From the journal RSC Chemical Biology Peer review history

04-Dec-2020

Dear Dr Ueda:

Manuscript ID: CB-COM-11-2020-000204
TITLE: Rational design of a stapled JAZ9 peptide inhibiting protein-protein interaction of plant transcription factor

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to accept your manuscript for publication after revisions.

Please revise your manuscript to fully address the reviewers’ comments. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.

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Please note: to support increased transparency, RSC Chemical Biology offers authors the option of transparent peer review. If authors choose this option, the reviewers’ comments, authors’ response and editor’s decision letter for all versions of the manuscript are published alongside the article. Reviewers remain anonymous unless they choose to sign their report. We will ask you to confirm whether you would like to take up this option at the revision stages.

I look forward to receiving your revised manuscript.

Yours sincerely,
Prof Seung Bum Park

Associate Editor, RSC Chemical Biology

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Reviewer 1

In this manuscript, Suzuki et al. describe the rational design of stapled peptidic inhibitors for MYCs based on the crystal structure of JAZ9 bound to MYC3. The results of AlphaScreen inhibitory assay demonstrated that the doubly stapled peptide 4 exhibits apparent inhibitory activity against JAZ9-MYC3 interaction. The high affinity for MYC3 was further supported by the results of fluorescent polarization binding assay. Analysis of the expression of JA-responsive marker genes showed that 4 suppresses the MYC related gene expression in plants. Chemical biological techniques enabling signaling manipulation in plants are important to solve problems in plant biology and may contribute to address the food production issues. I think this manuscript is concisely written, and suitable for publication in RSC Chemical Biology after some minor points have been resolved or corrected.

Reviewer 2

In this paper authors reported the design, development, and testing of the first stapled peptide to regulate plant hormone signaling. Their JAZ stapled peptide was designed based on the crystal structure of complex of JAZ-regulator transcription factor (TF) MYC, and was shown to selectively inhibit the MYCs. The work is sound, properly written. I recommend publication.

Dear Editor and Referees,

Thank you for your kind responses.
I would like to answer the comments from each referee.

Referee1:
Thank you for your kind comments.
It will be highly appreciated if you will let me know some minor points to be resolved or corrected.
We will improve them soon.

Referee2:
Thank you for your positive comment.

10-Dec-2020

Dear Dr Ueda:

Manuscript ID: CB-COM-11-2020-000204.R1
TITLE: Rational design of a stapled JAZ9 peptide inhibiting protein-protein interaction of plant transcription factor

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to accept your manuscript for publication after revisions.

Please revise your manuscript to fully address the reviewers’ comments. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.

Please submit your revised manuscript as soon as possible using this link :

*** PLEASE NOTE: This is a two-step process. After clicking on the link, you will be directed to a webpage to confirm. ***

https://mc.manuscriptcentral.com/rsccb?link_removed

(This link goes straight to your account, without the need to log in to the system. For your account security you should not share this link with others.)

Alternatively, you can login to your account (https://mc.manuscriptcentral.com/rsccb) where you will need your case-sensitive USER ID and password.

You should submit your revised manuscript as soon as possible; please note you will receive a series of automatic reminders. If your revisions will take a significant length of time, please contact me. If I do not hear from you, I may withdraw your manuscript from consideration and you will have to resubmit. Any resubmission will receive a new submission date.

Supporting our community through Covid-19
While our publishing services are running as usual, we also know that this is a very challenging time for everyone, for many different reasons. If any aspect of the publishing process is worrying you – for example you think you may struggle to meet a pre-determined deadline – please let us know, and we will work out an answer together.

RSC Chemical Biology strongly encourages authors of research articles to include an ‘Author contributions’ section in their manuscript, for publication in the final article. This should appear immediately above the ‘Conflict of interest’ and ‘Acknowledgement’ sections. I strongly recommend you use CRediT (the Contributor Roles Taxonomy from CASRAI, https://casrai.org/credit/) for standardised contribution descriptions. All authors should have agreed to their individual contributions ahead of submission and these should accurately reflect contributions to the work. Please refer to our general author guidelines http://www.rsc.org/journals-books-databases/journal-authors-reviewers/author-responsibilities/ for more information.

The Royal Society of Chemistry requires all submitting authors to provide their ORCID iD when they submit a revised manuscript. This is quick and easy to do as part of the revised manuscript submission process. We will publish this information with the article, and you may choose to have your ORCID record updated automatically with details of the publication.

Please also encourage your co-authors to sign up for their own ORCID account and associate it with their account on our manuscript submission system. For further information see: https://www.rsc.org/journals-books-databases/journal-authors-reviewers/processes-policies/#attribution-id

Please note: to support increased transparency, RSC Chemical Biology offers authors the option of transparent peer review. If authors choose this option, the reviewers’ comments, authors’ response and editor’s decision letter for all versions of the manuscript are published alongside the article. Reviewers remain anonymous unless they choose to sign their report. We will ask you to confirm whether you would like to take up this option at the revision stages.

