The PAR2 signal peptide prevents premature receptor cleavage and activation

Belinda Liu; Grace Lee; Jiejun Wu; Janise Deming; Chester Kuei; Anthony Harrington; Lien Wang; Jennifer Towne; Timothy Lovenberg; Changlu Liu; Siquan Sun

doi:10.1371/journal.pone.0222685

Peer Review History

Original SubmissionAugust 30, 2019
4 Dec 2019 Decision Letter - Hubert Vaudry, Editor PONE-D-19-23622 The PAR2 signal peptide prevents premature receptor cleavage and activation PLOS ONE Dear Dr. Liu, 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. Your manuscript has been reviewed by two referees who are recognized exoerts in this field. Both reviewers made reasonable recommendations that you should carefully consider in order to revise your manuscript accordingly. We would appreciate receiving your revised manuscript by Jan 18 2020 11:59PM. When you are 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 change the online submission form on your behalf. Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests [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: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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: Yes Reviewer #2: No ****** 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: General Comments: This manuscript presents interesting work on the functional importance of signal peptide on the GPCR sequences. As it is well known that only 10% of the modern GPCRs have a signal peptide on their gene sequences, therefore, their importance and function has always been questioned. In this work, the role of the signal peptide region in PAR2 is explored. It is presented with evidences that the prime role of PAR2 signal sequence is to inhibit intracellular protease from unintended and premature activation. Overall the article is very well written and raises interesting points about the role of signal peptide in this particular and other GPCRs Concerns: 1) Author’s suggests that the change in the EC50 of Trypsin and artificial ligand PAR2-AP observed on PAR2ΔSP when compared to PAR2 (WT) receptor is evidence for less number of PAR2ΔSP receptor on cell surface (Line 309). Since, EC50 is a measure of agonist concentration to produce half of the maximal response, hence its relationship with number of receptors, usually measured by the maximal response, must be explained in detail. 2) line-308: the mention of Super-Pharmacology Phenomenon cited the paper: “Differences between natural and recombinant G protein-coupled receptor systems with varying receptor/G protein stoichiometry”, that does not talk about “Super-Pharmacology Phenomenon”. 3) As the paper showed that the signal sequence of PAR2 is capable of functioning as a classical signal peptide (Fig 2), the authors however subsequently mentioned that the signal peptide property is not important while its main effects are to prevent premature activation in ER and golgi (Fig 5, 7). Can the signal peptide do both at the same time? As the signal peptide is release after entering the ER, how can it protect the receptor? what is the mechanism and explanation for the observed result? 4. The text quality of Fig 4(F), Fig 8, Fig10(B) must be improved. Reviewer #2: The present manuscript entitled "The PAR2 signal peptide prevents premature receptor cleavage and activation”, described the role of signal peptide presents in PAR2 in expression and activation of this receptor. Experiments based on detection of wild-type and mutant receptors at the surface level cells associated with functional analysis of PAR2 and its mutants are rigorously performed. However, one major point and some minor points should be clarified: - As demonstrated by authors, the deletion of the signal peptide induces the sequestration of PAR2 into the cell. This sequestration was modulated by the presence or not of tethered ligand region. Authors concluding that signal peptide prevents the protease action into the cell. However, as mentioned by authors the signal peptide sequence was coded by one exon suggesting that the expression of signal peptide deleted receptors might be possible. The question is: what is the functional role of this signal peptide deleted intracellular receptor? Could be it activated in sequestered intracellular localization? This aspect should be investigated and/or discussed. Minor points: Line 219 “immune-staining” could be changed by “immunofluorescence-staining” Lines 219-225, this paragraph was very unclear. More referencing to figure N°2 should be added to clarify the purpose. No statistical significance (or not) appears in the figure 2C, 2D, 4A, 9 and 10 ****** 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: Yes: Couvineau Alain [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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0222685.r001
Revision 1
