508-CFTR at the cell surface.
Biochemistry. 2010 Aug 3;49(30):6352-7., 2010-08-03 [PMID:20590134]

Abstract [show]
Deletion of Phe508 (DeltaF508) in the first nucleotide-binding domain (NBD1) of CFTR causes cystic fibrosis. The mutation severely reduces the stability and folding of the protein by disrupting interactions between NBD1 and the second transmembrane domain (TMD2). We found that replacement of Val510 with acidic residues (but not neutral or positive residues) promoted maturation of DeltaF508-CFTR with V510D more efficiently than V510E. Promotion of DeltaF508-CFTR maturation did not require NBD2 as introduction of V510D into a DeltaNBD2/DeltaF508-CFTR mutant restored maturation to levels similar to that of full-length protein. The V510D mutation increased the half-life of mature DeltaF508-CFTR at the cell surface by about 5-fold to resemble the half-life of wild-type CFTR. It was also observed that introduction of the V510R/R1070D mutations into DeltaF508-CFTR also promoted maturation whereas the V510D/R1070A mutations did not. We propose that the V510D mutation in NBD1 promotes maturation and stabilizes DeltaF508-CFTR at the cell surface through formation of a salt bridge with Arg1070 in TMD2.

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No. Sentence Comment
64 It was recently reported that rescue of ΔF508-CFTR byother suppressor mutations inNBD1(I539T,G550E,R553M, R555K) was drastically reduced in wild-type CFTR lacking NBD2 (ΔNBD2) (20). Login to comment
129 A similar effect was observed when the combination of four NBD1 suppressormutations(I539T,G550E,R553M,R555K) was introduced into ΔF508-CFTR (20). Login to comment

[hide] The cystic fibrosis-causing mutation deltaF508 aff... J Biol Chem. 2010 Nov 12;285(46):35825-35. Epub 2010 Jul 28. Thibodeau PH, Richardson JM 3rd, Wang W, Millen L, Watson J, Mendoza JL, Du K, Fischman S, Senderowitz H, Lukacs GL, Kirk K, Thomas PJ
The cystic fibrosis-causing mutation deltaF508 affects multiple steps in cystic fibrosis transmembrane conductance regulator biogenesis.
J Biol Chem. 2010 Nov 12;285(46):35825-35. Epub 2010 Jul 28., 2010-11-12 [PMID:20667826]

Abstract [show]
The deletion of phenylalanine 508 in the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator is directly associated with >90% of cystic fibrosis cases. This mutant protein fails to traffic out of the endoplasmic reticulum and is subsequently degraded by the proteasome. The effects of this mutation may be partially reversed by the application of exogenous osmolytes, expression at low temperature, and the introduction of second site suppressor mutations. However, the specific steps of folding and assembly of full-length cystic fibrosis transmembrane conductance regulator (CFTR) directly altered by the disease-causing mutation are unclear. To elucidate the effects of the DeltaF508 mutation, on various steps in CFTR folding, a series of misfolding and suppressor mutations in the nucleotide binding and transmembrane domains were evaluated for effects on the folding and maturation of the protein. The results indicate that the isolated NBD1 responds to both the DeltaF508 mutation and intradomain suppressors of this mutation. In addition, identification of a novel second site suppressor of the defect within the second transmembrane domain suggests that DeltaF508 also effects interdomain interactions critical for later steps in the biosynthesis of CFTR.

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20 Subsequently, in a screen for suppressor mutations of the ⌬F508 defect, the original R553Q suppressor mutation was identified as were I539T, * Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealth NIDDK Grants 49835 (to P. J. T.) and 75302 (to G. L. L.). Login to comment

[hide] Intragenic suppressing mutations correct the foldi... J Biol Chem. 2010 Nov 19;285(47):36304-14. Epub 2010 Sep 13. Pagant S, Halliday JJ, Kougentakis C, Miller EA
Intragenic suppressing mutations correct the folding and intracellular traffic of misfolded mutants of Yor1p, a eukaryotic drug transporter.
J Biol Chem. 2010 Nov 19;285(47):36304-14. Epub 2010 Sep 13., 2010-11-19 [PMID:20837481]

Abstract [show]
ATP-binding cassette (ABC) transporters play pivotal physiological roles in substrate transport across membranes, and defective assembly of these proteins can cause severe disease associated with improper drug or ion flux. The yeast protein Yor1p is a useful model to study the biogenesis of ABC transporters; deletion of a phenylalanine residue in the first nucleotide-binding domain (NBD1) causes misassembly and retention in the endoplasmic reticulum (ER) of the resulting protein Yor1p-DeltaF670, similar to the predominant disease-causing allele in humans, CFTR-DeltaF508. Here we describe two novel Yor1p mutants, G278R and I1084P, which fail to assemble and traffic similar to Yor1p-DeltaF670. These mutations are located in the two intracellular loops (ICLs) that interface directly with NBD1, and thus disrupt a functionally important structural module. We isolated 2 second-site mutations, F270S and R1168M, which partially correct the folding injuries associated with the G278R, I1084P, and DeltaF670 mutants and reinstate their trafficking. The position of both corrective mutations at the cytoplasmic face of a transmembrane helix suggests that they restore biogenesis by influencing the behavior of the transmembrane domains rather than by direct restoration of the ICL1-ICL4-NBD1 structural module. Given the conserved topology of many ABC transporters, our findings provide new understanding of functionally important inter-domain interactions and suggest new potential avenues for correcting folding defects caused by abrogation of those domain interfaces.

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249 Remarkably, all suppressing mutations identified (I539T, G550E, R553M, and R555K) by this study are located within the NBD1 domain itself. Login to comment

[hide] The primary folding defect and rescue of DeltaF508... PLoS One. 2010 Nov 30;5(11):e15458. Hoelen H, Kleizen B, Schmidt A, Richardson J, Charitou P, Thomas PJ, Braakman I
The primary folding defect and rescue of DeltaF508 CFTR emerge during translation of the mutant domain.
PLoS One. 2010 Nov 30;5(11):e15458., [PMID:21152102]

Abstract [show]
In the vast majority of cystic fibrosis (CF) patients, deletion of residue F508 from CFTR is the cause of disease. F508 resides in the first nucleotide binding domain (NBD1) and its absence leads to CFTR misfolding and degradation. We show here that the primary folding defect arises during synthesis, as soon as NBD1 is translated. Introduction of either the I539T or G550E suppressor mutation in NBD1 partially rescues DeltaF508 CFTR to the cell surface, but only I539T repaired DeltaF508 NBD1. We demonstrated rescue of folding and stability of NBD1 from full-length DeltaF508 CFTR expressed in cells to isolated purified domain. The co-translational rescue of DeltaF508 NBD1 misfolding in CFTR by I539T advocates this domain as the most important drug target for cystic fibrosis.

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3 Introduction of either the I539T or G550E suppressor mutation in NBD1 partially rescues DF508 CFTR to the cell surface, but only I539T repaired DF508 NBD1. Login to comment
5 The co-translational rescue of DF508 NBD1 misfolding in CFTR by I539T advocates this domain as the most important drug target for cystic fibrosis. Login to comment
22 Teem and coworkers [19] identified two mutations, G550E and I539T, that both significantly increased plasma membrane levels of DF508 CFTR and improved channel activity [19,20,21]. Login to comment
26 Introduction of I539T, but not the G550E suppressor mutation, counteracted all folding defects within NBD1, whereas both mutations rescue CFTR trafficking to the cell surface. Login to comment
101 Only I539T but not G550E suppresses the DF508 phenotype in NBD1 As the folding defect in DF508 CFTR arose in NBD1, we asked whether this defect could be rescued in NBD1 as well. Login to comment
102 Teem and colleagues identified two suppressor mutations (G550E, I539T) in NBD1 that were located in the same subdomain as F508 (Figure 3B), and that each partially rescued DF508 CFTR from the ER [19]. Login to comment
103 We therefore examined whether the I539T mutation stabilized purified DF508 NBD1 (Figure 3A), and found that I539T completely rescued thermal stability of DF508 NBD1, and improved stability of wild-type NBD1 as well. Login to comment
105 Indeed, thermal stability curves of mouse NBD1s were indistinguishable from those of the corresponding human I539T NBD1s (Figure 3A). Login to comment
106 To establish whether the in vitro suppression of DF508 by I539T leads to improved in vivo stability of NBD1, we expressed wild-type and DF508 NBD1 with or without suppressor mutation I539T in CHO cells, and measured each protein`s half-life (Figure 2E). Login to comment
108 The two mutations exerted very different effects in that G550E did not affect stability of wild-type or DF508 NBD1, while I539T measurably stabilized the domain (Figure 3D). Login to comment
109 The amount of DF508 NBD1 remaining after 4 hours of chase improved from 10% to ,60% by insertion of I539T, whereas wild-type NBD1 improved from 30% to ,60%, consistent with the improved melting temperatures we measured. Login to comment
110 We concluded that the I539T suppressor mutation rescues the thermodynamic and biological stability of DF508 NBD1. Login to comment
111 I539T but not G550E fully restores the conformational defect in DF508 NBD1 To establish whether the improved stability of the I539T mutant was due to rescued conformation, we in vitro translated wild-type and DF508 NBD1 with or without suppressor mutations and monitored changes in proteolytic digestion (Figure 4A). Login to comment
112 Again, I539T, but not G550E, caused a dramatic effect on the proteinase K digest, particularly detectable at 5 mg/ml. Login to comment
113 Notably, the I539T mutation restored the wild-type NBD1 pattern in DF508: both protease resistant bands of ,25 and 27 kDa that had been lost in DF508 NBD1 returned upon mutation of I539 to Threonine (Figure 4A, b). Login to comment
114 Comparing longer exposures of the 100 mg/ml ProtK treatment of the NBD1 molecules revealed that only the I539T mutation restored the ,17 kDa protease resistant band that had been lost in DF508 NBD1, whereas G550E had no measurable impact (Figure 4B, N). Login to comment
115 Performing similar limited proteolysis experiments on purified DF508 NBD1 with or without the I539T mutation confirmed that this suppressor mutation specifically restored protease resistance of the 25 and 17 kDa fragments (data not shown). Login to comment
118 The I539T mutation itself slightly decreased electrophoretic mobility of NBD1 and CFTR (not shown), but also of the 25 kDa fragment, suggesting that this fragment contained the I539T mutation. Login to comment
119 We concluded that I539T but not G550E rescues the DF508 phenotype by completely restoring NBD1 conformation and stability. Login to comment
126 A similar experiment was done with I539T CFTR and DF508 I539T CFTR. Login to comment
127 Lane quantitation of the proteolytic fragments after 30 minutes of synthesis showed a protease resistant 25 kDa fragment for both wild-type and DF508 CFTR containing the additional I539T mutation (Figure 5C). Login to comment
128 These results show that the NBD1 conformation that leads to protection from limited proteolysis already formed co-translationally, and that DF508 CFTR was deficient in this process but was rescued by I539T during synthesis. Login to comment
129 Both I539T and G550E partially restore ''band C`` levels of DF508 CFTR. Login to comment
132 We found that only the I539T suppressor restored DF508 NBD1 domain stability suggesting that multiple mechanisms can contribute to rescue of DF508 CFTR. Login to comment
133 To analyze whether the I539T suppressor improved CFTR maturation like G550E [20,21] we used a pulse-chase approach to monitor both rate and efficiency of DF508 CFTR rescue. Login to comment
136 Introducing either G550E or I539T within DF508 CFTR partially countered misfolding and enhanced export from ER to Golgi (Figure 6). Login to comment
137 The I539T suppressor was much more effective than G550E in rescuing DF508 CFTR: the majority of CFTR molecules now reached the Golgi complex, whereas some loss of signal still occurred for G550E, implying some residual degradation. Login to comment
139 We conclude that, while both mutations rescue full-length CFTR to the plasma membrane, the I539T mutation rescues the DF508 phenotype within the isolated NBD1 domain already during its synthesis whereas G550E practically bypasses the folding defect in NBD1 and rescues via an alternative mechanism. Login to comment
143 The I539T suppressor mutation but not G550E in the same subdomain rescued the defect and restored NBD1 conformation to wild-type, Figure 3. Login to comment
144 Stability of DF508 NBD1 is restored by introducing I539T. Login to comment
162 Rescue of NBD1 conformation by the I539T suppressor mutation. Login to comment
163 (A) Wild-type and DF508 NBD1 (top panel) mRNAs containing the G550E (middle panel) or I539T (bottom panel) mutation were in vitro translated in the presence of 35 S-labeled methionine and cysteine and analyzed by 15% SDS-PAGE after proteinase K treatment. Login to comment
165 (B) Longer exposure of the 100 mg/ml proteinase K digest of in vitro translated NBD1, from same experiment as shown in B, showing the rescue of the 17 kDa band by the I539T but not by the G550E mutation. Login to comment
168 The arrowhead indicates the 25 kDa fragment, which has slightly decreased mobility when the I539T mutation is present. Login to comment
190 (C) Similar experimental conditions as in B but with the I539T mutation in wild-type and DF508 CFTR background. Login to comment
201 Both 25 kDa and 17 kDa fragments did show increased protease susceptibility in the DF508 background, which was rescued completely by the I539T suppressor mutation, not only in terms of protease susceptibility but also its in vivo stability and function. Login to comment
203 Reverting the DF508 phenotype While the I539T mutation rescued NBD1, G550E hardly affected the isolated DF508 NBD1 domain. Login to comment
214 The I539T mutation, by contrast, increases chloride channel activity at the plasma membrane (Eric J. Sorscher, Birmingham, personal communication) by increasing the number of CFTR molecules at the cell surface [19] (and Gergely Lukacs, Toronto, personal communication). Login to comment
216 The crystal structure of NBD1 and the structural model of CFTR provides no obvious hint towards the molecular mechanism of rescue by the I539T mutation. Login to comment
239 The deletion of F508 and the reverting mutations G550E and I539T were introduced both in full-length CFTR and NBD1 by side directed mutagenesis using primers (amino acid change underlined, I539T: 59-CCAAGTTTGCAGAGAAAGACAATACCG- TTCTTGGAGAAGGTGGAATC-39 G550E: 59-GGAGAAGG- TGGAATCACACTGAGTGAGGGTCAACGAGCAAGAATT- TCTTTAGC-39 DF508: 59-GGCACCATTAAAGAAAATATC- ATTGGTGTTTCCTATGATGAATATAG-39) and all constructs were sequence verified. Login to comment

