PONE-D-23-43062cAmbly, a non-toxic cell-penetrating peptide (CPP) derived from Amblyomin-X, targeting   mitochondrial and cytoplasmic proteinsPLOS ONE

Dear Dr. Chudzinski-Tavassi,

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Parvez Alam, PhD

Academic Editor

PLOS ONE

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 [This research was funded by “Grant 2015/50040-4, São Paulo Research Foundation and GlaxoSmithKline”, “Grant 2020/13139-0, São Paulo Research Foundation and GlaxoSmithKline”, and Grant number 2013/07467-1 (FAPESP-CETICs). A.M.C.-T. is a recipient of CNPq-PQ grant number 303197/2017-0 and Fundação Butantan-PQ grants. R.N.G. is a recipient of FAPESP grant number 2018/20469-7. A.M.A is a recipient of FAPESP grant number 2018/10937-3.].  

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Additional Editor Comments:

The manuscript by Buri et al., titled "cAmbly, a non-toxic cell-penetrating peptide (CPP) derived from Amblyomin-X, targeting mitochondrial and cytoplasmic proteins," covers a very interesting topic for a wider audience in the field. However, it is not accepted in its current form due to several reasons. It can only be considered for publication after major revision.

1.The manuscript is poorly written and does not follow the flow of information. It should be clearly rewritten, and the rationale for each experiment should be explained very clearly.

2.The purpose of cAmbly CPP targeting mitochondrial proteins should be explained clearly, both in the abstract and introduction. What kind of pathological conditions can it help in targeting, and how?

3.The quality of confocal images is not up to standard and makes it hard to interpret the data. The experiment needs to be repeated or the data should be reanalyzed.

4.Results and discussions should be carefully written to allow readers to understand the importance of this study.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: No

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors of the manuscript " cAmbly, a non-toxic cell-penetrating peptide (CPP) derived from Amblyomin-X, targeting mitochondrial and cytoplasmic proteins" have used array of methods to demonstrate the cellular internalization of a derived peptide. Overall, the work is very interesting. However, the authors need to address the following concerns:

1.The authors are encouraged to include computational analysis of the designed peptide, encompassing aspects like three-dimensional structure prediction and amino acid parameters. This would enrich the presentation of their work with a comprehensive perspective.

2.The quality of the figures can be improved. Although it can be a pdf conversion issue.

Reviewer #2: The overall manuscript is loosely written the structure and clarity of results and their implications is lacking.

Major points:

1.The abstract provides a good overview of the study however it could benefit from improved clarity and emphasis on the significance of the findings. The abstract should clearly state the significance of the finding for example, colocalizing with mitochondria, with preference for targeting mitochondrial proteins. The importance could be emphasized more. Also,the broader implications of the findings which is use of C-terminal of cAmbly for coupling molecules inside cells should be presented with more clarity and its potential impact on the field.

2.Line 64-66: Clarify which native and modified molecules were synthesized (e.g., specify the precise sequences of each peptide and there extensions in a figure. It may be helpful to provide a brief description of the properties of the native cAmbly sequence.

3.The MTT assay section is clear but it should be supplemented with how cell viability was calculated based on spectrophotometric readings and any controls used in the experiment. Also, consider specifying the number of replicates conducted for each assay to ensure the results' reliability and reproducibility.

4.Consider specifying the solvent control concentration Line 146: Provide more detail on how cell viability was calculated and how normalization was performed. Explain the significance of the lack of cytotoxicity for cAmbly. Line 149-151: The increase in activity of reducing enzymes in cells treated with cAmbly is an interesting finding. However, the implications of this result should be explained further. For instance, why is this increase significant? Does it suggest a potential benefit of cAmbly treatment? The reducing enzymes could be explained more to specify which reducing enzymes and their relevance. Provide more context for the results, particularly in relation to existing literature or expectations. How do these results compare to similar studies or to known effects of other CPPs?

5.Specify whether the UV light exposure is meant to crosslink the peptides with their targets in the cells or on the cell surface. provide information on how long the UV light exposure lasted. Also, consider adding more details about the fixation process (duration and temperature). Mention the concentrations of Hoechst, Phalloidin conjugated to Alexa Fluor 647, and streptavidin conjugated to Alexa Fluor 488 used for labeling. Consider specifying the number of replicates.

6.Mention the concentration of cAmbly-FITC and Mitotracker Deep Red used in the Mitochondrial Colocalization assay. Also, specify the duration of treatment. Provide details on the confocal microscopy imaging conditions, such as settings used for each fluorophore, and how the images were analyzed.

7.Specify the concentrations of native and modified cAmbly peptides used in the assay. Include a description of the controls used in the chemoproteomic assay, such as untreated cells or cells treated with non-modified peptides. Specify the concentration or volume of trypsin used for protein digestion. Provide more information about TMT-tagging, such as the specific reagents or kits used. Clarify the desalinization process using SDB-XC stage-tips (e.g., duration, flow rate). Include more details about the qExactivePlus mass spectrometer settings, including any specific parameters used for data acquisition and analysis. Clarify the methods used for protein quantification, such as specific algorithms or statistical models.

