ABCMdb

ABCC7 p.Gln1071Pro

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PMID: 16417523 [PubMed] Loo TW et al: "The chemical chaperone CFcor-325 repairs folding defects in the transmembrane domains of CFTR-processing mutants."

No. Sentence Comment

4 To test if CFcor-325 promoted correct folding of the TMDs (TM domains), we selected two of the CL4 mutants (Q1071P and H1085R) for disulphide cross-linking analysis. Login to comment
5 Pairs of cysteine residues that were cross-linked in mature wild-type CFTR were introduced into mutants Q1071P and H1085R. Login to comment
6 The cross-linking patterns of the Q1071P or H1085R double cysteine mutants rescued with CFcor-325 were similar to those observed with mature wild-type double cysteine proteins. Login to comment
21 EXPERIMENTAL Construction and expression of mutants The cDNAs of wild-type and CL4 mutants (H1054D, G1061R, L1065P, R1066H, Q1071P, L1077P, H1085R and W1098R) were inserted into pcDNA3 vector (Invitrogen, Oakville, ON, Canada) as described previously [2]. Login to comment
23 Site-directed mutagenesis was used to introduce the Q1071P or H1085R mutations into the double cysteine mutants as described previously [11]. Login to comment
31 Disulphide cross-linking analysis The cDNAs of double cysteine mutants M348C(TM6)/T1142C- (TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/ W1145C(TM12) constructed in wild-type, mutant Q1071P or mutant H1085R backgrounds were expressed in HEK-293 cells in the presence or absence of 3 µM CFcor-325. Login to comment
37 Briefly, cells expressing wild-type, mutant Q1071P or H1085R CFTR were grown with or without 3 µM CFcor-325. Login to comment
51 Accordingly, HEK-293 cells were transfected with mutants H1054D, G1061R, L1065P, R1066H, Q1071P, L1077P, H1085R or W1098R cDNAs. Login to comment
57 Expression of mutants H1054D, G1061R, R1066H, Q1071P, L1077P, H1085R and W1098R in the presence of 3 µM CFcor-325, however, induced expression of the 190 kDa mature protein. Login to comment
59 The chemical chaperone most efficiently rescued mutants R1066H, Q1071P, H1085R and W1098R as the amount of mature CFTR was approx. Login to comment
73 Two CL4 mutants, Q1071P and H1085R, were selected for cross-linking analysis because they were efficiently rescued with CFcor-325. Login to comment
74 Expression of mutants Q1071P or H1085R in the presence of CFcor-325 increased the amount of mature CFTR from less than 5% (in the absence of corrector) of total to more than 30% (Figures 2A and 2B). Login to comment
77 Mature 190 kDa CFTR, however, contains complex carbohydrate groups that are resistant to endo H but not to PNGase F. Accordingly, cells expressing wild-type, mutant Q1071P or mutant H1085R CFTR and grown in the presence or absence of CFcor-325 were extracted with SDS and then treated with endoglycosidases. Login to comment
78 Figure 3 shows that the 170 kDa immature CFTRs but not the 190 kDa mature CFTRs of wild-type, mutant Q1071P or mutant H1085R were sensitive to digestion with endo H. Login to comment
79 Both the 190 kDa mature and 170 kDa immature CFTRs were sensitive to digestion with PNGase F. Therefore expression of mutant Q1071P and H1085R in the presence of CFcor-325 promoted maturation and trafficking of the proteins from the ER to the Golgi where complex carbohydrates are added to the protein. Login to comment
81 Cells expressing wild-type, mutant Q1071P or mutant H1085R CFTRs were grown in the presence or absence of 3 µM CFcor-325. Login to comment
82 The cells Figure 3 Endoglycosidase digestion Whole cell SDS extracts of HEK-293 cells expressing wild-type, mutant Q1071P or mutant H1085R in the absence (-) or presence (+) of 3 µM CFcor-325 were treated with endo H (H), PNGase F (F) or no endoglycosidase (-). Login to comment
85 Figure 4 Cell surface labelling of CFTR (A) BHK cells (Control) or BHK cells stably expressing wild-type (Wild-type), mutant Q1071P or mutant H1085R CFTRs were grown in the presence (+) or absence (-) of 3 µM CFcor-325. Login to comment
