Phylogenetic analysis of the MCL1 BH3 binding groove and rBH3 sequence motifs in the p53 and INK4 protein families

Anna McGriff; William J. Placzek

doi:10.1371/journal.pone.0277726

Peer Review History

Original SubmissionOctober 31, 2022
22 Nov 2022 Decision Letter - Klaus Roemer, Editor PONE-D-22-30013Phylogenetic analysis of the MCL1 BH3 binding groove and rBH3 sequence motifs in the p53 and INK4 protein familiesPLOS ONE Dear Dr. Placzek, 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. Although both referees suggest wet lab support of the presented in silico data, we feel that this should be subject of further studies. However, it might be useful to combine figures 3, 4, and 5 as well as figures 6, 7 and 8, as suggested by referee 2. Please submit your revised manuscript by Jan 06 2023 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. 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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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: N/A ****** 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). 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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: Understanding the evolution of the apoptotic machinery and how it is linked to cell cycle regulators and the p53 family is of fundamental interest and importance. This study by McGriff and Placzek adds a few pieces of data to the overall picture. They make a careful analysis of the reverse BH3 motif and its presence in p53 family members and INK4 members in various animals. The overall conclusions states that MCL1 interaction with rBH3 motifs is conserved in jawed vertebrates. I find the manuscript well written, the introduction is perhaps a bit too long and rather detailed. There is an option to condense it a bit, while putting the study in an even bigger context. As always with this type of study, one may wonder if a predicted motif actually is a functional. Many times the deduced presence of a motif has failed to be confirmed experimentally, sometimes because certain amino acids in the vicinity has marked negative impact on the binding motif. And I guess this is one thing with evolution, functional motifs may evolve from dysfunctional motifs that turns functional. So in this sense I miss experimental testing but of course this is not easy and somewhat beyond the scope of the present investigation. I would also like to ask the authors if they may have an alternative file format or extension on some jvl files in the supplement. I could not access 3 of those files on my MacBookAir, despite looking for some appropriate software. If it is possible. Reviewer #2: In their work submitted to PLOS ONE, McGriff & Placzek apply established tools for in silico protein sequence analyses to study the phylogenetic conservation of the BH3 binding grove in the anti-apoptotic Bcl-2 family member MCL1 and the reverse BH3 (rBH3) motif in the p53 (p53, p63, p73) and INK4 (p15, p16, p18, p19) families. Placzek and colleagues had shown before that rBH3 motifs in p73 and p18 bind to the MCL1 binding grove, and that this interaction interferes with p18 function by stimulating its degradation and prevents tetramerization of p73. Based on the identification of conserved binding motifs in vertebrates, the authors predict that another member of the p53 family – p63 – can interact with MCL1, but that the phylogenetically youngest family member p53 has lost this potential. Comparable predictions were made regarding the INK4 protein family: While a putative rBH3 motif can be found in p19 - which together with p18 represents the more ancestral members of the INK4 family - the more recent duplications p15 and p16 have lost the domains that contain the rBH3 motifs. The authors conclude that both the BH3 binding grove in MCL1 and the rBH3 motif in p63/p73 and p18/p19 are conserved in many jawed vertebrates, which suggests that the interaction of the proteins is important for vertebrate cell signaling. They further state that the absence of rBH3 motifs in the important tumor suppressors p53 and p16 – which evolved more recently than the more ancient family members – “shows a unique mode of regulation in which MCL1 is able to interact with and influence general homeostatic family function, while not interfering with acute stress signaling”. Studying the mechanisms underlying cell cycle progression and apoptosis is critical for understanding oncogenesis and developing treatments for cancer. Thus, analyses of how the antiapoptotic protein MCL1 interacts with p53 and INK4 family members are of interest to a broad readership. However, even though it is well-written, and the presented data satisfy the criteria for publication in PLOS ONE, I am not very enthusiastic about the manuscript submitted by McGriff and Placzek. To me, the provided data appear to be rather preliminary and would be a good starting point to study whether their predictions are supported by wet lab data. Some rather simple experiments that validate the predicted interaction between MCL1 and p63/p19 and that show that mutation of the rBH3 motif in these proteins disrupts binding would add valuable data to the manuscript. Furthermore, without such data, much about the suggested mechanisms remains speculative, and if the authors are to make statements as the one cited above (lines 404-408 in the manuscript), they must include experimental results to support them. As a minor comment, I suggest combining figures 3, 4, and 5 as well as figures 6, 7, and 8. ****** 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: No Reviewer #2: 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. https://doi.org/10.1371/journal.pone.0277726.r001
Revision 1
