Biochemical, Biophysical and Functional Characterization of an Insoluble Iron Containing Hepcidin–Ferritin Chimeric Monomer Assembled Together with Human Ferritin H/L Chains at Different Molar Ratios

Round 1

Reviewer 1 Report

The paper from Mohamed Boumaiza et al in the Arosio and Marzouki groups in Tunis the makes the unexpected and elegant observation supporting that H-ferritin and hepcidin, which are small proteins (182 AA for ferritin and 25 AA for mature cleaved hepcidin), can be fused so that hepcidin can assemble into ferritin shells. The experiments follow on from a previous paper citated in their Ref 12 in 2017. Their experiments were certainly are performed in a rigorous manner.

The paper should be accepted with the clarification:

1. that their system is an artificial setup towards protein-based therapy rather than a report of evidence for hepcidin vs. H-ferritin multimers occurring in nature.
2. How is the new constructed protein heteropolymer going to be delivered in the future?  Also, how will this new protein be delivered to treat conditions iron overload better?

Author Response

Response to Reviewer 1 comments

We are grateful to the reviewer for his positive comments and for his deep and thorough review. We have revised our present research paper and we hope our revision has improved the manuscript. Comments and specific responses are as follows.

 

Comments and Suggestions for Author:

“The work of Boumaiza et al, analyzing a chimera formed by hepcidin-H ferritin (HepcH:FTH) produced in E. coli in an insoluble form, and its denaturation/renaturation to produce various heteropolymeric shells with the L-chain. Authors used mass spectrometry to distinguish between folded and unfolded heteropolymers assembled with different molar ratio. A proteomic profile was generated for each HepcH-FTH/FTL heteropolymer assembled with a specific molar ratios. It is not very clear why authors decided to proceed to produce this chimera. If drug delivery agent is the purpose should be clarified.”

 

Major comments

Comment 1: “The abstract should be rewritten in order to clarify the aims of this work, why this fused molecule is important. As presented it is focused on the methodology/results and technical details and only in the last sentence an explanation is given to the reviewer.”

Response to comment 1: As suggested the abstract was rewritten in the revised manuscript.

Comment 2: “In Results the sentence “Here, we demonstrate that after fusing the two proteins a bifunctional polymer is obtained with enhanced potential as iron storage molecule as well as iron homeostasis regulator through ferroportin degradation in human hepatocyte (HepG2 cells) and murine macrophagic cells (J774 cells)” should be re-written. It is not clear what authors suggested.”

Response to comment 2: As suggested by the reviewer the sentence was reformulated as follow:

“Here, we demonstrate that after fusing the two proteins a bifunctional polymer is obtained with  an enhanced iron storage potential and  a  binding and internalization capability through ferroportin degradation in both human hepatocyte (HepG2 cells) and murine macrophagic cells (J774 cells)”.

Comment 3: “Why is important to use “both mouse J774 macrophage cells as well as the human HepG2 cell line”?

Response to comment 3: This is important because human and mouse cells have different receptors for the H-ferritin, in human it is the transferrin receptor 1, while in mouse it is TIM2.  The finding that the chimera binds to both cell types in similar manner supports the hypothesis that the interaction is due to the hepcidin moiety.

Comment 4: “In 3.1 “was shown to isolate” should be “to coagulate/precipitate”??”

Response to comment 4:  As suggested by the reviewer the sentence “was shown to isolate” was replaced by “was shown to precipitate”.

Comment 5: “Fig.1 and Fig.2 Please provide information about how many times experiments were done.”

 Response to comment 5: As suggested number of replicate was indicated in Fig.1 and Fig.2 legends (N=3).

Comment 6: “Fig. 3: Please provide MW values. Indicate the correct product”.

Response to comment 6: As suggested the MW was included in Fig. 3 and the correct products was noted.

Comment 7: “In 3.5 “J774 cells were also treated with cycloheximide”. Please give the reason behind using cyclohexamide to your experiment”.

Response to comment 7: Cycloheximide is a well-known inhibitor of protein synthesis (Baliga et al., 1969) and we used it to verify how suppression of ferroportin expression in J744 cells affected the binding of human hepcidin.

 

Baliga, B. S., Pronczuk, A. W. & Munro, H. N. Mechanism of cycloheximide inhibition of protein synthesis in a cell-free system prepared from rat liver. J Biol Chem 244, 4480–4489 (1969).

