SYVN1, NEDD8, and FBXO2 Proteins Regulate ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Ubiquitin-mediated Proteasomal Degradation

Abstract

We previously reported that delivery of a microRNA-138 mimic or siRNA against SIN3A to cultured cystic fibrosis (ΔF508/ΔF508) airway epithelia partially restored ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR)-mediated cAMP-stimulated Cl^- conductance. We hypothesized that dissecting this microRNA-138/SIN3A-regulated gene network would identify individual proteins contributing to the rescue of ΔF508-CFTR function. Among the genes in the network, we rigorously validated candidates using functional CFTR maturation and electrolyte transport assays in polarized airway epithelia. We found that depletion of the ubiquitin ligase SYVN1, the ubiquitin/proteasome system regulator NEDD8, or the F-box protein FBXO2 partially restored ΔF508-CFTR-mediated Cl^- transport in primary cultures of human cystic fibrosis airway epithelia. Moreover, knockdown of SYVN1, NEDD8, or FBXO2 in combination with corrector compound 18 further potentiated rescue of ΔF508-CFTR-mediated Cl^- conductance. This study provides new knowledge of the CFTR biosynthetic pathway. It suggests that SYVN1 and FBXO2 represent two distinct multiprotein complexes that may degrade ΔF508-CFTR in airway epithelia and identifies a new role for NEDD8 in regulating ΔF508-CFTR ubiquitination.

Keywords: E3 ubiquitin ligase; airway epithelia; cystic fibrosis; endoplasmic-reticulum-associated protein degradation (ERAD); neddylation; proteostasis; ubiquitylation (ubiquitination).

FIGURE 1.

Prioritizing candidate genes by an integrated network analysis. A, flowchart describing identification of 13 candidate genes for the functional screen. DEG, differentially expressed gene; ↑, up-regulated; ↓, down-regulated. B, 67 of the 144 co-regulated differentially expressed genes demonstrate protein/protein interactions. STRING-based predicted protein/protein interactome for 67 genes is shown. C, STRING-based predicted protein/protein interactome for 13 candidate genes.

FIGURE 2.

ΔF508-CFTR biosynthesis is regulated by SYVN1 and NEDD8. A, HeLa-ΔF508-CFTR-HA cells were transfected with the DsiRNAs targeting each candidate gene. 24 h post-transfection, ΔF508-CFTR surface display was measured using an anti-HA antibody. As negative controls, cells were transfected with siScr or left untreated (no treatment (NoT)). As positive controls, SIN3A expression was depleted with a DsiRNA, or cells were treated with the corrector compound C18 for 24 h (6 μm). -Fold increase and significance are plotted relative to siScr transfection. C18 (6 μm) was administered for 24 h. n = 48. B, representative immunoblot depicting ΔF508-CFTR expression in CFBE cells. CFBE cells were transfected with the DsiRNAs targeting each candidate gene. As negative controls, cells were transfected with siScr, or left untreated (no treatment (NoT)). As positive controls, SIN3A expression was depleted with a DsiRNA, or cells were treated with the corrector compound C18 for 24 h (6 μm). Protein was harvested 72 h post-treatment. Densitometry represents -fold increase of ΔF508-CFTR bands C and B in CFBE cells relative to siScr. n = 4. C18 (6 μm) was administered for 24 h. C, change in I_t in response to forskolin and IBMX (F&I) treatment in polarized ALI cultures of CFBE cells. n = 8 (minimum) cultures per treatment. C18 (6 μm) was administered basolaterally 24 h prior to electrophysiology study. A and B represent mean with error bars indicating S.E. C, box and whisker plot (minimum-maximum) represents median. Statistical significance was determined by one-way ANOVA with Holm-Bonferroni correction: *, p < 0.01 (relative to siScr); #, p < 0.01.

FIGURE 3.

SYVN1 or NEDD8 depletion in concert with C18 or low temperature enhance functional rescue of ΔF508-CFTR. A, ΔF508-CFTR ubiquitination measured 72 h after the indicated treatments. CFTR was immunoprecipitated with anti-HA antibody, and ubiquitin was detected with anti-ubiquitin antibody. CFTR protein levels were detected using the anti-HA antibody. C18 (6 μm) and 27 °C were administered 24 h prior to harvesting protein. Densitometry is relative to siScr. n = 4. B, surface display of ΔF508-CFTR in HeLa cells measured by cell surface ELISA 72 h after the indicated treatments. -Fold increase and significance relative to siScr transfection are shown. C18 (6 μm) was administered for 24 h; low temperature (27 °C) was administered for 24 h. n = 18. C and D, membrane stability of ΔF508-CFTR in HeLa cells measured by pulse-chase cell surface ELISA 72 h after the indicated treatments. Chase was performed at 37 °C. n = 18. E, representative immunoblot depicting ΔF508-CFTR expression in CFBE cells. Protein was harvested 72 h post-treatment. Densitometry represents -fold increase of ΔF508-CFTR bands C and B relative to siScr in CFBE cells. n = 4. F, change in I_t in response to forskolin and IBMX (F&I) treatment in polarized ALI cultures of CFBE cells. n = 10 (minimum) cultures per treatment. C18 (6 μm) and 27 °C treatment were for 24 h prior to electrophysiology study. A and B represent mean with error bars indicating S.E. F, box and whisker plot (minimum-maximum) represents median. Statistical significance was determined by one-way ANOVA with Holm-Bonferroni correction: *, p < 0.01 (relative to siScr); #, p < 0.01. WB, Western blotting; Ab, antibody; NoT, no treatment; Denat, denatured; Ub, ubiquitin; PM, plasma membrane.

FIGURE 4.

