From the journal RSC Chemical Biology Peer review history

19-Sep-2023

Dear Dr Quintanar:

Manuscript ID: CB-REV-08-2023-000145
TITLE: Copper binding and protein aggregation: A journey from the brain to the human lens

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Cai-Guang Yang, Ph.D.
Associate Editor/RSC Chemical Biology
Professor/Shanghai Institute of Materia Medica, CAS
Phone: +86-021-50806029
Email: yangcg@simm.ac.cn

************

Reviewer 1

This is a really interesting topic for an Opinion text: the role of metals, copper specifically, in disorders involving amyloid formation. Clearly, there is data out there that shows importance and relevance, but few scientists consider the connection between protein aggregation and metal homeostasis.
The authors take a molecular approach and discuss binding sites and aggregation reactions. Of course, an opinion text should be short, but I would want to see more details and there are some issues where conflicting data exist that should be discussed. Also, more connection to disease progression and copper levels in patients must be included. For example, most meta studies show less Cu in the brain, but higher Cu in the blood, in Alzheimers patients as compared to normal persons. Also, the role of oxidation and oxidative stress must be included, as well as Cu's two redox state and what is 'normal' redox state in cells.
Also, i would request some more 'opinions'. As it is now, there is only 2 sentences in the end that suggest future structural work. I would want some more insights to what this additional modulating factor - clearly shown here to be important - means for future research around amyloid diseases.
As for additional points:
The authors draw the conclusion of competition between b-sheet stability and metal ion coordination. I find this link vauge. It does not hold true for Abeta, only IAPP and crystallins. And there is too little data on copper binding to amyloids of other amyloidogenic proteins. This is merely a speculation as of now.
Table 1. insert references to facts given.
Diversity of amyloids folds. I think it should be clear too, that in vitro amyloid structures vary a lot. Thus variations within in vitro experiments and between in vitro and in vivo and between two different patients.
For proteins in Table 1, it would be very interesting to know for which ones Cu binding to the amyloid state has been identified, characterized.
In both Alzheimers, and Parkinsons, Cu levels in brain go down when sick, but still we think Cu ions can promote aggregation. This is so puzzling to me.
In contrast to the text, one study showed Cu2+ and Cu1+ to have opposite effect on Abeta aggregation.
https://pubmed.ncbi.nlm.nih.gov/32570820/
It would be useful with Cu-binding affinities given to the monomeric forms of the proteins (or at least where known, also for amyloid forms).
Would it be possible to add a section on Parkinson's alpha-synuclein? There is a lot of data on Cu binding to the monomer and effects on aggregation. It would complement the Alzheimer's part. Or at least mention some aspects. See for example:
https://pubmed.ncbi.nlm.nih.gov/35757906/
For crystallins, it is stated that they reduce Cu2+ to Cu1+ in absence of reducing agent. so spontaneous. Does it mean Cu2+ has a binding affinity for the protein? Or it is truly only Cu1+ that finds to the protein with an affinity? Can this affinity be measured? What is oxidized in the protein when Cu is reduced? This is really cool.
Taken together, I feel some more important aspects should be incorporated to really put this extremely important molecular-mechanistic topic in its right place within biology and pathology. Also, some more elaborate future directions and possible consequences should be given.
It was very nice to read. Thank you for writing this opinion. I will cite it.
Finally, check for typos here and there.

Reviewer 2

This perspective article describes three cases of copper ion-associated protein aggregation and discusses them in the context of protein aggregation diseases. Table 1 is a useful and visually compelling summary of the state of the literature on some of these proteins. The discussion of the role of metal ions in inducing polymorphism is particularly insightful.

Overall, this paper is clear and well-written and the descriptions of the individual cases are
highly detailed and informative, but some statements in the introduction are overly general or omit important references. In the introduction the authors state that the oligomers are the most toxic species, but this is still controversial and has not been definitively demonstrated, particularly when generalizing to all proteinopathies. The description of amyloid structures on p. 3-5 suggests that all such structures have been solved by TEM/cryo-EM, when in fact many of these structures have been first determined by solid-state NMR (this is acknowledged in Figure 1, but the text focuses almost exclusively on EM). It would be appropriate to cite some of these structures. Several excellent reviews have also been written on this topic. I would also soften the language around these proteins being IDPs that undergo LLPS from “most” to “many” or similar, because numerous counterexamples are also known.

This text has been copied from the PDF response to reviewers and does not include any figures, images or special characters:

October 7th, 2023.

