Membrane Transporters Involved in Iron Trafficking: Physiological and Pathological Aspects

Round 1

Reviewer 1 Report

General:

The strength of this review are the biochemical and bioinformatics aspects of discussed transporters. Unfortunately, the first section is incomplete, biased and seems to have heavily relied on one review (ref. 15) rather than on a thorough screening of the literature. Part 1 (1. General iron metabolism and trafficking) needs to be completely rewritten (including Figure 1) and original literature should be cited rather than 1-2 reviews with a more or less biased focus.

 

Specific:

1. The authors claim that LTCC transport Fe. This is based on reference 15 (a review by M.D. Knutson). Yet this review cites another more detailed review by the same author (M.D. Knutson, Non-transferrin-bound iron transporters, Free Radic. Biol. Med. 133 (2019) 101–111), which is also much more accurate than ref. 15 and describes that both L- type and T-type Ca channels transport Fe. The latter channel is not mentioned in this manuscript (acknowledge for instance the original work by K.V. Lopin et al. Mol. Pharmacol. 82 (6) (2012) 1194–1).
2. The authors claim that the only lysosomal Fe transporter are ZIP8/14 and DMT1. These transporters may indeed be present in lysosomal membranes. However, they overlook an important Fe transporter, namely TRPML1 (see for instance X.-P. Dong et al. Nature 455(7215):992-6 (2008)).
3. The authors claim that mitochondrial inner membranes express mitoferrin and outer membranes ZIP8. The evidence for the latter is based on one older reference only (ref. 82). Yet several recent publications provide biochemical and functional evidence that DMT1 is also expressed in the mitochondrial membrane and transports both Fe and Mn. This is neglected by the authors (see for instance Wolff N.A. et al. Sci Rep. 2018 Jan 9;8(1):211, Channels (Austin). 2014;8(5):458-66, FASEB J. 2014 May;28(5):2134-45). This work should be acknowledged.

 

Moderate editing of english language required.

Author Response

Reviewer #1

The strength of this review are the biochemical and bioinformatics aspects of discussed transporters. Unfortunately, the first section is incomplete, biased and seems to have heavily relied on one review (ref. 15) rather than on a thorough screening of the literature. Part 1 (1. General iron metabolism and trafficking) needs to be completely rewritten (including Figure 1) and original literature should be cited rather than 1-2 reviews with a more or less biased focus.

Specific:

1. The authors claim that LTCC transport Fe. This is based on reference 15 (a review by M.D. Knutson). Yet this review cites another more detailed review by the same author (M.D. Knutson, Non-transferrin-bound iron transporters, Free Radic. Biol. Med. 133 (2019) 101–111), which is also much more accurate than ref. 15 and describes that both L- type and T-type Ca channels transport Fe. The latter channel is not mentioned in this manuscript (acknowledge for instance the original work by K.V. Lopin et al. Mol. Pharmacol. 82 (6) (2012) 1194–1).
2. The authors claim that the only lysosomal Fe transporter are ZIP8/14 and DMT1. These transporters may indeed be present in lysosomal membranes. However, they overlook an important Fe transporter, namely TRPML1 (see for instance X.-P. Dong et al. Nature 455(7215):992-6 (2008)).
3. The authors claim that mitochondrial inner membranes express mitoferrin and outer membranes ZIP8. The evidence for the latter is based on one older reference only (ref. 82). Yet several recent publications provide biochemical and functional evidence that DMT1 is also expressed in the mitochondrial membrane and transports both Fe and Mn. This is neglected by the authors (see for instance Wolff N.A. et al. Sci Rep. 2018 Jan 9;8(1):211, Channels (Austin). 2014;8(5):458-66, FASEB J. 2014 May;28(5):2134-45). This work should be acknowledged.

