ABCMdb

ABCC7 p.Ile539Thr

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PMID: 12110684 [PubMed] DeCarvalho AC et al: "Mutations in the nucleotide binding domain 1 signature motif region rescue processing and functional defects of cystic fibrosis transmembrane conductance regulator delta f508."

No. Sentence Comment

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

PMID: 15765539 [PubMed] Amaral MD et al: "Processing of CFTR: traversing the cellular maze--how much CFTR needs to go through to avoid cystic fibrosis?"

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

PMID: 20233947 [PubMed] He L et al: "Restoration of domain folding and interdomain assembly by second-site suppressors of the DeltaF508 mutation in CFTR."

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

PMID: 20551307 [PubMed] Da Paula AC et al: "Folding and rescue of a cystic fibrosis transmembrane conductance regulator trafficking mutant identified using human-murine chimeric proteins."

No. Sentence Comment

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

PMID: 20590134 [PubMed] Loo TW et al: "The V510D suppressor mutation stabilizes DeltaF508-CFTR at the cell surface."

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

PMID: 20667826 [PubMed] Thibodeau PH et al: "The cystic fibrosis-causing mutation deltaF508 affects multiple steps in cystic fibrosis transmembrane conductance regulator biogenesis."

No. Sentence Comment

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

PMID: 20837481 [PubMed] Pagant S et al: "Intragenic suppressing mutations correct the folding and intracellular traffic of misfolded mutants of Yor1p, a eukaryotic drug transporter."

No. Sentence Comment

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

PMID: 21152102 [PubMed] Hoelen H et al: "The primary folding defect and rescue of DeltaF508 CFTR emerge during translation of the mutant domain."

No. Sentence Comment

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

PMID: 21594797 [PubMed] Schmidt A et al: "Biochemical and biophysical approaches to probe CFTR structure."

No. Sentence Comment

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

PMID: 21182301 [PubMed] Loo TW et al: "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."

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

PMID: 22680785 [PubMed] Liu X et al: "Thermal instability of DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) channel function: protection by single suppressor mutations and inhibiting channel activity."

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

PMID: 22406676 [PubMed] Aleksandrov AA et al: "Allosteric modulation balances thermodynamic stability and restores function of DeltaF508 CFTR."

No. Sentence Comment

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

PMID: 22210114 [PubMed] Dong Q et al: "Human-mouse cystic fibrosis transmembrane conductance regulator (CFTR) chimeras identify regions that partially rescue CFTR-DeltaF508 processing and alter its gating defect."

No. Sentence Comment

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

PMID: 22701530 [PubMed] Coppinger JA et al: "A chaperone trap contributes to the onset of cystic fibrosis."

No. Sentence Comment

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

PMID: 19896304 [PubMed] Edelman A et al: "Twenty years after cystic fibrosis gene identification: Where are we and what are we up to?"

No. Sentence Comment

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

PMID: 18555011 [PubMed] Liu Y et al: "Mild processing defect of porcine DeltaF508-CFTR suggests that DeltaF508 pigs may not develop cystic fibrosis disease."

No. Sentence Comment

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

PMID: 22265409 [PubMed] Mendoza JL et al: "Requirements for efficient correction of DeltaF508 CFTR revealed by analyses of evolved sequences."

No. Sentence Comment

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

PMID: 23055971 [PubMed] Molinski S et al: "Functional Rescue of F508del-CFTR Using Small Molecule Correctors."

No. Sentence Comment

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

PMID: 23060795 [PubMed] Pedemonte N et al: "Pharmacological Correctors of Mutant CFTR Mistrafficking."

No. Sentence Comment

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

PMID: 23104983 [PubMed] He L et al: "Correctors of DeltaF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein."

No. Sentence Comment

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

PMID: 23248597 [PubMed] Kim SJ et al: "Mechanisms of CFTR Folding at the Endoplasmic Reticulum."

No. Sentence Comment

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

PMID: 23378596 [PubMed] Hunt JF et al: "Cystic fibrosis transmembrane conductance regulator (ABCC7) structure."

No. Sentence Comment

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

PMID: 23380248 [PubMed] Hanrahan JW et al: "Novel pharmacological strategies to treat cystic fibrosis."

No. Sentence Comment

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

PMID: 23457292 [PubMed] Chong PA et al: "Dynamics intrinsic to cystic fibrosis transmembrane conductance regulator function and stability."

No. Sentence Comment

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

PMID: 23835419 [PubMed] Loo TW et al: "Corrector VX-809 stabilizes the first transmembrane domain of CFTR."

No. Sentence Comment

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

PMID: 24058550 [PubMed] Dawson JE et al: "Allosteric coupling between the intracellular coupling helix 4 and regulatory sites of the first nucleotide-binding domain of CFTR."

No. Sentence Comment

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

PMID: 24412276 [PubMed] Loo TW et al: "The cystic fibrosis V232D mutation inhibits CFTR maturation by disrupting a hydrophobic pocket rather than formation of aberrant interhelical hydrogen bonds."

No. Sentence Comment

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

PMID: 24685677 [PubMed] Pranke IM et al: "Biosynthesis of cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

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

PMID: 24949105 [PubMed] Cebotaru L et al: "Complement yourself: Transcomplementation rescues partially folded mutant proteins."

No. Sentence Comment

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

PMID: 24970227 [PubMed] Wang XR et al: "Decoding F508del misfolding in cystic fibrosis."

No. Sentence Comment

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

PMID: 25083918 [PubMed] He L et al: "Restoration of NBD1 thermal stability is necessary and sufficient to correct F508 CFTR folding and assembly."

No. Sentence Comment

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

PMID: 25120007 [PubMed] Tripathi R et al: "Biophysical characterisation of calumenin as a charged F508del-CFTR folding modulator."

No. Sentence Comment

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

PMID: 25148434 [PubMed] Liu X et al: "Cystic fibrosis transmembrane conductance regulator (CFTR) potentiators protect G551D but not DeltaF508 CFTR from thermal instability."

No. Sentence Comment

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

PMID: 26149808 [PubMed] Chong PA et al: "Deletion of Phenylalanine 508 in the First Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator Increases Conformational Exchange and Inhibits Dimerization."

No. Sentence Comment

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

PMID: 26384709 [PubMed] Aleksandrov LA et al: "Thermal stability of purified and reconstituted CFTR in a locked open channel conformation."

No. Sentence Comment

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

PMID: 26517912 [PubMed] Bose SJ et al: "Exploiting species differences to understand the CFTR Cl- channel."

No. Sentence Comment

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