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

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors provided strong evidence to conclude that UBE3C plays a pivotal role in the ERAD of misfolded CFTR and ABCB1, potentially also contributing to the peripheral quality control of CFTR. The studies in this manuscript have been well-designed and executed with precision. Please consider addressing the following minor issues:

For the benefit of general readers, it would be helpful to include information about how UBE3C's contribution compares to other ubiquitin E3 ligases in regulating protein quality, particularly in the context of CFTR or beyond.

Could you clarify why the replication number in the studies for Fig. 4 (B) was less than three, whereas most others were conducted with a minimum of three replications?

Would it be possible to include a brief discussion about the clinical relevance or implications of the major findings in the present study?

Author Response

Firstly, we would like to express our gratitude for your valuable feedback. We have thoroughly reviewed your comments and have crafted appropriate responses to address them.

1. For the benefit of general readers, it would be helpful to include information about how UBE3C's contribution compares to other ubiquitin E3 ligases in regulating protein quality, particularly in the context of CFTR or beyond.

As recommended, we have incorporated the following descriptions concerning this aspect into the discussion section of the revised manuscript (page 16, lines 609-615).

“Based on our experimental findings, UBE3C's role in ∆F508-CFTR ERAD appears to be partial compared to RNF5/185, showing a similarity in contribution to the cytoplasmic E3 ligase HERC3 (24). However, given UBE3C's substantial impact on ∆Y490-ABCB1 ERAD, it suggests that UBE3C's involvement varies based on the misfolding region or characteristics of the ERAD substrate. In forthcoming investigations, it is imperative to elucidate UBE3C's substrate recognition mechanism to discern the structural abnormalities it identifies. This exploration may offer valuable insights into UBE3C's role in ERAD.”

2. Could you clarify why the replication number in the studies for Fig. 4 (B) was less than three, whereas most others were conducted with a minimum of three replications?

 The experiments displayed in Figures 4A and 4B were conducted to establish the conditions for the primary experiment presented in Figure 4D; hence, they were evaluated with a sample size of n=2.

3. Would it be possible to include a brief discussion about the clinical relevance or implications of the major findings in the present study?

As recommended, we have incorporated the following descriptions concerning this aspect into the discussion section of the revised manuscript (page 17, lines 657-662).

"Although further investigation is needed to explore the potential adverse effects resulting from UBE3C inhibition, it's evident that such inhibition suppresses ERAD and peripheral degradation of ∆F508-CFTR, consequently leading to an increase in functional CFTR channels. This observation suggests that targeting UBE3C could be a viable strategy in CF treatment, addressing both ER and peripheral CFTR quality control mechanisms."

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, the authors reported a non-canonical role of UBE3C in promoting the ERAD of misfolded CFTR. By using cellular degradation and biochemical assays, they demonstrated that UBE3C knockdown increases the abundance of ΔF508-CFTR by inhibiting its ERAD. Moreover, this effect seems independent of the ubiquitin ligase activity of UBE3C or the RNF5/185 pathway. Although the molecular mechanism of how UBE3C regulates the ERAD of CFTR remains unclear, this study contains a lot of new data and provides insights into a novel regulatory axis involved in the ERAD of misfolded proteins. Below I have several technical comments.

1.      The way the authors show the statistical significance in figures 2, 4 and 5 is very uncommon and hard to understand. Please consider present them consistently and clearly.

2.      Line 391. Can the authors define the “initial degradation portion” and provide rationale for how it is determined. It is hard to see the difference in the degradation rate between siNC and siUBE3C group in WT cells.

3.      Line 414. Can the authors show some immunofluorescence evidence confirming the colocalization of CFTR and UBE3C at the ER?

4.      Figure 3A. why transfection of C1051A mutant greatly increases the level of CFTR in the 0.125 ug group?

5.      Line 545-547. How is the half-life of immature CFTR determined in the provided references? If different methods were used then it is not reasonable to compare directly.

6.      Please double-check the column panel of Figure 6A. the data points shown in the siNC group seem all below the mean.

7.      Is it possible to perform in vitro ubiquitination reaction to more rigorously investigate the function of UBE3C on CFTR turnover?

Comments on the Quality of English Language

The language is generally OK.

Author Response

Firstly, we would like to express our gratitude for your valuable feedback. We have thoroughly reviewed your comments and have crafted appropriate responses to address them.

 

1. The way the authors show the statistical significance in figures 2, 4 and 5 is very uncommon and hard to understand. Please consider present them consistently and clearly.

The statistical analyses for the results (Fig.2A-D, 4D-E, and 5A-C) were performed using a two-way ANOVA. This analysis was utilized to evaluate the significance of each main effect, such as UBE3C KD or RNF5/185 DKO, and to determine if there is an interaction between these factors (P _int). To enhance reader understanding, we have included supplementary details in the figure legend to clarify these statistical interpretations. Regarding Figure 4D, errors in the statistical analysis results have been corrected (P_int).

 

2. Line 391. Can the authors define the “initial degradation portion” and provide rationale for how it is determined. It is hard to see the difference in the degradation rate between siNC and siUBE3C group in WT cells.

We have rectified the description concerning this matter in the revised manuscript. The degradation rate was calculated by fitting the curve within the timeframes of 0 to 180 minutes (A, C) or 0 to 480 minutes (D).

As noted, the difference in the ERAD rate between the siNC and siUBE3C groups in WT cells was small; however, statistical analysis revealed a significant difference (Fig. 2A).

 

3. Line 414. Can the authors show some immunofluorescence evidence confirming the colocalization of CFTR and UBE3C at the ER?

As suggested, we performed additional experiments showing FLAG-His-UBE3C was partially colocalized with HBH-∆F508-CFTR-3HA in 293MSR cells (Fig.3C). This result supports the interaction of UBE3C with ∆F508-CFTR at the ER.

 

4. Figure 3A. why transfection of C1051A mutant greatly increases the level of CFTR in the 0.125 ug group?

Since we haven't conducted a quantitative analysis to confirm whether ∆F508-CFTR increases due to the 0.125 µg C1051A transfection, it is unclear whether such an increase occurs. We suspect that the observed increase in CFTR depicted in Figure 3A on the blotting might be due to experimental error.

 

5. Line 545-547. How is the half-life of immature CFTR determined in the provided references? If different methods were used then it is not reasonable to compare directly.

We calculated the half-lives of both immature and mature ∆F508-CFTR using the CFTR-Nluc degradation assay and quantified them using the same method (Taniguchi et al, Front Mol Biosci. 2022, ref 23).

 

6. Please double-check the column panel of Figure 6A. the data points shown in the siNC group seem all below the mean.

Thank you for pointing out. Now the data was replaced with the corrected one.

 

7. Is it possible to perform in vitro ubiquitination reaction to more rigorously investigate the function of UBE3C on CFTR turnover?

Due to the constraints of the revision period, we did not evaluate your points this time. It would be interesting to evaluate the points you have raised in future research.