 

Authors’ reply

We wish to thank the Reviewer for highlighting these points that we overlooked in our original manuscript. Details and related references regarding L- type and T-type Ca channels, TRMPL1 and DMT1 localization and function on the outer mitochondrial membrane have been included in the introductory part of the review and in the section on DMT1. Figure 1 has also been modified accordingly. All changes are highlighted in the “marked manuscript” file.

Reviewer 2 Report

This review manuscript summarizes current knowledge on five proteins transporting iron into and out of cell. The manuscript is well structured and clearly written. Importantly, the authors have their own publications in the field of iron transport, qualifying them as experts in the field. Approximately one third of the references are to publications of the last five years. Altogether, this will a worthy guide for both beginners and experts in the field.

I have only minor remarks:

Lines 36-37: it is unusual to consider solubility of a cation alone. This property usually refers to a salt, base, or acid formed by a cation and anion.

Lines 81-82: please note that a reference number (like [14]) cannot be a sentence member. A simple test: sentence meaning should not be lost without the reference number. It may be better to use “see reference 14 for a review”.

Figures 4-6 would benefit from brighter colors, like in Figure 2.

Some paragraphs consist of only one sentence, which is a bad style.

Author Response

Reviewer #2

This review manuscript summarizes current knowledge on five proteins transporting iron into and out of cell. The manuscript is well structured and clearly written. Importantly, the authors have their own publications in the field of iron transport, qualifying them as experts in the field. Approximately one third of the references are to publications of the last five years. Altogether, this will a worthy guide for both beginners and experts in the field.

I have only minor remarks:

Lines 36-37: it is unusual to consider solubility of a cation alone. This property usually refers to a salt, base, or acid formed by a cation and anion.

Authors’ reply

The sentences have been rephrased as follows: “In fact, the largely prevalent form in the aerobic environment is the ferric ion Fe3+. However, Fe3+ salts are very poorly soluble in a polar solvent like water.”

 

Lines 81-82: please note that a reference number (like [14]) cannot be a sentence member. A simple test: sentence meaning should not be lost without the reference number. It may be better to use “see reference 14 for a review”.

Authors’ reply

The text has been corrected as suggested by the Reviewer.

 

Figures 4-6 would benefit from brighter colors, like in Figure 2.

Authors’ reply

Colours in Figure 4,5 and 6 have been changed as suggested by the Reviewer.

 

Some paragraphs consist of only one sentence, which is a bad style.

Authors’ reply

We modified the text formatting to avoid single-sentence paragraphs.

All changes are highlighted in the “marked manuscript” file.

Reviewer 3 Report

This review article provides a comprehensive overview of the currently characterized membrane transporters involved in iron trafficking, offering valuable insights based on available structural information or modeling of variant pathogenicity. While the article is well-written, there are some suggestions for revisions:

 

1.     It is strongly recommended that the authors adhere to the standard nomenclature for gene and protein names. Human genes should be designated using their HGNC IDs in uppercase italics, and human proteins should follow the same format but with regular style (e.g., replace hFnp and hMfrn1/2 with FPN and MFRN1/2, respectively).

2.     In the discussion about the mammalian iron exporter FPN (aka SLC40A1), it is important to mention its recent identification as a component of red blood cell membrane (PMID: 30213870; PMID: 29599243) and its implications for human health and physiology.

3.     In Figure 1, for consistency with the main text, ZIP8 and ZIP14 should be listed without the hyphen. Additionally, numbers should be included for PCBP (e.g., PCBP1/2 given that both have been shown to deliver iron to ferritin). Transferrin, transferrin receptor 1, and transferrin receptor 2 should be labeled as TF, TFRC, and TFR2, respectively. It would be helpful to slightly adjust the position of the ceruloplasmin/hephaestin-mediated oxidation of Fe(II) to Fe(III) in the graphic representation to clearly indicate that the ferroxidase associated with FPN is responsible for the reaction.

