A Nanoenzyme Constructed from Manganese and Strandberg-Type Phosphomolybdate with Versatility in Antioxidant and Modulating Conformation of Aβ Protein Misfolding Aggregates In Vitro

Round 1

Reviewer 1 Report

 

1. There are a lot of sentence framing errors in the introduction (e.g 45, 51-53, 67-69, 168 etc) – Correct this. Also, wherever the work of a different lab/professor is cite please use et.al instead of Prof. XX reported ……!

2. Give a brief description of what a stranberg type polyphospolybdate is?

3. Give a brief description of your finding from the current work in the concluding section of the introduction so as to create an interest in the readers for the work been presented.

4. Considering the effects of MnPM on fibrils, have the authors investigated the effects of MnPM on prefibrillar oligomers in AD? This is very important since prefibrillar oligomers are one of the most toxic species in AD pathogenesis.

5. Throughout the manuscript the authors describe how MnPM inhibits and/or reduces the misfolded Abeta aggregation and ROS through different experiments. While there are many interesting findings from these experiments, this manuscript would be incomplete without showing the actual graphical representation of how MnPM associates/interacts with the fibrils –show this in the results section!

6. Materials and method section is a complete disarray! Rewrite this section with clear headings of what experiments are being discussed, the concentration of the protein, MnPM and fibrils being used in the respective experiments in addition to details regarding the instrumentation and the experimental procedure.

7. Apart from introduction there are a lot of sentence framing errors throughout the manuscript – check and correct this.

 

 

Author Response

Reviewer: #1

Thank you for your positive comments. We have revised our manuscript according to the suggestions.

 

Comment (C) 1: There are a lot of sentence framing errors in the introduction (e.g 45, 51-53, 67-69, 168 etc)-Correct this. Also, wherever the work of a different lab/professor is cite please use et.al instead of Prof. XX reported ……!

Answer (A) 1: Thanks a lot. The corresponding text has been revised as follows:

Line 45:  In addition, it is well known that the reactive oxygen species (ROS) caused by metal ions-Aβ aggregates are also causative factor of the neuronal dysfunction.

Line 51-53: Furthermore, polyoxometalates (POMs), a class of metal-oxygen clusters, have also been recognized as modulator against protein misfolding.

Line 67-69: Herein we report the structure and multifunctional property of a newly designed manganese-substituted polyphosphomolybdate, (H₂en)₃[Mn(H₂O)₄][Mn(H₂O)₃]₂[P₂Mo₅O₂₃]₂·14.5H₂O (abbreviated as MnPM, en = ethanediamine). MnPM can not only inhibit the β-sheet transformation of Aβ peptide, but also act as nanoenzyme to suppress ROS generation, which as a result it can reduce the toxicity of misfolding Aβ species to cells in vitro.

“Dr. Qu et al. and Dr. Liu et al.” have been used instead of “Prof. Qu and Prof. Liu”.

 

 

 

C 2: Give a brief description of what a stranberg type polyphospolybdate is ?

A 2: Thanks for your suggestion. The description of Strandberg-type polyphosphomolybdate has been added into both manuscript and ESI:

 As shown in Figure R1, the Strandberg-type POM fragment is obviously different from other types of POMs, which there is no derivative relationship from the others. In a brief, the structure of Strandberg-type cluster can be viewed as a puckered ring of five nearly coplanar corner-sharing/edge-sharing distorted MoO₆ octahedra with two capping PO₄ tetrahedra on both poles of the {Mo₅O₂₁} ring centers.

 

Figure R1. View of classic Polyoxometalate building blocks with bioactive ability. Oxygen atoms: red (The big red balls represent the effective coordination site). (A) α-Keggin-type fragment; (B) ε-Keggin-type fragment; (C) Anderson-type fragment; (D) Strandberg-type fragment; (E) Sandwich-type fragment; (F) Dawson-type fragment.

 

C3: Give a brief description of your finding from the current work in the concluding section of the introduction so as to create an interest in the readers for the work been presented.

A3: Thank you for your suggestion. The brief description as follows has been added into the introduction section.

Our work has focused on the development of complexes with novel structure for anti-protein misfolding. In 2018, we have designed a modified POM, {[CoL(H₂O)]₂[CoL]₂[HAs^VMo^V₆Mo^VI₆O₄₀]}·2.5H₂O, which can intervene with the β-sheet aggregates through modulating the conformation of Aβ based on non-covalent strategy. Next, two complexes, 2-{2-[(1H-benzoimidazol-2-yl)methoxy]phenyl}benzothiazole and K₁₀Na₂[Ca₆P₆O₁₂(H₂O)₆][PMo₆O₂₈]₂·24H₂O, with β-sheet-rich conformation modulation activity based on π-π stacking and electrostatic interaction have been synthesized successfully in 2020 and 2021, respectively. 

