PONE-D-24-15873Implications of tissue specificSTING protein flux and abundance on the development of therapeutics.PLOS ONE

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: No

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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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?

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Reviewer #1: Yes

Reviewer #2: 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: In this manuscript, Thomas Angel et al. use 2H2O to measure protein fractional synthesis rates (FSR) of STING across different tissues in vivo. The authors reached a conclusion that the FSR of STING varies from 0.027 in muscle to 0.731 in lung per day, and the half-life of STING varies from 3.8 to 25.4 days. Even unexpectedly, the half-life of STING in WT mice and STING-driven auto-immune TREX1D18N/D18N mice are largely comparable. Though the authors used cutting-edge techniques in the experiments, the conclusion is still not fully convincing, mainly due to very limited data provided and one single approach used.

1. The authors show the half-life of activated STING is 2 hours in Fig 1a, and this is consistent with most previous studies. The authors should use this widely-used system to compare with 2H2O-based techniques used in this study to verify the latter approach indeed works.

2. The authors also need more supportive data to verify their approach. For example, comparing their 2H2O-based techniques with standard CHX chase experiments to further validate their study. CHX chase experiments can at least be done in vitro using some culturable tissue or cells from WT and TREX1D18N/D18N mice.

3. The authors conclude the half-life of STING is at least 3.8 days, which is quite longer than the observed static STING half-life from CHX chase experiments in previous literature. Method-wise, mice were injected with 2H2O once followed by enriched drinking water (8% 2H2O) for the remainder of the experiment. Is this approach sufficient to make any amino acids in the mice incorporated with 2H2O during the whole experiment? If only a fraction of amino acids in the cell is incorporated with 2H2O and the unlabeled amino acids, for example, recycled from digested protein by the proteasome, can still be used in synthesizing STING, then the techniques used cannot detect the actual synthesis rate of STING, resulting in lower than the actual rate. This needs to be thoroughly addressed.

Reviewer #2: In this manuscript from researchers at GlaxoSmithKline, the authors document the protein turn-over of STING in wildtype (WT) C57Bl/6 mice and those carrying the Acardi-Goutiéres syndrome (AGS)-associated mutant isoform of Trex1 (D18N homozygous). The authors demonstrate that STING protein half-life is tissue specific and similar between WT and AGS mice, with the shortest half-life in colon and lymph node and longest in skeletal muscle. Overall, the experiments are well executed, and this study constitutes important but rather specialist information that will be useful to those researchers working on STING drug development.

Major Comments

1) The authors need to explicitly state where they obtained the Trex1 [D18N/D18N] model, as the details are not clear in the methods. Was this generated by the authors, did they receive it from a laboratory? What specific model is it? The original Trex1[D18N/D18N] model was in the 129S6/SvEvTac mouse background, not C57Bl/6. Did the authors obtain the mice already in the C57Bl/6 background, or did they do the backcrossed themselves. Really, much more detail is needed here to ensure future reproducibility and establish the provenance of the mice.

2) This is a rather limited manuscript in scope, and of VERY specialist interest. To move beyond specialist interest for drug development, the authors should provide correlation data on interferon stimulated gene (ISG) regulation down-stream of STING relative to protein turn-over and abundance in the tissues tested and between WT and AGS mice. This could be done using a basket of ISGs and a qPCR panel, or if budget allows via RNA-seq. This information would greatly increase the impact of the current study and interest for a broader range of researchers and in particular immunologists.

Minor Comments

1) Despite the misuse of human gene and protein (ALL CAPS) nomenclature by the original creators of the Trex1[D18N/D18N] mouse – the authors throughout the manuscript should use the correct gene and protein nomenclature for the mouse Trex1, which is capitalized first letter and then lowercase.

2) In the Author contributions please clarify what “…in-life TREX [D18N/D18N]…” means?

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Reviewer #1: No

Reviewer #2: No

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Implications of tissue specific STING protein flux and abundance on inflammation and the development of targeted therapeutics

PONE-D-24-15873R1

Dear Dr. Pesiridis,

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Additional Editor Comments (optional):

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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 #2: All comments have been addressed

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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 #2: Yes

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

Reviewer #2: 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 #2: 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 #2: 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 #2: (No Response)

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Reviewer #2: No

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