93 Figure 4(A) Figure 5 Measurement of cAMP-stimulated iodide efflux activities Iodide efflux assays were performed on BHK cells stably expressing wild-type, mutant Q1071P or mutant H1085R CFTRs that were grown in the presence (+CFcor-325) or absence (untreated) of CFcor-325 as described in the Experimental section. Login to comment
94 The activity of mutant H1085R grown without CFcor-325 was similar to that of mutant Q1071P (untreated) and is omitted for clarity. Login to comment
97 Similarly, labelled mature 190 kDa CFTR proteins were detected in mutants Q1071P and H1085R only after rescue with CFcor-325. Login to comment
98 Figure 4(B) shows that there was good correlation between the amount of biotinylated CFTR proteins in mutants Q1071P and H1085R with the amount of mature CFTRs detected in Figure 2(B) (63 and 50% of wild-type respectively). Login to comment
99 Next, we tested whether mutant Q1071P or H1085R was active at the cell surface after rescue with CFcor-325. Login to comment
100 Mutants Q1071P and H1085R CFTRs were stably expressed in BHK cells because adherent cells are essential for the iodide efflux assays. Login to comment
102 The BHK cells stably expressing mutants Q1071P or H1085R were treated for 48 h with or without 3 µM CFcor-325 and then used in iodide efflux assays. Login to comment
106 In contrast, BHK cells stably expressing CFTR mutants Q1071P or H1085R demonstrated iodide efflux activity only when grown in the presence of CFcor-325 (Figure 5). Login to comment
108 Therefore mutants Q1071P and h1085R were used for cross-linking studies. Login to comment
109 Accordingly, M348C(TM6)/T1142C(TM12), T351C(TM6)/ T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutations were introduced into a Q1071P or H1085R CFTR background. Login to comment
110 Mutants Q1071P or H1085R containing M348C(TM6)/T1142C- (TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/ W1145C(TM12) mutations were then transiently expressed in HEK-293 cells in the presence or absence of 3 µM CFcor-325 for 48 h and then treated with the homobifunctional cross-linkers M5M, M8M or M17M. Login to comment
115 Mature mutant Q1071P/ M348C(TM6)/T1142C(TM12) protein was cross-linked with Figure 6 Disulphide cross-linking analysis of CFTR processing mutants HEK-293 cells expressing mutants Q1071P/M348C(TM6)/T1142C(TM12), Q1071P/T351C- (TM6)/T1142C(TM12) and Q1071P/W356C(TM6)/W1145C(TM12) (A), mutants H1085R/ M348C(TM6)/T1142C(TM12), H1085R/T351C(TM6)/T1142C(TM12) and H1085R/W356C- (TM6)/W1145C(TM12) (B) or wild-type, mutant Q1071P or mutant H1085R (C) were incubated for 48 h with (+) or without (-) 3 µM CFcor-325. Login to comment
119 Mature mutant Q1071P/T351C- (TM6)/T1142C(TM12) protein was cross-linked with M8M and to a lesser extent with M17M, while the mature mutant Q1071P/W356C(TM6)/W1145C(TM12) protein was cross-linked with M5M, M8M and M17M (Figure 6A). Login to comment
122 In contrast, wild-type, mature mutant Q1071P or H1085R CFTRs lacking the introduced cysteine residues was not cross-linked by M5M, M8M or M17M (Figure 6C). Login to comment
123 The immature 170 kDa mutant proteins did not 7 Effect of dithiothreitol on immature CFTR treated with cross-linker HEK-293 cells expressing mutant Q1071P/M348C(TM6)/T1142C(TM12) in the absence of CFcor-325 were treated with 0.2 mM M5M, M8M or M17M cross-linker for 15 min at 20◦C. Samples were then incubated with (+DTT) or without (-DTT) 30 mM dithiothreitol for 5 min at 20◦C. Following SDS/PAGE on a 5.5% gel, the stacking and separating gels were transferred on to a sheet of nitrocellulose and CFTR protein was detected by immunoblot analysis. Login to comment
126 An example of aggregation is shown for mutant Q1071P/M348C(TM6)/ T1142C(TM12) (Figure 7). Login to comment
144 The processing mutation Q1071P traps the protein in the ER in a loosely folded conformation because of altered TM packing (B). Login to comment
152 The presence of a processing mutation such as Q1071P introduces a thermodynamic hurdle during folding so that the protein is trapped in an immature conformation (Figure 8B). Login to comment