[hide] Biochemical and biophysical approaches to probe CF... Methods Mol Biol. 2011;741:365-76. Schmidt A, Mendoza JL, Thomas PJ
Biochemical and biophysical approaches to probe CFTR structure.
Methods Mol Biol. 2011;741:365-76., [PMID:21594797]

Abstract [show]
The cystic fibrosis transmembrane regulator (CFTR) is a multi-domain integral membrane protein central to epithelial fluid secretion (see Chapter 21). Its activity is defective in the recessive genetic disease cystic fibrosis (CF). The most common CF-causing mutation is F508del in the first nucleotide binding domain (NBD1) of CFTR. This mutation is found on at least one allele of more than 90% of all CF patients. It is known to interfere with the trafficking/maturation of CFTR through the secretory pathway, leading to a loss-of-function at the plasma membrane. Notably, correction of the trafficking defect by addition of intragenic second-site suppressor mutations, or the alteration of bulk solvent conditions, such as by reducing the temperature or adding osmolytes, leads to appearance of functional channels at the membrane--thus, the rescued F508del-CFTR retains measurable function. High-resolution structural models of NBD1 from X-ray crystallographic data indicate that F508 is exposed on the surface of the domain in a position predicted by homologous ABC transporter structures to lie at the interface with the intracellular loops (ICLs) connecting the transmembrane spans. Determining the relative impact of the F508del mutation directly on NBD1 folding or on steps of domain assembly or both domain folding and assembly requires methods for evaluating the structure and stability of the isolated domain.

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30 Subsequently, in a screen for suppressor mutations of the F508del defect, the original R553Q suppressor mutation was identified as were I539T, G550E, R553Q, and R555K (18, 19). Login to comment

[hide] The W232R suppressor mutation promotes maturation ... Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11. Loo TW, Bartlett MC, Clarke DM
The W232R suppressor mutation promotes maturation of a truncation mutant lacking both nucleotide-binding domains and restores interdomain assembly and activity of P-glycoprotein processing mutants.
Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11., 2011-02-08 [PMID:21182301]

Abstract [show]
ATP-binding cassette (ABC) proteins contain two nucleotide-binding domains (NBDs) and two transmembrane (TM) domains (TMDs). Interdomain interactions and packing of the TM segments are critical for function, and disruption by genetic mutations contributes to disease. P-glycoprotein (P-gp) is a useful model to identify mechanisms that repair processing defects because numerous arginine suppressor mutations have been identified in the TM segments. Here, we tested the prediction that a mechanism of arginine rescue was to promote intradomain interactions between TM segments and restore interdomain assembly. We found that suppressor W232R(TM4/TMD1) rescued mutants with processing mutations in any domain and restored defective NBD1-NBD2, NBD1-TMD2, and TMD1-TMD2 interactions. W232R also promoted packing of the TM segments because it rescued a truncation mutant lacking both NBDs. The mechanism of W232R rescue likely involved intradomain hydrogen bond interactions with Asn296(TM5) since only N296A abolished rescue by W232R and rescue was only observed when Trp232 was replaced with hydrogen-bonding residues. In TMD2, suppressor T945R(TM11) also promoted packing of the TM segments because it rescued the truncation mutant lacking the NBDs and suppressed formation of alternative topologies. We propose that T945R rescue was mediated by interactions with Glu875(TM10) since T945E/E875R promoted maturation while T945R/E875A did not.

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No. Sentence Comment
122 In a recent study of four of the CFTR suppressor mutations located in NBD1 (I539T, G550E, R553M, and R555K), it was found that they only restored maturation of mutants that had processing mutations in NBD1 but not those that had processing mutations in other domains such as NBD2 (N1303K) or TMD2 (L1065P or R1066C) (66). Login to comment
329 It appears that the ΔF508 mutation inhibits folding of NBD1 and its ability to stably associate with other domains resulting in altered CFTR-chaperone interactions, ER retention,andenhanceddegradation(65).Second-sitesuppressor mutations in NBD1 (such as I539T/G550E/R553M/R555K) can restore interdomain assembly (65, 66) to yield a more stable ΔF508-CFTR molecule (64, 66). Login to comment

[hide] Thermal instability of DeltaF508 cystic fibrosis t... Biochemistry. 2012 Jun 26;51(25):5113-24. Epub 2012 Jun 15. Liu X, O'Donnell N, Landstrom A, Skach WR, Dawson DC
Thermal instability of DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) channel function: protection by single suppressor mutations and inhibiting channel activity.
Biochemistry. 2012 Jun 26;51(25):5113-24. Epub 2012 Jun 15., [PMID:22680785]

Abstract [show]
Deletion of Phe508 from cystic fibrosis transmembrane conductance regulator (CFTR) results in a temperature-sensitive folding defect that impairs protein maturation and chloride channel function. Both of these adverse effects, however, can be mitigated to varying extents by second-site suppressor mutations. To better understand the impact of second-site mutations on channel function, we compared the thermal sensitivity of CFTR channels in Xenopus oocytes. CFTR-mediated conductance of oocytes expressing wt or DeltaF508 CFTR was stable at 22 degrees C and increased at 28 degrees C, a temperature permissive for DeltaF508 CFTR expression in mammalian cells. At 37 degrees C, however, CFTR-mediated conductance was further enhanced, whereas that due to DeltaF508 CFTR channels decreased rapidly toward background, a phenomenon referred to here as "thermal inactivation." Thermal inactivation of DeltaF508 was mitigated by each of five suppressor mutations, I539T, R553M, G550E, R555K, and R1070W, but each exerted unique effects on the severity of, and recovery from, thermal inactivation. Another mutation, K1250A, known to increase open probability (P(o)) of DeltaF508 CFTR channels, exacerbated thermal inactivation. Application of potentiators known to increase P(o) of DeltaF508 CFTR channels at room temperature failed to protect channels from inactivation at 37 degrees C and one, PG-01, actually exacerbated thermal inactivation. Unstimulated DeltaF508CFTR channels or those inhibited by CFTR(inh)-172 were partially protected from thermal inactivation, suggesting a possible inverse relationship between thermal stability and gating transitions. Thermal stability of channel function and temperature-sensitive maturation of the mutant protein appear to reflect related, but distinct facets of the DeltaF508 CFTR conformational defect, both of which must be addressed by effective therapeutic modalities.

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No. Sentence Comment
5 Thermal inactivation of ΔF508 was mitigated by each of five suppressor mutations, I539T, R553M, G550E, R555K, and R1070W, but each exerted unique effects on the severity of, and recovery from, thermal inactivation. Login to comment
13 Consistent with this hypothesis, low-temperature rescued ΔF508 CFTR channels exposed to 37 °C exhibit a markedly reduced metabolic half-life (t1/2 < 4 h versus t1/2 > 24 h for wt CFTR14-17,21 ) and rapid thermal inactivation of chloride channel function.5,22 ΔF508 CFTR folding defects can also be suppressed to varying degrees by a variety of second-site mutations in NBD1.4,8,18,23-30 I539T, occurring naturally in many CFTR orthologs, improved the maturation of ΔF508 CFTR at 37 °C,4,25,30 but actually reduced open probability (Po) determined in detached patches.9 Another, R555K, modestly improved protein processing but also increased Po of ΔF508 CFTR channels in detached patches. Login to comment
18 We identified unique functional signatures for five second-site mutations, four in NBD1 (I539T, G550E, R553M, and R555K) and one in the fourth intracellular loop (ICL4, R1070W), and also investigated the relation of thermal stability to variations in channel gating brought about by intracellular cAMP, CFTR potentiators, and CFTR inhibitors. Login to comment
19 Consistent with previous studies, ΔF508 CFTR-mediated conductance, rescued by incubating oocytes at room temperature, decreased rapidly at 37 °C.5,22 When ΔF508 CFTR was expressed in the context of single, second site mutations, however, results ranged from complete protection from thermal inactivation at 37 °C (R553M) to profound inactivation that was fully reversed upon returning the bath to room temperature (I539T). Login to comment
141 (D) I539T/ΔF508 CFTR (n = 5). Login to comment
144 ΔF508 CFTR and I539T/ΔF508 CFTR, but in both cases the conductance decrease at 37 °C was followed by complete recovery at 22 °C. Login to comment
153 After two exposures to the elevated temperature, recovery, although evident, was much slower than that seen with G550E/ΔF508 or I539T/ΔF508 CFTR. Login to comment
155 In contrast, combining ΔF508 with R1070W and I539T resulted in channels that could not sustain the elevated conductance seen immediately after warming to 37 °C but were nevertheless able to sustain a substantial conductance at 37 °C (Figure 7C). Login to comment
171 (C) Following stimulation, an oocyte expressing I539T/R1070W/ΔF508 CFTR (n = 4) was warmed to 37 °C for 10 min twice. After cooling to 22 °C, the oocyte was exposed to 10 μM CF172. Login to comment
248 In contrast, I539T, which also slightly improved ΔF508 CFTR maturation in mammalian cells, failed to protect ΔF508 CFTR channels from profound thermal inactivation at 37 °C, although the double mutant recovered fully when returned to room temperature. Login to comment
256 A fourth NBD1 suppressor mutation, I539T, in contrast to G550E, R553M, and R555K, is predicted to lie within an unstructured linker connecting two α-helical portions of NBD1. Login to comment
259 Recovery from partial inactivation was also seen with R555K and G550E, but unlike I539T, both of these second-site mutations also resulted in persistent, steady-state conductance at 37 °C. Login to comment
260 The basis for this difference may be apparent in the gating behavior of I539T/ΔF508 channels. Login to comment
261 Recently, Dong et al.9 reported that the open probability of I539T/ΔF508 channels, studied at room temperature in patches detached from HeLa cells, was only about 20% of that of ΔF508 channels, due primarily to prolonged interburst intervals. Login to comment
262 This result indicates that I539T, although it appears to significantly improve the folding of ΔF508 NBD1 in a cell-based assay,6 actually further reduces open probability of the double mutant channel, even at room temperature. Login to comment
263 This observation is consistent with the previous report of DeCarvalho et al.25 that I539T/ΔF508, although it exhibited somewhat improved protein processing, was actually inferior to ΔF508 CFTR in a forskolin dose-response assay. Login to comment
268 Mendoza et al.6 also reported that combining R1070W with I539T, which alone increased the cellular yield of NBD1 to about 80% of wt, resulted in a yield of the I539T/R1070W/ ΔF508 protein that was 76% of the wt level. Login to comment
269 We found that these channels, although apparently more thermostable that ΔF508 CFTR, nevertheless failed to exhibit full, wt-like thermostability of channel function, a result that is consistent with the adverse effect of the I539T second-site mutation on open probability.9 Channel Gating and Functional Stability of ΔF508 CFTR. Login to comment
277 In addition, the slowing of the time course of stimulation of ΔF508 CFTR channels (lacking any second-site mutation) following a pre-stimulation thermal challenge is consistent with a model in which, at 37 °C, unstimulated ΔF508 CFTR channels that are rarely if ever transitioning to an open state, can nevertheless be driven into an inactivated state, but like I539T/ΔF508 CFTR, one from which they can recover spontaneously under stimulating conditions at 22 °C. Login to comment