 

C4: Considering the effects of MnPM on fibrils, have the authors investigated the effects of MnPM on prefibrillar oligomers in AD? This is very important since prefibrillar oligomers are one of the most toxic species in AD pathogenesis.

A4: Thanks a lot. That’s a very good question. The aggregation process of Aβ and the inhibition effect of MnPM have been tracked with the ThT fluorescent probe from 0 to 24 h. As shown in Figure R2, the fluorescence intensity of Aβ alone group maintained at a low level before 20 h and began to climb gradually after 20 h, which indicated that Aβ underwent nucleation and rapid transformed into β-sheet within 24 h. In the presence of MnPM, the fluorescence intensity of Aβ + MnPM is very weak during the first 16 h. After 16 h, the fluorescence intensity seems to ascend, which imply that increasing amounts of Aβ were transformed into the β-sheet. However, the fluorescence intensity of Aβ + MnPM keeps weak, indicating that the conformational transformation is largely suppressed. The fluorescence increases obviously when Aβ was incubated with Zn²⁺/Cu²⁺, especially with Zn²⁺, which indicates that Zn²⁺ and Cu²⁺ can promote the formation of β-sheet-rich aggregates and the promotive effect of Zn²⁺ is stronger than that of Cu²⁺. By contrast, in the presence of MnPM, the increasing slope of the fluorescence intensity of Zn²⁺- or Cu²⁺-Aβ solution maintains at a low level, which the quenching of the fluorescence indicates that MnPM may inhibit the prefibrillar oligomers of Aβ induced by the both β-sheet misfolding in the presence of metal ions and the self-β-sheet-transformation of Aβ. These results are consistent with the conclusion of CD spectrum experiments. 

  

Figure R2. ThT fluorescence intensity (λ_ex = 415 nm) of Aβ40 (20 μM) solutions in the absence and presence of Zn²⁺ or Cu²⁺ after incubation with or without MnPM at 37 °C and pH 7.4 for 0−1440 min ([Aβ40]: [metal ion]: [MnPM] = 1: 2: 1) 

 

C5: Throughout the manuscript the authors describe how MnPM inhibits and/or reduces the misfolded Abeta aggregation and ROS through different experiments. While there are many interesting findings from these experiments, this manuscript would be incomplete without showing the actual graphical representation of how MnPM associates/interacts with the fibrils-show this in the results section!

A5: Thank you for your work and suggestion. According to the results of the ¹H-NMR experiment, the binding site of the Aβ peptides to MnPM is most likely to be the imidazole residues. As for MnPM, its coordination sites were explored by using EPR methods [1, 2]. As shown in Figure R3, there are two lines centered at 3200 and 3750 G, which indicated a typical single electron paramagnetic effect of Mn²⁺ [3]. However, after incubated with Aβ peptides, the single is showing signs of weakening, which may suggest the number of spin electrons is decreasing. It known that Mn²⁺ has 3d⁵ electron shell structure. When the coordination atoms in ligands are all weak field ones, such as O atoms, following Hund's rule, its five d electrons filled five d orbitals respectively to form a high spin complex (t_2g³e_g²) with spin quantum number m_s = +5/2. When the O ligands are replaced by the strong field ligands, such as N ligands, it's going to squeeze all of its d electrons into the t_2g orbitals, forming low spin complex (t_2g⁵e_g⁰) with m_s = +1/2, which the paramagnetism intensity of the compound will decline. Therefore, it can be inferred that Mn ions in MnPM are important sites that interacts with Aβ peptides. However, as interactions between macromolecules, it well known that it is very difficult to describe the exact mechanism of action. First of all, as shown in Table R1, the mode of interaction is not limited to coordination. By calculation, it is found that there also are van der Waals energy (E_v), desolvo energy (E_d), hydrogen bond energy (E_H) and electrostatic energy (E_e) between MnPM and Aβ. Furthermore, although we believe that coordination is the strongest, it still has various modes, which is the dominant configuration depends on many factors. Considering that this part can be written independently, and adding it to the text would be distracting, we do not describe it in details in this manuscript. 

Table R1. Lowest Docking Energy (kJ mol⁻¹) for the β-Sheet Conformation of Aβ in the Reaction with MnPM Calculated by the Autodock Program

conformer	EHVda	Ee	Eimb	Etc	ΔGd
β-Sheet	−38.95	−0.44	−39.39	+6.46	−32.93
a EHVd = EH + EV + Ed; b Eim = EHVd + Ee; c torsional energy; d ΔG = Eim + Et

 

 

Figure R3. EPR spectra of the MnPM incubated with or without Aβ complexes.

