The SARM1 TIR domain produces glycocyclic ADPR molecules as minor products

Jeremy Garb; Gil Amitai; Allen Lu; Gal Ofir; Alexander Brandis; Tevie Mehlman; Philip J. Kranzusch; Rotem Sorek

doi:10.1371/journal.pone.0302251

Peer Review History

Original SubmissionOctober 4, 2023
6 Feb 2024 Decision Letter - Asif Ali, Editor PONE-D-23-31863The SARM1 TIR domain produces glycocyclic ADPR molecules as minor productsPLOS ONE Dear Dr. Garb, 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 Mar 22 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. 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We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript. 4. Thank you for stating the following financial disclosure: "We thank A. Yaron, O. Abraham, N. Pursotham, and S. Hobbs for fruitful discussions on SARM1 activity and biochemical analysis of gcADPR signaling, as well as the Sorek and Kranzusch lab members for comments on the manuscript. R.S. was supported, in part, by the European Research Council (grant no. ERC-AdG GA 101018520), Israel Science Foundation (MAPATS Grant 2720/22), the Deutsche Forschungsgemeinschaft (SPP 2330, Grant 464312965), the Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine, Dr. Barry Sherman Institute for Medicinal Chemistry, Miel de Botton, the Andre Deloro Prize, and the Knell Family Center for Microbiology. P.J.K. was supported, in part, by the Pew Biomedical Scholars program and The Mathers Foundation. 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Additional Editor Comments: The manuscript titled "The SARM1 TIR domain produces glycocyclic ADPR molecules as minor products" by Garb et al. presents significant findings related to gcADPR molecules during Wallerian degeneration by the SARM1 TIR domain. Reviewer #1 highlights the need for statistical details, raw data in supplementary materials, and an exploration of evolutionary differences between Drosophila and human SARM1 TIR domains. Reviewer #2 expresses concern about the lack of evidence regarding the role of gcADPR molecules in inducing cell death, seeks an explanation for the Drosophila's higher catalytic activity, and recommends including stastical significance values for figures. Addressing these comments will enhance the manuscript's rigor and suitability for publication. [Note: HTML markup is below. Please do not edit.] 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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: No ****** 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: No Reviewer #2: Yes ****** 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: Yes ****** 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: Review Comments The manuscript "The SARM1 TIR domain produces glycocyclic ADPR molecules as minor products" by Garb et al. is a good discovery about gcADPR molecules during Wallerian degeneration by SARM1 TIR domain. This research article provides importance of small molecules during stress conditions. This manuscript is of interest to the neurobiology and human biology researchers, as well as different biology researchers and I expect that the article will be well-cited. I have the following minor comments to consider. 1. Authors add statistical details in their figure’s legend and statistical section in materials and methods. 2. Authors should provide raw data of figures in supplementary with details? 3. In both figures, the Drosophila SARM1 TIR domain is more catalytically active than the human SARM1I, therefore I recommend that the authors consider your results from an evolutionary standpoint as well. Is there any difference in protein sequence in the Drosophila and human catalytic sites? Would like to give some insights about that in manuscript discussions? Reviewer #2: The manuscript titled “The SARM1 TIR domain produces glycolytic ADPR molecules as minor products” discusses the generation of 1''-2 and 1''-3 glycolytic ribose molecules as subordinate products of TIR domains in both human and Drosophila SARM1. The authors, through well-defined experiments, have preliminarily concluded that these molecules are minor reaction products, yet they also suggest that these byproducts may play a role in SARM1-induced programmed axonal death in animals. However, they have not provided evidence for the latter part. Despite this, the novel findings are noteworthy and could be considered for acceptance once the following concerns are addressed. 1. In an invitro setting, it is unclear whether the 1”-2 and 1”-3 gcADPR molecules (in concentrations ranging from high nanomolar to low micromolar) alone or in combination with ADPR or cADPR induce cell death of cultured axons. 2. The inclusion of a mutated TIR domain protein in the mass spectrometry analysis presented in Figure 2 would enhance the study. 3. An explanation for the increased activity of the Drosophila TIR domain compared to the human TIR domain would be informative. 4. Please include the significance values of the figures. ****** 6. 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: Pawan Kumar Reviewer #2: Yes: Suvranil Ghosh ******** [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 < Attachments Attachment Submitted filename: PlosOne Review TIR domain SARM1.docx https://doi.org/10.1371/journal.pone.0302251.r001
Revision 1
