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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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Reconstitution of CFTR ubiquitination. (A) Ubiquitination of gst–NBD1–R. (B) Ubc6 and Ubc7 cannot cooperate with Hsc70 and CHIP to ubiquitinate gst–NBD1–R. (C) CHIP K30A is defective in polyubiquitination. (D) UbcH5a C85A cannot ubiquitinate Gst–NBD–R. Ubiquitination of Gst–NBD–R (1 μM) was performed at 37°C and where indicated the following components were present: UbcH5a (4 μM), UbcH5a C85A (4 μM), Ubc6 (4 μM), Ubc7 (4 μM), CHIP (3 μM), CHIP K30A (3 μM), Hsc70 (2 μM), and Hdj2 (4 μM). In 1B-D Hsc70 and Hdj-2 were in all reactions except for lane 1. Incubations in A, C, and D were for 2 h. Ubiquitination of Gst–NBD–R was determined by Western blot with α-R domain antibody. In the x-ray films shown in A–D the band that corresponds to Gst–NBD–R is overexposed to allow for the visualization of its ubiquitinated forms. Quantitation of shorter exposures of these x-ray films indicates that >85% of Gst–NBD–R is polyubiquitinated by the joint action of UbcH5a, Hsc70, Hdj-2, and CHIP (A, lane 5). When Hsc70 or Hdj-2 were absent (A, lanes 2 and 3), UbcH5a and CHIP ubiquitinated <3% of Gst–NBD–R.
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fig1: Reconstitution of CFTR ubiquitination. (A) Ubiquitination of gst–NBD1–R. (B) Ubc6 and Ubc7 cannot cooperate with Hsc70 and CHIP to ubiquitinate gst–NBD1–R. (C) CHIP K30A is defective in polyubiquitination. (D) UbcH5a C85A cannot ubiquitinate Gst–NBD–R. Ubiquitination of Gst–NBD–R (1 μM) was performed at 37°C and where indicated the following components were present: UbcH5a (4 μM), UbcH5a C85A (4 μM), Ubc6 (4 μM), Ubc7 (4 μM), CHIP (3 μM), CHIP K30A (3 μM), Hsc70 (2 μM), and Hdj2 (4 μM). In 1B-D Hsc70 and Hdj-2 were in all reactions except for lane 1. Incubations in A, C, and D were for 2 h. Ubiquitination of Gst–NBD–R was determined by Western blot with α-R domain antibody. In the x-ray films shown in A–D the band that corresponds to Gst–NBD–R is overexposed to allow for the visualization of its ubiquitinated forms. Quantitation of shorter exposures of these x-ray films indicates that >85% of Gst–NBD–R is polyubiquitinated by the joint action of UbcH5a, Hsc70, Hdj-2, and CHIP (A, lane 5). When Hsc70 or Hdj-2 were absent (A, lanes 2 and 3), UbcH5a and CHIP ubiquitinated <3% of Gst–NBD–R.

Mentions: Gst–NBD1–R was incubated with different combinations of chaperones and ubiquitination enzymes and its ubiquitination was monitored by analyzing the retardation of its mobility on SDS-PAGE gels (Fig. 1 A). E1 and UbcH5a were unable to facilitate the ubiquitination of gst–NBD1–R and the addition of CHIP lead to the formation of a small quantity of ubiquitinated gst–NBD1–R. The presence of Hsc70 or Hdj-2 in combination with CHIP further stimulated the ubiquitination by UbcH5a, but the efficiency of this reaction remained low with <3% of gst–NBD1–R being detected as a mono-, di-, or triubiquitinated species. However, >85% of gst–NBD1–R was polyubiquitinated when E1, UbcH5a, Hsc70, Hdj-2, and CHIP were jointly present. Mutation of conserved residues in CHIPs U box, H260A and P269A, greatly reduced gst–NBD1–R polyubiquitination. In addition, mutation of K30A in the TPR repeat domain reduced CHIPs ubiquitination activity to levels observed when CHIP and Hdj-2 were present, but Hsc70 was omitted (Fig. 1, A and C). CHIP cooperates with UbcH5a, Hsc70, and Hdj-2 in a TPR repeat and U box–dependent manner to facilitate CFTR polyubiquitination.