 

Comment 8: “In 3.5 “however hepcidin induction by BMP2” please provide more details”.
Response to comment 8: To avoid any confusion, the sentence “however hepcidin induction by BMP2 was affected after cycloheximide treatment“ has been deleted in the revised version of the manuscript as it refer to the work done by Kanamori et al. 2016 (Reference 15 in the manuscript).

Comment 9: In Fig.7 the authors should provide an average quantification of all their western blots experiments. How many times experiment was done? Is this western blot a representative of all experiments?

Response to comment 9: As suggested by the reviewer, quantification of all the blots of figure 7 was performed, using Image J software, and given in the Excel file. The experiments was performed 3 times in J744 cells and one time in HepG2 cells. This western blot is a representation of a single experiment.

Comment 10: “Extensive editing of English language and style required.”

Response to comment 10: As suggested English corrections are in red color in the revised manuscript.

 

Minor comments:

1. In lane 2 abstract “to human ferritin H-chain (HepcH) that expressed” should be “that is expressed”

Response to comment 1: Corrected in the revised manuscript

 

1. In introduction “with a tissues specific distribution” should be tissue

Response to comment 2: corrected

 

1. In introduction “without protein degradation” type space

Response to comment 3: corrected

 

1. In introduction “via its binding to its receptor, the iron exporter ferroportin” should be “binding to the iron exporter ferroportin, its receptor”.

Response to comment 4: corrected

 

1. In materials section “Native and denaturing” should be native

Response to comment 5: corrected

 

1. In 2.7 section “Hhepcidin-Ferritin” should be Hepcidin

Response to comment 6: corrected

 

1. In 3.5 “The mouse J774 human HepcG2 cells” add “and”

Response to comment 7: corrected

 

Author Response File: Author Response.docx

Reviewer 2 Report

The work of Boumaiza et al, analyzing  a chimera formed by hepcidin-H ferritin (HepcH:FTH) produced in E. coli in an insoluble form, and its denaturation/renaturation to produce various heteropolymeric shells with the L-chain. Authors used mass spectrometry to distinguish between folded and unfolded heteropolymers assembled with different molar ratio. A proteomic profile was generated for each HepcH-FTH/FTL heteropolymer assembled with a specific molar ratios. It is not very clear why authors decided to proceed to produce this chimera. If drug delivery agent is the purpose should be clarified.

Major comments

1. The abstract should be rewritten in order to clarify the aims of this work, why this fused molecule is important. As presented it is focused on the methodology/results and technical details and only in the last sentence an explanation is given to the reviewer.
2. In Results the sentence “Here, we demonstrate that after fusing the two proteins a bifunctional polymer is obtained with enhanced potential as iron storage molecule as well as iron homeostasis regulator through ferroportin degradation in human hepatocyte (HepG2 cells) and murine macrophagic cells (J774 cells)” should be re-written.It is not clear what authors suggested.
3. Why is important to use “both mouse J774 macrophage cells as well as the human HepG2 cell line”?
4. In 3.1 “was shown to isolate” should be “to coagulate/precipitate”??
5. Fig.1 and Fig.2 Please provide information about how many times experiments were done.
6. Fig. 3 : Please provide MW values. Indicate the correct product.
7. In 3.5 “J774 cells were also treated with cycloheximide”. Please give the reason behind ising cyclohexamide to your experiment
8. In 3.5 “however hepcidin induction by BMP2” please provide more details.
9. In Fig.7 the authors should provide an average quantification of all their western blots experiments. How many times experiment was done? Is this western blot a representative of all experiments?

Minor comments:

1. In lane 2 abstract “to human ferritin H-chain (HepcH) that expressed” should be “that is expressed”
2. In introduction “with a tissues specific distribution” should be tissue
3. In introduction “withoutprotein degradation” type space
4. In introduction “via its binding to its receptor, the iron exporter ferroportin” should be “binding to the iron exporter ferroportin, its receptor”.
5. In materials section “Native and denaturing” should be native
6. In 2.7 section “Hhepcidin-Ferritin” should be Hepcidin
7. In 3.5 “The mouse J774 human HepcG2 cells” add “and”

Author Response

Response to Reviewer 2 comments

We are very thankful to the reviewer for these excellent suggestions that should enhance the manuscript quality. We have revised our present research paper and we hope our revision has improved the paper. Comments and specific responses are as follows.

