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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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Triton X-100–soluble CFTR and CFTRΔF508 degradation intermediates accumulate in response to overexpression of UbcH5a C85A. (A and B) Analysis of the solubility of CFTR or CFTRΔF508. HEK293 were transiently tranfected with vectors that express the indicated proteins. As indicated, the proteasome inhibitor ALLN (200 μM) was added to the growth medium 4 h preceding cell lysis. (C) Coexpression of CHIP and UbcH5a C85A blocks CFTR folding and causes its biogenic intermediates to aggregate. CFTR and CFTRΔF508 were detected by Western blot. The fractionation of cell extracts into Triton X-100–soluble and –insoluble material was performed as described in the Materials and methods. The upper panel represents Triton X-100–soluble material, where as the lower part represents Triton X-100–insoluble material. The immaturely glycosylated B form of CFTR and CFTRΔF508, and maturely glycosylated C form of CFTR are denoted.
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fig4: Triton X-100–soluble CFTR and CFTRΔF508 degradation intermediates accumulate in response to overexpression of UbcH5a C85A. (A and B) Analysis of the solubility of CFTR or CFTRΔF508. HEK293 were transiently tranfected with vectors that express the indicated proteins. As indicated, the proteasome inhibitor ALLN (200 μM) was added to the growth medium 4 h preceding cell lysis. (C) Coexpression of CHIP and UbcH5a C85A blocks CFTR folding and causes its biogenic intermediates to aggregate. CFTR and CFTRΔF508 were detected by Western blot. The fractionation of cell extracts into Triton X-100–soluble and –insoluble material was performed as described in the Materials and methods. The upper panel represents Triton X-100–soluble material, where as the lower part represents Triton X-100–insoluble material. The immaturely glycosylated B form of CFTR and CFTRΔF508, and maturely glycosylated C form of CFTR are denoted.

Mentions: CFTR and CFTRΔF508 biogenic intermediates appear to be aggregation prone and therefore are selected for ubiquitination and proteasomal degradation. When the proteasome is inhibited, polyubiquitinated CFTRΔF508 accumulates in detergent-insoluble aggregates (Ward et al., 1995), but nonubiquitinated CFTRΔF508 biogenic intermediates are not well characterized. To investigate the aggregation state of nonubiquitinated CFTRΔF508, we modulated the activity of the Hsc70–CHIP E3 and determined the detergent solubility of the CFTR and CFTRΔF508 biogenic intermediates that accumulated (Fig. 4). Overexpression of UbcH5a and CHIP caused an overall decrease in the pool of CFTR and CFTRΔF508 and none of the B form was detected in the Triton X-100–insoluble fraction. When UbcH5a C85A was overexpressed, severalfold more of the B and C form of CFTR accumulated in the Triton X-100–soluble fraction. To our surprise, we also observed UbcH5a C85A overexpression to drive a severalfold increase in the quantity of the B form of CFTRΔF508 that accumulated in a Triton X-100–soluble state. Under these experimental conditions, we also observed a small quantity of the C form of CFTR in the detergent-insoluble fraction. Because the C form of CFTR is typically soluble in Triton X-100, this material appears to represent a minor contamination of the detergent-insoluble fraction with detergent soluble material.


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)

Triton X-100–soluble CFTR and CFTRΔF508 degradation intermediates accumulate in response to overexpression of UbcH5a C85A. (A and B) Analysis of the solubility of CFTR or CFTRΔF508. HEK293 were transiently tranfected with vectors that express the indicated proteins. As indicated, the proteasome inhibitor ALLN (200 μM) was added to the growth medium 4 h preceding cell lysis. (C) Coexpression of CHIP and UbcH5a C85A blocks CFTR folding and causes its biogenic intermediates to aggregate. CFTR and CFTRΔF508 were detected by Western blot. The fractionation of cell extracts into Triton X-100–soluble and –insoluble material was performed as described in the Materials and methods. The upper panel represents Triton X-100–soluble material, where as the lower part represents Triton X-100–insoluble material. The immaturely glycosylated B form of CFTR and CFTRΔF508, and maturely glycosylated C form of CFTR are denoted.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172621&req=5

fig4: Triton X-100–soluble CFTR and CFTRΔF508 degradation intermediates accumulate in response to overexpression of UbcH5a C85A. (A and B) Analysis of the solubility of CFTR or CFTRΔF508. HEK293 were transiently tranfected with vectors that express the indicated proteins. As indicated, the proteasome inhibitor ALLN (200 μM) was added to the growth medium 4 h preceding cell lysis. (C) Coexpression of CHIP and UbcH5a C85A blocks CFTR folding and causes its biogenic intermediates to aggregate. CFTR and CFTRΔF508 were detected by Western blot. The fractionation of cell extracts into Triton X-100–soluble and –insoluble material was performed as described in the Materials and methods. The upper panel represents Triton X-100–soluble material, where as the lower part represents Triton X-100–insoluble material. The immaturely glycosylated B form of CFTR and CFTRΔF508, and maturely glycosylated C form of CFTR are denoted.
Mentions: CFTR and CFTRΔF508 biogenic intermediates appear to be aggregation prone and therefore are selected for ubiquitination and proteasomal degradation. When the proteasome is inhibited, polyubiquitinated CFTRΔF508 accumulates in detergent-insoluble aggregates (Ward et al., 1995), but nonubiquitinated CFTRΔF508 biogenic intermediates are not well characterized. To investigate the aggregation state of nonubiquitinated CFTRΔF508, we modulated the activity of the Hsc70–CHIP E3 and determined the detergent solubility of the CFTR and CFTRΔF508 biogenic intermediates that accumulated (Fig. 4). Overexpression of UbcH5a and CHIP caused an overall decrease in the pool of CFTR and CFTRΔF508 and none of the B form was detected in the Triton X-100–insoluble fraction. When UbcH5a C85A was overexpressed, severalfold more of the B and C form of CFTR accumulated in the Triton X-100–soluble fraction. To our surprise, we also observed UbcH5a C85A overexpression to drive a severalfold increase in the quantity of the B form of CFTRΔF508 that accumulated in a Triton X-100–soluble state. Under these experimental conditions, we also observed a small quantity of the C form of CFTR in the detergent-insoluble fraction. Because the C form of CFTR is typically soluble in Triton X-100, this material appears to represent a minor contamination of the detergent-insoluble fraction with detergent soluble material.

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