-->PONE-D-24-29025-->-->The Impact of Mutations on TP53 Protein and MicroRNA Expression in HNSCC: Novel Insights for Diagnostic and Therapeutic Strategies-->-->PLOS ONE

Dear Dr. Mohamed,

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.

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We look forward to receiving your revised manuscript.

Kind regards,

Taj Mohammad, Ph.D.

Academic Editor

PLOS ONE

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Additional Editor Comments:

After evaluating and considering the feedback provided by two reviewers, I found several points that need attention to improve the overall quality and clarity of the manuscript.

1.The manuscript contains too many figures, many of which are of poor quality. The clarity and resolution of these figures need significant improvement to ensure that they convey the data accurately.

2.The manuscript employs 100 ns simulations to analyze the structural and dynamic impacts of TP53 mutations. However, this simulation time is insufficient to capture all the relevant conformational changes in the system. I recommend extending the simulation time to at least 200–300 ns.

3.The results section contains a substantial amount of information, but it lacks concise explanations.

4.The results are not interpreted in-depth in the context of HNSCC biology and clinical relevance. Authors should expand the discussion to analyze the biological implications of the mutations, particularly how they may inform diagnostics and treatment strategies in a clinical setting.

5.Survival data appear to be underexplained. Provide more comprehensive statistical methods and interpretations.

6.The authors have not fully explored how PTMs impact TP53’s interaction with other proteins or the regulation of its activity.

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Reviewers' comments:

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Reviewer #1: Partly

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: #Reviewers Comments

Sofia B Mohamed et al. authored an article titled “The Impact of Mutations on TP53 Protein and MicroRNA Expression in HNSCC: Novel Insights for Diagnostic and Therapeutic Strategies.” M.ID: PONE-D-24-29025. The study aimed to explore how mutations affect the structure and function of the TP53 protein and the expression of microRNAs (miRNAs) through computational analysis. Genomic data from patients with HNSCC was sourced from the TCGA database, and the impact of TP53 gene mutations was analyzed using various bioinformatics tools.

The study found that TP53 mutations led to an increase in TP53 expression levels in HNSCC, correlating with a poorer prognosis. Additionally, the authors demonstrated that the expression level of has-mir-133b was significantly reduced in TP53-mutated samples, which had a notable impact on the survival of HNSCC patients. Six mutations—R273C, G105C, G266E, Q136H/P, and R280G—were identified as deleterious, carcinogenic, driver mutations that are highly conserved and exposed. These mutations were located within the P53 domain, and PTM analysis revealed that R280G and R273C are at a methylation site, while R273C, Q136H/P, and R280G are located in the protein pocket. Docking studies indicated that these mutations decreased DNA-binding affinity, with R273C, R280G, G266E, and G105C showing the most significant differences. Molecular dynamics analysis suggested that the R280G, Q136H, and G105C mutations conferred a gain of function by stabilizing the TP53-substrate complex.

The research findings suggest that TP53 mutations influence protein and miRNA expression, as well as the development, survival, and progression of HNSCC patients. Additionally, has-mir-133b could serve as a promising novel biomarker for monitoring HNSCC progression. The authors assert that G105C and Q136H/P, identified as novel mutations, impact the structure and function of proteins involved in HNSCC, making them potential subjects for further investigation, diagnostics, and therapeutic strategies. This research provides new insights into the mechanisms by which these mutations contribute to cancer development.

The work by Sofia B Mohamed et al. is suitable for acceptance, provided the authors address the following major and minor revisions as outlined in the comments.

Minor comments:

1. There are some grammatical mistakes and few words in the manuscript are merged, correct them.

2.Most of the figures shown are not clear, please show them in perfect resolution.

3.Conclusion: This study indicated that the mutations in the tp53 protein linked to HNSCC have: Correct TP53, and should be in capital.

Major comments:

1.The research begins with a strong introduction, highlighting the significance of TP53 mutations in various human malignancies, particularly HNSCC. However, a brief mention of why TP53 is considered a "tumor suppressor" and its general role in cancer biology could further strengthen the context.

2.Whole work is done and investigated using computational analysis, it would be better and complete if in vitro/ in vivo studies were also provided in this research for validation.

3.If there is any related in vitro study done so far, please mention and cite.

4.Identifying specific mutations (R273C, G105C, G266E, etc.) as harmful, carcinogenic, and conserved is well-presented. However, it would be beneficial to explain the significance of these terms briefly (e.g., what makes a mutation "deleterious" or "carcinogenic"?).

5.The reduced expression of has-mir-133b in TP53 and its impact on survival is an important finding. It may be useful to elaborate on the role of this specific miRNA in cancer, or why its downregulation is particularly significant.

6.The discovery of G105C and Q136H/P as novel mutations and their implications is a key highlight. Highlighting why these mutations are novel or different from previously known mutations could add more value to the findings.

7.If it can be taken into account as per the methodology used then the research paper is well written and the methodology is fine, though for validation of this work, bioinformatics is not alone enough if the journal doesn’t focus on bioinformatics tools.

Accept the manuscript after the authors have addressed the queries and responded to the comments. It should be accepted for publication, particularly if the journal or issue to which this manuscript was submitted focuses more on computational studies (data is enough and in detail with statistical data in supplementary) than on in vitro research.

Reviewer #2: 1) The authors must mention the study limitations, strength, novelty, and future prospects of the current study.

2) The quality of figure can be improved at a higher resoultion.

3) The figure legends needs to be elaborated.

4) There are too many grammatical errors throughout the manuscript. Please proof-read via a native english speaker or a professional english editing software.

5) Please mention the thresholds used for PPI network construction.

6) Please mention a detailed protocol of how preprocessing of TCGA datasets was performed along with batch-correction and statistical analysis.

7) What was the need for using GEPIA when the TCGA data was already available. Please justify.

8) It would be nice if the authors could validate their findings in an external cohort.

9) The title of study needs to be written as per the study objectives.

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Reviewer #1: Yes:  ZAHOOR AHMAD PARRAY

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Reviewers Comments.docx

-->PONE-D-24-29025R1-->-->The Impact of Mutations on TP53 Protein and MicroRNA Expression in HNSCC: Novel Insights for Diagnostic and Therapeutic Strategies-->-->PLOS ONE

Dear Dr. Mohamed,

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 Jan 29 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.

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,

Taj Mohammad, Ph.D.

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.

Additional Editor Comments:

I could not find the authors's response to Reviewer 2.

Moreover, I noticed significant differences in the MD simulation parameters between the earlier 100 ns simulations and the current 200 ns simulations. Could the authors clarify the source of this inconsistency? Were the simulations reinitiated, or were the previous 100 ns simulations extended? Additionally, what is the origin of the prominent peak observed in the RMSD and Rg of Apo-TP53? Furthermore, why was there a significant difference in the intermolecular hydrogen bonds between the 100 ns and 200 ns trajectories?

To ensure the robustness and reproducibility of the findings, I suggest performing MD simulations in triplicate.

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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.-->

The Impact of Mutations on TP53 Protein and  MicroRNA Expression in HNSCC: Novel Insights for Diagnostic and Therapeutic Strategies

PONE-D-24-29025R2

Dear Dr. Mohamed,

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.

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Kind regards,

Taj Mohammad, Ph.D.

Academic Editor

PLOS ONE

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