 

Comments and Suggestions for Authors:

“The authors used a chimeric hepcidin-ferritin molecule for their structural and functional studies. They proved that this molecule binds more iron than ferritin by itself. They could express this molecule in E. coli and use it in cell culture to prove that this molecular structure is able to bind ferroportin like hepcidin alone. The paper is well structured, the experiments are correctly described. I have some questions about the theory of the paper.”

My questions:

Comment 1: “Why is camel hepcidin used instead of the human? “

Response to comment 1: This is a relevant point of discussion, indeed, we demonstrated in a previous work (Boumaiza et al, 2014, J. Pept. Sci. 2014; 20: 680–688) that camel hepcidin has less tendency to precipitate that its human counterpart. Thus we choosed the mature camel hepcidin which differ by only 2 amino acids with the human hepcidin.

Comment 2: “How did the idea come to generate this chimeric molecule?”

Response to comment 2:  Hepcidin and ferritins and major molecules of iron metabolism, and also remarkably different. One is a small peptide with a beta strand structure stabilised by sulfide bridges, the other is multimeric nanocage.  The presence of multiple hepcidin on the surface of a molecule may increase the avidity binding to cells expressing ferroportin.

Comment 3: “The structure and the potential drug binding activity is not clear for me. In reduced state, it is assumed that iron-sulfur clusters could be generated in the chimeric molecule. It is very hard to imagine, because in folded structure the disulfide bridges of hepcidin are essential for the physiological activity.”

Response to comment 3: This point of discussion is relevant, indeed, it was shown that only the correctly folded structure of hepcidin can bind ferroportin via its N-terminal domain, and to attain it the formation of the 4 sufide bridges is necessary. Thus, we reduced the molecule to facilitate co-assembly with ferritin subunits, and oxidized it for correct hepcidin folding. 

Comment 4: “Why do the authors think that the chimera can be used for drug delivery?”  

Response to comment 4: The use of ferritin for drug delivery is a topic of increasing interest, the attachment of a moiety with a well-defined target for cell binding and incorporation would increase it potential use.

Comment 5: “If it binds large amount of iron, where can the drug be bound? Drugs are usually hydrophobic molecules, and not every type of protein can carry them.”

Response to comment 5: This point of discussion is relevant. In fact, after removing iron, molecules can be incorporated inside the ferritin cavity either by diffusion, or by performing the reassembly in the presence of the drug. This has been described in a number of papers (Karimi et al., 2017 Nanoscale 9(4): 1356–1392; Palombarini et al., 2020, Molecules. 25(4): 825; Zang et al., 2021 Advanced Drug Delivery Reviews 176 (2021) 113892).

 

Author Response File: Author Response.docx

Reviewer 3 Report

The authors used a chimeric hepcidin-ferritin molecule for their structural and functional studies. They proved that this molecule binds more iron than ferritin by itself. They could express this molecule in E. coli and use it in cell culture to prove that this molecular structure is able to bind ferroportin like hepcidin alone. The paper is well structured, the experiments are correctly described. I have some questions about the theory of the paper.

My questions: Why is camel hepcidin used instead of the human? How did the idea come to generate this chimeric molecule? The structure and the potential drug binding activity is not clear for me. In reduced state, it is assumed that iron-sulfur clusters could be generated in the chimeric molecule. It is very hard to imagine, because in folded structure the disulfide bridges of hepcidin are essential for the physiological activity. Why do the authors think that the chimera can be used for drug delivery? If it binds large amount of iron, where can the drug be bound? Drugs are usually hydrophobic molecules, and not every type of protein can carry them.

Author Response

Response to Reviewer 3 comments

We are very thankful to the reviewer for these excellent suggestions that should enhance the manuscript quality. Comments and specific responses are as follows.

 

“The paper from Mohamed Boumaiza et al in the Arosio and Marzouki groups in Tunis makes the unexpected and elegant observation supporting that H-ferritin and hepcidin, which are small proteins (182 AA for ferritin and 25 AA for mature cleaved hepcidin), can be fused so that hepcidin. Can assemble into ferritin shells. The experiments follow on from a previous paper citated in their Ref 12 in 2017. Their experiments were certainly are performed in a rigorous manner. The paper should be accepted with the clarification:”

 

Comment 1: “That their system is an artificial setup towards protein-based therapy rather than a report of evidence for hepcidin vs. H-ferritin multimers occurring in nature.”