6 Jan 2023 Author Response We would like to thank both reviewers for their time and suggestions. We have attempted to address all of your comments and suggestions. Referee #1 (Remarks to the Author): Understanding the evolution of the apoptotic machinery and how it is linked to cell cycle regulators and the p53 family is of fundamental interest and importance. This study by McGriff and Placzek adds a few pieces of data to the overall picture. They make a careful analysis of the reverse BH3 motif and its presence in p53 family members and INK4 members in various animals. The overall conclusions states that MCL1 interaction with rBH3 motifs is conserved in jawed vertebrates. I find the manuscript well written, the introduction is perhaps a bit too long and rather detailed. There is an option to condense it a bit, while putting the study in an even bigger context. - As suggested, we have attempted to streamline the introduction somewhat, but also want to cover a lot of information to make the manuscript as accessible to people outside of the Bcl2 community. As always with this type of study, one may wonder if a predicted motif actually is a functional. Many times the deduced presence of a motif has failed to be confirmed experimentally, sometimes because certain amino acids in the vicinity has marked negative impact on the binding motif. And I guess this is one thing with evolution, functional motifs may evolve from dysfunctional motifs that turns functional. So in this sense I miss experimental testing but of course this is not easy and somewhat beyond the scope of the present investigation. - We agree that our findings provide a lot of stepping off points for analysis of MCL1 regulation and further that wet-lab studies would be excellent in future studies, though they are outside the scope of the present manuscript. I would also like to ask the authors if they may have an alternative file format or extension on some jvl files in the supplement. I could not access 3 of those files on my MacBookAir, despite looking for some appropriate software. If it is possible. - We apologize that these were not more accessible. We have updated the file format for all supplementary data to FASTA format that should make these much easier to interact with. Referee #2 (Remarks to the Author): In their work submitted to PLOS ONE, McGriff & Placzek apply established tools for in silico protein sequence analyses to study the phylogenetic conservation of the BH3 binding grove in the anti-apoptotic Bcl-2 family member MCL1 and the reverse BH3 (rBH3) motif in the p53 (p53, p63, p73) and INK4 (p15, p16, p18, p19) families. Placzek and colleagues had shown before that rBH3 motifs in p73 and p18 bind to the MCL1 binding grove, and that this interaction interferes with p18 function by stimulating its degradation and prevents tetramerization of p73. Based on the identification of conserved binding motifs in vertebrates, the authors predict that another member of the p53 family – p63 – can interact with MCL1, but that the phylogenetically youngest family member p53 has lost this potential. Comparable predictions were made regarding the INK4 protein family: While a putative rBH3 motif can be found in p19 - which together with p18 represents the more ancestral members of the INK4 family - the more recent duplications p15 and p16 have lost the domains that contain the rBH3 motifs. The authors conclude that both the BH3 binding grove in MCL1 and the rBH3 motif in p63/p73 and p18/p19 are conserved in many jawed vertebrates, which suggests that the interaction of the proteins is important for vertebrate cell signaling. They further state that the absence of rBH3 motifs in the important tumor suppressors p53 and p16 – which evolved more recently than the more ancient family members – “shows a unique mode of regulation in which MCL1 is able to interact with and influence general homeostatic family function, while not interfering with acute stress signaling”. Studying the mechanisms underlying cell cycle progression and apoptosis is critical for understanding oncogenesis and developing treatments for cancer. Thus, analyses of how the antiapoptotic protein MCL1 interacts with p53 and INK4 family members are of interest to a broad readership. However, even though it is well-written, and the presented data satisfy the criteria for publication in PLOS ONE, I am not very enthusiastic about the manuscript submitted by McGriff and Placzek. To me, the provided data appear to be rather preliminary and would be a good starting point to study whether their predictions are supported by wet lab data. Some rather simple experiments that validate the predicted interaction between MCL1 and p63/p19 and that show that mutation of the rBH3 motif in these proteins disrupts binding would add valuable data to the manuscript. Furthermore, without such data, much about the suggested mechanisms remains speculative, and if the authors are to make statements as the one cited above (lines 404-408 in the manuscript), they must include experimental results to support them. -We agree that this dry-lab analysis provides a number of novel insights that are definitely of interest for further study, but we believe this to be outside the scope of the current manuscript. -We do agree that the statement in lines 404-408 was too strong and have tempered this to highlight that this is only a model that could be suggested by the current study. As a minor comment, I suggest combining figures 3, 4, and 5 as well as figures 6, 7, and 8. -We agree and have made this suggested change. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0277726.r002
10 Jan 2023 Decision Letter - Klaus Roemer, Editor Phylogenetic analysis of the MCL1 BH3 binding groove and rBH3 sequence motifs in the p53 and INK4 protein families PONE-D-22-30013R1 Dear Dr. Placzek, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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, Klaus Roemer Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0277726.r003
Formally Accepted
16 Jan 2023 Acceptance Letter - Klaus Roemer, Editor PONE-D-22-30013R1 Phylogenetic analysis of the MCL1 BH3 binding groove and rBH3 sequence motifs in the p53 and INK4 protein families Dear Dr. Placzek: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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. Klaus Roemer Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0277726.r004

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