19 Mar 2024 Author Response A response to reviewers letter is included in this submission. We thank the referees for their thoughtful comments and suggestions. Our point-to-point response to each of the referees’ comments is detailed below. Reviewer #1: Review Comments The manuscript "The SARM1 TIR domain produces glycocyclic ADPR molecules as minor products" by Garb et al. is a good discovery about gcADPR molecules during Wallerian degeneration by SARM1 TIR domain. This research article provides importance of small molecules during stress conditions. This manuscript is of interest to the neurobiology and human biology researchers, as well as different biology researchers and I expect that the article will be well-cited. Answer: Thank you for acknowledging the importance of our study! I have the following minor comments to consider. 1. Authors add statistical details in their figure’s legend and statistical section in materials and methods. Answer: We thank the reviewer for this valuable comment. We now added statistical details to figures, and explain the statistical analyses in the figure legends. 2. Authors should provide raw data of figures in supplementary with details? Answer: We thank the reviewer for this comment. We now included the raw data for figures 1 and 2 in a new Supplementary Table S1. 3. In both figures, the Drosophila SARM1 TIR domain is more catalytically active than the human SARM1I, therefore I recommend that the authors consider your results from an evolutionary standpoint as well. Is there any difference in protein sequence in the Drosophila and human catalytic sites? Would like to give some insights about that in manuscript discussions? Answer: We thank the reviewer for this comment. We want to emphasize that the proteins were tested in conditions other than their natural conditions in neuronal cells – we either expressed them in bacteria (Figure 1B, 1C, 1D) or in vitro (Figure 2). For this reason, we would caution against interpretations regarding different enzymatic rates between the human and Drosophila SARM1 TIRs, due to the non-native environment. To clarify this point, we modified the Discussion text and it now reads: “Notably, we observed production of gcADPR when SARM1 TIR domains were overexpressed in bacterial cells, and it is possible that the full length SARM1 protein does not generate these molecules when expressed at physiological levels in the native animal cells.” Reviewer #2: The manuscript titled “The SARM1 TIR domain produces glycolytic ADPR molecules as minor products” discusses the generation of 1''-2 and 1''-3 glycolytic ribose molecules as subordinate products of TIR domains in both human and Drosophila SARM1. The authors, through well-defined experiments, have preliminarily concluded that these molecules are minor reaction products, yet they also suggest that these byproducts may play a role in SARM1-induced programmed axonal death in animals. However, they have not provided evidence for the latter part. Despite this, the novel findings are noteworthy and could be considered for acceptance once the following concerns are addressed. Answer: We thank the reviewer for acknowledging the novelty of our findings! 1. In an invitro setting, it is unclear whether the 1”-2 and 1”-3 gcADPR molecules (in concentrations ranging from high nanomolar to low micromolar) alone or in combination with ADPR or cADPR induce cell death of cultured axons. Answer: as indicated by the reviewer in their summary above, in this study we did not test whether the molecules produced by SARM1 affect cell death in axons. We hope that our discoveries will prompt neurobiologists to examine this possibility, and hence our motivation in publishing the current manuscript. To make this point clear to the reader, our text in the Discussion section reads: “The gcADPR molecules revealed in this study as minor products of SARM1 TIR domains might be related to additional processes that could operate downstream of SARM1. Whether these molecules indeed accumulate in axons during Wallerian degeneration, and whether they have a biological function in axonal death, remains to be determined by future studies.” 2. The inclusion of a mutated TIR domain protein in the mass spectrometry analysis presented in Figure 2 would enhance the study. Answer: We thank the reviewer for this comment, but we believe we addressed these mutations already in Figure 1. We performed our experiments including controls for active-site mutations when the SARM1 TIR was expressed in bacterial cells (Figure 1). The mutations included substitution of the active site glutamate in both the human and Drosophila SARM1 TIR, and in both cases the enzymatic activity was clearly and completely eliminated (Figure 1B, 1C, 1D). We feel that these mutations are already very convincing. 3. An explanation for the increased activity of the Drosophila TIR domain compared to the human TIR domain would be informative. Answer: We thank the reviewer for this comment. We want to emphasize that the proteins were tested in conditions other than their natural conditions in human neuronal cells – we either expressed them in bacteria (Figure 1B, 1C, 1D) or in vitro (Figure 2). For this reason, we would caution against interpretations regarding different enzymatic rates between the human and Drosophila SARM1 TIRs, due to the non-native environment. To clarify this point, we modified the Discussion text and it now reads: “Notably, we observed production of gcADPR when SARM1 TIR domains were overexpressed in bacterial cells, and it is possible that the full length SARM1 protein does not generate these molecules when expressed at physiological levels in the native animal cells.” 4. Please include the significance values of the figures. Answer: We thank the reviewer for this valuable comment. We have added statistical details to figures and explain the statistical analyses in the revised figure legends. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0302251.r002
1 Apr 2024 Decision Letter - Asif Ali, Editor The SARM1 TIR domain produces glycocyclic ADPR molecules as minor products PONE-D-23-31863R1 Dear Dr. Garb, 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. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Asif Ali Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0302251.r003
Formally Accepted
7 Apr 2024 Acceptance Letter - Asif Ali, Editor PONE-D-23-31863R1 PLOS ONE Dear Dr. Garb, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Asif Ali Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0302251.r004

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