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. 2009 Sep;20(18):4059-69.
doi: 10.1091/mbc.e08-09-0929. Epub 2009 Jul 22.

VSports在线直播 - Mechanisms for rescue of correctable folding defects in CFTRDelta F508

Diane E Grove  1 , Meredith F N Rosser, Hong Yu Ren, Anjaparavanda P Naren, Douglas M Cyr
Affiliations

Mechanisms for rescue of correctable folding defects in CFTRDelta F508

Diane E Grove (VSports app下载) et al. Mol Biol Cell. 2009 Sep.

Abstract

Premature degradation of CFTRDeltaF508 causes cystic fibrosis (CF). CFTRDeltaF508 folding defects are conditional and folding correctors are being developed as CF therapeutics. How the cellular environment impacts CFTRDeltaF508 folding efficiency and the identity of CFTRDeltaF508's correctable folding defects is unclear. We report that inactivation of the RMA1 or CHIP ubiquitin ligase permits a pool of CFTRDeltaF508 to escape the endoplasmic reticulum VSports手机版. Combined RMA1 or CHIP inactivation and Corr-4a treatment enhanced CFTRDeltaF508 folding to 3-7-fold greater levels than those elicited by Corr-4a. Some, but not all, folding defects in CFTRDeltaF508 are correctable. CHIP and RMA1 recognize different regions of CFTR and a large pool of nascent CFTRDeltaF508 is ubiquitinated by RMA1 before Corr-4a action. RMA1 recognizes defects in CFTRDeltaF508 related to misassembly of a complex that contains MSD1, NBD1, and the R-domain. Corr-4a acts on CFTRDeltaF508 after MSD2 synthesis and was ineffective at rescue of DeltaF508 dependent folding defects in amino-terminal regions. In contrast, misfolding caused by the rare CF-causing mutation V232D in MSD1 was highly correctable by Corr-4a. Overall, correction of folding defects recognized by RMA1 and/or global modulation of ER quality control has the potential to increase CFTRDeltaF508 folding and provide a therapeutic approach for CF. .