8 Jan 2020 Author Response Responses to Reviewers Authors responses to reviewer#1 Reviewer #1: General Comments: This manuscript presents interesting work on the functional importance of signal peptide on the GPCR sequences. As it is well known that only 10% of the modern GPCRs have a signal peptide on their gene sequences, therefore, their importance and function has always been questioned. In this work, the role of the signal peptide region in PAR2 is explored. It is presented with evidences that the prime role of PAR2 signal sequence is to inhibit intracellular protease from unintended and premature activation. Overall the article is very well written and raises interesting points about the role of signal peptide in this particular and other GPCRs Authors’ responses: The authors thank the reviewer’s compliments and we believe the results will help us for the better understanding of the functions of signal peptides for GPCRs, particularly for PARs, and for signal peptides in general. Concerns: 1) Author’s suggests that the change in the EC50 of Trypsin and artificial ligand PAR2-AP observed on PAR2ΔSP when compared to PAR2 (WT) receptor is evidence for less number of PAR2ΔSP receptor on cell surface (Line 309). Since, EC50 is a measure of agonist concentration to produce half of the maximal response, hence its relationship with number of receptors, usually measured by the maximal response, must be explained in detail. Authors’ responses: The Authors thank the reviewer for the critical points. In our revised manuscript, we added more discussions to help explain this phenomenon. It is true that when receptor expression level is very low, an increase of receptor expression often leads to an increased maximum response (Emax value). However, when receptor expression level exceeds certain level, which is often true for the recombinantly expressed receptors, the Emax value will not increase further due to reaching the capacity of cell signal transduction. In contrast, increased receptor expression will lead to increased apparent potency. In our revised manuscript, we added these discussions (Page 14, lines 322-328). To help explain this phenomenon, we changed reference#15: “Analysis of the role of receptor number in defining the intrinsic activity and potency of partial agonists in neuroblastoma x glioma hybrid NG108-15 cells transfected to express differing levels of the human beta 2-adrenoceptor. Mol. Pharmacol. 1995; 48:316-325” which provided very detailed studies for the receptor expression level and the apparent potencies. 2) line-308: the mention of Super-Pharmacology Phenomenon cited the paper: “Differences between natural and recombinant G protein-coupled receptor systems with varying receptor/G protein stoichiometry”, that does not talk about “Super-Pharmacology Phenomenon”. Authors’ responses: We thank the reviewer for pointing this out. We now changed the sentence to “This is likely due to the over-expression of the recombinant receptor causing a phenomenon and displaying increased potency of the ligand to the receptor (ref. 15)”. Please see page 14, lines 322-328. Page 23, lines 594-598. 3) As the paper showed that the signal sequence of PAR2 is capable of functioning as a classical signal peptide (Fig 2), the authors however subsequently mentioned that the signal peptide property is not important while its main effects are to prevent premature activation in ER and golgi (Fig 5, 7). Can the signal peptide do both at the same time? As the signal peptide is release after entering the ER, how can it protect the receptor? what is the mechanism and explanation for the observed result? Authors’ responses: We apologize for the confusions. For PAR2 with the protease cleavage/activation site, the signal peptide is very critical for PAR2 cell surface expression. However, when the cleavage site is blocked (by mutation, or protease inhibitors), or removed, then the signal peptide is not critical for PAR2 cell surface expression. It is not clear whether the signal peptide can do both jobs at the same time, or do the two jobs sequentially. Although the signal peptide is cleaved and released after entering ER, it is still possible that the signal peptide reignition particle (or some of signal peptide reignition particle proteins) may still bind to PAR2 protein and physically block the protease cleavage site. Alternatively, the recognition of the signal peptide may sequester PAR2 protein into a zone where proteases do not function, or where is free of proteases. We added these discussions in our revised manuscript (page 21, lines 516-521). The similar mechanism may also apply to secreted proteases, which are often secreted first as pro-enzymes that lack protease activities. After secretion, they are activated by extracellular factors (other protease, pH, etc.) and the activated protease can do self-activation of the pro-enzymes. Taking trypsin as an example, trypsin is made as trypsinogen by pancreas. Upon secretion, trypsinogen is then activated by enterokinase and the active trypsin can activate trypsinogen and the activation cascade starts on. Inside the pancreas cells, trypsinogen has to be kept inactivated. Otherwise, a very small amount activation of trypsinogen, even by accident, will lead to tremendous amount of trypsin activation, which will eventually lyse the trypsin producing cells. This never happened and suggest that trypsinogen is protected from protease cleavage/activation inside the cells. 