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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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Characterization of the CFTRΔF508 biogenic intermediate that accumulates when UbcH5a C85A is overexpressed. (A) Analysis of CFTRΔ508 levels when its proteasomal degradation is inhibited by different methods. HEK293 cells were transfected with the indicated expression vectors and harvested in SDS sample buffer 24 h later. ALLN (200 μM) was added to culture media 4 h before cell harvest. CFTRΔF508 levels were determined by Western blot. Ubiquitinated CFTRΔF508 runs as a high molecular smear on SDS-PAGE gels and is denoted. (B) Complex formation between Hsc70 and CFTRΔF508. Cells were transfected with the indicated expression plasmids and 24 h after incubation they were radiolabeled for 30 min with 35S-translabel and harvested. Half of each cell pellet was lysed under denaturing or native buffer conditions and immunoprecipitations were performed and analyzed by fluorography. (C) CFTRΔF508 that accumulates in the presence of UbcH5a C85A is glycosylated. Cells were harvested and half of each respective lysate was treated with endoglycosidase H whereas the other half served as the control. A and B denote the mobility of nonglycosylated and immaturely glycosylated forms of CFTRΔF508, respectively.
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fig5: Characterization of the CFTRΔF508 biogenic intermediate that accumulates when UbcH5a C85A is overexpressed. (A) Analysis of CFTRΔ508 levels when its proteasomal degradation is inhibited by different methods. HEK293 cells were transfected with the indicated expression vectors and harvested in SDS sample buffer 24 h later. ALLN (200 μM) was added to culture media 4 h before cell harvest. CFTRΔF508 levels were determined by Western blot. Ubiquitinated CFTRΔF508 runs as a high molecular smear on SDS-PAGE gels and is denoted. (B) Complex formation between Hsc70 and CFTRΔF508. Cells were transfected with the indicated expression plasmids and 24 h after incubation they were radiolabeled for 30 min with 35S-translabel and harvested. Half of each cell pellet was lysed under denaturing or native buffer conditions and immunoprecipitations were performed and analyzed by fluorography. (C) CFTRΔF508 that accumulates in the presence of UbcH5a C85A is glycosylated. Cells were harvested and half of each respective lysate was treated with endoglycosidase H whereas the other half served as the control. A and B denote the mobility of nonglycosylated and immaturely glycosylated forms of CFTRΔF508, respectively.

Mentions: To investigate the nature of the detergent-soluble CFTRΔF508 degradation intermediate that accumulated when UbcH5a C85A was overexpressed, we compared its ubiquitination state to that of degradation intermediates that accumulate in response to proteasome inhibition by ALLN, or overexpression of a dominant negative form of p97 (p97 QQ; Ye et al., 2003). P97 is a cytosolic chaperone that extracts polyubiquitinated proteins from the ER and participates in CFTRΔF508 degradation (Ye et al., 2003; Dalal et al., 2004). Nonubiquitinated CFTRΔF508 migrates on SDS-PAGE gels with the same mobility as its B form, where as polyubiquitinated CFTRΔF508, which accumulated when its degradation was blocked by ALLN or p97 QQ, migrates on SDS-PAGE gels as a high molecular weight smear (Fig. 5 A). CFTRΔF508 that accumulated in response to UbcH5a C85A overexpression did not migrate as a high molecular smear and, therefore, represents a nonubiquitinated species. This result is consistent with the notion that UbcH5a C85A inhibits CFTRΔF508 ubiquitination and thereby blocks its degradation.


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)

Characterization of the CFTRΔF508 biogenic intermediate that accumulates when UbcH5a C85A is overexpressed. (A) Analysis of CFTRΔ508 levels when its proteasomal degradation is inhibited by different methods. HEK293 cells were transfected with the indicated expression vectors and harvested in SDS sample buffer 24 h later. ALLN (200 μM) was added to culture media 4 h before cell harvest. CFTRΔF508 levels were determined by Western blot. Ubiquitinated CFTRΔF508 runs as a high molecular smear on SDS-PAGE gels and is denoted. (B) Complex formation between Hsc70 and CFTRΔF508. Cells were transfected with the indicated expression plasmids and 24 h after incubation they were radiolabeled for 30 min with 35S-translabel and harvested. Half of each cell pellet was lysed under denaturing or native buffer conditions and immunoprecipitations were performed and analyzed by fluorography. (C) CFTRΔF508 that accumulates in the presence of UbcH5a C85A is glycosylated. Cells were harvested and half of each respective lysate was treated with endoglycosidase H whereas the other half served as the control. A and B denote the mobility of nonglycosylated and immaturely glycosylated forms of CFTRΔF508, respectively.
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Related In: Results  -  Collection

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fig5: Characterization of the CFTRΔF508 biogenic intermediate that accumulates when UbcH5a C85A is overexpressed. (A) Analysis of CFTRΔ508 levels when its proteasomal degradation is inhibited by different methods. HEK293 cells were transfected with the indicated expression vectors and harvested in SDS sample buffer 24 h later. ALLN (200 μM) was added to culture media 4 h before cell harvest. CFTRΔF508 levels were determined by Western blot. Ubiquitinated CFTRΔF508 runs as a high molecular smear on SDS-PAGE gels and is denoted. (B) Complex formation between Hsc70 and CFTRΔF508. Cells were transfected with the indicated expression plasmids and 24 h after incubation they were radiolabeled for 30 min with 35S-translabel and harvested. Half of each cell pellet was lysed under denaturing or native buffer conditions and immunoprecipitations were performed and analyzed by fluorography. (C) CFTRΔF508 that accumulates in the presence of UbcH5a C85A is glycosylated. Cells were harvested and half of each respective lysate was treated with endoglycosidase H whereas the other half served as the control. A and B denote the mobility of nonglycosylated and immaturely glycosylated forms of CFTRΔF508, respectively.
Mentions: To investigate the nature of the detergent-soluble CFTRΔF508 degradation intermediate that accumulated when UbcH5a C85A was overexpressed, we compared its ubiquitination state to that of degradation intermediates that accumulate in response to proteasome inhibition by ALLN, or overexpression of a dominant negative form of p97 (p97 QQ; Ye et al., 2003). P97 is a cytosolic chaperone that extracts polyubiquitinated proteins from the ER and participates in CFTRΔF508 degradation (Ye et al., 2003; Dalal et al., 2004). Nonubiquitinated CFTRΔF508 migrates on SDS-PAGE gels with the same mobility as its B form, where as polyubiquitinated CFTRΔF508, which accumulated when its degradation was blocked by ALLN or p97 QQ, migrates on SDS-PAGE gels as a high molecular weight smear (Fig. 5 A). CFTRΔF508 that accumulated in response to UbcH5a C85A overexpression did not migrate as a high molecular smear and, therefore, represents a nonubiquitinated species. This result is consistent with the notion that UbcH5a C85A inhibits CFTRΔF508 ubiquitination and thereby blocks its degradation.

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