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V体育2025版 - Figures

Figure 1.
Figure 1.
Knockdown of ER quality control checkpoints enhance CFTRΔF508 folding and Corr-4a activity. (A) Chemical structure of small molecule corrector Corr-4a. (B) Transfections with siRNA oligos and CFTR or CFTRΔF508 were performed as described in Materials and Methods. The siRNA samples were treated with DMSO or Corr-4a as indicated. Twenty-four hours later the samples were lysed in 2× SDS sample buffer and normalized to total amount of protein, and Western blot analysis was used to determine the steady-state levels of either CFTR or CFTRΔF508. Bands B and C represent the immature and maturely glycosylated forms of CFTR, respectively. Tubulin blots were used as loading controls. The B- and C-band levels for each reaction were quantified by densitometry and were normalized to the DMSO-treated reaction which contained the siRNA control duplex (cont-siRNA). Results shown are representative of one experiment, but trends were identical when the experiment was repeated three individual times. (C) A monoclonal RMA1 antibody and a polyclonal CHIP antibody were used to monitor the impact of RNAi addition on endogenous RMA1 and CHIP levels, respectively.
Figure 2.
Figure 2.
Modulation of the cellular folding environment by siRNA and Corr-4a affects the folding efficiency of CFTR and stability of CFTRΔF508. Transfections with siRNA oligos and CFTR (A) or CFTRΔF508 (B) were performed as described in Materials and Methods. The siRNA reactions were incubated with DMSO or Corr-4a for 2 h before being pulse-labeled with [35S]methionine and chased for the indicated time periods in the presence of chemical treatment. Cell lysates were normalized to total amount of protein, immunoprecipitated with a polyclonal CFTR N-terminal antibody, and visualized by SDS-PAGE analysis and autoradiography. Asterisk denotes a background band. Results were quantified by densitometry and normalized relative to the amount of B-band at t = 0 min for each condition. Similar results were observed upon three repeats of the pulse-chase experiment.
Figure 3.
Figure 3.
Corr-4a appears to act on CFTR and CFTRΔF508 biogenic intermediates after synthesis of MSD2. (A) A schematic depicting the amino acid position in CFTR where stop codons were introduced to generate the CFTR biogenic intermediates utilized in B and C. HEK293 cells were transiently transfected with 1 μg of the indicated CFTR constructs. (B) Corr-4a or DMSO was added to the cells 18 h after transfection, and the cells were incubated with the chemicals for 24 h. The samples were subjected to Western blot analysis with an antibody that specifically recognizes the N-terminus of CFTR to determine steady-state levels of the CFTR fragments. Bands B and C represent the immature and maturely glycosylated forms of CFTR, respectively. Tubulin blots were performed to indicate loading controls. The B- and C-band levels for each reaction were quantified by densitometry and were normalized to the DMSO-treated control. (C) For the pulse-chase reactions, 18 h after transfection the cells were pretreated for 2 h with the indicated chemicals, labeled with [35S]methionine, and chased for the indicated amounts of time in the presence of chemical treatment. The isolated cells were lysed, soluble cell lysates were normalized to contain the same total amount of protein, and CFTR was immunoprecipitated with a polyclonal N-terminal CFTR antibody. The immunoprecipitates were visualized by SDS-PAGE analysis and autoradiography. Results were quantified by densitometry and normalized to the amount of B-band at t = 0 for each CFTR construct and chemical treatment. For each CFTR fragment, the steady-state (B) and pulse-chase (C) results are consistent and the observed trends reproducible.
Figure 4.
Figure 4.
Folding defects caused by CF disease-related mutations in different CFTR subdomains exhibit varied degrees of correction. (A) HEK293 cells were transiently transfected with 1 μg of the indicated CF disease-causing mutants. The transfected cells were allowed to recover for 18 h before addition of Corr-4a or DMSO. The cells were incubated with chemicals for 24 h before using Western blot analysis to determine the steady-state levels of the mutant CFTR proteins. The immature and maturely glycosylated forms of CFTR are designated as B- and C-bands, respectively. Tubulin is used as a loading control. The B- and C-band levels of each CFTR point mutant were quantified by densitometry and were normalized to the DMSO-treated samples for each mutant. The affect of Corr-4a on the studied CF disease causing mutants was reproducible (n = 3). (B) Corr-4a increases the biosynthetic maturation of CFTR V232D. HEK293 cells transfected with CFTR V232D (1 μg) were preincubated with Corr-4a or DMSO for 2 h, labeled with [35S]methionine, and chased for the indicated amounts of time in the continuous presence of chemical treatment. The cells were lysed, soluble cell lysates were normalized to contain the same total amount of protein, and CFTR was immunoprecipitated with a polyclonal N-terminal CFTR antibody. The reactions were visualized by SDS-PAGE analysis and autoradiography. Results were quantified by densitometry and graphed.
Figure 5.
Figure 5.
Corr-4a stabilizes the MSDs of CFTR. (A) Domain schematic of CFTR halves: CFTR 837X and CFTR 837-1480. When CFTR 837-1480 is expressed alone it accumulates as an immaturely glycosylated species. Coexpression of the two halves of wild-type CFTR promotes the formation of a maturely glycosylated form of CFTR 837-1480. (B) HEK293 cells transfected individually with CFTR 837X (1 μg), CFTR 837XΔF508 (1 μg), CFTR 837X V232D (1 μg), or CFTR 837-1480 (1 μg) were cultured for 18 h before addition of Corr-4a or DMSO. Cells were incubated with chemicals for 24 h. Steady-state levels were then determined by Western blot analysis using CFTR N-terminal or NBD2-specific antibodies. Loading consistency was indicated with tubulin blots. The B-band levels for each reaction were quantified by densitometry and were normalized to the DMSO-treated control. (C) Corr-4a increases the stability of CFTR 837X V232D and CFTR 837-1480. HEK293 cells transfected with either CFTR 837X V232D or CFTR 837-1480 were preincubated with Corr-4a or DMSO for 2 h, labeled with [35S]methionine, and chased for the indicated amounts of time in the continuous presence of chemical treatment. Soluble cell lysates were normalized to contain the same total amount of protein, and CFTR was immunoprecipitated with a CFTR N-terminal– or NBD2–specific antibody. The reactions were visualized by SDS-PAGE analysis and autoradiography. Results were quantified by densitometry and normalized to the amount of B-band at t = 0 for each CFTR construct and chemical treatment. (D) HEK293 cells were transfected with either CFTR 837-1480, CFTR 837X and CFTR 837-1480, CFTR 837XΔF508 and CFTR 837-1480, or CFTR 837X V232D and CFTR 837-1480. Transfected cells were cultured and incubated with chemicals as in B. Steady-state levels of CFTR 837-1480 were determined by Western blot using a NBD2 specific antibody, whereas the levels of the N-terminal fragments were analyzed with a CFTR N-terminal antibody. Maturely glycosylated CFTR 837-1480 is designated as C-band, whereas the immaturely glycosylated form is indicated as B-band. Tubulin is used as a loading control. Results shown are representative of one experiment; however, the reported observations were repeatedly observed.
Figure 6.
Figure 6.
Enhancement of CFTRΔF508 folding by combined use of Corr-4a and suppressor mutations. (A) HEK293 cells were transfected with 1 μg of either CFTRΔF508, CFTRΔF508 V510D, CFTR 1172XΔF508, or CFTR 1172XΔF508 V510D. Chemicals Corr-4a or DMSO were added to the cells 18 h after transfection, and the cells were incubated with the chemicals for 24 h. Steady-state levels were then determined by Western blot analysis. Tubulin serves as a loading control. The B- and C-band levels for each reaction were quantified by densitometry and were normalized to the DMSO-treated control. (B) HEK293 cells transfected with the indicated CFTR plasmids were incubated with Corr-4a or DMSO for 2 h. Next, the cells were pulse-labeled with [35S]methionine and chased for the indicated time periods in the presence of chemical treatment. Soluble cell lysates were normalized to total amount of protein, immunoprecipitated with a CFTR N-terminal antibody, and visualized by SDS-PAGE analysis and autoradiography. Results were quantified by densitometry and normalized to the amount of B-band at t = 0 min for each condition. Similar trends were observed upon repetition of the experiments shown in A and B.
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