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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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Insensitivity of TCRα and ApoB48 turnover to inhibition of the Hsc70–CHIP E3. (A) TCRα degradation. (B) ApoB48 turnover. HEK293 cells were transiently transfected with expression plasmids for the indicated proteins. 24 h after transfection, cells were labeled for 20 min with 35S-translabel. A chase period was initiated by the addition of cycloheximide and, at the indicated times, cells were harvested and lysed. TCRα or ApoB48 were immunoprecipitated from cell extracts and detected by SDS-PAGE and autoradiography. (C) Quantitation of TCRα and Apob48 levels, the relative amount of each protein present at t = 0 is expressed as 100% of control.
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fig3: Insensitivity of TCRα and ApoB48 turnover to inhibition of the Hsc70–CHIP E3. (A) TCRα degradation. (B) ApoB48 turnover. HEK293 cells were transiently transfected with expression plasmids for the indicated proteins. 24 h after transfection, cells were labeled for 20 min with 35S-translabel. A chase period was initiated by the addition of cycloheximide and, at the indicated times, cells were harvested and lysed. TCRα or ApoB48 were immunoprecipitated from cell extracts and detected by SDS-PAGE and autoradiography. (C) Quantitation of TCRα and Apob48 levels, the relative amount of each protein present at t = 0 is expressed as 100% of control.

Mentions: To ascertain whether or not UbcH5a C85A overexpression generally inhibited ERAD or specifically blocked CFTR degradation, its effect on the degradation of the T cell receptor α subunit (TCRα) was examined (Fig. 3 A). TCRα is a transmembrane protein that exposes a large extracellular domain in the ER lumen whose unassembled form is degraded via an ERAD pathway that uses the E2s Ubc6 and Ubc7 (Tiwari and Weissman, 2001; Lenk et al., 2002). Pulse chase analysis revealed that TCRα had a half-life of around 1 h in absence or presence of UbcH5a C85A (Fig. 3, A and C). In addition, the overexpression of CHIP was not observed to influence the rate of TCRα turnover (unpublished data). In contrast, overexpression of either Ubc6 C91S or Ubc7 C89S led to an increase in the half-life of TCRα from 1 h to 1.5 and 2 h, respectively. Thus, UbcH5a C85A overexpression does not detectably hinder the turnover of a transmembrane ERAD substrate whose degradation can be blocked by interference with the action of Ubc6 and Ubc7.


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)

Insensitivity of TCRα and ApoB48 turnover to inhibition of the Hsc70–CHIP E3. (A) TCRα degradation. (B) ApoB48 turnover. HEK293 cells were transiently transfected with expression plasmids for the indicated proteins. 24 h after transfection, cells were labeled for 20 min with 35S-translabel. A chase period was initiated by the addition of cycloheximide and, at the indicated times, cells were harvested and lysed. TCRα or ApoB48 were immunoprecipitated from cell extracts and detected by SDS-PAGE and autoradiography. (C) Quantitation of TCRα and Apob48 levels, the relative amount of each protein present at t = 0 is expressed as 100% of control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172621&req=5

fig3: Insensitivity of TCRα and ApoB48 turnover to inhibition of the Hsc70–CHIP E3. (A) TCRα degradation. (B) ApoB48 turnover. HEK293 cells were transiently transfected with expression plasmids for the indicated proteins. 24 h after transfection, cells were labeled for 20 min with 35S-translabel. A chase period was initiated by the addition of cycloheximide and, at the indicated times, cells were harvested and lysed. TCRα or ApoB48 were immunoprecipitated from cell extracts and detected by SDS-PAGE and autoradiography. (C) Quantitation of TCRα and Apob48 levels, the relative amount of each protein present at t = 0 is expressed as 100% of control.
Mentions: To ascertain whether or not UbcH5a C85A overexpression generally inhibited ERAD or specifically blocked CFTR degradation, its effect on the degradation of the T cell receptor α subunit (TCRα) was examined (Fig. 3 A). TCRα is a transmembrane protein that exposes a large extracellular domain in the ER lumen whose unassembled form is degraded via an ERAD pathway that uses the E2s Ubc6 and Ubc7 (Tiwari and Weissman, 2001; Lenk et al., 2002). Pulse chase analysis revealed that TCRα had a half-life of around 1 h in absence or presence of UbcH5a C85A (Fig. 3, A and C). In addition, the overexpression of CHIP was not observed to influence the rate of TCRα turnover (unpublished data). In contrast, overexpression of either Ubc6 C91S or Ubc7 C89S led to an increase in the half-life of TCRα from 1 h to 1.5 and 2 h, respectively. Thus, UbcH5a C85A overexpression does not detectably hinder the turnover of a transmembrane ERAD substrate whose degradation can be blocked by interference with the action of Ubc6 and Ubc7.

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