[hide] Allosteric modulation balances thermodynamic stabi... J Mol Biol. 2012 May 25;419(1-2):41-60. Epub 2012 Mar 8. Aleksandrov AA, Kota P, Cui L, Jensen T, Alekseev AE, Reyes S, He L, Gentzsch M, Aleksandrov LA, Dokholyan NV, Riordan JR
Allosteric modulation balances thermodynamic stability and restores function of DeltaF508 CFTR.
J Mol Biol. 2012 May 25;419(1-2):41-60. Epub 2012 Mar 8., [PMID:22406676]

Abstract [show]
Most cystic fibrosis is caused by a deletion of a single residue (F508) in CFTR (cystic fibrosis transmembrane conductance regulator) that disrupts the folding and biosynthetic maturation of the ion channel protein. Progress towards understanding the underlying mechanisms and overcoming the defect remains incomplete. Here, we show that the thermal instability of human DeltaF508 CFTR channel activity evident in both cell-attached membrane patches and planar phospholipid bilayers is not observed in corresponding mutant CFTRs of several non-mammalian species. These more stable orthologs are distinguished from their mammalian counterparts by the substitution of proline residues at several key dynamic locations in first N-terminal nucleotide-binding domain (NBD1), including the structurally diverse region, the gamma-phosphate switch loop, and the regulatory insertion. Molecular dynamics analyses revealed that addition of the prolines could reduce flexibility at these locations and increase the temperatures of unfolding transitions of DeltaF508 NBD1 to that of the wild type. Introduction of these prolines experimentally into full-length human DeltaF508 CFTR together with the already recognized I539T suppressor mutation, also in the structurally diverse region, restored channel function and thermodynamic stability as well as its trafficking to and lifetime at the cell surface. Thus, while cellular manipulations that circumvent its culling by quality control systems leave DeltaF508 CFTR dysfunctional at physiological temperature, restoration of the delicate balance between the dynamic protein's inherent stability and channel activity returns a near-normal state.

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5 Introduction of these prolines experimentally into full-length human ΔF508 CFTR together with the already recognized I539T suppressor mutation, also in the structurally diverse region, restored channel function and thermodynamic stability as well as its trafficking to and lifetime at the *Corresponding author. Login to comment
40 This low-level maturation of rabbit ΔF508 CFTR may reflect primarily the I539T substitution (see below), Fig. 2. Login to comment
104 Among the differences, in addition to the I539T substitution present in many non-human species, we noted a pattern where proline residues replaced other residues in the maturing compared to the non- maturing species (Fig. 5a). Login to comment
108 Since chicken CFTR appeared least influenced by the F508 deletion among the species studied, we tested the effect of its four proline substitutions on human ΔF508 CFTR with and without the I539T mutation. Login to comment
110 However, the impact of the I539T substitution was much more dramatic when combined with proline replacements at the four positions at which they normally occur in the chicken sequence. Login to comment
111 Although introduction of all four prolines in the absence of I539T does not promote the maturation of ΔF508 human protein, they have a major effect in its presence, increasing maturation to near the WT level (Fig. 5c). Login to comment
112 Combination of introduction of a proline into the Q-loop at position 492 together with I539T to generate ΔF/PT was sufficient to allow a high level of maturation even without further addition of the substitutions in the I539 loop (A534P) to generate ΔF/2PT or also in the RI (S422P and S434P) to generate ΔF/4PT. Login to comment
115 Figure 5d shows that the immature forms of all these variants decayed at similar rates (upper graph), consistent with earlier studies of WT and mutant human CFTR.24 However, formation of the mature species was least with I539T alone (ΔF/T) and increased incrementally with inclusion of one, two, and four proline substitutions as judged by the amounts formed by 2 and 4 h (lower graph). Login to comment
120 With just the I539T (ΔF/T) substitution, a T1/2 of ~6 h was observed. Login to comment
122 Mimicking chicken CFTR (prolines and I539T) restores maturation and lifetime of ΔF508 CFTR. Login to comment
125 The common I539T replacement is also indicated. Login to comment
128 (c) Western blot of WT and ΔF508 CFTR expressed in HEK-293 cells and ΔF508 modified with I539T (ΔF/T), S422P/S434P/S492P/A534P (ΔF/4P), I539T/S492P (ΔF/PT), I539T/S492P/A534P (ΔF/2PT), and I539T/S422P/S434P/S492P/A534P (ΔF/4PT). Login to comment
136 Thermostable binding of the nucleotide by the WT, which is lost in ΔF508 CFTR,19 was not restored after rescue in cells grown at 27 °C [(r)ΔF panel], but was to some extent by I539T (ΔF/T panel), to a greater extent when S492P was added (ΔF/PT panel), still further with A534P also added (ΔF/2PT panel), and to near the WT level with proline replacements at residues S492 and A534 as well (ΔF/4PT panel). Login to comment
139 At a fixed sub-physiological temperature (35 °C), the I539T/ΔF508 protein exhibited predominantly a fast flickering mode (ffm) with only rare full-conductance channel openings (Fig. 6a). Login to comment
151 Thus, both the constant and varying temperature experiments demonstrated a requirement for a balance between thermal stability and channel activity in CFTR, consistent with considerations of such a relationship between stability and catalytic function of proteins in general.28 NBD1 stabilization restores an NBD1-CL4 interface In addition to destabilizing NBD1, deletion of F508 also disrupts interdomain contacts including the interface between the NBD1 surface and the cytoplasmic loop (CL)4 in MSD2 in which the residue normally participates.29 Specific second-site mutations on either side of this interface (e.g., R1070W or V510D) have been shown to promote maturation of ΔF508 CFTR.27,30,31 The current observations that the ΔF508 protein with NBD1 strongly stabilized by the proline and I539T substitutions had channel activity similar to the WT at physiological temperature suggested either that the NBD1-CL4 interface is not important for function or that it is adequately restored by NBD1 stabilization. Login to comment
176 Both the RI and the SDR have been hypothesized to influence quaternary assembly of NBD1 with the rest of CFTR.29,33 Strikingly, substitution of S492 to proline in the context of the I539T mutation Fig. 7. Login to comment
177 Restoration of the ΔF508 CFTR NBD1-CL4 interface by proline and I539T mutations. Login to comment
178 HEK293 cells were transiently transfected with Cys-less CFTR or Cys-less ΔF508-CFTR in the presence or absence of the 4PT mutations (S422P/S434P/S492P/A534P/I539T), with the Cys pair V510C/G1069C introduced at the CL4/NBD1 interface. Login to comment
182 Avian ΔF509 CFTR is destabilized by reversal of proline and I539T substitutions. Login to comment
188 Such stabilization of the SDR was not observed in simulations with the I539T substitution alone (Fig. 9a). Login to comment
195 Thus, while I539T alone did not appear to significantly improve the stability of ΔF508 NBD1, stabilization of the SDR via incorporation of prolines at different positions increased the stability of ΔF508 NBD1. Login to comment
216 Introduction of a proline at S492 in the context of I539T increases the folding transition temperature of ΔF508 NBD1 to ~327 K, consistent with the observed decrease in thermal fluctuations upon S492P substitution (Fig. 9b, orange broken line). Login to comment
237 A striking feature of the strong stabilizing effect of the proline substitutions was the essentially absolute dependence on the I539T substitution. This dependence contrasts the positive effects on ΔF508 CFTR maturation of other second site changes that are not wholly dependent on I539 T, such as those near the NBD1 signature sequence (G550E/R553M/R555K) and the RI. Login to comment
238 I539T has an additive effect with the 550/ 553/555 set.33 The I539T substitution alone promotes a low level of human ΔF508 CFTR maturation, and its normal presence in murine CFTR probably contributes to the partial maturation of the mouse ΔF508 variant15,21 as well as that of the rabbit (Fig. 2a). Login to comment

[hide] Human-mouse cystic fibrosis transmembrane conducta... Proc Natl Acad Sci U S A. 2012 Jan 17;109(3):917-22. Epub 2011 Dec 30. Dong Q, Ostedgaard LS, Rogers C, Vermeer DW, Zhang Y, Welsh MJ
Human-mouse cystic fibrosis transmembrane conductance regulator (CFTR) chimeras identify regions that partially rescue CFTR-DeltaF508 processing and alter its gating defect.
Proc Natl Acad Sci U S A. 2012 Jan 17;109(3):917-22. Epub 2011 Dec 30., [PMID:22210114]

Abstract [show]
The DeltaF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is the most common cause of cystic fibrosis. The mutation disrupts biosynthetic processing, reduces channel opening rate, and decreases protein lifetime. In contrast to human CFTR (hCFTR)-DeltaF508, mouse CFTR-DeltaF508 is partially processed to the cell surface, although it exhibits a functional defect similar to hCFTR-DeltaF508. To explore DeltaF508 abnormalities, we generated human-mouse chimeric channels. Substituting mouse nucleotide-binding domain-1 (mNBD1) into hCFTR partially rescued the DeltaF508-induced maturation defect, and substituting mouse membrane-spanning domain-2 or its intracellular loops (ICLs) into hCFTR prevented further DeltaF508-induced gating defects. The protective effect of the mouse ICLs was reverted by inserting mouse NBDs. Our results indicate that the DeltaF508 mutation affects maturation and gating via distinct regions of the protein; maturation of CFTR-DeltaF508 depends on NBD1, and the DeltaF508-induced gating defect depends on the interaction between the membrane-spanning domain-2 ICLs and the NBDs. These appear to be distinct processes, because none of the chimeras repaired both defects. This distinction was exemplified by the I539T mutation, which improved CFTR-DeltaF508 processing but worsened the gating defect. Our results, together with previous studies, suggest that many different NBD1 modifications improve CFTR-DeltaF508 maturation and that the effect of modifications can be additive. Thus, it might be possible to enhance processing by targeting several different regions of the domain or by targeting a network of CFTR-associated proteins. Because no one modification corrected both maturation and gating, perhaps more than a single agent will be required to correct all CFTR-DeltaF508 defects.