Cai-Guang Yang, Ph.D., Associate Editor/RSC Chemical Biology
Alexander Whiteside (Royal Society of Chemistry, UK), Assistant Editor


In reference to: “Copper binding and protein aggregation: A journey from the brain to the human lens”, by Y.
Posadas, C. Sánchez-López, L. Quintanar


Dear Editors,

Thank you for considering our Opinion Article for publication in the RSC Chemical Biology themed collection on ‘Chemical Biology of Metals’. We thank all reviewers for their constructive comments that have helped us revise and improve our manuscript. We have now addressed all reviewer´s concerns and attached you will find a point-by-point response to the reviewer´s comments.
While discussing three case studies to address the question of how metal ion binding to a peptide or protein may impact its conformation, folding, and aggregation, and how this may be relevant in understanding the polymorphic nature of the aggregates related to disease, we have added more references to our manuscript and now mention some areas where there are current controversies in the field. We consider these questions of great importance to understand the role of metal ions in proteinopathies, such as Alzheimer´s disease, diabetes type-2 and cataract disease.

We hope you will find the revised version of our opinion article now suitable for publication at the RSC
Chemical Biology. On behalf of all authors, I thank you for your consideration and for handling this manuscript.

Sincerely,
Prof. Liliana Quintanar
Department of Chemistry, and Center for Research in Aging, Center for Research and Advanced Studies (Cinvestav) lilianaq@cinvestav.mx

RESPONSE TO REVIEWERS

Referee: 1

This is a really interesting topic for an Opinion text: the role of metals, copper specifically, in disorders involving amyloid formation. Clearly, there is data out there that shows importance and relevance, but few scientists consider the connection between protein aggregation and metal homeostasis. Response: We thank the referee for the kind comments.

The authors take a molecular approach and discuss binding sites and aggregation reactions. Of course, an opinion text should be short, but I would want to see more details and there are some issues where conflicting data exist that should be discussed.
Response: Indeed, we have length constraints for the manuscript, but we have now referred briefly to the conflicting reports on the effect of copper in Abeta aggregation, including the reference that this reviewer has suggested (see below) and a recent Acc. Chem. Res. article on the topic. In the case of IAPP, while there are no conflicting reports on the impact of copper ions in its amyloid aggregation, the different proposals on how Cu(II) binds to IAPP are now mentioned in the text.

Also, more connection to disease progression and copper levels in patients must be included. For example, most meta studies show less Cu in the brain, but higher Cu in the blood, in Alzheimers patients as compared to normal persons.
Response: We have now mentioned how Cu homeostasis is affected in Alzheimer’s disease, as well as in diabetes type 2.

Also, the role of oxidation and oxidative stress must be included, as well as Cu's two redox state and what is 'normal' redox state in cells.
Response: The role of oxidation and oxidative stress is in itself a big topic, but we have now referred the reader to a couple of reviews on the subject, and we have specified the two redox states of Cu in cells.

Also, i would request some more 'opinions'. As it is now, there is only 2 sentences in the end that suggest future structural work. I would want some more insights to what this additional modulating factor - clearly shown here to be important - means for future research around amyloid diseases.
Response: We have added a paragraph at the end commenting on potential applications of this research to design therapeutic strategies that target metal ions in degenerative diseases.

As for additional points:
The authors draw the conclusion of competition between b-sheet stability and metal ion coordination. I find this link vauge. It does not hold true for Abeta, only IAPP and crystallins. And there is too little data on copper binding to amyloids of other amyloidogenic proteins. This is merely a speculation as of now. Response: We agree with the reviewer that the competition between beta-sheet stability and metal ion coordination only holds for IAPP and crystallins. In fact, this is why we contrast those two cases towards the end of the manuscript. Being an opinion article, we think it is not out of place to point out this possible competition. Clearly, more work is needed to study it, specially in a quantitative way, i.e. how do the thermodynamics of metal ion coordination and the formation of a beta-sheet structure compare? We mention this as a perspective for future work in the last section of the manuscript.

Table 1. insert references to facts given.
Response: References have been added.

Diversity of amyloids folds. I think it should be clear too, that in vitro amyloid structures vary a lot. Thus variations within in vitro experiments and between in vitro and in vivo and between two different patients. Response: Indeed, in vitro amyloid structures may apparently be diverse, mainly due to different methods of preparation (peptide length, peptide purity, metal:peptide ratio, etc) and/or techniques to study them; we have added a reference to a review on this.

For proteins in Table 1, it would be very interesting to know for which ones Cu binding to the amyloid state has been identified, characterized.
Response: We have added this.

In both Alzheimers, and Parkinsons, Cu levels in brain go down when sick, but still we think Cu ions can promote aggregation. This is so puzzling to me.
Response: Indeed, this is very puzzling! However, when referring to Cu levels in the brain, ideally a distinction should be made regarding distribution and location of the metal ion, i.e. cerebrospinal fluid vs. tissue vs. amyloid plaques. It could be that, while total Cu levels in the brain decrease, the metal ion concentrates at the plaques; indeed, this has been demonstrated by Lisa et al.. Metallomics, 2020, 12, 539 in beta-amyloid plaques, and it is also discussed in J. Biol. Chem. (2021) 296 100105. We have added a sentence on this regard in the revised manuscript. Finally, we would like to point out that for Parkinson’s, a copper deficiency is reported for substantia nigra, while there seems to be no differences in serum and cerebrospinal fluid levels; we have added this to the text as well.

