Regulated Cell Death in Fungi, the Role of Metacaspases and Assay Techniques

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a lengthy and comprehensive review of plant and fungi proteases related to mammalian caspases.  It discusses their putative roles in death and survival.  The review has merit, but there are several issues that detract from possible impact as described below.  Moreover, it is not apparent why this review would be considered by an issue dedicated to Methods and Protocols.

 

Main points.

1. This topic has been discussed many times in the literature most recently in the article not cited in the review: Salas-Gómez M, Ruiz-Solaní N, Armengot L, Coll NS. The role of plant metacaspases in cell death and survival. Cell Death Differ. 2026 Feb;33(2):259-266. doi: 10.1038/s41418-025-01555-y. Epub 2025 Aug 5. PMID: 40764672; PMCID: PMC12881520.
2. The topic hardly loves up to the hyperbolic title. A “great debate” may be on the use of RNA-derived vaccines, or the origin of life for example, but whether an obscure family of poorly named proteases constitute such an inflated description is highly dubious.  Remove the first phrase of the title to yield “Cell Death in Fungi and the Role of Metacaspases”
3. Since plants and fungi contain walls around their cells it is very misleading to suggest that they can undergo the kind of apoptotic cell death illustrated in Fig 1.
4. I found no mention of the mammalian inflammatory caspases and their role in pyroptotic cell death. This is strange because if there was a form of cell death mediated by metacaspases it would be more analogous to pyroptosis than apoptosis
5. The author should reference the protease-centric MEROPS database, where the relatives of caspases are designated as Clan CD. https://www.ebi.ac.uk/merops/cgi-bin/clansum?clan=CD

 

Other point.

Line 645.  Correct spelling (karbobenzoxy-

Author Response

Replies to reviewers

First of all, I should mention that I have added a section (Chapter 6: Assay methods for detecting fungal RCD as I had originally intended to include protocols in the manuscript to make it more suitable for the journal. I have now done this with descriptions in Chapter 6 and the protocols (with citations) in the appendix.

Reviewer 1

Main points.

1. This topic has been discussed many times in the literature most recently in the article not cited in the review: Salas-Gómez M, Ruiz-Solaní N, Armengot L, Coll NS. The role of plant metacaspases in cell death and survival. Cell Death Differ. 2026 Feb;33(2):259-266. doi: 10.1038/s41418-025-01555-y. Epub 2025 Aug 5. PMID: 40764672; PMCID: PMC12881520.

 

I thank the reviewer for these constructive comments. I agree, there are many great reviews available. I noted, however, that reviews of cell death in fungi tended to cover only a few examples and I want to put together a more comprehensive review of fungal regulated cell death. I deliberately steered clear of the plant and protist reviews as there just wasn’t space to incorporate these topics, which are better researched than fungal RCD.

 

2. The topic hardly loves up to the hyperbolic title. A “great debate” may be on the use of RNA-derived vaccines, or the origin of life for example, but whether an obscure family of poorly named proteases constitute such an inflated description is highly dubious. Remove the first phrase of the title to yield “Cell Death in Fungi and the Role of Metacaspases”

 

Again, I agree. One of my original intentions was to focus on the changing opinions about the roles of metacaspases in fungal RCD but I gradually moved towards covering a broader topic. I have now changed the title.

 

3. Since plants and fungi contain walls around their cells it is very misleading to suggest that they can undergo the kind of apoptotic cell death illustrated in Fig 1.

 

Yes, the cell wall does change many things, including calling into question phosphatidylserine externalization as a marker of RCD. Figure 1 is purely to illustrate cell death in mammals.

 

4. I found no mention of the mammalian inflammatory caspases and their role in pyroptotic cell death. This is strange because if there was a form of cell death mediated by metacaspases it would be more analogous to pyroptosis than apoptosis

 

I thank the reviewer for pointing this out. I was not as familiar with inflammatory forms of cell death and with the view that fungal RCD probably resembles pyroptosis and/or necroptosis rather then apoptosis. I have now included inflammatory caspases in figure 3 and I added a summary of the different types of regulated cell death, recognized by the NCCD (2.6). I also added a few sentences to the end of the section in 3.2 concerning heterokaryon incompatibility and a new section 8.2, discussing similarities between fungal cell death and pyroptosis as well as a mention at the end of the conclusions chapter.