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8 This distinction was exemplified by the I539T mutation, which improved CFTR- ΔF508 processing but worsened the gating defect. Login to comment
52 Previous reports indicated that an I539T mutation partially improved hCFTR-ΔF508 processing (18-20). Login to comment
54 In addition, we found that combining I539T with human-mouse RE (hmRE) caused hCFTR-ΔF508 to produce more band C than either substitution alone. Login to comment
55 Moreover, the proportion of band C in the chimera containing hmRE plus I539T was similar to that obtained when the entire mNBD1- ΔF508 replaced the human domain. Login to comment
62 Of all the chimeras, only hmMSD2 prevented B C D * * * * ** † C B Wild-type ΔF508 mCFTR hmNBD1 hCFTR hmNBD2 hmMSD2 hmMSD1 hmRI1 hmRI2 hmCenter hmRE hmRI1/RE I539T hmRE/I539T A Chimeras hCFTR Boundary hmNBD1 389-671 hmNBD2 1169-1480 hmMSD1 58-388 hmMSD2 837-1177 hmRI1 389-432 hmRI2 404-436 hmRE 633-671 hmRI1/RE 389-432, 633-671 hmCenter 434-632 WT ΔF WT ΔF WT ΔF WT ΔF WT ΔF WT ΔF WT ΔF WT ΔF WT ΔF WT ΔF WT ΔF WT ΔF WT ΔF Fig. 1. Login to comment
74 † Difference in hmRE/I539T compared with hmRE and I539T (P < 0.05) (hCFTR, n = 15; mCFTR, n = 13; hmNBD1, n = 11; hmNBD2, n = 6; hmMSD1, n = 3; hmMSD2, n = 7; hmRI1, n = 3; hmRI2, n = 2; hmRE, n = 7; hmRI1/RE, n = 5; human-mouse Center, n = 6; hI539T, n = 4; hmRE/I539T, n = 4). Login to comment
77 Interestingly, the I539T mutation, which minimized the effect of ΔF508 on processing, actually accentuated the ΔF508-induced gating defect (Fig. 2A). Login to comment
94 If that were the case, we rea- hmNBD1 hmRE hmRE/I539T hmRI1/RE mCFTR hCFTR I539T hmRI1 hmRI2 hmCenter Wild-type ΔF508 A C B hmNBD2 hmMSD1 hmMSD2 Fig. 2. Login to comment
117 However, mutating I539 to the mouse sequence also partially rescued processing, and the mouse RE and I539T were additive in their effects. Login to comment
120 (i) A genetic approach identified second-site suppressor mutations, including I539T, G550E, R553M/Q, and R555K (18-21, 25, 26). Login to comment
153 This distinction was exemplified by the I539T mutation. Login to comment
154 I539T improved processing; however, it not only failed to prevent the ΔF508 gating defect but actually further prolonged the interburst interval. Login to comment
178 First, finding that I539T enhanced ΔF508-CFTR processing but reduced its channel activity suggests that a drug screening strategy that only detects cell surface CFTR- ΔF508 might miss adverse consequences for channel function. Login to comment
181 In addition, finding that hmRE and I539T together improved CFTR- ΔF508 processing more than either alone suggests that simultaneously targeting more than one NBD1 site might be beneficial. Login to comment

[hide] A chaperone trap contributes to the onset of cysti... PLoS One. 2012;7(5):e37682. doi: 10.1371/journal.pone.0037682. Epub 2012 May 31. Coppinger JA, Hutt DM, Razvi A, Koulov AV, Pankow S, Yates JR 3rd, Balch WE
A chaperone trap contributes to the onset of cystic fibrosis.
PLoS One. 2012;7(5):e37682. doi: 10.1371/journal.pone.0037682. Epub 2012 May 31., [PMID:22701530]

Abstract [show]
Protein folding is the primary role of proteostasis network (PN) where chaperone interactions with client proteins determine the success or failure of the folding reaction in the cell. We now address how the Phe508 deletion in the NBD1 domain of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein responsible for cystic fibrosis (CF) impacts the binding of CFTR with cellular chaperones. We applied single ion reaction monitoring mass spectrometry (SRM-MS) to quantitatively characterize the stoichiometry of the heat shock proteins (Hsps) in CFTR folding intermediates in vivo and mapped the sites of interaction of the NBD1 domain of CFTR with Hsp90 in vitro. Unlike folding of WT-CFTR, we now demonstrate the presence of DeltaF508-CFTR in a stalled folding intermediate in stoichiometric association with the core Hsps 40, 70 and 90, referred to as a 'chaperone trap'. Culturing cells at 30 C resulted in correction of DeltaF508-CFTR trafficking and function, restoring the sub-stoichiometric association of core Hsps observed for WT-CFTR. These results support the interpretation that DeltaF508-CFTR is restricted to a chaperone-bound folding intermediate, a state that may contribute to its loss of trafficking and increased targeting for degradation. We propose that stalled folding intermediates could define a critical proteostasis pathway branch-point(s) responsible for the loss of function in misfolding diseases as observed in CF.

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147 This stable core structure is never achieved in DF508-NBD1, unless the I539T and/or other suppressor mutations are introduced [20,21,23,29,30]. Login to comment
148 Interestingly, peptide 4 spans residues 525-532 of NBD1, which places it in close proximity to the I539T mutation and within the stable core of the WT protein. Login to comment
149 This stable core structure is never achieved in DF508-NBD1, unless the I539T and/or other suppressor mutations are introduced [20,21,23,29,30]. Login to comment
150 Interestingly, peptide 4 spans residues 525-532 of NBD1, which places it in close proximity to the I539T mutation and within the stable core of the WT protein. Login to comment

[hide] Twenty years after cystic fibrosis gene identifica... Pathol Biol (Paris). 2011 Jun;59(3):131-3. Epub 2009 Nov 5. Edelman A, Fritsch J, Ollero M
Twenty years after cystic fibrosis gene identification: Where are we and what are we up to?
Pathol Biol (Paris). 2011 Jun;59(3):131-3. Epub 2009 Nov 5., [PMID:19896304]

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58 Moreover, it has been observed by mutagenesis followed by heterologous expression of CFTR, that replacing glycine at position 550 by a glutamic acid residue (G550E) or isoleucine 539 by threonine (I539T), in cis in F508del-NBD1 leads to the delivery of functional F508del-CFTR to the plasma membrane. Login to comment

[hide] Mild processing defect of porcine DeltaF508-CFTR s... Biochem Biophys Res Commun. 2008 Aug 15;373(1):113-8. Epub 2008 Jun 12. Liu Y, Wang Y, Jiang Y, Zhu N, Liang H, Xu L, Feng X, Yang H, Ma T
Mild processing defect of porcine DeltaF508-CFTR suggests that DeltaF508 pigs may not develop cystic fibrosis disease.
Biochem Biophys Res Commun. 2008 Aug 15;373(1):113-8. Epub 2008 Jun 12., [PMID:18555011]

Abstract [show]
Recent efforts have made significant progress in generating transgenic pigs with the DeltaF508-CFTR mutation to model the lung and pancreatic disease of human cystic fibrosis. However, species differences in the processing and function of human, pig and mouse DeltaF508-CFTR reported recently raise concerns about the phenotypic consequence of the gene-targeted pig model. The purpose of the present study was to characterize the DeltaF508 mutant of porcine CFTR to evaluate the severity of its processing defect. Biochemical and immunofluorescence analysis in transfected COS7 and FRT cells indicated that pig DeltaF508-CFTR efficiently targets to the plasma membrane and is present mainly as the mature glycosylated protein. Functional characterization in stably transfected FRT cells by fluorometric and electrophysiological assays supported efficient plasma membrane targeting of pig DeltaF508-CFTR. The mild cellular processing defect of pig DeltaF508-CFTR suggests that its gene-targeted pig model may not develop the lung and pancreatic phenotypes seen in CF patients.

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149 The identified revertant mutations I539T, G550E, and R555K each partially res- Fig. 4. Login to comment
148 The identified revertant mutations I539T, G550E, and R555K each partially res- Fig. 4. Login to comment

[hide] Requirements for efficient correction of DeltaF508... Cell. 2012 Jan 20;148(1-2):164-74. doi: 10.1016/j.cell.2011.11.023. Mendoza JL, Schmidt A, Li Q, Nuvaga E, Barrett T, Bridges RJ, Feranchak AP, Brautigam CA, Thomas PJ
Requirements for efficient correction of DeltaF508 CFTR revealed by analyses of evolved sequences.
Cell. 2012 Jan 20;148(1-2):164-74. doi: 10.1016/j.cell.2011.11.023., [PMID:22265409]

Abstract [show]
Misfolding of DeltaF508 cystic fibrosis (CF) transmembrane conductance regulator (CFTR) underlies pathology in most CF patients. F508 resides in the first nucleotide-binding domain (NBD1) of CFTR near a predicted interface with the fourth intracellular loop (ICL4). Efforts to identify small molecules that restore function by correcting the folding defect have revealed an apparent efficacy ceiling. To understand the mechanistic basis of this obstacle, positions statistically coupled to 508, in evolved sequences, were identified and assessed for their impact on both NBD1 and CFTR folding. The results indicate that both NBD1 folding and interaction with ICL4 are altered by the DeltaF508 mutation and that correction of either individual process is only partially effective. By contrast, combination of mutations that counteract both defects restores DeltaF508 maturation and function to wild-type levels. These results provide a mechanistic rationale for the limited efficacy of extant corrector compounds and suggest approaches for identifying compounds that correct both defective steps.

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18 Additional second-site revertant mutations I539T, G550E, R553M, and R555K, within the portion of CFTR NBD1 included in the chimera, were also identified (DeCarvalho et al., 2002; Teem et al., 1993, 1996). Login to comment
19 The R553M, I539T, and the combination of G550E-R553M-R555K (3M) mutations correct the folding and stability defects of the DF508 NBD1 domain in isolation (DeCarvalho et al., 2002; Hoelen et al., 2010; Pissarra et al., 2008; Qu et al., 1997; Thibodeau et al., 2010) but only partially restore maturation of the full-length mutant protein (Hoelen et al., 2010; Pissarra et al., 2008; Thibodeau et al., 2010). Login to comment
127 The surface view A B I539T G550E R553M R555K 3M WT F S466T L475Y F490L W496V Y517I C524A L526A D529F D537F Y563V A566P S573E P574A F575T E583G H609T 0 1 2 3 Relative Yield NBD1 ( -gal.) 25 30 35 40 45 0.0 0.5 1.0 Temperature (C ) Relative Turbitity 0 1 2 3 4 -5 0 5 10 WT F I539T I539T F S573E R555K D529F Relative Yield NBD1 ( -gal.) Tm Figure 3. Login to comment
158 Mirroring the maturation results, correction of either the NBD1 defect (I539T, R555K, red bars) or the ICL4-NBD1 defect (R1070W, white bar) alone provided only modest improvements of function. Login to comment
166 B C A B 0 1 2 3 0 1 2 Relative Yield NBD1 (b2;-gal.) Relative Yield CFTR (ELISA) WT ࢞F WT ƊF S466T L475Y F490L W496V Y517I C524A L526A D529F D537F Y563V A566P S573E P574A F575T E583G H609T 0 1 2 Relative Yield CFTR (ELISA) I539T G550E R553M R555K 3M Figure 4. Login to comment
172 See also Figure S5. (B) The influence of the 508-coupled mutations (green circles), four second-site suppressor mutations (I539T, G550E, R553M, and R555K) and three suppressors in combination (G550E, R553M, and R555K) (orange circles) on F508 background on relative maturation of full-length CFTR and relative NBD1 folding yield is correlated (green line, m = 0.75, R = 0.85). Login to comment
185 Previously identified second-site suppressor (I539T, G550E, R553M, R555K, and 3M) but not the 508-coupled mutants (D529F and S573E) increase the yield of DF508 NBD1. Login to comment
187 See also Table S2. (C) F508K, F508R, and F508K in combination with I539T, G550E, R553M, R555K, and 3M mutations increase folding yield of NBD1, but exhibit no corresponding increase in CFTR maturation yield (dark blue circles and line, m = 0.03, R = 0.40) (&#b1;SEM, n = 9 along x axis and n = 3 along y axis). Login to comment
219 When R1070W is combined with mutations that improve DF508 NBD1 folding yield, I539T, G550E, R553M, R555K, and 3M (open triangles), the correlation between NBD1 folding and CFTR maturation in the wild-type protein is restored (m = 0.77, R = 0.47, black line) (&#b1;SEM). Login to comment
224 A representative trace of the corrected mutant, DF508-I539T-R1070W CFTR (cyan triangles) is more like wild-type (filled circles) than DF508 (filled triangles) (inset). Login to comment

[hide] Functional Rescue of F508del-CFTR Using Small Mole... Front Pharmacol. 2012 Sep 26;3:160. doi: 10.3389/fphar.2012.00160. eCollection 2012. Molinski S, Eckford PD, Pasyk S, Ahmadi S, Chin S, Bear CE
Functional Rescue of F508del-CFTR Using Small Molecule Correctors.
Front Pharmacol. 2012 Sep 26;3:160. doi: 10.3389/fphar.2012.00160. eCollection 2012., [PMID:23055971]

Abstract [show]
High-throughput screens for small molecules that are effective in "correcting" the functional expression of F508del-CFTR have yielded several promising hits. Two such compounds are currently in clinical trial. Despite this success, it is clear that further advances will be required in order to restore 50% or greater of wild-type CFTR function to the airways of patients harboring the F508del-CFTR protein. Progress will be enhanced by our better understanding of the molecular and cellular defects caused by the F508del mutation, present in 90% of CF patients. The goal of this chapter is to review the current understanding of defects caused by F508del in the CFTR protein and in CFTR-mediated interactions important for its biosynthesis, trafficking, channel function, and stability at the cell surface. Finally, we will discuss the gaps in our knowledge regarding the mechanism of action of existing correctors, the unmet need to discover compounds which restore proper CFTR structure and function in CF affected tissues and new strategies for therapy development.

