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. 2023 Nov 30;12(23):2741.
doi: 10.3390/cells12232741.

UBE3C Facilitates the ER-Associated and Peripheral Degradation of Misfolded CFTR

Affiliations

UBE3C Facilitates the ER-Associated and Peripheral Degradation of Misfolded CFTR

Yuka Kamada et al. Cells. .

Abstract

The ubiquitin E3 ligase UBE3C promotes the proteasomal degradation of cytosolic proteins and endoplasmic reticulum (ER) membrane proteins. UBE3C is proposed to function downstream of the RNF185/MBRL ER-associated degradation (ERAD) branch, contributing to the ERAD of select membrane proteins. Here, we report that UBE3C facilitates the ERAD of misfolded CFTR, even in the absence of both RNF185 and its functional ortholog RNF5 (RNF5/185). Unlike RNF5/185, UBE3C had a limited impact on the ubiquitination of misfolded CFTR. UBE3C knockdown (KD) resulted in an additional increase in the functional ∆F508-CFTR channels on the plasma membrane when combined with the RNF5/185 ablation, particularly in the presence of clinically used CFTR modulators. Interestingly, although UBE3C KD failed to attenuate the ERAD of insig-1, it reduced the ERAD of misfolded ∆Y490-ABCB1 and increased cell surface expression. UBE3C KD also stabilized the mature form of ∆F508-CFTR and increased the cell surface level of T70-CFTR, a class VI CFTR mutant. These results suggest that UBE3C plays a vital role in the ERAD of misfolded CFTR and ABCB1, even within the RNF5/185-independent ERAD pathway, and it may also be involved in maintaining the peripheral quality control of CFTR.