SYVN1 regulates ΔF508-CFTR biosynthesis in part via the RNF5/AMFR pathway. A, representative immunoblots depicting rescue of ΔF508-CFTR maturation in CFBE cells. Protein was harvested 72 h post-treatment. Densitometry represents -fold increase of ΔF508-CFTR bands C and B relative to siScr in CFBE cells. n = 3. B, surface display of ΔF508-CFTR in HeLa cells measured by cell surface ELISA 72 h after the indicated treatments. -Fold increase and significance are relative to siScr transfection. n = 18. C, representative immunoblot depicting ΔF508-CFTR expression in CFBE cells. Protein was harvested 72 h post-treatment. Densitometry represents -fold increase of ΔF508-CFTR bands C and B relative to siScr in CFBE cells. n = 4. D, change in I_t in response to forskolin and IBMX (F&I) treatment in polarized ALI cultures of CFBE cells. n = 6. E, ΔF508-CFTR ubiquitination measured 72 h after the indicated treatments. CFTR was immunoprecipitated with anti-HA antibody, and ubiquitin was detected with anti-ubiquitin antibody. Densitometry is relative to siScr. n = 4. A, B, C, and E represent mean with error bars indicating S.E. D, box and whisker plot (minimum-maximum) represents median. Statistical significance was determined by one-way ANOVA with Holm-Bonferroni correction: *, p < 0.01 (relative to siScr); #, p < 0.01. WB, Western blotting; Ab, antibody; NoT, no treatment; Ub, ubiquitin.

FIGURE 5.

Catalytic domain of SYVN1 is important to regulate ΔF508-CFTR biosynthesis. A, co-immunoprecipitation of AMFR, SYVN1, and ΔF508-CFTR after immunoprecipitation of AMFR (anti-AMFR antibody), SYVN1 (anti-SYVN1 antibody), or ΔF508-CFTR (anti-HA antibody). B, co-immunoprecipitation of AMFR with ΔF508-CFTR (immunoprecipitated with anti-HA antibody) measured 72 h after the indicated treatments. Densitometry is relative to siScr. n = 3. C, surface display of ΔF508-CFTR in HeLa cells measured by cell surface ELISA 72 h after the indicated treatments. -Fold increase and significance are relative to siScr transfection. n = 18. D, ΔF508-CFTR ubiquitination measured 72 h after the indicated treatments. CFTR was immunoprecipitated with anti-HA antibody, and ubiquitin was detected with anti-ubiquitin antibody. CFTR protein levels were detected using the anti-HA antibody. Densitometry is relative to siScr. n = 4. B–D represent mean with error bars indicating S.E. Statistical significance was determined by one-way ANOVA with Holm-Bonferroni correction: *, p < 0.01 (relative to siScr); #, p < 0.01. WB, Western blotting; Ab, antibody; NoT, no treatment; Ub, ubiquitin; Denat, denatured; dnSYVN1, dominant-negative SYVN1; SYVN1exp, exogenously expressed wild-type SYVN1; Neat, total protein.

FIGURE 6.

NEDD8 and FBXO2 exhibit overlapping action in regulating ΔF508-CFTR biosynthesis. A, representative immunoblots depicting rescue of ΔF508-CFTR maturation in CFBE cells. Protein was harvested 72 h post-treatment. Densitometry represents -fold increase of ΔF508-CFTR bands C and B relative to siScr in CFBE cells. n = 3. B, surface display of ΔF508-CFTR in HeLa cells measured by cell surface ELISA 72 h after the indicated treatments. -Fold increase and significance are relative to siScr transfection. n = 18. C, representative immunoblot depicting ΔF508-CFTR expression in CFBE cells. Protein was harvested 72 h post-treatment. Densitometry represents -fold increase of ΔF508-CFTR bands C and B relative to siScr in CFBE cells. n = 4. D, change in I_t in response to forskolin and IBMX (F&I) treatment in polarized ALI cultures of CFBE cells. n = 6. E, ΔF508-CFTR ubiquitination measured 72 h after the indicated treatments. CFTR was immunoprecipitated with anti-HA antibody, and ubiquitin was detected with anti-ubiquitin antibody. Densitometry is relative to siScr. n = 4. A, B, C, and E represent mean with error bars indicating S.E. D, box and whisker plot (minimum-maximum) represents median. Statistical significance was determined by one-way ANOVA with Holm-Bonferroni correction: *, p < 0.01 (relative to siScr); #, p < 0.01; ns, not significant. WB, Western blotting; Ab, antibody; NoT, no treatment; Ub, ubiquitin; Denat, denatured.

FIGURE 7.

Inhibition of SYVN1, NEDD8, or FBXO2 partially restores ΔF508-CFTR function in primary CF airway epithelial cells. A, change in I_t in response to forskolin and IBMX (F&I) treatment in polarized primary airway epithelial cell cultures. n = 4 (minimum) cultures per donor; all treatments were replicated in six donors or more; FBXO2 treatment was replicated in three donors. B, LDH levels measured in the airway surface liquid and basolateral medium of primary air-liquid interface non-CF airway epithelial cultures every 4 days for a period of 28 days post-transfection with the noted reagents. n = 3 donors (three cultures per donor). Error bars indicate S.E. C, cell morphology was assessed by hematoxylin and eosin (H&E) staining and examination of sections of primary non-CF airway epithelial cell cultures at days 14 and 28 post-transfection with the noted interventions. A well differentiated morphology was maintained across all interventions. n = 3 donors (three cultures per donor). A, box and whisker plot (minimum-maximum) represents median. Statistical significance was determined by one-way ANOVA with Holm-Bonferroni correction: *, p < 0.01 (relative to siScr); #, p < 0.01. NoT, no treatment.