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60 Similarly, second site mutations in unique, flexible regions of NBD1 (i.e., I539T) partially correct the processing defect in F508del-CFTR. Login to comment
62 Employing biophysical methods, including circular dichroism, dynamic light scattering,and fluorescence,both groups confirmed that the introduction of "stabilizing mutations" residing in the ABC b1;-helical subdomain (G550E, R553M, R555K) and the structural diverse region (I539T), fully corrects defects in kinetic and thermal stability of the isolated F508del-NBD1 domain. Login to comment

[hide] Pharmacological Correctors of Mutant CFTR Mistraff... Front Pharmacol. 2012 Oct 5;3:175. doi: 10.3389/fphar.2012.00175. eCollection 2012. Pedemonte N, Galietta LJ
Pharmacological Correctors of Mutant CFTR Mistrafficking.
Front Pharmacol. 2012 Oct 5;3:175. doi: 10.3389/fphar.2012.00175. eCollection 2012., [PMID:23060795]

Abstract [show]
The lack of phenylalanine 508 (DeltaF508 mutation) in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl(-) channel represents the most frequent cause of CF, a genetic disease affecting multiple organs such as lung, pancreas, and liver. DeltaF508 causes instability and misfolding of CFTR protein leading to early degradation in the endoplasmic reticulum and accelerated removal from the plasma membrane. Pharmacological correctors of mutant CFTR protein have been identified by high-throughput screening of large chemical libraries, by in silico docking of virtual compounds on CFTR structure models, or by using compounds that affect the whole proteome (e.g., histone deacetylase inhibitors) or a single CFTR-interacting protein. The presence of multiple defects of the CFTR protein caused by the DeltaF508 mutation and the redundancy of quality control mechanisms detecting DeltaF508-CFTR as a defective protein impose a ceiling to the maximal effect that a single compound (corrector) may obtain. Therefore, treatment of patients with the most frequent CF mutation may require the optimized combination of two drugs having additive or synergic effects.

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144 The first type of mutation,such as I539T or R555K,increases the stability of NBD1. Login to comment

[hide] Correctors of DeltaF508 CFTR restore global confor... FASEB J. 2013 Feb;27(2):536-45. doi: 10.1096/fj.12-216119. Epub 2012 Oct 26. He L, Kota P, Aleksandrov AA, Cui L, Jensen T, Dokholyan NV, Riordan JR
Correctors of DeltaF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.
FASEB J. 2013 Feb;27(2):536-45. doi: 10.1096/fj.12-216119. Epub 2012 Oct 26., [PMID:23104983]

Abstract [show]
Most cystic fibrosis is caused by the deletion of a single amino acid (F508) from CFTR and the resulting misfolding and destabilization of the protein. Compounds identified by high-throughput screening to improve DeltaF508 CFTR maturation have already entered clinical trials, and it is important to understand their mechanisms of action to further improve their efficacy. Here, we showed that several of these compounds, including the investigational drug VX-809, caused a much greater increase (5- to 10-fold) in maturation at 27 than at 37 degrees C (<2-fold), and the mature product remained short-lived (T(1/2) approximately 4.5 h) and thermally unstable, even though its overall conformational state was similar to wild type, as judged by resistance to proteolysis and interdomain cross-linking. Consistent with its inability to restore thermodynamic stability, VX-809 stimulated maturation 2-5-fold beyond that caused by several different stabilizing modifications of NBD1 and the NBD1/CL4 interface. The compound also promoted maturation of several disease-associated processing mutants on the CL4 side of this interface. Although these effects may reflect an interaction of VX-809 with this interface, an interpretation supported by computational docking, it also rescued maturation of mutants in other cytoplasmic loops, either by allosteric effects or via additional sites of action. In addition to revealing the capabilities and some of the limitations of this important investigational drug, these findings clearly demonstrate that DeltaF508 CFTR can be completely assembled and evade cellular quality control systems, while remaining thermodynamically unstable. He, L., Kota, P., Aleksandrov, A. A., Cui, L., Jensen, T., Dokholyan, N. V., Riordan, J. R. Correctors of DeltaF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.

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43 Metabolic pulse chase BHK cells stably expressing èc;F508 CFTR with or without I539T were treated with VX-809 (3 òe;M; Vertex Pharmeceuti- cals, Cambridge, MA, USA) for 24 h at 27&#b0;C. Cells expressing wild-type (WT) CFTR were grown at 37&#b0;C. Long-term pulse-chase experiments were performed in the absence (WT CFTR) or the presence of VX-809 (èc;F508 and èc;F508/I539T CFTR) to follow the lifetime of mature CFTR (14). Login to comment
44 Briefly, BHK cells stably expressing WT CFTR, èc;F508 CFTR, and èc;F508/I539T CFTR were grown in 60-mm-diameter dishes and labeled for 8 h in 1.5 ml methionine-free medium supplemented with 10% normal growth medium, 10% FBS, and 66 òe;Ci/ml 35 S-methionine (Perkin-Elmer, Waltham, MA), at 27 or 37&#b0;C. Cells were then washed 2 times and chased at 37&#b0;C with growth medium supplemented with 10 mM unlabeled methionine. Login to comment
62 The transport capacity of the structural unit ᐹॹᐺ for èc;F508/I539T CFTR with unstable gating kinetics and variable conductive state at 35&#b0;C was estimated as total charge transported in 10 min (area under the trace), divided by the potential difference applied and normalized per second so as to be an exact analog of ॹPo used for the channels with stable and well-defined open state. Login to comment
77 Furthermore, even when the modestly effective second-site suppressor mutation, I539T (22, 23) also was present in VX-809-treated èc;F508-CFTR-ex- Figure 1. Login to comment
82 While these lifetimes as reflections of the stability of the protein in cells do not necessarily equate to the functional thermal stability of its ion channel activity, only very transient channel activity with low open probability was observed in èc;F508/I539T-CFTR-expressing cells treated with VX-809 (Fig. 2B). Login to comment
83 Thus, the shortened functional lifetime of the èc;F508-CFTR channel described previously (14, 25, 26) is not extended by the I539T substitution. Login to comment
84 We used the èc;F508/I539T- CFTR variant as it, in contrast to the èc;F508 CFTR (14), has reasonably stable functional activity at 25&#b0;C, although it inactivates at higher temperatures (24). Login to comment
86 For VX-809 treatment, the èc;F508/I539T CFTR variant was continuously exposed to the compound during cell growth, membrane vesicle isolation, and channel assay. Login to comment
88 This behavior strongly contrasted that of the èc;F508/I539T variant with the stabilizing S492P mutation added, where transport capacity increased, and full conductance state persisted up to 35&#b0;C (compare tracings in Fig. 2Biii, iv). Login to comment
92 We employed this assay to compare the folded state of èc;F508 CFTR that had matured under the influence of the strongly NBD1-stabilizing 4PT modification (prolines introduced at 4 mobile sites: S422, S434, S492, and A534 plus I539T); or exposure of cells to VX-809 at 27&#b0;C. Login to comment
96 A) BHK cells stably expressing èc;F508 CFTR with or without I539T were treated with VX-809 (3 òe;M) for 24 h at 27&#b0;C. Cells expressing WT CFTR were grown at 37&#b0;C. Long-term pulse-chase experiments were performed in the absence of VX-809 (WT CFTR) or presence of VX-809 (èc;F508 and èc;F508/I539T CFTR) to follow the lifetime of CFTR, as described in Materials and Methods. Login to comment
98 B) Single-channel recordings of èc;F508/I539T CFTR (èc;F/T) rescued by 3 òe;M VX-809 at 35&#b0;C (i) and 25&#b0;C (ii) and of èc;F508/I539T/S492P CFTR (èc;F/PT) as an example of an already known (24) alternative type of èc;F508/I539T, thermally stabilized by proline substitutions at 35&#b0;C (iii) and 25&#b0;C (iv). Login to comment
99 Six independent experiments of 38 min total time were used to estimate transport capacity ᐹॹᐺ afd; 1.46 afe; 0.28 for èc;F508/I539T at 25&#b0;C; data are shown as means afe; se. Login to comment
100 Five independent experiments of 34 min total time were used to estimate ᐹॹᐺ afd; 0.35 afe; 0.14 for èc;F508/I539T at 35&#b0;C. Login to comment
101 Two sets of 4 independent experiments of 35 and 38 min total time were used to estimate transport capacity ᐹॹᐺ afd; 0.85 afe; 0.26 at 25&#b0;C (iii) and ᐹॹᐺ afd; 3.14 afe; 0.32 at 35&#b0;C (iv) for èc;F508/I539T/S492P CFTR. Login to comment
122 4PT, S422P/S434P/S492P/ A534P/I539T. Login to comment
134 VX-809 treatment of the combined NBD1 signature suppressor mutations together with the I539T substitution (èc;F/4S) caused substantial further enhancement of maturation at both temperatures. Login to comment
135 With the strongly stabilizing regulatory insertion deletion (èc;RI), the compound caused large increments that were of equivalent magnitude at both temperatures, and a similar effect was observed with the proline insertions in the context of I539T variant (4PT). Login to comment
148 A) èc;F508 with NBD1-stabilizing mutations: 4S, I539T/G550E/R553M/R555K; èc;RI, deletion of amino acid residues 404-435; 4PT, S422P/S434P/S492P/A534P/I539T. Login to comment

[hide] Mechanisms of CFTR Folding at the Endoplasmic Reti... Front Pharmacol. 2012 Dec 13;3:201. doi: 10.3389/fphar.2012.00201. eCollection 2012. Kim SJ, Skach WR
Mechanisms of CFTR Folding at the Endoplasmic Reticulum.
Front Pharmacol. 2012 Dec 13;3:201. doi: 10.3389/fphar.2012.00201. eCollection 2012., [PMID:23248597]

Abstract [show]
In the past decade much has been learned about how Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) folds and misfolds as the etiologic cause of cystic fibrosis (CF). CFTR folding is complex and hierarchical, takes place in multiple cellular compartments and physical environments, and involves several large networks of folding machineries. Insertion of transmembrane (TM) segments into the endoplasmic reticulum (ER) membrane and tertiary folding of cytosolic domains begin cotranslationally as the nascent polypeptide emerges from the ribosome, whereas posttranslational folding establishes critical domain-domain contacts needed to form a physiologically stable structure. Within the membrane, N- and C-terminal TM helices are sorted into bundles that project from the cytosol to form docking sites for nucleotide binding domains, NBD1 and NBD2, which in turn form a sandwich dimer for ATP binding. While tertiary folding is required for domain assembly, proper domain assembly also reciprocally affects folding of individual domains analogous to a jig-saw puzzle wherein the structure of each interlocking piece influences its neighbors. Superimposed on this process is an elaborate proteostatic network of cellular chaperones and folding machineries that facilitate the timing and coordination of specific folding steps in and across the ER membrane. While the details of this process require further refinement, we finally have a useful framework to understand key folding defect(s) caused by DeltaF508 that provides a molecular target(s) for the next generation of CFTR small molecule correctors aimed at the specific defect present in the majority of CF patients.