Keywords: ABCB1; CFTR; ERAD; RNF185; RNF5; UBE3C; protein quality control; ubiquitin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
UBE3C limits the abundance of ∆F508-CFTR. (A) Western blotting shows HBH-∆F508-CFTR-3HA level in 293MSR cells transfected with 25 nM siNC, siUBE3C #1, or siUBE3C #3. Cells were treated with or without Trikafta (3 µM VX-661, 1 µM VX-445, 1 µM VX-770) for 1 day. The quantities of immature (B band) and mature (C band) ΔF508-CFTR were quantified using densitometry and presented as a percentage relative to the control. Cell lysates were prepared 4 days post-transfection. Ponceau staining was used as a loading control. (B) RT-qPCR measured UBE3C KD in 293MSR cells at 2 days post-transfection of siRNA (25 nM). Statistical significance was evaluated using a one-way repeated-measures (RM) ANOVA alongside Dunnett’s multiple comparison tests (n = 3). (C) Western blotting shows HBH-∆F508-CFTR-3HA level in RNF5/185 DKO cells as performed as A. Ablation of RNF5 and RNF185 was confirmed by an anti-RNF185 antibody that recognizes both RNF5 and RNF185 and the lysate from 293MSR cells (WT) was employed to confirm the presence of bands corresponding to RNF185 and RNF5. (D) PM level of ∆F508-CFTR-Nluc(Ex) in 293MSR WT and RNF5/185 DKO cells with 25 nM siNC (-) or siUBE3C #1 (+) (n = 3). Cells were treated with Trikafta (3 µM VX-661, 1 µM VX-445, 1 µM VX-770) for 1 day. A two-way RM ANOVA with Holm-Sidak multiple comparison tests demonstrated a significant interaction between UBE3C KD and RNF5/185 DKO (Pint < 0.05). (E) The channel function of ∆F508-CFTR-3HA in CFBE Teton cells transfected with siRNA indicated and treated with Trikafta (3 µM VX-661, 1 µM VX-445, 1 µM VX-770) for 2 days was evaluated using a YFP quenching assay. The rate of initial YFP quenching was measured and quantified as the CFTR function (right, n = 10–12). The total siRNA concentration was adjusted to 125 nM (25 nM siUBE3C #1, 50 nM siRNF5 pool, and 50 nM siRNF185 pool) for all samples. The assay was conducted 4 days post-transfection. Statistical significance was assessed by a one-way ANOVA with Dunnett’s multiple comparison tests. Data represent mean ± SE. * p < 0.05, ** p < 0.01, ns, not significant.
Figure 2
Figure 2
UBE3C attenuates ∆F508-CFTR ERAD. (A) The degradation kinetics of ∆F508-CFTR-HiBiT(CT) in 293MSR WT and RNF5/185 KO cells transfected with 25 nM siNC or siUBE3C #1. Luminescence levels were consistently observed for 180 min while cells were exposed to 100 µg/mL CHX. The data were plotted after normalization to the luminescence levels of untreated cells. The ERAD rate of ∆F508-CFTR-HiBiT(CT) was determined by fitting individual kinetic degradation curves (right, n = 3). Two-way ANOVA demonstrated a notable primary impact of UBE3C KD or RNF5/185 DKO. However, it did not reveal any interaction between the two factors (Pint). (B) UBE3C KD in 293MSR WT and RNF5/185 cells was validated using RT-qPCR (n = 3). Two-way ANOVA demonstrated a significant main effect of UBE3C KD but not of RNF5/185 DKO, with no observed interaction between the two factors (Pint). (C,D) Kinetic degradation of Insig-1-HiBiT(CT) ((C), n = 3) and N1303K-CFTR-HiBiT(CT) ((D), n = 3) in 293MSR WT and RNF5/185 KO cells transfected with 25 nM siNC or siUBE3C #1. The ERAD rate was calculated by fitting each kinetic degradation curve (right). Two-way ANOVA demonstrated a significant main effect of UBE3C KD or RNF5/185 DKO but no interaction between the two factors (Pint). Each biological replicate (n) is color-coded: the averages from 4 technical replicates are shown in triangles (A,C,D). Statistical significance was assessed by a two-way RM ANOVA (A,C,D) or two-way ANOVA (B). Data represent mean ± SE. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.
Figure 3
Figure 3
UBE3C OE reduces ∆F508-CFTR abundance independently of its E3 ligase activity. (A) Western blotting shows ∆F508-CFTR-3HA level in 293MSR cells co-transfected with ∆F508-CFTR-3HA and FLAG-His-UBE3C WT or C1051A (0.5, 0.25, or 0.125 µg). Cells were treated with 2 mM NaB for 1 day before cell lysis. The immature ΔF508-CFTR (B band) in cells transfected with 0.5 µg FLAG-His-UBE3C WT or C1051A (CA) was quantified using densitometry (n = 3–4). Ponceau staining was used as a loading control. Statistical significance was assessed by one-way ANOVA. Data represent mean ± SE. * p < 0.05, ** p < 0.01. (B) The interaction of FLAG-His-UBE3C WT or C1051A mutant with HBH-ΔF508-CFTR-3HA in 293MSR WT and RNF5/185 DKO cells was analyzed by NA pull-down and Western blotting. Cells were treated with 2 mM NaB for 1 day and 10 µM MG-132 for 1 h before cell lysis. The asterisk shows a non-specific band. (C) Cellular localization of HBH-∆F508-CFTR-3HA and FLAG-His-UBE3C in 293MSR cells transfected with FLAG-His-UBE3C. Cells were treated with 10 µM MG-132 for 1 h before fixation. The nucleus was stained with DAPI.
Figure 4
Figure 4