4.     Lanes 161 to 166 should be rephrased to improve clarity for readers who are not experts in the field. The following is an attempt:

“The FPN transport cycle occurs through a process known as alternating access mechanism, where conformational changes between different states (inward-open, occluded, and outward-open) take place. This process relies on the rigid-body relative rotation of the N-terminal and C-terminal domains of FPN, as depicted in Figure 3 [34,35]. During this process, the first set of helices (helices 1, 4, 7, and 10) and second set of helices (helices 2, 5, 8, and 11) within each repeat of the protein, which interact along the cytoplasmic ends in the inward-open state, undergo a conformational change and switch their interaction from the cytoplasmic ends to the extracellular ends of the protein.

The alternating access mechanism, driven by the rotation and conformational changes of the N-terminal and C-terminal domains, enables the movement of substrates (such as iron) across the cell membrane.”

5.     In Lane 172, "an uniport" should be corrected to "a uniport."

6.     In Lanes 238-239, the sentence regarding PCBP2 can be misleading. Instead, clarify that PCBP2 has been characterized as a dual-function protein, acting both as an RNA binding protein and an iron chaperone.

7.     Lane 394- “Gly204Cys is located at the interface with the bilayer membrane lipids (Figure 5), and it is not clear whether it could be detrimental for the protein function [89]”. Gly204 is not highlighted in Figure 5.

8.     Lanes 398-399 discuss a polymorphism in ZIP8. Residue 391 is labeled as Cys in Figure 5, which contradicts the authors' discussion of an Ala391 to threonine substitution. Clarify this inconsistency.

9.     Residues Cys74 and Asp443 are also not shown in Figure 5 contrary to the indication made in the main text.

 

Author Response

Reviewer #3

This review article provides a comprehensive overview of the currently characterized membrane transporters involved in iron trafficking, offering valuable insights based on available structural information or modeling of variant pathogenicity. While the article is well-written, there are some suggestions for revisions:

 

1. It is strongly recommended that the authors adhere to the standard nomenclature for gene and protein names. Human genes should be designated using their HGNC IDs in uppercase italics, and human proteins should follow the same format but with regular style (e.g., replace hFnp and hMfrn1/2 with FPN and MFRN1/2, respectively).

Authors’ reply

            The format of human genes and human proteins name has been corrected throughout the text.

 

2. In the discussion about the mammalian iron exporter FPN (aka SLC40A1), it is important to mention its recent identification as a component of red blood cell membrane (PMID: 30213870; PMID: 29599243) and its implications for human health and physiology.

Authors’ reply

            In the FPN section of the review, the identification of this protein as a component of the red blood cells membrane and its relevance in maintaining systemic iron homeostasis and in protecting red blood cells from oxidative stress has been discussed, and the relevant literature properly cited.

 

3. In Figure 1, for consistency with the main text, ZIP8 and ZIP14 should be listed without the hyphen. Additionally, numbers should be included for PCBP (e.g., PCBP1/2 given that both have been shown to deliver iron to ferritin). Transferrin, transferrin receptor 1, and transferrin receptor 2 should be labeled as TF, TFRC, and TFR2, respectively. It would be helpful to slightly adjust the position of the ceruloplasmin/hephaestin-mediated oxidation of Fe(II) to Fe(III) in the graphic representation to clearly indicate that the ferroxidase associated with FPN is responsible for the reaction.

Authors’ reply

            Figure 1 has also been modified according to the Reviewer’s suggestions.

 

4. Lanes 161 to 166 should be rephrased to improve clarity for readers who are not experts in the field. The following is an attempt:

“The FPN transport cycle occurs through a process known as alternating access mechanism, where conformational changes between different states (inward-open, occluded, and outward-open) take place. This process relies on the rigid-body relative rotation of the N-terminal and C-terminal domains of FPN, as depicted in Figure 3 [34,35]. During this process, the first set of helices (helices 1, 4, 7, and 10) and second set of helices (helices 2, 5, 8, and 11) within each repeat of the protein, which interact along the cytoplasmic ends in the inward-open state, undergo a conformational change and switch their interaction from the cytoplasmic ends to the extracellular ends of the protein.

