ABCMdb

PMID: 19176754

Du K, Lukacs GL
Cooperative assembly and misfolding of CFTR domains in vivo.
Mol Biol Cell. 2009 Apr;20(7):1903-15. Epub 2009 Jan 28., [PubMed]

Sentences

No. Mutations Sentence Comment

37 ABCC7 p.Asn1303Lys ABCC7 p.Gly91Arg ABCC7 p.Leu1093Pro ABCC7 p.Leu346Pro MATERIALS AND METHODS Cell Lines Baby hamster kidney (BHK) cells, stably expressing the wt and mutant (G91R, L346P, L1093P, N1303K, ⌬F508, 4D, 1218X, 1158X, and 823X) CFTR with three tandem hemagglutinin (HA)-epitope (3HA) inserted into the fourth extracellular loop, were isolated and maintained as described previously (Sharma et al., 2004; Du et al., 2005). Login to comment 53 ABCC7 p.Leu570Asp ABCC7 p.Ile601Asp ABCC7 p.Leu571Asp ABCC7 p.Leu602Asp After the insertion of the 2D (I601D/L602D) mutations, the L570D/L571D mutations were introduced into the 2D CFTR-3HA by PCR mutagenesis. Login to comment 54 ABCC7 p.Asn1303Lys The cytosolic domains (NBD1, NBD1-R, and NBD2) and their mutant counterparts (⌬F508, 4D, and N1303K) were PCR amplified with specified amino acid boundaries (Supplemental Table S1) flanked with the XmaI/XbaI sites and fused in frame to the C terminus of the transmembrane segment of the CD4T (CD4T-N1, CD4T-N1R, CD4T-N2, etc. Login to comment 113 ABCC7 p.Leu69Gly The L57G/L69G mutations decreased the melting temperature of the recombinant ␭ (molecular mass ϳ12 kDa) from 52 to 28°C (Pakula et al., 1986). Login to comment 121 ABCC7 p.Leu69Gly CD4T-␭ and CD4T-␭m contain the wt or the mutant (L57C/L69G) ␭ repressor, respectively. Login to comment 146 ABCC7 p.Asn1303Lys Four, introducing the ⌬F508 or the N1303K (Gregory et al., 1991) mutation in the NBD1 and NBD2, respectively, resulted in a small but significant decrease in the cell surface expression of CD4T-N1⌬F, CD4T-N1*⌬F, and CD4T-N2K compared with their wt counterparts (Figure 3, a and b, and Figure 3. Login to comment 152 ABCC7 p.Asn1303Lys ABCC7 p.Leu570Asp ABCC7 p.Ile601Asp ABCC7 p.Leu571Asp ABCC7 p.Leu602Asp Abbreviations: N1⌬F, NBD1⌬F508; N14D, NBD1(L570D/L571D/ I601D/L602D); N2K, NBD2(N1303K); N1R, NBD1-R; N1⌬R, ⌬F508-NBD1-R (also see Supplemental Table S1). Login to comment 155 ABCC7 p.Phe429Ser ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg The N1*⌬F and N1*4D contains the same mutations as defined in a. N1*3S incorporates the F429S, F494N, and Q637R solubilization mutations. Login to comment 170 ABCC7 p.Leu570Asp ABCC7 p.Ile601Asp ABCC7 p.Leu571Asp ABCC7 p.Leu602Asp Similar results were obtained upon replacing four hydrophobic core residues with aspartic acids (4D: L570D/L571D/I601D/ L602D) to disrupt the NBD1 folding (Figure 3, a and b, and Supplemental Figure S2b). Login to comment 171 ABCC7 p.Asn1303Lys The 4D mutation, as the ⌬F508 and N1303K, impaired the biosynthetic processing and global conformation of CFTR, according to immunoblotting, cell surface anti-HA Ab binding and limited proteolysis (Figure 3d and Supplemental Figure S3). Login to comment 174 ABCC7 p.Phe429Ser ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Five, remarkably, introducing three solubilization mutations (F429S, F494N, and Q637R) that were required to produce soluble, recombinant NBD1 in bacteria (Lewis et al., 2005), significantly increased the steady-state cell surface expression of the CD4Tl-N1*-3S at 37°C and suppressed