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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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Degradation of CFTR and CFTRΔF508 is inhibited by overexpression of UbcH5A C85A. (A) Western blot analysis of CFTR levels upon overexpression of the E2s UbcH5A, Ubc6, and Ubc7. HEK293 cells were transfected transiently with expression plasmids that encode the indicated proteins. Total cell extracts were prepared 24 h after transfection in SDS-PAGE sample buffer. Nitrocellulose was probed with α-CFTR and developed. The immaturely glycosylated ER localized B form and maturely glycosylated plasma membrane associated C form of CFTR are denoted as B and C, respectively. (B and C) UbcH5A C85A overexpression slows the rate of CFTR and CFTRΔF508 degradation. Cells were labeled for 20 min with 35S-translabel and a chase period was initiated by the addition of cycloheximide. At the indicated times, 35S-CFTR or 35S-CFTRΔF508 was immunoprecipitated from cell extracts. CFTR or CFTRΔF508 isolated in this manner was detected by SDS-PAGE and fluorography. (D and E) Graphs illustrate the processing efficiency and half-life of CFTR and CFTRΔF508. Relative CFTR and CFTRΔF508 levels were quantitated by laser densitometry of the x-ray films shown in B and C, respectively. Values were normalized to the quantity of the B form of CFTR and CFTRΔF508 present at t = 0 under the indicated experimental condition levels. t = 0 values for the B form of CFTR were 1.1 and 1.8 OD, in the absence and presence of UbcH5a C85A, respectively. t = 0 values for the B form of CFTRΔF508 were 2.3 and 7.0 OD, in the absence and presence of UbcH5a C85A, respectively. (F) CHIP and UbcH5a jointly reduce levels of CFTRΔF508 in Western blots.
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fig2: Degradation of CFTR and CFTRΔF508 is inhibited by overexpression of UbcH5A C85A. (A) Western blot analysis of CFTR levels upon overexpression of the E2s UbcH5A, Ubc6, and Ubc7. HEK293 cells were transfected transiently with expression plasmids that encode the indicated proteins. Total cell extracts were prepared 24 h after transfection in SDS-PAGE sample buffer. Nitrocellulose was probed with α-CFTR and developed. The immaturely glycosylated ER localized B form and maturely glycosylated plasma membrane associated C form of CFTR are denoted as B and C, respectively. (B and C) UbcH5A C85A overexpression slows the rate of CFTR and CFTRΔF508 degradation. Cells were labeled for 20 min with 35S-translabel and a chase period was initiated by the addition of cycloheximide. At the indicated times, 35S-CFTR or 35S-CFTRΔF508 was immunoprecipitated from cell extracts. CFTR or CFTRΔF508 isolated in this manner was detected by SDS-PAGE and fluorography. (D and E) Graphs illustrate the processing efficiency and half-life of CFTR and CFTRΔF508. Relative CFTR and CFTRΔF508 levels were quantitated by laser densitometry of the x-ray films shown in B and C, respectively. Values were normalized to the quantity of the B form of CFTR and CFTRΔF508 present at t = 0 under the indicated experimental condition levels. t = 0 values for the B form of CFTR were 1.1 and 1.8 OD, in the absence and presence of UbcH5a C85A, respectively. t = 0 values for the B form of CFTRΔF508 were 2.3 and 7.0 OD, in the absence and presence of UbcH5a C85A, respectively. (F) CHIP and UbcH5a jointly reduce levels of CFTRΔF508 in Western blots.

Mentions: To access whether or not UbcH5a is an in vivo component of Hsc70–CHIP E3, the effect that UbcH5a and UbcH5a C85A overexpression in HEK293 cells had on CFTR biogenesis was determined (Fig. 2 A). In addition, we compared the effect of UbcH5a or UbcH5a C85A overexpression on CFTR biogenesis to that of wild-type and dominant negative mutant forms of the human E2s Ubc6 and Ubc7 (Fig. 2 A). Elevation of UbcH5a levels caused a decrease in the accumulation of the immaturely glycosylated ER localized B form and the maturely glycosylated plasma membrane localized C form of CFTR. In contrast, overexpression of UbcH5a C85A led to a severalfold increase in the steady-state level of the B and C form of CFTR. Ubc6 and Ubc6 C91S overexpression were also observed to influence CFTR expression levels, but the effect that Ubc6 C91S had on the accumulation of the B form of CFTR was modest when compared with results obtained with UbcH5a C85A. On the other hand, Ubc7 and Ubc7 C89S overexpression did not cause a detectable change in the steady-state level of CFTR and CFTRΔF508. Because Ubc6 does not appear to interact with CHIP (Fig. 1), the effect that its overexpression has on CFTR biogenesis appears to result from its ability to function with additional quality control factors that monitor the folded state of CFTR (Gnann et al., 2004).