1. Wu, A.J.; Penner-Hahn, J.E.; Pecoraro, V.L. Structural, spectroscopic, and reactivity models for the manganese catalases. Rev.2004, 104, 903–938.
2. Smirnova, T.I.; Smirnov, A.I. Dynamic molecular oxygen accessibility to a buried Mn²⁺protein site: A high-field EPR experiment. Phys. Chem. B. 2003, 107, 7212–7215.
3. Antharavally, B.S.; Poyner, R.R.; Ludden, P.W. EPR spectral evidence for a binuclear Mn(II) center in dinitrogenase reductase-activating glycohydrolase from rhodospirillum rubrum. Am. Chem. Soc.1998, 120, 8897–8898.   

 

C6: Materials and method section is a complete disarray! Rewrite this section with clear headings of what experiments are being discussed, the concentration of the protein, MnPM and fibrils being used in the respective experiments in addition to details regarding the instrumentation and the experimental procedure.

A6: Thank you for your suggestion. The experimental section were reorganized, which the headings and labels were added. The details of the experiment were refined.     

 

C7: Apart from introduction there are a lot of sentence framing errors throughout the manuscript -check and correct this.

A7: Thank you for your suggestion. We have carefully revised the manuscript.

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

The authors report the effect of an inorganic compound on modulating the toxicity and the aggregation pathway of Abeta peptide into amyloid fibrils. They also show the same compound to reduce ROS species.

The presented results are interesting and presented overall clearly, although with some notable exception (see below). 

Major comments

- the authors should comment on the reason why they chose Abeta40 and not Abeta42 for their experiments. Abeta42 is known to be more amyloidogenic and more toxic than Abeta40 so it would make for a more supportive test.

- The discussion in Section 2.3 is quite confusing in my opinion, since the authors seem to overlook the fact that the unsoluble stable fibrillar species are not the toxic ones (although they state something similar at lines 252-254), wheres the toxic species are known to be transient soluble beta-sheet rich oligomeric species. The authors seem not to consider important the distinction between soluble and not soluble species. For example the sentence at page 6, lines 200-201, about the beta-sheet rich conformations of Abeta40 having been possibly "destroyed" by MnPM, does not take into account that although fibrils disappeared in TEM images thanks to MnPM, this might in principle lead to an increase of the soluble toxic oligomers. Similarly, the authors a couple of times (lines 197-198 and  233-234) wrongly connect the evidence for the presence of fibrils (from TEM images or from ThT assay) to the abundance of beta-sheet rich conformation in solution. In this respect, the data from CD, obtained in solution, actually allow the authors to support those statements, as well as the similar one at lines 260-261. Conversely, the statement at lines 258-259, about the absence of beta-sheet rich aggregates after incubation with MnPM is not supported by the authors data, in my opinion: in fact, ThT is known to be a specific binder for the cross-beta structure of fibrils, so it may not be sensitive to other kind of beta-sheet aggregates. From the perspective of toxicity this is not a relevant point however.

- one key result is shown by the authors in Fig. 7, that is the ability of MnPM to disaggregate already formed amyloid fibrils; yet, the authors do not state how long Abeta40 is incubated with Zn++ before adding MnPM in the different concentrations shown in Fig. 7. The authors need to state the duration of that incubation.

- at the end of the introduction the authors state that to their knowledge MnPM is the first inorganic compound capable of both modulating the Abeta peptide aggregation activity and of antioxidant activity; in section 2.4 (line 288) they state that "most of mono-functional modulators .... do not possess antioxidant activity". Given they say "most" and not "all", the doubt remains in the reader whether MnPM is really the first modulator to possess antioxidant activity or not.

 

Minor comments

- page 9, line 273-274: in discussing Fig. 8 data, the authors state that "the peaks of Abeta40 with MnPM are much more simpler than that without MnPm, which may indicate that MnPM can suppress the aggregation of advanced misfolding of Abeta". Leaving aside the unclear meaning of "aggregation of advances misfolding", the authors should explain why peaks with a simpler structure should imply a suppression of the misfolding/aggregation pathway.

- page 2, line 68: is polyphospolybdate a typo for polyposphomolybdate?

- page 3, line 97: B as defined in the equation for bond valence sum is a length, so setting B=0.37 should also include the info about the length unit measure

- there are several acronyms which are never defined throughout the text: en ligands;  TGA; cal. (in the steps for weight loss); SOD;

- page 5, line 166: I do not get what "dove derivatives" are

 

Author Response

Reviewer: #2

Thank you for your careful reviews and positive comments.

 

Comment (C) 1: The authors should comment on the reason why they chose Abeta40 and not Abeta42 for their experiments. Abeta42 is known to be more amyloidogenic and more toxic than Abeta40 so it would make for a more supportive test.