Response to comment 1: We showed that the produced hybrid heteropolymer can bind specifically both mouse and human cells via the hepcidin-ferroportin pathway with iron chelation potential. The biological activity of this molecule have to be tested also in-vivo using for example mouse models of HH (Hfe-/-) and β-thalassemia (Hbb^th3/+) with iron overload disorder (Finberg et al. 2011; Nai et al., 2012).

Finberg, K.E.; Whittlesey, R.L.; Andrews, N.C. Tmprss6 is a genetic modifier of the Hfe hemochromatosis phenotype in mice. Blood 2011, 117, 4590–4599, doi:10.1182/blood-2010-10-315507.

Nai, A.; Pagani, A.; Mandelli, G.; Lidonnici, M.R.; Silvestri, L.; Ferrari, G.; Camaschella, C. Deletion of TMPRSS6 attenuates the phenotype in a mouse model of beta-thalassemia. Blood 2012, 119, 5021–5029, doi:10.1182/blood-2012-01-401885.

Comment 2. How is the new constructed protein heteropolymer going to be delivered in the future? Also, how will this new protein be delivered to treat conditions iron overload better than the strategy of simply adjusting endogenous ferritin and hepcidin levels in living systems.

Response to comment 2: This point of discussion is relevant, Indeed, in humans, iron overload can be caused by several diseases such as hereditary hemochromatosis (HH): primary hemochromatosis (a genetic mutation); Juvenile hemochromatosis (results from defects in a gene called HJV) and secondary hemochromatosis that can result from some kinds of anemia, such as thalassemia, or chronic liver disease, such as chronic hepatitis C infection or alcohol-related liver disease. The standard of care for HH is therapeutic phlebotomy or iron chelation therapy using iron chelating drugs such as desferrioxamine (DFO). However, DFO is poorly absorbed in the gastrointestinal tract and has a short (~20 min) plasma half-life. Thus, to reach effective pharmacological concentrations the drug is administered parenterally with the aid of a portable infusion pump at least 4–5 days per week for 8- 10 h each time. The cumbersome procedure reduces compliance and significantly compromises the quality of patients’ life. The use of iron chelating drugs is associated with the risk of side effects, such as neutropenia/agranulocytosis, skin rash, gastrointestinal disturbances, retinopathy or bone abnormalities. Another problem is that these drugs cannot prevent dietary iron absorption, which is highly induced in patients with iron-loading anemias, due to erythropoietic suppression of hepcidin.  Thus, hepcidin replacement therapy could provide an etiologic cure of HH, and correct aggravating dietary iron absorption in transfusional iron overload (Katsarou and Kostas Pantopoulos, 2018, Pharmaceuticals 2018, 11, 127; doi:10.3390/ph11040127). Hepcidin agonists, or inducers of hepcidin expression, could also be used for the treatment of these highly prevalent disorders. These leads to the importance of developing new hepcidin agonist molecules such as the recombinant HepcH-FTH. Finally we did not planned yet how the HepcH-FTH will be delivered in the future.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Manuscript was improved.

Please provide molecular weight markers in Fig 3 (no indications of MW in the corresponding lane ) and Fig. 7 (not only the arrow 75kDa)

Please check the folder named 'original gels/blots'. Authors should provide the original gels not the ppt figures.

Author Response

We are grateful to the reviewer for his positive comments and for his deep and thorough review. We have revised our present research paper and we hope our revision has improved the manuscript. Comments and specific responses are as follows.

 

Comments and Suggestions for Authors

Manuscript was improved.

Comment 1 “Please provide molecular weight markers in Fig 3 (no indications of MW in the corresponding lane) and Fig. 7 (not only the arrow 75kDa)”

Response to comment 1 As suggested by the reviewer the molecular weight markers was given the revised Fig 3 and 7.

 

Comment 2 Please check the folder named 'original gels/blots'. Authors should provide the original gels not the ppt figures.

Response to comment 2 We thank the reviewer to ask for the non-edited and original photos of the western blots. Indeed, we have only the edited version of the blots (in PowerPoint) and we didn’t fount the original ones in our discs.

Author Response File: Author Response.docx