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)

Degradation of CFTR and CFTRΔF508 is inhibited by overexpression of UbcH5A C85A. (A) Western blot analysis of CFTR levels upon overexpression of the E2s UbcH5A, Ubc6, and Ubc7. HEK293 cells were transfected transiently with expression plasmids that encode the indicated proteins. Total cell extracts were prepared 24 h after transfection in SDS-PAGE sample buffer. Nitrocellulose was probed with α-CFTR and developed. The immaturely glycosylated ER localized B form and maturely glycosylated plasma membrane associated C form of CFTR are denoted as B and C, respectively. (B and C) UbcH5A C85A overexpression slows the rate of CFTR and CFTRΔF508 degradation. Cells were labeled for 20 min with 35S-translabel and a chase period was initiated by the addition of cycloheximide. At the indicated times, 35S-CFTR or 35S-CFTRΔF508 was immunoprecipitated from cell extracts. CFTR or CFTRΔF508 isolated in this manner was detected by SDS-PAGE and fluorography. (D and E) Graphs illustrate the processing efficiency and half-life of CFTR and CFTRΔF508. Relative CFTR and CFTRΔF508 levels were quantitated by laser densitometry of the x-ray films shown in B and C, respectively. Values were normalized to the quantity of the B form of CFTR and CFTRΔF508 present at t = 0 under the indicated experimental condition levels. t = 0 values for the B form of CFTR were 1.1 and 1.8 OD, in the absence and presence of UbcH5a C85A, respectively. t = 0 values for the B form of CFTRΔF508 were 2.3 and 7.0 OD, in the absence and presence of UbcH5a C85A, respectively. (F) CHIP and UbcH5a jointly reduce levels of CFTRΔF508 in Western blots.
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fig2: Degradation of CFTR and CFTRΔF508 is inhibited by overexpression of UbcH5A C85A. (A) Western blot analysis of CFTR levels upon overexpression of the E2s UbcH5A, Ubc6, and Ubc7. HEK293 cells were transfected transiently with expression plasmids that encode the indicated proteins. Total cell extracts were prepared 24 h after transfection in SDS-PAGE sample buffer. Nitrocellulose was probed with α-CFTR and developed. The immaturely glycosylated ER localized B form and maturely glycosylated plasma membrane associated C form of CFTR are denoted as B and C, respectively. (B and C) UbcH5A C85A overexpression slows the rate of CFTR and CFTRΔF508 degradation. Cells were labeled for 20 min with 35S-translabel and a chase period was initiated by the addition of cycloheximide. At the indicated times, 35S-CFTR or 35S-CFTRΔF508 was immunoprecipitated from cell extracts. CFTR or CFTRΔF508 isolated in this manner was detected by SDS-PAGE and fluorography. (D and E) Graphs illustrate the processing efficiency and half-life of CFTR and CFTRΔF508. Relative CFTR and CFTRΔF508 levels were quantitated by laser densitometry of the x-ray films shown in B and C, respectively. Values were normalized to the quantity of the B form of CFTR and CFTRΔF508 present at t = 0 under the indicated experimental condition levels. t = 0 values for the B form of CFTR were 1.1 and 1.8 OD, in the absence and presence of UbcH5a C85A, respectively. t = 0 values for the B form of CFTRΔF508 were 2.3 and 7.0 OD, in the absence and presence of UbcH5a C85A, respectively. (F) CHIP and UbcH5a jointly reduce levels of CFTRΔF508 in Western blots.
Mentions: To access whether or not UbcH5a is an in vivo component of Hsc70–CHIP E3, the effect that UbcH5a and UbcH5a C85A overexpression in HEK293 cells had on CFTR biogenesis was determined (Fig. 2 A). In addition, we compared the effect of UbcH5a or UbcH5a C85A overexpression on CFTR biogenesis to that of wild-type and dominant negative mutant forms of the human E2s Ubc6 and Ubc7 (Fig. 2 A). Elevation of UbcH5a levels caused a decrease in the accumulation of the immaturely glycosylated ER localized B form and the maturely glycosylated plasma membrane localized C form of CFTR. In contrast, overexpression of UbcH5a C85A led to a severalfold increase in the steady-state level of the B and C form of CFTR. Ubc6 and Ubc6 C91S overexpression were also observed to influence CFTR expression levels, but the effect that Ubc6 C91S had on the accumulation of the B form of CFTR was modest when compared with results obtained with UbcH5a C85A. On the other hand, Ubc7 and Ubc7 C89S overexpression did not cause a detectable change in the steady-state level of CFTR and CFTRΔF508. Because Ubc6 does not appear to interact with CHIP (Fig. 1), the effect that its overexpression has on CFTR biogenesis appears to result from its ability to function with additional quality control factors that monitor the folded state of CFTR (Gnann et al., 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.

Show MeSH
Related in: MedlinePlus