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A foldable CFTR{Delta}F508 biogenic intermediate accumulates upon inhibition of the Hsc70-CHIP E3 ubiquitin ligase.

Younger JM, Ren HY, Chen L, Fan CY, Fields A, Patterson C, Cyr DM - J. Cell Biol. (2004)

Bottom Line: CHIP is demonstrated to function as a scaffold that nucleates the formation of a multisubunit E3 ubiquitin ligase whose reconstituted activity toward CFTR is dependent upon Hdj2, Hsc70, and the E2 UbcH5a.Inactivation of the Hsc70-CHIP E3 leads CFTRDeltaF508 to accumulate in a nonaggregated state, which upon lowering of cell growth temperatures, can fold and reach the cell surface.Inhibition of CFTRDeltaF508 ubiquitination can increase its cell surface expression and may provide an approach to treat CF.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT
CFTRDeltaF508 exhibits a correctable protein-folding defect that leads to its misfolding and premature degradation, which is the cause of cystic fibrosis (CF). Herein we report on the characterization of the CFTRDeltaF508 biogenic intermediate that is selected for proteasomal degradation and identification of cellular components that polyubiquitinate CFTRDeltaF508. Nonubiquitinated CFTRDeltaF508 accumulates in a kinetically trapped, but folding competent conformation, that is maintained in a soluble state by cytosolic Hsc70. Ubiquitination of Hsc70-bound CFTRDeltaF508 requires CHIP, a U box containing cytosolic cochaperone. CHIP is demonstrated to function as a scaffold that nucleates the formation of a multisubunit E3 ubiquitin ligase whose reconstituted activity toward CFTR is dependent upon Hdj2, Hsc70, and the E2 UbcH5a. Inactivation of the Hsc70-CHIP E3 leads CFTRDeltaF508 to accumulate in a nonaggregated state, which upon lowering of cell growth temperatures, can fold and reach the cell surface. Inhibition of CFTRDeltaF508 ubiquitination can increase its cell surface expression and may provide an approach to treat CF.

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Related in: MedlinePlus

Localization of GFP–CFTRΔF508 in HEK293 cells. Cells grown on glass coverslips were transiently transfected with GFP–CFTR or GFP–CFTRΔF508 with or without UbcH5a C85A and cultured for 24 h at 37°C. Where indicated 25 μg/ml cyclohexamide and/or brefeldin A (10 μM) was added to culture media. After 4 h of culture at 26°C these cells were fixed and the coverslips were mounted on glass slides. Images were collected and processed as described in the Materials and methods section.
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fig7: Localization of GFP–CFTRΔF508 in HEK293 cells. Cells grown on glass coverslips were transiently transfected with GFP–CFTR or GFP–CFTRΔF508 with or without UbcH5a C85A and cultured for 24 h at 37°C. Where indicated 25 μg/ml cyclohexamide and/or brefeldin A (10 μM) was added to culture media. After 4 h of culture at 26°C these cells were fixed and the coverslips were mounted on glass slides. Images were collected and processed as described in the Materials and methods section.

Mentions: To demonstrate that the CFTRΔF508 that was converted to the C form during the 26°C chase incubation was trafficked to the cell surface the localization of GFP–CFTRΔF508 was examined under these experimental conditions and compared with that of GFP–CFTR (Fig. 7). At 37°C, GFP–CFTR was detected both at the cell surface and in a perinuclear location that corresponds to the ER (Moyer et al., 1998). At 37°C, in the presence or absence of UbcH5a C85A, GFP–CFTRΔF508 was only detected in its soluble ER form. However, when UbcH5a C85A-transfected cells were cultured for 24 h at 37°C, treated with cycloheximide, and then incubated at 26°C for 4 h, a pool of GFP–CFTRΔF508 accumulated, in a BFA-sensitive fashion, at the cell surface.


A foldable CFTR{Delta}F508 biogenic intermediate accumulates upon inhibition of the Hsc70-CHIP E3 ubiquitin ligase.

Younger JM, Ren HY, Chen L, Fan CY, Fields A, Patterson C, Cyr DM - J. Cell Biol. (2004)

Localization of GFP–CFTRΔF508 in HEK293 cells. Cells grown on glass coverslips were transiently transfected with GFP–CFTR or GFP–CFTRΔF508 with or without UbcH5a C85A and cultured for 24 h at 37°C. Where indicated 25 μg/ml cyclohexamide and/or brefeldin A (10 μM) was added to culture media. After 4 h of culture at 26°C these cells were fixed and the coverslips were mounted on glass slides. Images were collected and processed as described in the Materials and methods section.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172621&req=5

fig7: Localization of GFP–CFTRΔF508 in HEK293 cells. Cells grown on glass coverslips were transiently transfected with GFP–CFTR or GFP–CFTRΔF508 with or without UbcH5a C85A and cultured for 24 h at 37°C. Where indicated 25 μg/ml cyclohexamide and/or brefeldin A (10 μM) was added to culture media. After 4 h of culture at 26°C these cells were fixed and the coverslips were mounted on glass slides. Images were collected and processed as described in the Materials and methods section.
Mentions: To demonstrate that the CFTRΔF508 that was converted to the C form during the 26°C chase incubation was trafficked to the cell surface the localization of GFP–CFTRΔF508 was examined under these experimental conditions and compared with that of GFP–CFTR (Fig. 7). At 37°C, GFP–CFTR was detected both at the cell surface and in a perinuclear location that corresponds to the ER (Moyer et al., 1998). At 37°C, in the presence or absence of UbcH5a C85A, GFP–CFTRΔF508 was only detected in its soluble ER form. However, when UbcH5a C85A-transfected cells were cultured for 24 h at 37°C, treated with cycloheximide, and then incubated at 26°C for 4 h, a pool of GFP–CFTRΔF508 accumulated, in a BFA-sensitive fashion, at the cell surface.

Bottom Line: CHIP is demonstrated to function as a scaffold that nucleates the formation of a multisubunit E3 ubiquitin ligase whose reconstituted activity toward CFTR is dependent upon Hdj2, Hsc70, and the E2 UbcH5a.Inactivation of the Hsc70-CHIP E3 leads CFTRDeltaF508 to accumulate in a nonaggregated state, which upon lowering of cell growth temperatures, can fold and reach the cell surface.Inhibition of CFTRDeltaF508 ubiquitination can increase its cell surface expression and may provide an approach to treat CF.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT
CFTRDeltaF508 exhibits a correctable protein-folding defect that leads to its misfolding and premature degradation, which is the cause of cystic fibrosis (CF). Herein we report on the characterization of the CFTRDeltaF508 biogenic intermediate that is selected for proteasomal degradation and identification of cellular components that polyubiquitinate CFTRDeltaF508. Nonubiquitinated CFTRDeltaF508 accumulates in a kinetically trapped, but folding competent conformation, that is maintained in a soluble state by cytosolic Hsc70. Ubiquitination of Hsc70-bound CFTRDeltaF508 requires CHIP, a U box containing cytosolic cochaperone. CHIP is demonstrated to function as a scaffold that nucleates the formation of a multisubunit E3 ubiquitin ligase whose reconstituted activity toward CFTR is dependent upon Hdj2, Hsc70, and the E2 UbcH5a. Inactivation of the Hsc70-CHIP E3 leads CFTRDeltaF508 to accumulate in a nonaggregated state, which upon lowering of cell growth temperatures, can fold and reach the cell surface. Inhibition of CFTRDeltaF508 ubiquitination can increase its cell surface expression and may provide an approach to treat CF.

Show MeSH
Related in: MedlinePlus