Pleiotropy of Progesterone Receptor Membrane Component 1 in Modulation of Cytochrome P450 Activity^†

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review of jox-2941949, “Pleiotropy of Progesterone Receptor Membrane Component 1 in modulation of cytochrome P450 activity”

Authors: Isabel S. Barata, José Rueff , Michel Kranendonk, and Francisco Esteves

Manuscript Summary:

This manuscript is a review of the role of Progesterone Receptor Membrane Component 1 (PRGMC1) in the modulation of cytochrome P450 (CYP) activity. PRGMC1 is a small (~15 kDa) protein that is involved in the modulation of certain human CYPs. However, the mechanism(s) behind this modulation are not well understood. This is important because this protein can impact metabolism not only of drugs, but also important endogenous substrates such as steroids and other hormones. The review takes a comprehensive approach, focusing on nine main topics that include the most relevant aspects of past and current PGRMC1 research, particularly in regard to its role in CYP-mediated drug metabolism.

General Assessment:

Overall, this is a very well-written and nicely organized review article on a timely subject. The figures are well-done and enhance the text of the manuscript. I particularly like the fact that each section of the article logically builds on the previous section. This organizational component is especially important when considering such a large and expansive topic such as this. Kudos to the authors for their brilliant organization. I have only a few minor critiques, as noted below.

Minor:

1. There could be a few more figures included to break up the monotony of the text or to illustrate some specific points better. Three figures for an ~20 page review seems a little light.

1. Line 48: change “vitamins” to “vitamin”; plurality is implied.

2. Line 77: change “firstly” to “first”.

Author Response

We would like to thanks the reviewer’s comments and suggestions.

1. In response to the reviewer’s initial minor concern, regarding illustration of specific points, we added one figure (Figure 1. – page 2, line 68), containing information about the main classes of compounds metabolized by CYPs and isoforms involved in their biotransformation. In the new figure we were able to pinpoint human CYP isoforms for which binding or enzyme activity modulation by PGRMC1 has been reported, which we think is a critical point of the review.
2. Page 2, line 48: changed “vitamins” to “vitamin”.
3. Page 3, line 82: changed “firstly” to “first”.

The references ([261]-[269]) were updated after the introduction of a new reference of a very recent publication describing intermolecular disulfide bonding as another route for PGRMC1 dimer formation, which we found pertinent to include (after the update reference [261]).

Reviewer 2 Report

Comments and Suggestions for Authors

This review is devoted to the most obscure and controversial protein partner of cytochromes P450, cytochrome-b5-alike membrane-associated progesterone receptor PGRMC1. To my knowledge, there are only two quite outdated reviews on this topic (Rohe et al. 2009, ref 155, and Ryu et al 2017, ref. 115). The current understanding of the effects of PGRMC1 on the P450 system is limited and the function of PGRMC1 in the cytochrome P450-centered machinery remains obscure. Thus, there is a foremost need for a comprehensive, up-to-date summarizing review on this subject. The present manuscript is very timeous and I agreed to review it in the hope that it will help me to better understand the mess of contradictory data on the function of PGRMC1 in the P450-centered machinery. Unfortunately, that did not happen. In my view, the review is far too lengthy, poorly structured, overloaded with irrelevant data, and lacks an integrating summary. For me, the only useful section of the manuscript was “5. Current knowledge gaps, challenges, and future perspectives”, which is well-written and informative. The rest reads hard and does not add any “take home message” to my current knowledge. Here are my main concerns and suggestions:

1. The manuscript starts with an overview of the cytochrome P450 system, its organization, function, and physiological role. I agree that this kind of introduction may be of worth, but in its present state, it is far too lengthy and overloaded with irrelevant information. One introductory paragraph about the structure (heme-thiolate enzyme), widespread occurrence, and main biological functions followed by a paragraph on the organization of microsomal and mitochondrial monooxygenases and a paragraph about main protein-protein interactions (P450-b5, P450-reductase, P450-P450) with a focus on P450-b5 – it is all that may be needed to my view. Instead, the authors present a 4-page-long review most of which is not related to the subject of this manuscript and not used in its following main part. In particular, section 2.2 (Metabolic phenotypes and pathologies) seems completely irrelevant. The introductory section and sections 2.1 and 2.3 can be shortened to 1-2 paragraphs each. In the present state, the introduction may exhaust the patience of the reader even before he/she comes to the main, much more cumbersome part.
2. The main part of the manuscript is poorly structured. The information on interactions of PGRMC1 with P450 enzymes, its effects on their activity, and the expression level is spread out between different sections, where some parts of already presented data are repeated. I would recommend condensing all information on (a) PGRMC1-P450 interactions, (b) their effect on P450 activity, and (c) the effect of PGRMC 1 on P450 expression in separate, well-structured sections. The review would highly benefit from summarizing paragraphs at the end of each of these sections.