I look forward to receiving your revised manuscript.

Yours sincerely,
Prof Seung Bum Park

Associate Editor, RSC Chemical Biology

************

Reviewer 1

My previous comments to the author seem to have gotten cut-off for some reasons. I have pasted it again as follows:

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In this manuscript, Suzuki et al. describe the rational design of stapled peptidic inhibitors for MYCs based on the crystal structure of JAZ9 bound to MYC3. The results of AlphaScreen inhibitory assay demonstrated that the doubly stapled peptide 4 exhibits apparent inhibitory activity against JAZ9-MYC3 interaction. The high affinity for MYC3 was further supported by the results of fluorescent polarization binding assay. Analysis of the expression of JA-responsive marker genes showed that 4 suppresses the MYC related gene expression in plants. Chemical biological techniques enabling signaling manipulation in plants are important to solve problems in plant biology and may contribute to address the food production issues. I think this manuscript is concisely written, and suitable for publication in RSC Chemical Biology after some minor points have been resolved or corrected.

1) Fig. 1a: What is the reference for the structure of JAZ1 overlayed with JAZ9? The PDB number should be provided.
2) It is interesting that, although peptide 3 possesses low helices, according to the CD spectra (Fig 1d), it exhibits high affinity to MYCs with the Kd values which are at a similar level to those of peptide 4. It would be helpful for readers if authors discuss this inconsistency.
3) The stapled peptides show similar affinity to all MYC isoforms. Are these the expected results? Are the binding grooves of MYC for a JAZ peptide highly conserved one another? Authors should comment on that in the text.
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Dear Editor and Referees,

Thank you for your kind responses.
I would like to submit the revised version of our manuscript. According to the suggestions from referees, we corrected all points raised by referees. We believe that this revised manuscript is now suitable for publication in RSC Chemical Biology.

Best regards,

Minoru Ueda, Ph. D.
Professor
Department of Chemistry
Graduate School of Science, Tohoku University
6-3 Aramaki-aza Aoba, Aoba-ku, Sendai 980-8578, Japan.
Tel&Fax: +81-22-795-6557
E-mail: minoru.ueda.d2@tohoku.ac.jp


Referee1:
1) Fig. 1a: What is the reference for the structure of JAZ1 overlayed with JAZ9? The PDB number should be provided.

Answer: Thank you for the valuable comment. PDB number of JAZ1 complexed with COI1 and JA-Ile is 3OGL. It is shown in the figure legend in the revised manuscript.

2) It is interesting that, although peptide 3 possesses low helices, according to the CD spectra (Fig 1d), it exhibits high affinity to MYCs with the Kd values which are at a similar level to those of peptide 4. It would be helpful for readers if authors discuss this inconsistency.

Answer: Thank you for the valuable comment. We also agree that the affinity of peptide 3 to MYCs was relatively high although it possesses low helices. It remains unclear why the peptide 3 has high affinity compare to peptide 2, which has similar helicity with peptide 3. However, the affinity of peptide 3 was significantly lower than that of peptide 4 (8.8-fold), maybe because of the increased helix contents. To clarify this, we newly added the following information in the revised manuscript:

P. 2, right column, line 8:
In contrast, the Kd value of 4 was 0.10 µM, which is 50-fold, 24-fold, or 8.8-fold lower than that of 1, 2 or 3, respectively (Table 1).

3) The stapled peptides show similar affinity to all MYC isoforms. Are these the expected results? Are the binding grooves of MYC for a JAZ peptide highly conserved one another? Authors should comment on that in the text.

Answer: Thank you for the valuable comment. As the referee suggested, the binding grooves of MYCs are highly conserved. We newly added the following sentences and the homology analysis of MYCs were shown in Figure S5 in the revised manuscript. Along with that, the figure numbers (Figure S6 and later) shift one by one.

P. 2, right column, line 13:
Moreover, the stapled peptide 4 showed similar affinity to all MYC isoforms (0.10, 0.16, or 0.88 nM for MYC3, MYC2, or MYC4, respectively), which is consistent to the sequence homology analysis of the binding sites of these three MYCs for a JAZ (Figure S5).

14-Dec-2020

Dear Dr Ueda:

Manuscript ID: CB-COM-11-2020-000204.R2
TITLE: Rational design of a stapled JAZ9 peptide inhibiting protein-protein interaction of plant transcription factor

Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, I am pleased to accept your manuscript for publication in its current form on the basis of my own evaluation.

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Prof Seung Bum Park

Associate Editor, RSC Chemical Biology


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