PMID: 17132688 [PubMed] Wang Y et al: "Modulating the folding of P-glycoprotein and cystic fibrosis transmembrane conductance regulator truncation mutants with pharmacological chaperones."

No. Sentence Comment

55 Cell surface labeling of ⌬NBD2/Q1071P CFTR was performed as described previously (Loo et al., 2006c). Login to comment
93 The position of the Q1071P mutation in ⌬NBD2 CFTR is indicated. Login to comment
139 Therefore, the Q1071P processing mutation was introduced into the ⌬NBD2 CFTR. Login to comment
140 The Q1071P processing mutation was selected because it is located in the fourth cytoplasmic loop (see Fig. 1B), and it was postulated to inhibit maturation because it disrupted NBD2-TMD2 interactions (Seibert et al., 1996). Login to comment
141 Because NBD2 is no longer present in ⌬NBD2 CFTR, we can now test whether the Q1071P mutation would still inhibit maturation. Login to comment
142 It was found that the processing mutant Q1071P could still inhibit maturation of CFTR lacking NBD2 (⌬NBD2/Q1071P, Fig. 5B). Login to comment
143 Therefore, the Q1071P mutation seems to interfere in the folding of TMD2. Login to comment
144 Maturation of the ⌬NBD2/Q1071P CFTR mutant could be promoted, however, when expression was carried out in the presence of VRT-325 or corr-4a (Fig. 5B). Login to comment
146 The conversion of ⌬NBD2/Q1071P CFTR to an endoglycosidase H-resistant protein after expression in the presence of correctors indicates that it reached the Golgi. Login to comment
147 To determine whether expression in the presence of correctors would increase the level of ⌬NBD2/Q1071P at the plasma membrane, cell surface labeling was carried out. Login to comment
148 HEK 293 cells transfected with ⌬NBD2/Q1071P cDNA containing an A52-epitope tag were expressed in the presence or absence of 10 ␮M VRT-325 or corr-4a. Login to comment
157 B, CFTR truncation mutants ⌬NBD2 CFTR or ⌬NBD2/Q1071P containing C-terminal A52 tags were expressed in the presence of 10 ␮M VRT-325 (325) or 10 ␮M corr-4a or no corrector (-) for 24 h. Whole-cell extracts were then subjected to immunoblot analysis with monoclonal antibody A52. C, the concentration-dependence of maturation of ⌬NBD2/Q1071P CFTR was determined by expressing the mutant in the indicated concentrations of corr-4a (4a) for 24 h followed by immunoblot analysis of whole-cell extracts. Login to comment
158 D, HEK 293 cells transfected with A52-tagged ⌬NBD2/Q1071P CFTR cDNA were grown in the presence (ϩ) or absence (-) of 10 ␮M VRT-325 (325) or 10 ␮M corr-4a (4a) for 24 h. Cell-surface labeling was then performed on whole cells using biotin-LC-hydrazide after oxidation of surface carbohydrates. Login to comment
164 Figure 5D shows that cell surface expression of ⌬NBD2/Q1071P was increased after expression in the presence of VRT-325 or corr-4a. Login to comment
165 A control immunoblot developed with monoclonal antibody A52 shows that similar levels of ⌬NBD2/Q1071P protein were recovered after immunoprecipitation (Fig. 5E). Login to comment
220 It is apparent, however, that NBD1-NBD2 or NBD2-TMD2 interactions are not essential for rescue with VRT-325 or corr-4a because both compounds could promote maturation of ⌬NBD2/Q1071P CFTR. Login to comment

PMID: 18193900 [PubMed] Cheung JC et al: "Misfolding of the cystic fibrosis transmembrane conductance regulator and disease."

No. Sentence Comment

90 In some additional examples, a number of mutations found in the fourth intracellular loop (H1054D, G1061R, L1065P, R1066C/H/L, Q1071P, L1077P, H1085R, W1098R, M1101K/ R) also affect the biosynthetic processing of CFTR (although function was not tested) (73); some intracellular loop 4 mutants (F1052V, K1060T, A1067T, G1069R, R1070Q/W) can process CFTR to the complex-glycosylated ("Band C") form but have altered channel activity compared to wild type. Login to comment

PMID: 21594776 [PubMed] Loo TW et al: "Repair of CFTR folding defects with correctors that function as pharmacological chaperones."

No. Sentence Comment

147 HEK 293 cells expressing the CFTR processing mutants, F508del, Q1071P, or H1085R, were incubated with media containing no corrector (-) or 0.05 mM VX-325 plus 0.015 mM corr-4a (+). Login to comment

PMID: 10649505 [PubMed] Yee K et al: "Novel Cystic Fibrosis mutation L1093P: functional analysis and possible Native American origin."

No. Sentence Comment

78 Other mutations in this region e.g. A1067T (Cotton et al. 1996; Seibert et al. 1996) and Q1071P (Seibert et al. 1996) are associated with pancreatic insufficiency, yet retain partial function when tested with efflux assays. Login to comment

PMID: 8662892 [PubMed] Seibert FS et al: "Disease-associated mutations in the fourth cytoplasmic loop of cystic fibrosis transmembrane conductance regulator compromise biosynthetic processing and chloride channel activity."

No. Sentence Comment

130 D: छ, WT; E, Q1071P; छϩ, W1098R; Ⅺ, H1085R; Ç, M1101K; µ, M1101R; Q, control; É, L1077P. Login to comment
136 D: L, WT; E, Q1071P; L 1, W1098R; M, H1085R; &#c7;, M1101K; &#b5;, M1101R; Q, control; &#c9;, L1077P. 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

258 Cross-linking analysis suggests that V232D causes incomplete packing of the TM segments Previous studies on processing mutations in NBD1 (DF508) or in the fourth intracellular loop connecting TM segments 10 and 11 (Q1071P or H1085R) showed that they trapped CFTR at an early folding step resulting in incomplete packing of the TM segments [17,50]. Login to comment
313 We predict that V232D inhibits CFTR maturation by a mechanism that is similar to that proposed for the Q1071P, H1085R [50] and DF508 [17,26] processing mutations (see Fig. 11). Login to comment