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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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Glycolytic maturation of CFTRΔF508 degradation intermediates that accumulate in response to UbcH5a C85A overexpression. (A) The CFTRΔF508 that accumulates when UbcH5a C85A is overexpressed becomes maturely glycosylated when cell growth temperatures are reduced to 26°C. HEK293 cells were transiently transfected with the indicated plasmids and were treated with cycloheximide (25 μg/ml) 24 h later. Cells were then either shifted to 26°C or maintained at 37°C for the indicated time.(B) Glycolytic processing of CFTRΔF508 in cells incubated at 26°C for 16 h is blocked by BFA and enhanced by the chemical chaperone TMAO (75 mM). (C) COS-7 cells are capable of maintaining CFTRΔF508 degradation intermediates in a folding competent state. This experiment was performed as described for panel A except that the chase time was for 24 h at 26°C. The immaturely glycosylated and maturely glycosylated forms of CFTRΔF508 are denoted as B and C, respectively.
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fig6: Glycolytic maturation of CFTRΔF508 degradation intermediates that accumulate in response to UbcH5a C85A overexpression. (A) The CFTRΔF508 that accumulates when UbcH5a C85A is overexpressed becomes maturely glycosylated when cell growth temperatures are reduced to 26°C. HEK293 cells were transiently transfected with the indicated plasmids and were treated with cycloheximide (25 μg/ml) 24 h later. Cells were then either shifted to 26°C or maintained at 37°C for the indicated time.(B) Glycolytic processing of CFTRΔF508 in cells incubated at 26°C for 16 h is blocked by BFA and enhanced by the chemical chaperone TMAO (75 mM). (C) COS-7 cells are capable of maintaining CFTRΔF508 degradation intermediates in a folding competent state. This experiment was performed as described for panel A except that the chase time was for 24 h at 26°C. The immaturely glycosylated and maturely glycosylated forms of CFTRΔF508 are denoted as B and C, respectively.

Mentions: To ascertain whether or not the CFTRΔF508 biogenic intermediate that accumulates in response to inhibition of Hsc70–CHIP E3 function is capable of folding we determined if it could be chased to its maturely glycosylated C form (Fig. 6 A). Transiently transfected HEK293 cells were grown for 24 h after transfection and then treated with cycloheximide to inhibit new protein synthesis. The fate of the accumulated CFTRΔF508 was then monitored by Western blot after the indicated chase incubation at 37° or 26°C (Fig. 6 A). The low-temperature chase incubation was incorporated into the design of this experiment because nascent CFTRΔF508 exhibits a temperature-sensitive folding defect and can fold to the native state and accumulate in its maturely glycosylated C form when cells are cultured at 26°C (Denning et al., 1992).


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)

Glycolytic maturation of CFTRΔF508 degradation intermediates that accumulate in response to UbcH5a C85A overexpression. (A) The CFTRΔF508 that accumulates when UbcH5a C85A is overexpressed becomes maturely glycosylated when cell growth temperatures are reduced to 26°C. HEK293 cells were transiently transfected with the indicated plasmids and were treated with cycloheximide (25 μg/ml) 24 h later. Cells were then either shifted to 26°C or maintained at 37°C for the indicated time.(B) Glycolytic processing of CFTRΔF508 in cells incubated at 26°C for 16 h is blocked by BFA and enhanced by the chemical chaperone TMAO (75 mM). (C) COS-7 cells are capable of maintaining CFTRΔF508 degradation intermediates in a folding competent state. This experiment was performed as described for panel A except that the chase time was for 24 h at 26°C. The immaturely glycosylated and maturely glycosylated forms of CFTRΔF508 are denoted as B and C, respectively.
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Related In: Results  -  Collection

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fig6: Glycolytic maturation of CFTRΔF508 degradation intermediates that accumulate in response to UbcH5a C85A overexpression. (A) The CFTRΔF508 that accumulates when UbcH5a C85A is overexpressed becomes maturely glycosylated when cell growth temperatures are reduced to 26°C. HEK293 cells were transiently transfected with the indicated plasmids and were treated with cycloheximide (25 μg/ml) 24 h later. Cells were then either shifted to 26°C or maintained at 37°C for the indicated time.(B) Glycolytic processing of CFTRΔF508 in cells incubated at 26°C for 16 h is blocked by BFA and enhanced by the chemical chaperone TMAO (75 mM). (C) COS-7 cells are capable of maintaining CFTRΔF508 degradation intermediates in a folding competent state. This experiment was performed as described for panel A except that the chase time was for 24 h at 26°C. The immaturely glycosylated and maturely glycosylated forms of CFTRΔF508 are denoted as B and C, respectively.
Mentions: To ascertain whether or not the CFTRΔF508 biogenic intermediate that accumulates in response to inhibition of Hsc70–CHIP E3 function is capable of folding we determined if it could be chased to its maturely glycosylated C form (Fig. 6 A). Transiently transfected HEK293 cells were grown for 24 h after transfection and then treated with cycloheximide to inhibit new protein synthesis. The fate of the accumulated CFTRΔF508 was then monitored by Western blot after the indicated chase incubation at 37° or 26°C (Fig. 6 A). The low-temperature chase incubation was incorporated into the design of this experiment because nascent CFTRΔF508 exhibits a temperature-sensitive folding defect and can fold to the native state and accumulate in its maturely glycosylated C form when cells are cultured at 26°C (Denning et al., 1992).

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