All reviews of published articles are made public. This includes manuscript files, peer review comments, author rebuttals and revised materials. Note: This was optional for articles submitted before 13 February 2023.
Peer reviewers are encouraged (but not required) to provide their names to the authors when submitting their peer review. If they agree to provide their name, then their personal profile page will reflect a public acknowledgment that they performed a review (even if the article is rejected). If the article is accepted, then reviewers who provided their name will be associated with the article itself.
Thanks for addressing the minor revisions requested. Now your manuscript is accepted in PeerJ.
[# PeerJ Staff Note - this decision was reviewed and approved by Pedro Silva, a PeerJ Section Editor covering this Section #]
Please take into consideration the reviewer’s comments and provide back a point-by-point rebuttal letter addressing those concerns.
The manuscript presented by Zagar T et al (ID 58778) is an interesting report on the effect of the destabilization of the EpCAM dimer by the TACE protease.
The topic i interesting and the results and conclusions are well argumented. The literature is sufficiently appropriate, exhaustive and centered.
The structure of the article is well planned, as well as figures. However, some colors are not well suitable, especially in figure 2. Then, page numbers are lacking.
The most important issue to be raised is about English language and the sloppiness of some terms.
Research is within the aims and scope of the Journal
Research question are well defined and meaningful
Rigorous investigation performed
Methods described sufficiently well, but some changes and improvements are required.
NO comment
The manuscript (ID 58778) is well designed and interesing, contributing valuable novelties in the field.
However, a revision of the style and of the English language are required.
Considering that no further experiments are needed in my opinion, my choice is a minor revision, but only if the English style and sloppiness are improved.
In particular, in the Methods:
1-The title of the firs paragraph should be amended to: "In silico construction and Molecular dynamic simulations of EpEX". Otherwise, is not easy to comprehend why is the first paragraph.
2- "Molecular weight analysis" should precede the "CD", that should in turn become "CD analysis".
3- RALLS and LALLS should be better explained in the methods, better than in the Results.
Then, please define better than at present the abbreviations: for example Molecular weight (Mw) should be defined the first time, then use always Mw! Also the acronym "TACE", should be better explained in line 39, as well as in the Abstract.
Again, the article "the" or "a" should be added before singular names. See line 23, before 'oligomeric state' and check throughout the whole manuscript.
Careful with the commas and full stops: line 146, pH 7.4, not 7,4. Check hroughout the whole manuscript.
Then, finally, the page numbers are lacking and some color figures are not well suitable (light green and yellow, Figure 2).
In general, if you could improve the resolution of all the figures should be better.
Other points:
Line 17, amend 'fully explained' with "fully understood".
Line 20, change 'mutant forms' with "mutants"
Line 41, before "secretase" the greek term is lacking.
Line 53, shift "only" after "take place".
Line 83-84, the sentence "After inizial minimization with protein fixed" is not very clear.
Line 147, ZnCl2: 2 must be underscored.
Line 242, change 'has' with "had".
Line 258, change to: "The peaks relative to... are symmetric, while the EpEX peak..."
Line 299, add "to be" after "appear"
Line 308, amend to: "challenging to be dected and accurately quantified"
Line 326, change to "a prolonged"
Line 331, change '9' with "nine"
Line 365, shift "for the first time" at the end of the sentence (line 366)
Line 373, delete "be" before "needed".
Lines 374 and 376: what is "CANDIS"?
Line 401, amend the sentence to "...oligomeric state either in solution or on the cell surface".
In this manuscript, the Authors designed, expressed, purified, and characterized two different mutants with distinct oligomeric states of EpCAM ectodomain (EpEX) protein. The purified recombinant proteins ( EpEX wt, dimeric and monomeric mutants, and TACE-EX) were used to study the role of the oligomeric state's effect on the proteolytic processing by TACE ectodomain. By comparing the proteolytic processing of EpEX wt, EpEX monomeric, and dimeric mutants, they concluded that EpCAM could only be efficiently cleaved as a monomer, indicating that these mutants are valuable tools to elucidate the role of the oligomeric state of EpCAM in the regulation of RIP.
The manuscript, in general, complies with the basic requirements of the journal; It is well written, clearly stated the aim of the study, the hypothesis and experimental evidence are consistent and interesting to the audience, figures and raw data are appropriate.
However, before publication, some minor observations might improve the manuscript.
1) Please correct the spelling error in lines 47 and 84
2) Line 69-7 …… confirming the role of EpCAM's oligomerization...
To confirm the role of oligomerization of EpCAM in the RIP, experiment with cells using these valuable mutants are needed. To be consistent with experimental evidence, discussion, and conclusions, please change "confirming" by something like "strongly indicating."
3) It is indeed surprising that Anti His Ab did not recognize the TACE-EX (Fig 3A). Since TACE-EX was also purified using the His6-tag, it will be desirable to provide some experimental evidence that this observation was because of the absence of His-tag in the purified protein.
4) To improve Fig1B, 2A, 2B, 2D, 3A,3B,3C, and 3D, is it possible to change the yellow-orange color?
All Alfanumeric in the figure labels should be in “black.”
The research question is clearly defined, experimental approach, and the methods are well described.
Their results, discusión, and conclusion are consistent with the aim of the study.
The data shown here with the recombinant proteins EpEX wt, dimeric and monomeric mutants contribute to advance the role of oligomerization of EpCAM in the RIP.
These monomeric and dimeric mutants will be valuable tools to confirm in cell experiments the EpCAM oligomeric state's role in this interesting molecule's biological function.
All text and materials provided via this peer-review history page are made available under a Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.