The alternating access mechanism, driven by the rotation and conformational changes of the N-terminal and C-terminal domains, enables the movement of substrates (such as iron) across the cell membrane.”

Authors’ reply

            We thank the Reviewer for his help in improving the clarity of the text, which has been rephrased following his/her advice.

 

4. In Lane 172, "an uniport" should be corrected to "a uniport."

Authors’ reply

    Corrected.

 

5. In Lanes 238-239, the sentence regarding PCBP2 can be misleading. Instead, clarify that PCBP2 has been characterized as a dual-function protein, acting both as an RNA binding protein and an iron chaperone.

Authors’ reply

Following the Reviewer’s suggestion, the sentence has been rephrased as follows: “Recently, it has been determined that PCBP2, originally described as poly(rC) RNA binding protein 2, is in fact a dual-function protein, acting also as an iron chaperone.”

 

7. Lane 394- “Gly204Cys is located at the interface with the bilayer membrane lipids (Figure 5), and it is not clear whether it could be detrimental for the protein function [89]”. Gly204 is not highlighted in Figure 5.
8. Lanes 398-399 discuss a polymorphism in ZIP8. Residue 391 is labeled as Cys in Figure 5, which contradicts the authors' discussion of an Ala391 to threonine substitution. Clarify this inconsistency.
9. Residues Cys74 and Asp443 are also not shown in Figure 5 contrary to the indication made in the main text.

 Authors’ reply

            Figure 5 has been modified to avoid inconsistencies with the text. In detail: Gly204 has been highlighted and labeled, and the wrong label C391 has been corrected in A391. Regarding Cys74 and Asp443, the residues are not shown because they are not mutated in the described diseases. Only residues whose mutation are causative of pathologies are shown in Figure 5.

 

All changes are highlighted in the “marked manuscript” file.

 

Round 2

Reviewer 1 Report

General:

Most comments have been addressed in an adequate manner. However, one precision is necessary.

 

Specific:

1. Both L- type and T-type Ca channels transport Fe. The authors cite S. Kumfu. et al. Europ. J. Haematol. 674 2011, 86, 156–166 as proof for involvement of TTCCs. This reference, however, provides only weak evidence (inhibition of Fe uptake by efonidipine), but no proof because eponidipine blocks both TTCCs and LTCCs (H. Tanaka, K. Shigenobu (2002). "Efonidipine hydrochloride: a dual blocker of L- and T-type ca(2+) channels". Cardiovascular Drug Reviews. 20 (1): 81–92). The first study so far demonstrating Fe currents through TTCCs is the study by K.V. Lopin et al. Mol. Pharmacol. 82 (6) (2012) 1194–1). This study, which provides definitive proof of Fe flux through TTCCs, needs to be cited.

Moderate editing of English language required.

Author Response

Reviewer #1

1. Both L- type and T-type Ca channels transport Fe. The authors cite S. Kumfu. et al. Europ. J. Haematol. 674 2011, 86, 156–166 as proof for involvement of TTCCs. This reference, however, provides only weak evidence (inhibition of Fe uptake by efonidipine), but no proof because eponidipine blocks both TTCCs and LTCCs (H. Tanaka, K. Shigenobu (2002). "Efonidipine hydrochloride: a dual blocker of L- and T-type ca(2+) channels". Cardiovascular Drug Reviews. 20 (1): 81–92). The first study so far demonstrating Fe currents through TTCCs is the study by K.V. Lopin et al. Mol. Pharmacol. 82 (6) (2012) 1194–1). This study, which provides definitive proof of Fe flux through TTCCs, needs to be cited.

Authors' reply

We wish to thank again the Reviewer for helping us to better support the statements reported in our manuscript with appropriate literature citations.

The article suggested by the Reviewer is now cited in the appropriate position in the text as ref. 23 of the latest manuscript version.

Further, the manuscript text has been revised thoroughly to ensure the best possible quality of the english language.