The role of UBE3C in the ∆F508-CFTR ubiquitination. (A) Polyubiquitination of HBH-∆F508-CFTR-3HA in BHK cells was detected by ELISA using an anti-K48-linked polyubiquitin antibody (αK48-Ub, Apu2 ZooMAb) or GST-TUBE and an anti-GST antibody (n = 2). The CFTR amount on the ELISA plate was also quantified using an anti-HA antibody (right). The signal from the sample containing HBH-∆F508-CFTR-3HA was calculated relative to the BG signal obtained from the sample derived from CFTR-non-expressing cells. The cells underwent treatment with 10 µM MG-132 for a duration of 3 h before undergoing cell lysis. (B) The GST-TUBE binding signal exhibited an increase in the case of HBH-∆F508-CFTR-3HA from BHK cells treated with 10 µM MG-132 for 3 h (n = 2). Cell lysates (150 µg) were used for analysis. (C) The signal detected by GST-TUBE was reduced by in vitro digestion by 5 µM USP21 for 1 h at 37°C prior to the GST-TUBE binding (n = 3). Cell lysates (150 µg) were used for analysis. (D) The ubiquitination levels of HBH-∆F508-CFTR-3HA in 293MSR WT and RNF5/185 DKO cells transfected with 25 nM siRNA indicated were measured by ELISA using GST-TUBE (n = 4). The cells underwent treatment with 10 µM MG-132 for a duration of 3 h before undergoing cell lysis. A two-way ANOVA demonstrated a significant main effect of RNF5/185 DKO but not of UBE3C KD, with no observed interaction between the two factors (Pint). (E) The ubiquitination level of HBH-∆F508-CFTR-3HA in 293MSR WT and RNF5/185 DKO cells transfected with 25 nM siNC (-) or siUBE3C #1 (+) was measured by NA pull-down and normalized for CFTR in precipitates. The cells underwent treatment with 10 µM MG-132 for a duration of 3 h before undergoing cell lysis. The levels of CFTR ubiquitination were assessed using densitometry and presented as a percentage relative to the control group (right, n = 3). Two-way ANOVA demonstrated a significant main effect of RNF5/185 DKO but not of UBE3C KD, with no observed interaction between the two factors (Pint). (F) Ubiquitination level of HBH-∆F508-CFTR-3HA in 293MSR cells transfected with Myc-Ub and FLAG-His-UBE3C WT or C1051A mutant was measured as E. Cells were treated with 2 mM NaB for 1 day and 10 µM MG-132 for 3 h before cell lysis. The levels of CFTR ubiquitination (Myc/HA) were quantified using densitometry and reported as a percentage in comparison to the control. Data represent the mean (A,B) or mean ± SE (C,D,E). Statistical significance was assessed by an unpaired t-test (C) or two-way ANOVA (D,E). ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant.
Figure 5
Figure 5
UBE3C facilitates the ERAD of misfolded ABCB1. (A) The HiBiT degradation assay quantified the ERAD of ΔY490-ABCB1-HiBiT(CT) in 293MSR WT and RNF5/185 DKO cells after transfection with either 25 nM siNC or siUBE3C #1. Luminescence was continually tracked for 420 min while CHX was present, and the data were graphed after normalization to the luminescence levels of non-treated cells. The ERAD rate of ΔY490-ABCB1-HiBiT(CT) was determined by fitting individual kinetic degradation curves (right, n = 3). Each biological replicate (n) is color-coded; the averages from 4 technical replicates are shown in triangles. Two-way ANOVA demonstrated a significant effect of UBE3C KD or RNF5/185 DKO but no interaction between the two factors (Pint). (B,C) PM levels of ∆Y490-ABCB1-HiBiT(Ex) in BEAS-2B cells transfected with 25 nM siUBE3C #1 and/or 50 nM siRNF5 and 50 nM siRNF185 (RNF5/185 DKD) as indicated (n = 6). (C) Cells were treated with 10 µM CLP-A for 24 h to facilitate cell surface expression. Two-way ANOVA demonstrated a significant effect of UBE3C KD or RNF5/185 DKO, as well as an interaction between the two factors (Pint). (D) Western blotting shows ∆Y490-ABCB1-HiBiT(Ex) level in BEAS-2B cells transfected with 25 nM siNC (-) or siUBE3C #1 (+), 100 nM siNT, or 50 nM siRNF5 and siRNF185 (si5/185). Cells were subjected to a 24-h treatment with or without 10 µM CLP-A. Additionally, cells were treated with 2 mM NaB for 24 h before cell lysis. The levels of immature (B band) and mature (C band) ∆Y490-ABCB1 were quantified using densitometry and presented as a percentage relative to the control. Cell lysates were prepared at 4 days post-transfection. Ponceau staining was employed as a loading control. The asterisk shows a non-specific band. Statistical significance was evaluated through a two-way RM ANOVA (A) or two-way ANOVA (B,C). Data represent mean ± SE. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant.
Figure 6
Figure 6
UBE3C participates in peripheral CFTR quality control. (A) Representative traces of mature ΔF508-CFTR-Nluc(CT) elimination in BEAS-2B cells transfected with 25 nM siNC or siUBE3C #1. Cells were pre-treated with 1 µg/mL Dox and Trikafta (3 μM VX-661, 1 μM VX-445, 1 μM VX-770) at 37 °C for 2 days. During the CHX chase, Trikafta was also treated. The degradation rate of ΔF508-CFTR-Nluc(CT) was determined by fitting individual kinetic degradation curves (right, n = 4). (B) Western blotting with CHX chase measured the stability of mature ∆F508-CFTR-3HA in 293MSR cells transfected with 25 nM siNC or siUBE3C #3. Cells were pre-treated with Trikafta (3 μM VX-661, 1 μM VX-445, 1 μM VX-770) and 2 mM NaB at 37 °C for 24 h but did not during the 100 µg/mL CHX chase. Levels of immature (B band) and mature (C band) ∆F508-CFTR were quantified using densitometry and reported as a percentage relative to the control. Cell lysates were prepared 4 days post-transfection. Ponceau staining was employed as a loading control. The asterisk shows a non-specific band. (C) PM levels of T70-CFTR-HiBiT(Ex) in BEAS-2B cells transfected with 25 nM siNC or siUBE3C #1 (n = 6). Statistical significance was assessed by an unpaired t-test (A,C). Data represent mean ± SE. * p < 0.05, **** p < 0.0001.

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