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)

Reconstitution of CFTR ubiquitination. (A) Ubiquitination of gst–NBD1–R. (B) Ubc6 and Ubc7 cannot cooperate with Hsc70 and CHIP to ubiquitinate gst–NBD1–R. (C) CHIP K30A is defective in polyubiquitination. (D) UbcH5a C85A cannot ubiquitinate Gst–NBD–R. Ubiquitination of Gst–NBD–R (1 μM) was performed at 37°C and where indicated the following components were present: UbcH5a (4 μM), UbcH5a C85A (4 μM), Ubc6 (4 μM), Ubc7 (4 μM), CHIP (3 μM), CHIP K30A (3 μM), Hsc70 (2 μM), and Hdj2 (4 μM). In 1B-D Hsc70 and Hdj-2 were in all reactions except for lane 1. Incubations in A, C, and D were for 2 h. Ubiquitination of Gst–NBD–R was determined by Western blot with α-R domain antibody. In the x-ray films shown in A–D the band that corresponds to Gst–NBD–R is overexposed to allow for the visualization of its ubiquitinated forms. Quantitation of shorter exposures of these x-ray films indicates that >85% of Gst–NBD–R is polyubiquitinated by the joint action of UbcH5a, Hsc70, Hdj-2, and CHIP (A, lane 5). When Hsc70 or Hdj-2 were absent (A, lanes 2 and 3), UbcH5a and CHIP ubiquitinated <3% of Gst–NBD–R.
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fig1: Reconstitution of CFTR ubiquitination. (A) Ubiquitination of gst–NBD1–R. (B) Ubc6 and Ubc7 cannot cooperate with Hsc70 and CHIP to ubiquitinate gst–NBD1–R. (C) CHIP K30A is defective in polyubiquitination. (D) UbcH5a C85A cannot ubiquitinate Gst–NBD–R. Ubiquitination of Gst–NBD–R (1 μM) was performed at 37°C and where indicated the following components were present: UbcH5a (4 μM), UbcH5a C85A (4 μM), Ubc6 (4 μM), Ubc7 (4 μM), CHIP (3 μM), CHIP K30A (3 μM), Hsc70 (2 μM), and Hdj2 (4 μM). In 1B-D Hsc70 and Hdj-2 were in all reactions except for lane 1. Incubations in A, C, and D were for 2 h. Ubiquitination of Gst–NBD–R was determined by Western blot with α-R domain antibody. In the x-ray films shown in A–D the band that corresponds to Gst–NBD–R is overexposed to allow for the visualization of its ubiquitinated forms. Quantitation of shorter exposures of these x-ray films indicates that >85% of Gst–NBD–R is polyubiquitinated by the joint action of UbcH5a, Hsc70, Hdj-2, and CHIP (A, lane 5). When Hsc70 or Hdj-2 were absent (A, lanes 2 and 3), UbcH5a and CHIP ubiquitinated <3% of Gst–NBD–R.
Mentions: Gst–NBD1–R was incubated with different combinations of chaperones and ubiquitination enzymes and its ubiquitination was monitored by analyzing the retardation of its mobility on SDS-PAGE gels (Fig. 1 A). E1 and UbcH5a were unable to facilitate the ubiquitination of gst–NBD1–R and the addition of CHIP lead to the formation of a small quantity of ubiquitinated gst–NBD1–R. The presence of Hsc70 or Hdj-2 in combination with CHIP further stimulated the ubiquitination by UbcH5a, but the efficiency of this reaction remained low with <3% of gst–NBD1–R being detected as a mono-, di-, or triubiquitinated species. However, >85% of gst–NBD1–R was polyubiquitinated when E1, UbcH5a, Hsc70, Hdj-2, and CHIP were jointly present. Mutation of conserved residues in CHIPs U box, H260A and P269A, greatly reduced gst–NBD1–R polyubiquitination. In addition, mutation of K30A in the TPR repeat domain reduced CHIPs ubiquitination activity to levels observed when CHIP and Hdj-2 were present, but Hsc70 was omitted (Fig. 1, A and C). CHIP cooperates with UbcH5a, Hsc70, and Hdj-2 in a TPR repeat and U box–dependent manner to facilitate CFTR polyubiquitination.

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