Besides these major concerns, I have several more specific comments:

1. The authors used a plethora of non- or semi-standard abbreviations hampering the reading. In some cases, it seems unnecessary. Thus, after its introduction at line 234, the abbreviation MAPR (membrane-associated progesterone receptor) was used only twice – at lines 334 and 397. Is it really necessary to force the reader to memorize the abbreviation for only two occurrences? I would also enjoy the use of “progesterone” instead of “P4” throughout the paper – the economy of space is not worth the inconvenience of reading for people unfamiliar with this abbreviation (like me). An important confusion for the reader is caused by the use of “IZA1” instead of “PGRMC1” at random places in the manuscript. This abbreviation is not introduced in its full form (on page 264 the authors introduce IZA, but not IZA1). In literature, IZA1 is used for rat homolog of human PGRMC1. However, in this manuscript random use of IZA1 and PGRMC is confusing. Why the authors could not write “rat PGRMC1” in the cases when the origin of the protein is important and simply “PGRMC1” when it is not?
2. In lines 258-264 the same sentence (“Moreover, PGRMC2 functions and modes of action...”) appears twice. Remove one of the occurrences.
3. In line 743 the authors cite the paper of “Szczesna-Skorupa and Kemper [130]”. However, the reference 130 points to the paper of Min and co-authors. The paper of Szczesna-Skorupa is under the number 134. I did not check other citations, but there may be more mistakes of the same kind. A careful check of all citations is needed.   

Author Response

We would like to thanks the Reviewer’s comments and suggestions. All comments were duly considered and appreciated. Taking into account the considerations from the other two reviewers (Reviewers 1 and 3), who find the manuscript well-organized, timely, and based on a comprehensive approach, we were not able to address all the criticisms raised by the Reviewer, as we would contradict/oppose the positive assessments of the other reviewers. Nevertheless, considering Reviewer 2 report, several issues were addressed.

The reviewer stated “The manuscript starts with an overview of the cytochrome P450 system, its organization, function, and physiological role. I agree that this kind of introduction may be of worth, but in its present state, it is far too lengthy and overloaded with irrelevant information.” Considering this issue, we deleted a paragraph regarding congenital adrenal hyperplasia, which the authors also considered not central to the manuscript scope, albeit informative on general role of CYP in steroidogenesis. Moreover, we added one figure (Figure 1. – page 2, line 68), containing summarized information on the main classes of compounds metabolized by CYPs and isoforms involved in their biotransformation. In the new figure we were able to pinpoint human CYP isoforms for which binding or enzyme activity modulation by PGRMC1 has been reported, which we think is a critical point of the review.

Also, in agreement with reviewer's suggestions, conclusion paragraphs were added to the direct and indirect effects sections, paragraphs 4.1 and 4.2, respectively (lines 515-522 and 684-691).

Regarding reviewer’s specific comments:

1. We removed abbreviations with few occurrences for easier reading, i.e., “P4” was replaced by “progesterone” throughout the manuscript and “IZA” by “Pgrmc1” or “PGRMC1”.
2. We deleted repeated sentence in page 6, lines 243-252 (“Moreover, PGRMC2 functions and modes of action...”).
3. We corrected reference in page 16, line 747 the authors previously written as [130] and when it corresponds to [134] and checked all citations.

Finally, references ([261]-[269]) were updated after the introduction of a new reference of a very recent publication describing intermolecular disulfide bonding as another route for PGRMC1 dimer formation, which we found pertinent to include (after the update reference [261]).

Reviewer 3 Report

Comments and Suggestions for Authors

This review article on the pleiotropic effects of PGRMC1 on cytochrome P450 activity is detailed and well written. There were some careless mistakes as shown below, so please correct them.

1. Page 5, lines 251-254: The same sentence as the previous sentence is repeated.

2. An explanation of the red molecule (probably part of haem) drawn in Figures 2(B) and 2(C) should be written in the figure legend.

3. Page 13, line 618: I think " ) " is missing between [158] and "could".

4. Page 14, line 694: [251] is written twice, so you should delete one.

Author Response

We would like to thanks the reviewer’s comments and suggestions. The following was corrected:

1. We deleted repeated sentence in page 6, lines 243-252 (“Moreover, PGRMC2 functions and modes of action...”).
2. Page 9: introduced in the legend of Figure 3 explanation of the red molecule as haem.
3. Page 13, lines 615: added ")" missing between [158] and "could".
4. Page 15, line 697: deleted repeated reference [251].

Additionally, the references ([261]-[269]) were updated after the introduction of a new reference of a very recent publication describing intermolecular disulfide bonding as another route for PGRMC1 dimer formation, which we found pertinent to include (after the update reference [261]).

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

To be honest, the authors' reply to my criticism disobliged me. The point of their reply is that since the two reviews were excellent, the third reviewer's criticism can be ignored.  Nonetheless, the authors took into account some of my comments and the manuscript reads easier now. It can be published in its current form after bringing the capitalization of cytochromes P450 and PGRMC1 into uniformity. Occurrences of "CYP2e1" or "CYP1a2", etc. (see lines 475 and 480 for instance) should be eliminated and replaced with "CYP2E1", "CYP1A2", etc. Likewise, occurrences of "Pgrmc1" (lines 276, 403, 406, and so on) should be replaced with "PGRMC1".

Author Response

We would like to thank the Reviewer comments and suggestions. In response to the reviewer’s minor concern, regarding capitalization of cytochromes P450 and PGRMC1 into uniformity, the authors carefully reviewed the entire manuscript and minor corrections were made, in order to standardize the nomenclature according to the criteria established by international convention (the gene symbols for rodent orthologues of human genes have the first letter capitalized followed by lower case letter; human genes have all letters in upper case; in both cases, proteins designations are the same as the gene symbol, but not italicized and all upper case):

Page 7: line 274;

Page 9: lines 401, 404, 408;

Page 10: lines 473, 476;

Page 11: lines 478, 479;

Page 13: line 616;

Page 14: lines 673, 675.