PONE-D-24-35176Investigating the functional and structural effect of non-synonymous single nucleotide polymorphisms in the cytotoxic T-lymphocyte antigen-4 gene: An in-silico studyPLOS ONE

Dear Dr. Hasan,

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Rajesh Kumar Pathak, Ph.D.

Academic Editor

PLOS ONE

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Additional Editor Comments:

The manuscript has been reviewed and found to be interesting. Comprehensive feedback addressing critical areas has been provided. Key revisions include updating dbSNP data, improving the molecular dynamics simulation results, and conducting secondary structure and PCA analyses. The authors should correct grammatical errors and vague phrasing, and ensure consistency in terminology. Expanding the discussion on the biological and clinical relevance of the findings will strengthen the impact of the 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: Partly

Reviewer #2: Yes

Reviewer #3: Partly

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

Reviewer #1: N/A

Reviewer #2: N/A

Reviewer #3: 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: Yes

Reviewer #3: Yes

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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: No

Reviewer #2: Yes

Reviewer #3: Yes

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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: Manuscript titled “Investigating the functional and structural effect of non-synonymous single nucleotide polymorphisms in the cytotoxic T-lymphocyte antigen-4 gene: An in-silico study’ presents good information about the nsSNPs in CTLA4. However, in my opinion following queris needs to be answered before accepting manuscript.

Major revision

1. dbSNP database (https://www.ncbi.nlm.nih.gov/snp/), respectively (accessed December, 2022) this is 1.5 years old data. What is the current status in this databse for the CTLA4?

Authors need to give current status also.

2. A 100 ns MD simulation analysis is not sufficient for the prediction, at least triplicate analysis should be done for better predicted results.

3. Line 280“The structural stability of native and four mutant proteins were calculated RMSD and 281 RMSF and compare to evaluate structural and functional change due to the mutation of the 282 corresponding protein” correct english

4. Manuscript needs to be checked with reference to english properly. There are many grammatical errors.

5. RMSD, RMSF, Rg values should be given in nm instead of A

6. Secondary structure analysis should be done for wild type and mutant structures.

7. PCA analysis should also be done to understand the structural impact of mutations.

Reviewer #2: The manuscript Titled: “Investigating the functional and structural effect of non-synonymous single nucleotide polymorphisms in the cytotoxic T-lymphocyte antigen-4 gene: An in-silico study” presents an in-silico analysis of the effects of non-synonymous single nucleotide polymorphisms (nsSNPs) in the cytotoxic T-lymphocyte antigen-4 (CTLA4) gene. The authors used various bioinformatics tools, including SIFT, PolyPhen-2, PROVEAN, and SNAP, to identify deleterious nsSNPs. Additionally, they evaluated protein stability, structural changes, and interactions using tools like ConSurf, I-Mutant, and molecular dynamics simulations.

The manuscript is timely and covers an important topic, as SNPs in the CTLA4 gene have been implicated in immune regulation and several diseases, including cancer and autoimmune disorders. Using computational tools for analyzing genetic variants is appropriate for this study, and the results may serve as a foundation for further experimental work. However, there are several areas where the manuscript can be strengthened in terms of clarity, depth of analysis, and presentation of results.

Major Comments:

The study presents a comprehensive computational analysis of nsSNPs in the CTLA4 gene. The significance of this work lies in its potential to shed light on the pathogenicity of certain SNPs that might contribute to disease susceptibility. However, the manuscript could benefit from stronger contextualization of its results with reference to previous experimental findings. While the authors mention autoimmune diseases and cancer, a more detailed discussion on the clinical relevance of the identified nsSNPs would enhance the paper’s impact.

The manuscript employs a wide array of computational tools, which is commendable. However, some methodological details are either missing or could be clarified:

Selection of SNPs: The criteria for selecting the 165 missense SNPs for further analysis are not fully explained. Were all missense variants analyzed, or was there a filtering process? Clarifying this would improve the transparency of the study.

Protein Modeling: The molecular dynamics simulation section is interesting, but further details are needed regarding the parameters used in these simulations. Specifically, what were the time steps and temperature conditions? Were multiple simulations conducted for each variant to ensure reproducibility?

The results provide a solid foundation for understanding how specific nsSNPs affect the structure and function of the CTLA4 protein. However:

The explanation of molecular dynamics simulation results could be expanded. The authors report RMSD and RMSF values but do not provide a thorough interpretation of what these fluctuations imply in terms of biological function.

The protein-protein interaction analysis using GeneMANIA and STRING is intriguing but lacks depth. A more detailed discussion of the relevance of interactions with proteins such as CD80 and CD86 in the context of immunological function would be beneficial.

A visual representation of the molecular dynamics simulation results would significantly enhance the clarity of the findings.

The discussion section should place more emphasis on how the findings can inform future in vitro or in vivo studies. The authors briefly mention the need for experimental validation, but they do not elaborate on how their results could be applied in a practical setting, for example, by guiding targeted mutagenesis experiments or developing therapeutic interventions.