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122 Mutations that increase NBD1 solubility and/or thermodynamic stability (I539T, G550E, R553Q, and others; Teem et al., 1993; DeCarvalho et al., 2002; Roxo-Rosa et al., 2006; Pissarra et al., 2008; Hoelen et al., 2010) and/or decrease backbone flexibility (Aleksandrov et al., 2012) can enhance both NBD1 folding yield in cells and trafficking efficiency of full length WT as well as ࢞F508 CFTR (Figure 3B). Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Cold Spring Harb Perspect Med. 2013 Feb 1;3(2):a009514. doi: 10.1101/cshperspect.a009514. Hunt JF, Wang C, Ford RC
Cystic fibrosis transmembrane conductance regulator (ABCC7) structure.
Cold Spring Harb Perspect Med. 2013 Feb 1;3(2):a009514. doi: 10.1101/cshperspect.a009514., [PMID:23378596]

Abstract [show]
Structural studies of the cystic fibrosis transmembrane conductance regulator (CFTR) are reviewed. Like many membrane proteins, full-length CFTR has proven to be difficult to express and purify, hence much of the structural data available is for the more tractable, independently expressed soluble domains. Therefore, this chapter covers structural data for individual CFTR domains in addition to the sparser data available for the full-length protein. To set the context for these studies, we will start by reviewing structural information on model proteins from the ATP-binding cassette (ABC) transporter superfamily, to which CFTR belongs.

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262 In contrast, the third recent paper showed essentially wild-type levels of maturation and stability in F508del-CFTR containing second-site suppressor mutations exclusively in NBD1 (i.e., the I539T mutation plus four proline substitutions found in chicken CFTR, which is naturally more thermostable than human CFTR) (Aleksandrov et al. 2012). Login to comment

[hide] Novel pharmacological strategies to treat cystic f... Trends Pharmacol Sci. 2013 Feb;34(2):119-25. doi: 10.1016/j.tips.2012.11.006. Hanrahan JW, Sampson HM, Thomas DY
Novel pharmacological strategies to treat cystic fibrosis.
Trends Pharmacol Sci. 2013 Feb;34(2):119-25. doi: 10.1016/j.tips.2012.11.006., [PMID:23380248]

Abstract [show]
Cystic fibrosis (CF) is a lethal disease caused by mutations in the CFTR gene. The most frequent mutation is deletion of a phenylalanine residue (DeltaF508) that results in retention of the mutant, but otherwise functional, protein in the endoplasmic reticulum (ER). There have been recent advances in the identification of chemically diverse corrector compounds that allow DeltaF508-CFTR protein to traffic from the ER to the plasma membrane. The most studied correctors fall into two categories, pharmacological chaperones that bind to the mutant protein and circumvent its recognition by the cellular protein quality control systems and proteostasis regulators that modify the cellular pathways responsible for protein quality control and trafficking. This review focuses on recent advances in the field, strategies for the development of drugs from corrector compounds for the treatment of CF, and identification of their targets and mechanism(s) of action.

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40 The suppressor mutations G550E and I539T restore the DF508-CFTR proteolytic pattern to that of wild type CFTR. Login to comment

[hide] Dynamics intrinsic to cystic fibrosis transmembran... Cold Spring Harb Perspect Med. 2013 Mar 1;3(3):a009522. doi: 10.1101/cshperspect.a009522. Chong PA, Kota P, Dokholyan NV, Forman-Kay JD
Dynamics intrinsic to cystic fibrosis transmembrane conductance regulator function and stability.
Cold Spring Harb Perspect Med. 2013 Mar 1;3(3):a009522. doi: 10.1101/cshperspect.a009522., [PMID:23457292]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) requires dynamic fluctuations between states in its gating cycle for proper channel function, including changes in the interactions between the nucleotide-binding domains (NBDs) and between the intracellular domain (ICD) coupling helices and NBDs. Such motions are also linked with fluctuating phosphorylation-dependent binding of CFTR's disordered regulatory (R) region to the NBDs and partners. Folding of CFTR is highly inefficient, with the marginally stable NBD1 sampling excited states or folding intermediates that are aggregation-prone. The severe CF-causing F508del mutation exacerbates the folding inefficiency of CFTR and leads to impaired channel regulation and function, partly as a result of perturbed NBD1-ICD interactions and enhanced sampling of these NBD1 excited states. Increased knowledge of the dynamics within CFTR will expand our understanding of the regulated channel gating of the protein as well as of the F508del defects in folding and function.

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202 Recently, substitution of proline residues at key positions in the Q-loop, the SDR, and the RI in context of the I539T suppressor mutation has been shown to restore channel function and thermostability to full-length F508del CFTR. Login to comment

[hide] Corrector VX-809 stabilizes the first transmembran... Biochem Pharmacol. 2013 Sep 1;86(5):612-9. doi: 10.1016/j.bcp.2013.06.028. Epub 2013 Jul 5. Loo TW, Bartlett MC, Clarke DM
Corrector VX-809 stabilizes the first transmembrane domain of CFTR.
Biochem Pharmacol. 2013 Sep 1;86(5):612-9. doi: 10.1016/j.bcp.2013.06.028. Epub 2013 Jul 5., [PMID:23835419]

Abstract [show]
Processing mutations that inhibit folding and trafficking of CFTR are the main cause of cystic fibrosis (CF). A potential CF therapy would be to repair CFTR processing mutants. It has been demonstrated that processing mutants of P-glycoprotein (P-gp), CFTR's sister protein, can be efficiently repaired by a drug-rescue mechanism. Many arginine suppressors that mimic drug-rescue have been identified in the P-gp transmembrane (TM) domains (TMDs) that rescue by forming hydrogen bonds with residues in adjacent helices to promote packing of the TM segments. To test if CFTR mutants could be repaired by a drug-rescue mechanism, we used truncation mutants to test if corrector VX-809 interacted with the TMDs. VX-809 was selected for study because it is specific for CFTR, it is the most effective corrector identified to date, but it has limited clinical benefit. Identification of the VX-809 target domain will help to develop correctors with improved clinical benefits. It was found that VX-809 rescued truncation mutants lacking the NBD2 and R domains. When the remaining domains (TMD1, NBD1, TMD2) were expressed as separate polypeptides, VX-809 only increased the stability of TMD1. We then performed arginine mutagenesis on TM6 in TMD1. Although the results showed that TM6 had distinct lipid and aqueous faces, CFTR was different from P-gp as no arginine promoted maturation of CFTR processing mutants. The results suggest that TMD1 contains a VX-809 binding site, but its mechanism differed from P-gp drug-rescue. We also report that V510D acts as a universal suppressor to rescue CFTR processing mutants.

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180 To test if the V232D or H1085R mutants could be rescued by suppressor mutations in other domains, suppressor mutations in NBD1 (I539T), the NBD1-TMD2 interface (V510D), or TMD2 (R1070W) (only V232D) locations were introduced into the mutants. Login to comment
184 The I539T and R1070W mutations only rescued DF508 CFTR. Login to comment
237 (C) Extracts of cells expressing processing mutants DF508, V232D, or H1085R with or without the V510D, I539T, or R1070W suppressor mutations were subjected to immunoblot analysis. Login to comment

[hide] Allosteric coupling between the intracellular coup... PLoS One. 2013 Sep 18;8(9):e74347. doi: 10.1371/journal.pone.0074347. eCollection 2013. Dawson JE, Farber PJ, Forman-Kay JD
Allosteric coupling between the intracellular coupling helix 4 and regulatory sites of the first nucleotide-binding domain of CFTR.
PLoS One. 2013 Sep 18;8(9):e74347. doi: 10.1371/journal.pone.0074347. eCollection 2013., [PMID:24058550]

Abstract [show]
Cystic fibrosis is caused by mutations in CFTR (cystic fibrosis transmembrane conductance regulator), leading to folding and processing defects and to chloride channel gating misfunction. CFTR is regulated by ATP binding to its cytoplasmic nucleotide-binding domains, NBD1 and NBD2, and by phosphorylation of the NBD1 regulatory insert (RI) and the regulatory extension (RE)/R region. These regulatory effects are transmitted to the rest of the channel via NBD interactions with intracellular domain coupling helices (CL), particularly CL4. Using a sensitive method for detecting inter-residue correlations between chemical shift changes in NMR spectra, an allosteric network was revealed within NBD1, with a construct lacking RI. The CL4-binding site couples to the RI-deletion site and the C-terminal residues of NBD1 that precede the R region in full-length CFTR. Titration of CL4 peptide into NBD1 perturbs the conformational ensemble in these sites with similar titration patterns observed in F508del, the major CF-causing mutant, and in suppressor mutants F494N, V510D and Q637R NBD1, as well as in a CL4-NBD1 fusion construct. Reciprocally, the C-terminal mutation, Q637R, perturbs dynamics in these three sites. This allosteric network suggests a mechanism synthesizing diverse regulatory NBD1 interactions and provides biophysical evidence for the allosteric coupling required for CFTR function.

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309 The mutations that can partially suppress the folding defects of F508del NBD1 are scattered across NBD1- V510D near CL4 in the channel, I539T directly opposite the NBD1:NBD2 interface, and Q637R near the start of the R region [12], hinting at the complex allosteric nature of the NBD1 folding landscape. Login to comment

[hide] The cystic fibrosis V232D mutation inhibits CFTR m... Biochem Pharmacol. 2014 Mar 1;88(1):46-57. doi: 10.1016/j.bcp.2013.12.027. Epub 2014 Jan 9. Loo TW, Clarke DM
The cystic fibrosis V232D mutation inhibits CFTR maturation by disrupting a hydrophobic pocket rather than formation of aberrant interhelical hydrogen bonds.
Biochem Pharmacol. 2014 Mar 1;88(1):46-57. doi: 10.1016/j.bcp.2013.12.027. Epub 2014 Jan 9., [PMID:24412276]

Abstract [show]
Processing mutations that inhibit folding and trafficking of CFTR are the main cause of cystic fibrosis. Repair of CFTR mutants requires an understanding of the mechanisms of misfolding caused by processing mutations. Previous studies on helix-loop-helix fragments of the V232D processing mutation suggested that its mechanism was to lock transmembrane (TM) segments 3 and 4 together by a non-native hydrogen bond (Asp232(TM4)/Gln207(TM3)). Here, we performed mutational analysis to test for Asp232/Gln207 interactions in full-length CFTR. The rationale was that a V232N mutation should mimic V232D and a V232D/Q207A mutant should mature if the processing defect was caused by hydrogen bonds. We report that only Val232 mutations to charged amino acids severely blocked CFTR maturation. The V232N mutation did not mimic V232D as V232N showed 40% maturation compared to 2% for V232D. Mutation of Val232 to large nonpolar residues (Leu, Phe) had little effect. The Q207L mutation did not rescue V232D because Q207L showed about 50% maturation in the presence of corrector VX-809 while V232D/Q207A could no longer be rescued. These results suggest that V232D inhibits maturation by disrupting a hydrophobic pocket between TM segments rather than forming a non-native hydrogen bond. Disulfide cross-linking analysis of cysteines W356C(TM6) and W1145C(TM12) suggest that the V232D mutation inhibits maturation by trapping CFTR as a partially folded intermediate. Since correctors can efficiently rescue V232D CFTR, the results suggest that hydrophilic processing mutations facing a hydrophobic pocket are good candidates for rescue with pharmacological chaperones.

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169 For example, V510D promotes maturation of mutants with processing mutations in TMD1 (V232D), TMD2 (H1085R) and NBD1 (DF508) whereas other suppressors such as I539T and R1070W promote maturation of DF508 CFTR but not mutants V232D or H1085R [19]. Login to comment

[hide] Biosynthesis of cystic fibrosis transmembrane cond... Int J Biochem Cell Biol. 2014 Jul;52:26-38. doi: 10.1016/j.biocel.2014.03.020. Epub 2014 Mar 28. Pranke IM, Sermet-Gaudelus I
Biosynthesis of cystic fibrosis transmembrane conductance regulator.
Int J Biochem Cell Biol. 2014 Jul;52:26-38. doi: 10.1016/j.biocel.2014.03.020. Epub 2014 Mar 28., [PMID:24685677]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride (Cl(-)) channel. Mutations of its gene lead to the disease of cystis fibrosis (CF) among which the most common is the deletion of phenylalanine at position 508 (Phe508del). CFTR is a multi-domain glycoprotein whose biosynthesis, maturation and functioning as an anion channel involve multi-level post-translational modifications of CFTR molecules and complex folding processes to reach its native, tertiary conformation. Only 20-40% of the nascent chains achieve folded conformation, while the remaining molecules are targeted for degradation by endoplasmic reticulum, lysosomes, or autophagy. A large number of mutations causing CF impair processing of CFTR. Growing knowledge of CFTR biosynthesis has enabled understanding the cellular basis of CF and has brought to light various potential targets for novel, promising therapies.