In contrast to the text, one study showed Cu2+ and Cu1+ to have opposite effect on Abeta aggregation. https://pubmed.ncbi.nlm.nih.gov/32570820/
Response: Thank you for pointing out this work to us. While we do not discuss in this opinion article Cu(I), we cite it in the context of ongoing controversy regarding the impact of copper ions in Abeta aggregation.

It would be useful with Cu-binding affinities given to the monomeric forms of the proteins (or at least where known, also for amyloid forms).
Response: For each case study, we have added a couple of sentences referring to Cu binding affinities to the proteins discussed in the text.

Would it be possible to add a section on Parkinson's alpha-synuclein? There is a lot of data on Cu binding to the monomer and effects on aggregation. It would complement the Alzheimer's part. Or at least mention some aspects. See for example: https://pubmed.ncbi.nlm.nih.gov/35757906/
Response: We agree that the alpha-synuclein system is a very important one; however, for this opinion article we chose only some case studies as model systems. The intention is not to do an exhaustive review of all systems, as has been done elsewhere. Still, alpha-synuclein is mentioned throughout the text, for example, it is included in Table 1 and it is cited as an example where the impact of Cu ions in its folding is known to contribute to its physiological function. We have also added a couple more sentences on alpha-synuclein in the introduction and in the Conclusions section, citing the provided reference.

For crystallins, it is stated that they reduce Cu2+ to Cu1+ in absence of reducing agent. so spontaneous. Does it mean Cu2+ has a binding affinity for the protein? Or it is truly only Cu1+ that finds to the protein with an affinity? Can this affinity be measured? What is oxidized in the protein when Cu is reduced? This is really cool.
Response: Gamma-crystallins do bind Cu2+ ions, as demonstrated directly by EPR. Cu2+ binding affinities of gamma-crystallins have been determined by isothermal titration calorimetry (ITC) and are best described with Kd values in the micromolar to nanomolar range. While Cu1+ binding constants have not been determined, they are estimated to be in the femtomolar range to assure a high reduction potential for the Cu2+ to Cu+ reaction to occur under aerobic conditions. We have added a sentence on this in the revised manuscript.

Taken together, I feel some more important aspects should be incorporated to really put this extremely important molecular-mechanistic topic in its right place within biology and pathology. Also, some more elaborate future directions and possible consequences should be given.
Response: We agree that the relevance of the topic within biology and pathology is perhaps much bigger than portrayed in this opinion article. We have now elaborated some more on future directions and the importance of the topic for the development of therapeutic strategies.

It was very nice to read. Thank you for writing this opinion. I will cite it.
Finally, check for typos here and there.
Response: We thank the referee for the nice and constructive comments.


Referee 2

This perspective article describes three cases of copper ion-associated protein aggregation and discusses them in the context of protein aggregation diseases. Table 1 is a useful and visually compelling summary of the state of the literature on some of these proteins. The discussion of the role of metal ions in inducing polymorphism is particularly insightful.
Response: We thank the referee for the positive comments.

Overall, this paper is clear and well-written and the descriptions of the individual cases are highly detailed and informative, but some statements in the introduction are overly general or omit important references. In the introduction the authors state that the oligomers are the most toxic species, but this is still controversial and has not been definitively demonstrated, particularly when generalizing to all proteinopathies.
Response: We have now modified the statement, mentioning current controversy regarding the toxicity of oligomers, and citing appropriate references.

The description of amyloid structures on p. 3-5 suggests that all such structures have been solved by TEM/cryo-EM, when in fact many of these structures have been first determined by solid-state NMR (this is acknowledged in Figure 1, but the text focuses almost exclusively on EM). It would be appropriate to cite some of these structures. Several excellent reviews have also been written on this topic.
Response: We apologize for the omission, we have now mentioned the fact that many amyloid structures were first determined by solid-state NMR and have cited an appropriate review (Chem. Soc. Rev., 2020, 49, 5473; Cell, 2021, 184, 4857) )

I would also soften the language around these proteins being IDPs that undergo LLPS from “most” to “many” or similar, because numerous counterexamples are also known.
Response: We have now changed the wording on this statement.

11-Oct-2023

Dear Dr Quintanar:

Manuscript ID: CB-REV-08-2023-000145.R1
TITLE: Copper binding and protein aggregation: A journey from the brain to the human lens

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Phone: +86-021-50806029
Email: yangcg@simm.ac.cn

Reviewer 1

The authors have addressed all my comments. Thank you. I think draft now is in good shape for publication. It is a nice study that will be of interest to many.