8.It was nowhere mentioned why mitochondria are of interest in this study. Consider stating the reason for focusing on mitochondrial colocalization specifically and why it is important. The lines 193-194 is not clear, present and explain the interpreted results more clearly. Provide more context for the results, such as how these findings compare to existing literature and discuss the implications of the colocalization results for potential therapeutic applications.

9.Chemoproteomic for cAmbly target discovery, explain briefly the mass spectrometry-based proteomics approach used, clarify the methods used for protein quantification. Explain the competitive assay in more detail. For example, describe how the concentrations of non-modified peptides were increased and what the expected outcomes were.

Minor points:

Line 14-15: "The delivery of drugs is a challenge when thinking about new molecules development" could be rephrased for clarity.

Line 16, “outstand this due its capacity" should be revised for clarity. Consider rephrasing to for example: "stand out due to their ability."

Line 25: "Delivery of some types of drugs are not always an easy task" could be improved for clarity. Consider rephrasing for example to "Delivering some types of drugs can be challenging."

Line 37: "small peptides, non-toxic, with up to 40 amino acids" could be improved for clarity. Consider rephrasing for example "small, non-toxic peptides consisting of up to 40 amino acids."

Line 57: "the Kunitz domain could not enter and kill the tumor cell" could gain from being more explicit about the interaction or mechanism involved.

Line 68-70: Explain the rationale for modifying the N-terminal or C-terminal of the peptide and how the design was expected to affect function.

Line 65: Rephrase "one native and three modified molecules of cAmbly peptide" to "one native cAmbly peptide and three modified variants."

Line 69: Rephrase "for purification and labeling purposes" to "for purification and labeling."

Line 73-77: The cell culture section provides the basic conditions for cell growth, also specify the number of cells seeded per well and any other relevant culture conditions.

Line 100: Mention the concentration of cAmbly-FITC and Mitotracker Deep Red used in the assay. Also, specify the duration of treatment.

Line 106: "At 100% confluency" could benefit from specifying the number of cells or density to provide more context on cell seeding.

Line 107: Specify the concentrations of native and modified cAmbly peptides used in the assay.

Line 115: Consider rephrasing "qExactivePlus mass spectrometer coupled to a nanoEasy 1200 chromatograph" for clarity.

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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

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Attachments

Attachment

Submitted filename: Revision.docx

PONE-D-23-43062R1cAmbly, a non-toxic cell-penetrating peptide (CPP) derived from Amblyomin-X, targeting   mitochondrial and cytoplasmic proteinsPLOS ONE

Dear Dr. Chudzinski-Tavassi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR  Authors have answered most of the reviweres comments. However, quality of figures are still need significant improvement in terms of resolution. It's hard to read figure axis in some places.

==============================

Please submit your revised manuscript by Sep 26 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Parvez Alam, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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[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.

PONE-D-23-43062R2cAmbly, a non-toxic cell-penetrating peptide (CPP) derived from Amblyomin-X, targeting   mitochondrial and cytoplasmic proteinsPLOS ONE

Dear Dr. Chudzinski-Tavassi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Dec 28 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Boyan Grigorov

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: N/A

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: No

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: No

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: I have the following comments concerning the manuscript of Marcus Buri et al.:

The manuscript needs to be proofread by a native speaker. Pluriels are missing (among other mistakes), e.g. Chemoproteomic(s), streptavidin bead(s)...

The Results section must be better organized. I was wondering if it were the Materials & Methods section that I was reading. Real explicative subheadings should be added.

Figures should appear in the Results section (actually, Fig.1 appears in Materials & Methods).

Concerning the mitochondria/cAmbly colocalization, include a wide field 2D image

to figure 6 (similar to Fig.4 & 5).

Concerning the discussion section, mention the potential delivery method of the peptides in vivo (systematic or local). What is the immunogenicity of this peptide?

In the conclusion, the authors argue that "cAmbly can potentially deliver mitochondria-targeted drugs...". Can you give examples for such drugs?

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7. PLOS authors have the option to publish the peer review history of their article (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 #3: 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.

PONE-D-23-43062R3cAmbly: A non-toxic cell-penetrating peptide derived from Amblyomin-X with targeted delivery to mitochondrial and cytoplasmic proteinsPLOS ONE

Dear Dr. Chudzinski-Tavassi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The authors should reply and include in the text the following points that have been raised by the reviewers:1. The "Results" must include explicative subheadings (titles) and not simply the method used.2. Discuss about the immonogenicity of the petide and its mode of delivery.3. Moderate the final sentence in the Conclusion: "Therefore, for optimal delivery efficiency, therapeutic molecules intended for mitochondrial targeting COULD be conjugated to the C-terminal of cAmbly." 

Please submit your revised manuscript by Feb 21 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

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Boyan Grigorov

Academic Editor

PLOS ONE

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cAmbly: A non-toxic cell-penetrating peptide derived from Amblyomin-X with targeted delivery to mitochondrial and cytoplasmic proteins

PONE-D-23-43062R4

Dear Dr. Chudzinski-Tavassi,

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Kind regards,

Boyan Grigorov

Academic Editor

PLOS ONE

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