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1442 Mutations located in NBD1, such as I539T, G550E, R553M/Q and R555K, as well as R1070W in CL4 of MSD2 promote Phe508del-CFTR maturation and trafficking to the cell surface and also restore channel activity (DeCarvalho et al., 2002; Teem et al., 1993, 1996; Thibodeau et al., 2010). Login to comment

[hide] Complement yourself: Transcomplementation rescues ... Biophys Rev. 2014 Mar 1;6(1):169-180. Cebotaru L, Guggino WB
Complement yourself: Transcomplementation rescues partially folded mutant proteins.
Biophys Rev. 2014 Mar 1;6(1):169-180., [PMID:24949105]

Abstract [show]
Cystic Fibrosis (CF) is an autosomal disease associated with malfunction in fluid and electrolyte transport across several mucosal membranes. The most common mutation in CF is an in-frame three-base pair deletion that removes a phenylalanine at position 508 in the first nucleotide-binding domain of the cystic fibrosis conductance regulator (CFTR) chloride channel. This mutation has been studied extensively and leads to biosynthetic arrest of the protein in the endoplasmic reticulum and severely reduced channel activity. This review discusses a novel method of rescuing DeltaF508 with transcomplementation, which occurs when smaller fragments of CFTR containing the wild-type nucleotide binding domain are co-expressed with the DeltaF508 deletion mutant. Transcomplementation rescues the processing and channel activity of DeltaF508 and reduces its rate of degradation in airway epithelial cells. To apply transcomplementation as a therapy would require that the cDNA encoding the truncated CFTR be delivered to cells. We also discuss a gene therapeutic approach based on delivery of a truncated form of CFTR to airway cells using adeno-associated viral vectors.

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80 For example, Hoelen (43), again using a limited proteolysis approach to assess domain stability, showed that an I539T second-site suppressor mutation, in combination with ƊF508 CFTR, restored NBD1 stability to the wt domain pattern, suggesting that correcting NBD1 stability will be critical for the development of a therapeutic for CF. Login to comment

[hide] Decoding F508del misfolding in cystic fibrosis. Biomolecules. 2014 May 6;4(2):498-509. doi: 10.3390/biom4020498. Wang XR, Li C
Decoding F508del misfolding in cystic fibrosis.
Biomolecules. 2014 May 6;4(2):498-509. doi: 10.3390/biom4020498., [PMID:24970227]

Abstract [show]
The functional deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR), a plasma membrane chloride channel, leads to the development of cystic fibrosis. The deletion of a phenylalanine at residue 508 (F508del) is the most common cause of CFTR misfolding leading to the disease. The F508del misfolding originates in the first nucleotide-binding domain (NBD1), which induces a global conformational change in CFTR through NBD1's interactions with other domains. Such global misfolding produces a mutant chloride channel that is impaired in exocytic trafficking, peripheral stability, and channel gating. The nature and atomic details of F508del misfolding have been subject to extensive research during the past decade. Current data support a central role for NBD1 in F508del misfolding and rescue. Many cis-acting NBD1 second-site mutations rescue F508del misfolding in the context of full-length CFTR. While some of these mutations appear to specifically counteract the F508del-induced misfolding, others release certain inherent conformational constraints of the human wild-type CFTR. Several small-molecule correctors were recently found to act on key interdomain interfaces of F508del CFTR. Potential rational approaches have been proposed in an attempt to develop highly effective small molecule modulators that improve the cell surface functional expression of F508del CFTR.

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62 I539T/4P refers to the combined I539T and four proline substitutions occurring in chicken CFTR. Login to comment
101 This is largely accomplished by the I539T substitution and four additional proline substitutions at residues 422, 434, 492, and 534 as the same substitutions made in human F508del CFTR restore its processing, peripheral stability, and channel gating [40]. Login to comment
104 Molecular dynamics simulation revealed that, in the presence of I539T, proline substitutions stabilize the structurally diverse regions of human F508del NBD1 [40]. Login to comment

[hide] Restoration of NBD1 thermal stability is necessary... J Mol Biol. 2015 Jan 16;427(1):106-20. doi: 10.1016/j.jmb.2014.07.026. Epub 2014 Jul 30. He L, Aleksandrov AA, An J, Cui L, Yang Z, Brouillette CG, Riordan JR
Restoration of NBD1 thermal stability is necessary and sufficient to correct F508 CFTR folding and assembly.
J Mol Biol. 2015 Jan 16;427(1):106-20. doi: 10.1016/j.jmb.2014.07.026. Epub 2014 Jul 30., [PMID:25083918]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) (ABCC7), unique among ABC exporters as an ion channel, regulates ion and fluid transport in epithelial tissues. Loss of function due to mutations in the cftr gene causes cystic fibrosis. The most common cystic-fibrosis-causing mutation, the deletion of F508 (DeltaF508) from the first nucleotide binding domain (NBD1) of CFTR, results in misfolding of the protein and clearance by cellular quality control systems. The DeltaF508 mutation has two major impacts on CFTR: reduced thermal stability of NBD1 and disruption of its interface with membrane-spanning domains (MSDs). It is unknown if these two defects are independent and need to be targeted separately. To address this question, we varied the extent of stabilization of NBD1 using different second-site mutations and NBD1 binding small molecules with or without NBD1/MSD interface mutation. Combinations of different NBD1 changes had additive corrective effects on F508 maturation that correlated with their ability to increase NBD1 thermostability. These effects were much larger than those caused by interface modification alone and accounted for most of the correction achieved by modifying both the domain and the interface. Thus, NBD1 stabilization plays a dominant role in overcoming the DeltaF508 defect. Furthermore, the dual target approach resulted in a locked-open ion channel that was constitutively active in the absence of the normally obligatory dependence on phosphorylation by protein kinase A. Thus, simultaneous targeting of both the domain and the interface, as well as being non-essential for correction of biogenesis, may disrupt normal regulation of channel function.

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45 2PT, S492P/A534P/I539T; 4PT, 2PT + S422P/S434P; 3SS, G550E/R553M/R555K; 4SS, 3SS + I539T; ƊRI, deletion of RI amino acids 404-435; combo, ƊRI + 2PT + 3SS. Login to comment
65 1-WT; 2-S492P; 3-I539T; 4. Login to comment
66 S492P/I539T; 5-G550E/R553Q/R555K; 6-combo. Login to comment
72 2PT, S492P/ A534P/I539T; 3PT, 2PT + S495P. Login to comment
95 S492P or I539T alone slightly increased the Tm of NBD1 (ƊTm = 2-3 &#b0;C) similar to the affect of the solubilization mutations F494N and Q637R combined. Login to comment
96 The S492P and I539T substitutions had additive affects such that ƊTm increased to 4.4 &#b0;C, and ƊTm was further increased to 8.4 &#b0;C when the additional mutations A534P/G550E/R553M/R555K were introduced. Login to comment
100 The effect of this single mutation was in contrast to that of S492P, which only increased maturation substantially when present together with I539T. Login to comment
101 Combination of I539T with S495P, however, did cause a further increase in maturation and the effects of the two proline substitutions also were additive. Login to comment
123 Figure 3e shows that both BIA and BEIA further strongly increased maturation of the NBD1 stabilized variant ƊF508/2PT (S492P/A534P/I539T). Login to comment

[hide] Biophysical characterisation of calumenin as a cha... PLoS One. 2014 Aug 13;9(8):e104970. doi: 10.1371/journal.pone.0104970. eCollection 2014. Tripathi R, Benz N, Culleton B, Trouve P, Ferec C
Biophysical characterisation of calumenin as a charged F508del-CFTR folding modulator.
PLoS One. 2014 Aug 13;9(8):e104970. doi: 10.1371/journal.pone.0104970. eCollection 2014., [PMID:25120007]

Abstract [show]
The cystic fibrosis transmembrane regulator (CFTR) is a cyclic-AMP dependent chloride channel expressed at the apical surface of epithelial cells lining various organs such as the respiratory tract. Defective processing and functioning of this protein caused by mutations in the CFTR gene results in loss of ionic balance, defective mucus clearance, increased proliferation of biofilms and inflammation of human airways observed in cystic fibrosis (CF) patients. The process by which CFTR folds and matures under the influence of various chaperones in the secretory pathway remains incompletely understood. Recently, calumenin, a secretory protein, belonging to the CREC family of low affinity calcium binding proteins has been identified as a putative CFTR chaperone whose biophysical properties and functions remain uncharacterized. We compared hydropathy, instability, charge, unfoldability, disorder and aggregation propensity of calumenin and other CREC family members with CFTR associated chaperones and calcium binding proteins, wild-type and mutant CFTR proteins and intrinsically disordered proteins (IDPs). We observed that calumenin, along with other CREC proteins, was significantly more charged and less folded compared to CFTR associated chaperones. Moreover like IDPs, calumenin and other CREC proteins were found to be less hydrophobic and aggregation prone. Phylogenetic analysis revealed a close link between calumenin and other CREC proteins indicating how evolution might have shaped their similar biophysical properties. Experimentally, calumenin was observed to significantly reduce F508del-CFTR aggregation in a manner similar to AavLEA1, a well-characterized IDP. Fluorescence microscopy based imaging analysis also revealed altered trafficking of calumenin in bronchial cells expressing F508del-CFTR, indicating its direct role in the pathophysiology of CF. In conclusion, calumenin is characterized as a charged protein exhibiting close similarity with IDPs and is hypothesized to regulate F508del-CFTR folding by electrostatic effects. This work provides useful insights for designing optimized synthetic structural correctors of CFTR mutant proteins in the future.

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307 Further insights into the design parameters for such peptides are gained by the observation that certain suppressor mutations such as G550E and I539T can partially rescue the F508del-CFTR to the cell surface [6]. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Biochemistry. 2014 Sep 9;53(35):5613-8. doi: 10.1021/bi501007v. Epub 2014 Aug 22. Liu X, Dawson DC
Cystic fibrosis transmembrane conductance regulator (CFTR) potentiators protect G551D but not DeltaF508 CFTR from thermal instability.
Biochemistry. 2014 Sep 9;53(35):5613-8. doi: 10.1021/bi501007v. Epub 2014 Aug 22., [PMID:25148434]

Abstract [show]
The G551D cystic fibrosis transmembrane conductance regulator (CFTR) mutation is associated with severe disease in approximately 5% of cystic fibrosis patients worldwide. This amino acid substitution in NBD1 results in a CFTR chloride channel characterized by a severe gating defect that can be at least partially overcome in vitro by exposure to a CFTR potentiator. In contrast, the more common DeltaF508 mutation is associated with a severe protein trafficking defect, as well as impaired channel function. Recent clinical trials demonstrated a beneficial effect of the CFTR potentiator, Ivacaftor (VX-770), on lung function of patients bearing at least one copy of G551D CFTR, but no comparable effect on DeltaF508 homozygotes. This difference in efficacy was not surprising in view of the established difference in the molecular phenotypes of the two mutant channels. Recently, however, it was shown that the structural defect introduced by the deletion of F508 is associated with the thermal instability of DeltaF508 CFTR channel function in vitro. This additional mutant phenotype raised the possibility that the differences in the behavior of DeltaF508 and G551D CFTR, as well as the disparate efficacy of Ivacaftor, might be a reflection of the differing thermal stabilities of the two channels at 37 degrees C. We compared the thermal stability of G551D and DeltaF508 CFTR in Xenopus oocytes in the presence and absence of CTFR potentiators. G551D CFTR exhibited a thermal instability that was comparable to that of DeltaF508 CFTR. G551D CFTR, however, was protected from thermal instability by CFTR potentiators, whereas DeltaF508 CFTR was not. These results suggest that the efficacy of VX-770 in patients bearing the G551D mutation is due, at least in part, to the ability of the small molecule to protect the mutant channel from thermal instability at human body temperature.