Minor Comments:

The manuscript is well-written overall, but there are several areas where the clarity of writing could be improved:

The abstract is dense and could benefit from more concise wording, particularly in the results section. Summarizing the key findings in a few clear sentences would make it more accessible to a broader audience.

In the results section, the use of terms such as “deleterious,” “probably damaging,” and “benign” are used without providing sufficient context for how these terms were determined by each computational tool. A short explanation in the methodology or results section about how these terms were defined would aid comprehension.

The manuscript contains a number of useful figures and tables. However, it would be helpful to:

Add more detailed captions for each figure and table. For example, in Table 4, providing more information on how the ΔΔG stability values were calculated and what these values mean in terms of biological relevance would enhance understanding.

Include a schematic that summarizes the workflow of the computational analyses performed. This would provide readers with a quick overview of the methodologies used.

The references are appropriate and up to date. However, the authors could strengthen the manuscript by citing more recent experimental studies that have validated nsSNPs in the CTLA4 gene, particularly in relation to cancer and autoimmune diseases.

Recommendations:

Clarify the methodology, particularly the selection process for the SNPs analyzed and the details of the molecular dynamics simulations.

Expand the discussion to include more implications for future experimental work and clinical relevance.

Enhance the figures and tables by adding more detailed captions and including a workflow schematic.

Improve the clarity of the writing, particularly in the abstract and results sections.

The manuscript presents valuable findings but requires minor revisions to improve clarity, detail, and depth of analysis. Once these revisions are made, the manuscript will strongly contribute to the field.

Reviewer #3: Title: Investigating the functional and structural effect of non-synonymous single nucleotide polymorphisms in the cytotoxic T-lymphocyte antigen-4 gene: An in-silico study

1. In section 3.1, Accession number of the sequence is missing, please provide it, in order to verify the result?

2. In line 219, You mention 5 nsSNPs for SNAP and 4 nsSNPs for PROVEAN, but it might be useful to explain why different numbers of nsSNPs were used for these analyses?

3. In line 227, Avoid Vague Phrasing: Instead of “notable consistency,” I used “strong concordance” to make the statement more specific.

4. Use the correct symbol for free energy change (ΔΔG instead of DDG) to maintain consistency with standard scientific terminology.

5. In Line 238 and 239, specify what a conservation score of 4, 5, and 9 indicates in terms of evolutionary conservation, as readers might not be familiar with the ConSurf scoring system.

6. Please correct the caption of Table 3.

7. In Line 246, can you please focus that why the size of amino acid residues is responsible for protein’s function disruption?

8. Alanine residue is generally an accepted single residue first choice for mutation, can you try to mutate the key residues to Alanine here and study the result?

9. Section 3.8 should be improved by explaining of how protein interactions can impact network function.

10. In Section 3.9, since you simulated all the systems for 100 ns, could you incorporate principal component analysis (PCA) to examine the atomic movements, particularly at the mutated residue sites?

11. In the Materials and Methods section, while you mention that molecular dynamics simulations were performed using the Desmond v6.3 program, there is no information provided regarding the number of steps for energy minimization, position restraints, or equilibration. Please include these details.

12. Since you simulated the systems in a water medium, you should also include solvent accessible surface area (SASA) analysis to examine the role of water around the mutated regions and compare it with the wild type. This is particularly relevant since you previously mentioned that the mutations were based on the shape of the amino acid residues.

13. How many simulation repeats did you performed?

14. Please correct the typographical and grammatical errors throughout the manuscript to improve its readability and clarity.

15. The plot in Figure 7 is unclear. Please add the average plot of hydrogen bonds for each system to make the data for each system more distinguishable.

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Reviewer #1: Yes: Monika Jain

Reviewer #2: No

Reviewer #3: Yes: Navaneet Chaturvedi, Amity Institute of Biotechnology, Amity University, Noida, 201313, Uttar Pradesh, India

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Investigating the functional and structural effect of non-synonymous single nucleotide polymorphisms in the cytotoxic T-lymphocyte antigen-4 gene: An in-silico study

PONE-D-24-35176R1

Dear Dr. Hasan,

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.

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Kind regards,

Rajesh Kumar Pathak, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The manuscript can be accepted for publication.

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 #1: All comments have been addressed

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 #1: Yes

Reviewer #3: Yes

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

Reviewer #1: N/A

Reviewer #3: Yes

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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 #1: Yes

Reviewer #3: Yes

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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 #1: Yes

Reviewer #3: Yes

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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 #1: Authors have answered almost all queries, except triplicate simulation, Manuscript can be accepted now

Reviewer #3: All responses have been thoroughly reviewed and are presented in a clear and coherent manner. I believe that the content meets the necessary criteria for academic rigor and relevance. Therefore, I recommend that the work be accepted for publication.

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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 #1: Yes: Monika Jain

Reviewer #3: No

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