Warburg-Cinotti disease variant p.Tyr740Cys enhances catalytic activity of DDR2 kinase

Ziteng Hao; Birgit Leitinger

doi:10.1371/journal.pone.0336895

Peer Review History

Original SubmissionJune 27, 2025
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Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Yes Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: The manuscript addresses an important topic and presents valuable data. After careful evaluation, I believe it can be accepted for publication pending minor revisions to clarify certain points and improve readability. The authors are encouraged to address the specific comments provided to enhance the overall quality and clarity of the manuscript. 1. Please clearly state in the introduction that the DDR2-L610P variant could not be biochemically characterized due to solubility problems, to set appropriate expectations for the results section. 2. Briefly mention any limitations of using DDR1 as a reference model for DDR2, despite structural similarity. 3. The hypothesis—that DDR2 mutations relieve autoinhibition and enhance kinase activity—should be clearly articulated at the end of the introduction to guide the reader. 4. Briefly explain or break down long, complex sentences to improve clarity and readability. a sahi hai kya? 5. Clarify and expand how the increased DDR2 kinase activity caused by mutations leads to the clinical symptoms of Warburg-Cinotti syndrome to strengthen the biological relevance. 6. Consider adding a brief discussion on the limitations of using HEK293 cells as a model for DDR2 activity, as these cells may not fully recapitulate physiological conditions. OR Please briefly discuss the limitations of using HEK293 cells as a model system for DDR2 activity, including possible differences from endogenous expression contexts. Reviewer #2: The study attempts to find the mechanistic basis behind Warburg-Cinotti syndrome caused by mutations in DDR2. There are valuable additions in the manuscript but few things need to be addressed before it's accepted for publication, includingg some clarifications and resolving what seems to be contradicting statements. The computational prediction using ELASPIC estimates a change in the stability of the L610P mutant (folding energy) of ΔΔG = -3 kcal•mol-1. The prediction cannot be verified due to the inability to purify an active construct. What do similar calculations show for Y740C mutant, which shows decreased stability? Showing the validity of the estimates with Y740C is needed to cement the predictions for L610P. S3 Fig. B, the localization is not clear in black and white. Was the fluorescence obtained quantified in the images? The cell cytometer data in panel C imply there are differences in cell population, if not at expression level, then maybe localization, with the mutated DDR2 variants. How did the authors deduce that there is comparable cell surface expression? The discussion section mentions (line 676) differential cellular trafficking, which contradicts their results! The ADP-Glo assay used is an endpoint assay that measures the total phosphorylation happening after time “t”, not a kinetic continuous assay, which should work to assess specific activity for the enzyme. I am not sure if it can be used accurately to measure Michaelis Menten kinetic constants. The measured activity might not represent the initial rate if the reaction has progressed beyond the linear phase of the reaction progress curve, where the rate is constant. The methods section mentions that the reaction was stopped at 10, 20, and 40 minutes, the earliest of which (10 min) far exceeds the 2 minutes that it takes the mutant and FP protein to fully phosphorylate the A loop per the WB data, hence not measuring the initial rates for sure. Thus, Table 1, results, and discussion should only copare the specific activity of the constructs. I am not sure what Figure 4B adds that is different than Figure 3B (other than using 100-fold of the enzyme concentrations, which I cannot justify), since they both seem to compare the time course of in vitro autophosphorylation of DDR1 and DDR1. (Lines 447 and 448: Are these the same two constructs, and are these the same ones in line 456/457?). If all the same, I suggest merging the native gel panel with Figure 3 for a more concise figure. In the discussion, the authors do a great job summarizing mutation effect in RTK, yet they need to acknowledge that because the constructs used lack some of the cytosolic components of the JM domain that were present in other studies, it’s harder to draw the conclusion that Src activation is not needed in a physiologically relevant environment. ****** what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: Yes: NA Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: Comments PLos.docx https://doi.org/10.1371/journal.pone.0336895.r001
Revision 1
3 Sep 2025 Author Response We thank the reviewers for their careful reading of the manuscript and constructive criticism. A point-by-point response is below. Reviewer #1 The manuscript addresses an important topic and presents valuable data. After careful evaluation, I believe it can be accepted for publication pending minor revisions to clarify certain points and improve readability. The authors are encouraged to address the specific comments provided to enhance the overall quality and clarity of the manuscript. 1. Please clearly state in the introduction that the DDR2-L610P variant could not be biochemically characterized due to solubility problems, to set appropriate expectations for the results section. Response: This information has now been added to the Introduction, lines 107-108. 2. Briefly mention any limitations of using DDR1 as a reference model for DDR2, despite structural similarity. Response: This information has now been added to the Introduction, lines 90-94. 3. The hypothesis—that DDR2 mutations relieve autoinhibition and enhance kinase activity—should be clearly articulated at the end of the introduction to guide the reader. Response: This information has now been added to the Introduction, lines 103-104. 4. Briefly explain or break down long, complex sentences to improve clarity and readability. a sahi hai kya? Response: We have carefully reviewed the manuscript text to check for issues with clarity and readability. 5. Clarify and expand how the increased DDR2 kinase activity caused by mutations leads to the clinical symptoms of Warburg-Cinotti syndrome to strengthen the biological relevance. Response: This information has now been added to the Discussion, lines 708 - 717. 