4. The text quality of Fig 4(F), Fig 8, Fig10(B) must be improved. Authors’ responses: The resolution of the figures decreased dramatically after the automatic conversion to PDF. We now enlarged the figures and hope they look better. Sorry for the inconvenience. Authors responses to reviewer#2 Reviewer #2: The present manuscript entitled "The PAR2 signal peptide prevents premature receptor cleavage and activation”, described the role of signal peptide presents in PAR2 in expression and activation of this receptor. Experiments based on detection of wild-type and mutant receptors at the surface level cells associated with functional analysis of PAR2 and its mutants are rigorously performed. However, one major point and some minor points should be clarified: - As demonstrated by authors, the deletion of the signal peptide induces the sequestration of PAR2 into the cell. This sequestration was modulated by the presence or not of tethered ligand region. Authors concluding that signal peptide prevents the protease action into the cell. However, as mentioned by authors the signal peptide sequence was coded by one exon suggesting that the expression of signal peptide deleted receptors might be possible. The question is: what is the functional role of this signal peptide deleted intracellular receptor? Could be it activated in sequestered intracellular localization? This aspect should be investigated and/or discussed. Authors responses Thanks to the reviewer for raising this very brain stimulating question. In our manuscript, we only created an artificial protein PAR2∆SP. It is possible that this receptor will be activated by intracellular protease located in ER and Golgi and causing cell signaling before reaching the cells surface, which may increase the basal activation of the cells without the extracellular proteases, achieving a raised tone for certain physiological status of the cells. Since the signal peptide coding region of PAR2 is on a separate exon in the PAR2 gene, alternative splicing, or transcription start (using a potential promotor in the intron region) could lead to a mRNA that encodes a PAR2 protein without the signal peptide. A blast search of NCBI Genbank showed that there is a single submission (XM_017009223.1) encoding a PAR2 without the signal peptide, suggesting that it remains a possibility that a natural PAR2 without the signal peptide may exist. In our revised manuscript, we add these discussions. Page 21, lines 537-543. Minor points: Line 219 “immune-staining” could be changed by “immunofluorescence-staining” Authors responses As suggested by reviewer, we changed “immune-staining” to “immunofluorescence-staining. Page 11, line 223. Lines 219-225, this paragraph was very unclear. More referencing to figure N°2 should be added to clarify the purpose. Authors responses In our revised manuscript, we added more referring of Fig 2A to help clarify the purposes. Page 11, lines 226-229; 236-237. No statistical significance (or not) appears in the figure 2C, 2D, 4A, 9 and 10 Authors responses We added statistical analysis to Figures 2C, 2D, 4A, 9 and 10 and added details for the statistical analysis in the legends respectively. Please see details for Figure 2 legend: Page 11, line 245 to page 12, line 254. Figure 4 legend: Page 13, lines 299 -302. Figure 9 legend: Page 18, lines 450-455. Figure 10 legend: Page 20, lines 492-497. Attachments Attachment Submitted filename: Responses to reviewers.docx https://doi.org/10.1371/journal.pone.0222685.r002
4 Feb 2020 Decision Letter - Hubert Vaudry, Editor The PAR2 signal peptide prevents premature receptor cleavage and activation PONE-D-19-23622R1 Dear Dr. Liu, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Hubert Vaudry Academic Editor PLOS ONE Additional Editor Comments (optional): 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 #2: All comments have been addressed ******** 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 #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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 #2: Yes ****** 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 #2: Yes ****** 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: The authors have addressed my previous concerns and I have no further comment. I think this is good article. Reviewer #2: The present revised manuscript entitled "The PAR2 signal peptide prevents premature receptor cleavage and activation”, described the role of signal peptide presents in PAR2 in expression and activation of this receptor. Experiments based on detection of wild-type and mutant receptors at the surface level cells associated with functional analysis of PAR2 and its mutants are rigorously performed. In this revised manuscript the authors have clearly replied to my comments. ****** 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: No Reviewer #2: Yes: COUVINEAU Alain https://doi.org/10.1371/journal.pone.0222685.r003
Formally Accepted
12 Feb 2020 Acceptance Letter - Hubert Vaudry, Editor PONE-D-19-23622R1 The PAR2 signal peptide prevents premature receptor cleavage and activation Dear Dr. Liu: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Hubert Vaudry Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0222685.r004

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