 

5. The author should reference the protease-centric MEROPS database, where the relatives of caspases are designated as Clan CD. https://www.ebi.ac.uk/merops/cgi-bin/clansum?clan=CD

 

The MEROPS citation is in the first paragraph of chapter 5. It is maybe not as prominent as it deserves to be.

 

Other point.

Line 645.  Correct spelling (karbobenzoxy-

Thank you for pointing this out. I have corrected the spelling.

 

In addition, the editors have requested more up-to-date references for the manuscript. I have now inserted new citations, dating from 2023 or later wherever possible. New citations are highlighted yellow in the manuscript, including in Table 1.

Reviewer 2 Report

Comments and Suggestions for Authors

The review article is original and could be very relevant for comparative medicine in the field of cellular and molecular biology.

Since it is well known that Caspases orchestrate metazoan apoptosis, regulating processes such as embryogenesis, the death of old and infected cells and immune tolerance, the author also proved that structural orthologs of caspases have been identified in bacteria, plants, protists and fungi and regulated cell death has been demonstrated in these organisms.

The methodology of the study is very good. The systematic review was conducted very logically, demonstrating precisely, step by step, the role of caspases in mammals and metazoa and the role of some metacaspases in protozoa.

The results could be very important for the antifungic treatment (very important in medicine), the author demonstrating that most metacaspases are activated by calcium whereas caspases are activated via proximity of caspase proteins and interaction between them. Metacaspases appear to be less specialized than caspases as cell death regulators and have pro-survival roles, including cell cycle regulation, growth, differentiation and protein regulations. Since metacaspases are certainly related to caspases and at least one substrate is conserved between humans and yeast. The substrate is cleaved at one specific residue (anaspartate) by caspase-3 but at 4 different residues (arginine or lysine) by MCII-Pa. However, it is believed that the ancestors of caspases and caspase orthologs resembled a metacaspase. So, the author consider that the most important future aims are to use metacaspase-specific substrates and inhibitors to study fungal cell death and associated protease activity and to identify the degradomes of fungal metacaspases and to elucidate the regulatory pathways in fungal Regulated Cell Death (RCD)

 

The conclusions are relevant for authors findings.

The references  are very adequated, demonstrating a very hard author s work.

Author Response

Replies to reviewers

First of all, I should mention that I have added a section (Chapter 6: Assay methods for detecting fungal RCD as I had originally intended to include protocols in the manuscript to make it more suitable for the journal. I have now done this with descriptions in Chapter 6 and the protocols (with citations) in the appendix.

Reviewer 2

The review article is original and could be very relevant for comparative medicine in the field of cellular and molecular biology.

Since it is well known that Caspases orchestrate metazoan apoptosis, regulating processes such as embryogenesis, the death of old and infected cells and immune tolerance, the author also proved that structural orthologs of caspases have been identified in bacteria, plants, protists and fungi and regulated cell death has been demonstrated in these organisms.

The methodology of the study is very good. The systematic review was conducted very logically, demonstrating precisely, step by step, the role of caspases in mammals and metazoa and the role of some metacaspases in protozoa.

The results could be very important for the antifungic treatment (very important in medicine), the author demonstrating that most metacaspases are activated by calcium whereas caspases are activated via proximity of caspase proteins and interaction between them. Metacaspases appear to be less specialized than caspases as cell death regulators and have pro-survival roles, including cell cycle regulation, growth, differentiation and protein regulations. Since metacaspases are certainly related to caspases and at least one substrate is conserved between humans and yeast. The substrate is cleaved at one specific residue (anaspartate) by caspase-3 but at 4 different residues (arginine or lysine) by MCII-Pa. However, it is believed that the ancestors of caspases and caspase orthologs resembled a metacaspase. So, the author consider that the most important future aims are to use metacaspase-specific substrates and inhibitors to study fungal cell death and associated protease activity and to identify the degradomes of fungal metacaspases and to elucidate the regulatory pathways in fungal Regulated Cell Death (RCD)

 

The conclusions are relevant for authors findings.