6. Consider adding a brief discussion on the limitations of using HEK293 cells as a model for DDR2 activity, as these cells may not fully recapitulate physiological conditions. OR Please briefly discuss the limitations of using HEK293 cells as a model system for DDR2 activity, including possible differences from endogenous expression contexts. Response: Further information about the use and limitations of HEK293 cells has been added into the manuscript, lines 373 – 376. Reviewer #2 The study attempts to find the mechanistic basis behind Warburg-Cinotti syndrome caused by mutations in DDR2. There are valuable additions in the manuscript but few things need to be addressed before it's accepted for publication, includingg some clarifications and resolving what seems to be contradicting statements. The computational prediction using ELASPIC estimates a change in the stability of the L610P mutant (folding energy) of ΔΔG = -3 kcal•mol-1. The prediction cannot be verified due to the inability to purify an active construct. What do similar calculations show for Y740C mutant, which shows decreased stability? Showing the validity of the estimates with Y740C is needed to cement the predictions for L610P. Response: We have now used the ELASPIC server to calculate the folding energies for both mutants. Frustratingly, the server now gives positive ΔΔG values for both proteins. Using a different server (DDGEMB) gives ΔΔG = -1.33 kcal•mol-1 for L610P, which is defined as destabilizing, and ΔΔG = -0.32 kcal•mol-1 for Y740C, which is defined as neutral. While the latter calculations are in line with our inability to produce the L610P construct while the Y740C could be obtained as a folded protein, we do not believe these calculations to be reliable and decided to remove the prediction of the folding energy of the L610P mutant and only describe experimentally obtained data in the manuscript with regard to protein stability. S3 Fig. B, the localization is not clear in black and white. Was the fluorescence obtained quantified in the images? The cell cytometer data in panel C imply there are differences in cell population, if not at expression level, then maybe localization, with the mutated DDR2 variants. How did the authors deduce that there is comparable cell surface expression? The discussion section mentions (line 676) differential cellular trafficking, which contradicts their results! Response: We now show quantification of the flow cytometry data in new Figure S3D. The data show somewhat reduced surface expression of the mutant DDR2 constructs, which aligns better with the comments about potential effects on cellular trafficking. The updated text is in the Results section, lines 396-398 and Discussion section, lines 687, 694 – 701. The ADP-Glo assay used is an endpoint assay that measures the total phosphorylation happening after time “t”, not a kinetic continuous assay, which should work to assess specific activity for the enzyme. I am not sure if it can be used accurately to measure Michaelis Menten kinetic constants. The measured activity might not represent the initial rate if the reaction has progressed beyond the linear phase of the reaction progress curve, where the rate is constant. The methods section mentions that the reaction was stopped at 10, 20, and 40 minutes, the earliest of which (10 min) far exceeds the 2 minutes that it takes the mutant and FP protein to fully phosphorylate the A loop per the WB data, hence not measuring the initial rates for sure. Thus, Table 1, results, and discussion should only copare the specific activity of the constructs. Response: The ADP-Glo assay is suitable to be used to determine Michaelis Menten kinetics because it measures ADP production over time (see also Zegzouti et al, Assay Drug Dev Technol. 2010). To clarify, we do not use this assay to measure autophosphorylation, as the reviewer seems to believe, but to measure phosphorylation of a model substrate peptide (Axltide peptide, also used in Iwai et al, Biochem J, 2013). We used the assay in the same way as we did in our previous study where we determined the Michale Menten kinetics for the DDR1 kinase (Sammon et al, PNAS, 2020). In both studies, ADP production was linear up to the last time point. This was checked in every assay. Furthermore, we have calculated ADP production at the highest activity (DDR2-KY740C incubated with 520 μM Axltide peptide) and found that only 21.6% of the peptide had been phosphorylated under these conditions. I am not sure what Figure 4B adds that is different than Figure 3B (other than using 100-fold of the enzyme concentrations, which I cannot justify), since they both seem to compare the time course of in vitro autophosphorylation of DDR1 and DDR1. (Lines 447 and 448: Are these the same two constructs, and are these the same ones in line 456/457?). If all the same, I suggest merging the native gel panel with Figure 3 for a more concise figure. Response: We have now given more rationale for using the two different concentrations of DDR constructs, lines 474 – 476. We considered merging the native gel with Figure 3 and adding the bottom panels of Figure 4 as a new Supplemental Figure. On careful reflection, we decided to keep the original Figures 3 and 4, as it would be difficult for the reader to follow the text (lines 476 – 487) that describes both the native gel and the Western blotting results if the Western blotting data were shown in a supplementary Figure, separate from the native gel. In the discussion, the authors do a great job summarizing mutation effect in RTK, yet they need to acknowledge that because the constructs used lack some of the cytosolic components of the JM domain that were present in other studies, it’s harder to draw the conclusion that Src activation is not needed in a physiologically relevant environment. Response: The Discussion has been expanded to include a comment about possible DDR2 activation by Src in cells, lines 649 – 653. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0336895.r002
2 Nov 2025 Decision Letter - Jianhong Zhou, Editor Warburg-Cinotti disease variant p.Tyr740Cys enhances catalytic activity of DDR2 kinase PONE-D-25-34746R1 Dear Dr. Leitinger, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support . If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Jianhong Zhou Staff Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: (No Response) Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: (No Response) Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: (No Response) Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: (No Response) Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: (No Response) Reviewer #2: Yes ****** Reviewer #1: (No Response) Reviewer #2: Thank you for addressing the comments. Please add the limitation to using ADP Global assay in determining the Michaelis Menten constant, the accuracy of the V0 is an approximation because the assay stops at a single point. ****** what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: No Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0336895.r003
Formally Accepted
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