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50 The relatively rapid and nearly complete recovery of the G551D channels was reminiscent of that seen with ƊF508 channels when the phenylalanine deletion was combined with a nearby, second-site suppressor mutation like I539T (ƊF508/I539T CFTR).7 Upon comparison to contemporaneous experiments with ƊF508 CFTR channels (see Figures 3 and 4), it also appeared that the extent of thermal deactivation was lower for G551D channels. Login to comment

[hide] Deletion of Phenylalanine 508 in the First Nucleot... J Biol Chem. 2015 Sep 18;290(38):22862-78. doi: 10.1074/jbc.M115.641134. Epub 2015 Jul 6. Chong PA, Farber PJ, Vernon RM, Hudson RP, Mittermaier AK, Forman-Kay JD
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 Sep 18;290(38):22862-78. doi: 10.1074/jbc.M115.641134. Epub 2015 Jul 6., [PMID:26149808]

Abstract [show]
Deletion of Phe-508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) results in destabilization of the domain, intramolecular interactions involving the domain, and the entire channel. The destabilization caused by F508del manifests itself in defective channel processing and channel gating defects. Here, we present NMR studies of the effect of F508del and the I539T stabilizing mutation on NBD1 dynamics, with a view to understanding these changes in stability. Qualitatively, F508del NMR spectra exhibit significantly more peak broadening than WT spectra due to the enhanced intermediate time scale (millisecond to microsecond) motions in the mutant. Unexpectedly, studies of fast (nanosecond to picosecond) motions revealed that F508del NBD1 tumbles more rapidly in solution than WT NBD1. Whereas F508del tumbles at a rate nearly consistent with the monomeric state, the WT protein tumbles significantly more slowly. Paramagnetic relaxation enhancement experiments confirm that NBD1 homodimerizes in solution in the expected head-to-tail orientation. NMR spectra of WT NBD1 reveal significant concentration-dependent chemical shift perturbations consistent with NBD1 dimerization. Chemical shift analysis suggests that the more rapid tumbling of F508del is the result of an impaired ability to dimerize. Based on previously published crystal structures and NMR spectra of various NBD1 mutants, we propose that deletion of Phe-508 affects Q-loop conformational sampling in a manner that inhibits dimerization. These results provide a potential mechanism for inhibition of channel opening by F508del and support the dimer interface as a target for cystic fibrosis therapeutics.

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6 Here, we present NMR studies of the effect of F508del and the I539T stabilizing mutation on NBD1 dynamics, with a view to understanding these changes in stability. Login to comment
27 WT, I539T and F508del NBD1 assignments have been deposited in the BMRB. Login to comment
47 Completion of b0e;82% of assignments for WT, F508del, and I539T NBD1 èc;RIèc;RE allowed extensive characterization of NBD1. Login to comment
50 Unexpectedly, studies of fast (nanosecond to picosecond) motions revealed that isolated F508del NBD1 èc;RIèc;RE tumbles more rapidly in solution than WT or I539T NBD1 èc;RIèc;RE. Login to comment
55 Experimental Procedures Protein Expression and Purification-WT, F508del, and I539T variants of human NBD1 èc;RIèc;RE (387-646, èc;405-436) were expressed and purified as described previously (32). Login to comment
105 NBD1 èc;RIèc;RE with the I539T-stabilizing mutation (23) was assigned first, with 91% completion of backbone 15 N, 13 Cb18;, and 1 HN resonances. Login to comment
106 Full assignment experiments were also recorded for WT and, together with the I539T assignments, enabled us to complete assignment of 89% of backbone 15 N, 13 Cb18;, and 1 HN resonances for WT. Login to comment
108 Comparison of WT, I539T, and F508del Spectra-Human NBD1 èc;RIèc;RE spectra, while significantly better than full-length mouse NBD1 spectra with the RI (13), still exhibit inhomogeneous peak intensities (Fig. 1). Login to comment
121 Comparison of WT, I539T, and F508del demonstrates overlapping unassigned regions clustered on or near the ॷ-subdomain, including most of the Q-loop, portions of helix 5 (H5), and the adjacent ABC signature sequence and residues immediately following the Walker B motif (Fig. 2). Login to comment
126 Comparison of WT, I539T, and F508del spectra demonstrate that the two mutations do not cause any major structural changes. Login to comment
128 We used the set of I539T chemical shift assignments for our prediction, because it was the most complete. Login to comment
136 FIGURE 2. a-c, ribbon diagrams of WT, I539T, and F508del NBD1 èc;RIèc;RE. Login to comment
144 F508del Increases Exchange and Reduces Dimerization 22866 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290ߦNUMBER 38ߦSEPTEMBER 18, 2015 at SEMMELWEIS UNIV OF MEDICINE on December 4, The similarity between WT, I539T, and F508del spectra strongly supports the conclusion that all share the same fold. Login to comment
152 NBD1 15 N Relaxation Studies-Because changes in NBD1 thermostability underlie F508del defects, we probed the changes in dynamics resulting either from deletion of Phe-508 or the I539T stabilizing mutation. Login to comment
153 We measured T1, T2, and heteronuclear NOE values, which are responsive to fast time scale (nanosecond to picosecond) motions, for WT, I539T, and F508del NBD1 èc;RIèc;RE. Login to comment
155 Plot of TALOSd19; chemical shift derived secondary structure for I539T NBD1 èc;RIèc;RE as a function of residue number. Login to comment
166 In contrast, the WT and I539T protein appear to be in exchange between the monomeric form and dimeric or higher order oligomeric forms resulting in higher ঄c values. Login to comment
170 The ঄c differences also complicate the quantitative interpretation of T1 and T2 in terms of fast time scale dynamics because it is likely that the WT and I539T samples, in particular, contain a mixture of monomers, dimers, and possibly higher order oligomers. Login to comment
190 Variant Sample concentration ঄c mM ns Predicted for monomer 16-20 Predicted for dimer 32-38 WT 0.6 29.4 afe; 2.5 F508del 0.9 19.9 afe; 1.1 I539T 1.5 27.3 afe; 1.5 F508del Increases Exchange and Reduces Dimerization 22868 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290ߦNUMBER 38ߦSEPTEMBER 18, 2015 at SEMMELWEIS UNIV OF MEDICINE on December , 547) (Fig. ). Login to comment
211 87ób;H) ribbon diagram plots for WT (a), F508del (b), and I539T NBD1 èc;RIèc;RE (c) are shown. Login to comment
213 d, spectral density functions at three different frequencies as a function of NBD1 residue number for WT (green), F508del (red), and I539T (blue). Login to comment
230 Notably, for I539T NBD1 èc;RIèc;RE, similar shift perturbations were confirmed for the indole of Trp-496 and the amides of Cys-491, Leu-571, and Glu-403 (Fig. 6e). Login to comment
231 For both WT and I539T, these chemical shift perturbations are modulated by concentration causing the peaks to "move" along a straight vector across the concentration series. Login to comment
363 Interestingly, the combined suppressor mutations I539T, G550E, R553M, and R555K have a bigger positive effect on F508del CFTR when NBD2 is present (58), suggesting the importance of the NBD interaction and hinting that these NBD1-stabilizing mutations may also improve the ability of F508del NBD1 to dimerize with NBD2. Login to comment
370 This conclusion is strongly supported by our NMR structural data, as well as studies of fast time scale dynamics in NBD1 èc;RIèc;RE indicating that overall patterns of flexibility are shared by the ground states of WT, F508del, and I539T NBD1. Login to comment

[hide] Thermal stability of purified and reconstituted CF... Protein Expr Purif. 2015 Dec;116:159-66. doi: 10.1016/j.pep.2015.09.018. Epub 2015 Sep 15. Aleksandrov LA, Jensen TJ, Cui L, Kousouros JN, He L, Aleksandrov AA, Riordan JR
Thermal stability of purified and reconstituted CFTR in a locked open channel conformation.
Protein Expr Purif. 2015 Dec;116:159-66. doi: 10.1016/j.pep.2015.09.018. Epub 2015 Sep 15., [PMID:26384709]

Abstract [show]
CFTR is unique among ABC transporters as the only one functioning as an ion channel and from a human health perspective because mutations in its gene cause cystic fibrosis. Although considerable advances have been made towards understanding CFTR's mechanism of action and the impact of mutations, the lack of a high-resolution 3D structure has hindered progress. The large multi-domain membrane glycoprotein is normally present at low copy number and when over expressed at high levels it aggregates strongly, limiting the production of stable mono-disperse preparations. While the reasons for the strong self-association are not fully understood, its relatively low thermal stability seems likely to be one. The major CF causing mutation, DeltaF508, renders the protein very thermally unstable and therefore a great deal of attention has been paid to this property of CFTR. Multiple second site mutations of CFTR in NBD1 where F508 normally resides and small molecule binders of the domain increase the thermal stability of the mutant. These manipulations also stabilize the wild-type protein. Here we have applied DeltaF508-stabilizing changes and other modifications to generate wild-type constructs that express at much higher levels in scaled-up suspension cultures of mammalian cells. After purification and reconstitution into liposomes these proteins are active in a locked-open conformation at temperatures as high as 50 degrees C and remain monodisperse at 4 degrees C in detergent or lipid for at least a week. The availability of adequate amounts of these and related stable active preparations of homogeneous CFTR will enable stalled structural and ligand binding studies to proceed.

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20 Abbreviations used: CFTR, cystic fibrosis transmembrane conductance regulator; ABC, ATP-binding cassette; NBD1, N-terminal nucleotide-binding domain; CF, cystic fibrosis; CHO, Chinese hamster ovary; HEK, human embryonic kidney; BHK, baby hamster kidney; Tm, melting temperature; Ti, inactivation temperature; RI, Regulatory Insertion (residues 404-435); 2PT, variant with NBD1 mutations S492P, A534P and I539T; Q loop, residues contacting the gamma-phosphate of ATP; SDR, structurally divers region; DMNG, Decyl Maltose Neopentyl Glycol; MALS, multi-angle light scattering analysis; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanola mine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPS, 1,2-dioleoyl-sn- glycero-3-phospho-L-serine; SUV, small unilamellar vesicles; LMV, large multilamellar vesicles; LULV, large unilamellar vesicles; PKA, protein kinase A; RIPA, radioimmunoprecipitation assay; ER, endoplasmic reticulum; RAMP, gradual increase with constant slope. Login to comment
107 As seen in Fig. 2a the ''2PT" variant with NBD1 mutations S492P, A534P and I539T and the DRI variant, from which the Regulatory Insertion (residues 404-435) was deleted both increased expression levels substantially compared to the wild type. Login to comment

[hide] Exploiting species differences to understand the C... Biochem Soc Trans. 2015 Oct 1;43(5):975-82. doi: 10.1042/BST20150129. Bose SJ, Scott-Ward TS, Cai Z, Sheppard DN
Exploiting species differences to understand the CFTR Cl- channel.
Biochem Soc Trans. 2015 Oct 1;43(5):975-82. doi: 10.1042/BST20150129., [PMID:26517912]

Abstract [show]
The anion channel cystic fibrosis transmembrane conductance regulator (CFTR) is a unique ATP-binding cassette (ABC) transporter. CFTR plays a pivotal role in transepithelial ion transport as its dysfunction in the genetic disease cystic fibrosis (CF) dramatically demonstrates. Phylogenetic analysis suggests that CFTR first appeared in aquatic vertebrates fulfilling important roles in osmosensing and organ development. Here, we review selectively, knowledge of CFTR structure, function and pharmacology, gleaned from cross-species comparative studies of recombinant CFTR proteins, including CFTR chimeras. The data argue that subtle changes in CFTR structure can affect strongly channel function and the action of CF mutations.

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120 Hypothesizing that structural differences between human and chicken CFTR account for the thermostability of F508del chicken CFTR, Aleksandrov et al. [41] demonstrated that the F508del revertant I539T and four proline residues at key positions within NBD1 (S422P, S434P, S492P and A534P) were responsible for rescuing the processing, plasma membrane stability and function of human CFTR. Login to comment