The references  are very adequated, demonstrating a very hard author s work.

Response: I thank the reviewer for these encouraging comments

Reviewer 3 Report

Comments and Suggestions for Authors

This is a very comprehensive review focusing on regulating cell death in fungi and the role  
of metacaspases. Overall, the review was well-organized and written in good English. The manuscript deserved to be published in MPs. I only have some minor comment on this manuscript as follows.

1. for figure1, the figure caption was not clear.
2. add an abbreviation list to help reading.
3. Since the figures used in this review were all adopted from other publications. I suggest to authors to draw the figures by himself and make some changes.
4. In the conclusion part, I suggest the authors to state the most important issues needed to be addressed in this field in our opinion as the authors have achieved a lot in this field.

Author Response

Replies to reviewers

First of all, I should mention that I have added a section (Chapter 6: Assay methods for detecting fungal RCD as I had originally intended to include protocols in the manuscript to make it more suitable for the journal. I have now done this with descriptions in Chapter 6 and the protocols (with citations) in the appendix.

Reviewer 3

This is a very comprehensive review focusing on regulating cell death in fungi and the role 

of metacaspases. Overall, the review was well-organized and written in good English. The manuscript deserved to be published in MPs. I only have some minor comment on this manuscript as follows.

1. for figure1, the figure caption was not clear.

 

       Thank you for pointing this out. The caption has now been rewritten to make it clearer

 

2. add an abbreviation list to help reading.

 

      There was a small list of abbreviations but I agree that this was inadequate and have expanded it

  

3. Since the figures used in this review were all adopted from other publications. I suggest to authors to draw the figures by himself and make some changes.

I would prefer not to make extensive changes after obtaining permissions to use these figures but I do think that many of the figures were not as specific as they could be to the manuscript. Therefore I have remade several of the figures: Figure 2 has been altered to include both single and double strand breaks to highlight the uncertainty regarding ds strand breaks in fungal RCD. Figure 3 has been altered to include the inflammatory caspases which, as reviewer 1 pointed out, were previously lacking. The figure has also been simplified. Figure 4 has also been simplified and made clearer. The perforin/granzyme pathway has been removed from Figure 5 since the text is concerned only with intrinsic and extrinsic apoptosis, specifically the DISC and apoptosome. Figure 6 has also been simplified, removing cFLIP, which was not needed. The text within parts of the DISC components was difficult to read so it has been removed and replaced by a better colour scheme and key. Irrelevant details regarding the mitochondrion in Figure 7 have been removed and an extra side view of the apoptosome has been added to improve understanding of the structure and mechanism. Figure 8 has been amended to better illustrate the formation of prion structures and the alteration of Het-S to yield pore-forming structures. Figure 9 is unchanged as it is simple and informative. In Figure 10, the schematic chromosomes have been replaced with simple representations of the cells with explosion shapes representing the killer or poison proteins. Figure 11 is unchanged as it seems to serve its purpose. Figures 12, 13 and 14 are micrographs. Figure 15 is already simple and clear so has not been changed. In Figure 16 the repetition of caspase structures has been removed as this is covered by an earlier figure. Figure 17 is new. It shows the molecular structures of the new generation of metacaspase inhibitors. Figure 18 needed to be expanded to show the dual roles of Esp1p and Mcd1p. This has now been done.

 

4. In the conclusion part, I suggest the authors to state the most important issues needed to be addressed in this field in our opinion as the authors have achieved a lot in this field.

 

      The conclusion has been extended to better address future goals. I thank the reviewer for useful

      suggestions, which have hopefully led to improved clarity in the manuscript.

 

In addition, the editors have requested more up-to-date references for the manuscript. I have now inserted new citations, dating from 2023 or later wherever possible. New citations are highlighted yellow in the manuscript, including in Table 1.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The author has responded adequately and elegantly to my comments.  I find the revised version very informative, especially the long list of primary citations.