the expression defect at 26°C (Figure 3, b and c). Login to comment 186 ABCC7 p.Arg555Lys ABCC7 p.Arg516Lys Replacement of critical Arg with Lys residues in the MSD1 (R29K) and NBD1 (R516K and R555K) failed to revert the processing defect of M1(RK) and M1-N1(3RK) (Supplemental Figure S5), whereas it partially rescued the ⌬F508 CFTR ER retention (Owsianik et al., 2003; Roxo-Rosa et al., 2006). Login to comment 252 ABCC7 p.Asn1303Lys ABCC7 p.Gly91Arg ABCC7 p.Leu1093Pro ABCC7 p.Leu346Pro Four CF mutations, G91R (Xiong et al., 1997), L346P (Choi et al., 2005), L1093P (Seibert et al., 1996), and N1303K (Gregory et al., 1991), localized to the transmembrane (TM) 1 and TM6 in MSD1, the cytosolic loop (CL) 4, and in the NBD2, respectively (Figure 7a). Login to comment 274 ABCC7 p.Gly91Arg ABCC7 p.Leu1093Pro ABCC7 p.Leu346Pro Remarkably, the G91R, L346P, ⌬F508, 4D, and L1093P mutations, regardless of their location, profoundly augmented the protease susceptibility of the NBD2 (ϳ30 kDa), probed with the M3A7 Ab (Figure 7g and Supplemental Figure S7c). Login to comment 291 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Phe429Asn In support of this hypothesis, severalfold increase in the cell surface expression of CD4T-NBD1* was documented in the presence of those solubilization mutations (F429N, F494N, and Q637R) that were required for the recombinant NBD1 expression (Figure 3, b and c) (Lewis et al., 2005). Login to comment 293 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Phe429Asn Indeed, the F429N, F494N, and Q637R mutations thermodynamically stabilized the isolated NBD1 in the absence of domain-domain interactions as indicated by the elevated melting temperature of the recombinant NBD1*-3S relative to its wt counterpart (Rabeh, Mulvihille and Lukacs, unpublished observations). Login to comment 301 ABCC7 p.Gly91Arg ABCC7 p.Leu1093Pro ABCC7 p.Leu346Pro One of the most important observations of this study is that the NBD2 conformational stability was dramatically impaired regardless the localization of mutations (G91R, L346P, ⌬F508, 4D, and L1093P) in the NBD1, TM1, TM6, or CL4 (Figure 6e). Login to comment 303 ABCC7 p.Asn1303Lys Nevertheless, considering that the N1303K mutation increased the protease susceptibility of both the MSD1 and MSD2 and deletion of the NBD2 modestly diminished the maturation efficiency of the CFTR-1218X as well as prevented the low-temperature rescue of the ⌬F508-CFTR-1218X-processing defect (Supplemental Figure S8), we propose that NBD2 contributes to the global stabilization CFTR despite that it is nonessential for the channel processing. Login to comment 309 ABCC7 p.Asn1303Lys 2) The N1303K NBD2 mutation disrupted the full-length CFTR export, as well as the MSD1 and MSD2 conformation, measured by the in situ protease susceptibility assay, emphasizing that the NBD2 stabilizes some of the N-terminal domains. Login to comment 314 ABCC7 p.Leu346Pro Cooperative misfolding is predicted to amplify relatively modest and localized structural defects caused by point mutations (e.g., L346P and ⌬F508). Login to comment 315 ABCC7 p.Leu346Pro The L346P mutation destabilizes the TM5/6 hairpin in vitro by decreasing intrahelical Hϩ bondings and the TM6 helix net hydrophobicity that could be reverted by a suppressor mutation (Choi et al., 2005). Login to comment