Answer (A) 1: Thanks a lot. That is a very good question. In fact, we are focusing on the effects of different Aβ types on the cause of Alzheimer’s disease. In 2020, we synthesized a fluorescent probe that can differentiate Aβ40 from Aβ42 [1]. It is reported that the immoderate increase of Aβ42 is a crucial inducement of early Aβ aggregation [2]. And the ratio of Aβ42 to Aβ40 is considered as an important biomarker for initial AD [3]. Since Aβ42 has two more hydrophobic amino acid residues, that is, Ile and Ala, at positions 41 and 42, Aβ tends to form hydrophobic β-sheet, with hydrophobic groups being folded outward; the additional Ile and Ala would make Aβ42 to form β-sheet more readily than Aβ40 [4], which is also the reason why Aβ42 is highly toxic in vivo [5]. However, although Aβ42 is more toxic, the composition of toxic β-sheet species accounted for a higher proportion of Aβ40, which  can reach 95 % [3]. Therefore, we believe that the contribution of Aβ40 misfolding to neurotoxicity should not be overlooked. In addition, the purpose of this manuscript is to describe a novel POM with conformational modulation and antioxidant functions. Previous studies on the interaction between POM and Aβ used Aβ40 as the research model [6–10]. As a proof of concept, we need sufficient data comparison, hence Aβ40 is chosen as the experimental model.   

 

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C2: The discussion in Section 2.3 is quite confusing in my opinion, since the authors seem to overlook the fact that the unsoluble stable fibrillar species are not the toxic ones (although they state something similar at lines 252-254), wheres the toxic species are known to be transient soluble beta-sheet rich oligomeric species. The authors seem not to consider important the distinction between soluble and not soluble species. For example the sentence at page 6, lines 200-201, about the beta-sheet rich conformations of Abeta40 having been possibly "destroyed" by MnPM, does not take into account that although fibrils disappeared in TEM images thanks to MnPM, this might in principle lead to an increase of the soluble toxic oligomers. Similarly, the authors a couple of times (lines 197-198 and  233-234) wrongly connect the evidence for the presence of fibrils (from TEM images or from ThT assay) to the abundance of beta-sheet rich conformation in solution. In this respect, the data from CD, obtained in solution, actually allow the authors to support those statements, as well as the similar one at lines 260-261. Conversely, the statement at lines 258-259, about the absence of beta-sheet rich aggregates after incubation with MnPM is not supported by the authors data, in my opinion: in fact, ThT is known to be a specific binder for the cross-beta structure of fibrils, so it may not be sensitive to other kind of beta-sheet aggregates. From the perspective of toxicity this is not a relevant point however.

A2: Thanks a lot for your commends. In fact, the view point we support is consistent with yours. We believe that the soluble Aβ oligomers with β-sheet conformation are the most toxic species. Meanwhile, the insoluble plaques can be thought of as a storage warehouse, which are in precipitation-dissolution equilibrium with the oligomers in solution. That is, when the concentration of oligomers in the solution reaches a critical value, the plaque will be deposited; Conversely, when the concentration of oligomers in the solution is reduced, the plaque will dissolve. Therefore, it can be inferred that when insoluble β-folded aggregates appear in the system, the soluble oligomers in the solution have reached their maximum solubility, which as the statements in lines 197-198 and 233-234.

Of course, it is clear that dissolving insoluble plaques in this case does pose a significant risk, as is the case with Benzbromarone used to dissolve uric acid crystals in patients with acute attacks of gout. However, as shown in Figure 6A, in the presence of MnPM, the β-sheet misfolding level of soluble species in the system was greatly inhibited, which may indicate that the conformations of most toxic β-sheet misfolding Aβ oligomer has been destroyed. Although ThT is a specific binder for the cross-beta structure of fibrils that can not be sensitive to other kinds of β-sheet misfolding species, the CD spectra assay is a powerful complement to the shortcomings of ThT experiment. At the same time, the cytotoxicity of Aβ incubated by MnPM was also verified by MTT assay, which proved the above experimental conclusions. We revised the manuscript to make this part more compact to better serve the readers.

 

C3: One key result is shown by the authors in Fig. 7, that is the ability of MnPM to disaggregate already formed amyloid fibrils; yet, the authors do not state how long Abeta40 is incubated with Zn++ before adding MnPM in the different concentrations shown in Fig. 7. The authors need to state the duration of that incubation.

A3: Thank you for your reminder. Details of this part of the experiment are as follows: Aβ40 (20 μM) in buffer solution (20 mM Tris-HCl/150 mM NaCl, 992 μL) was incubated with Zn(OAc)₂ (4μL, 10mM) at 37 °C for 24 h. MnPM solutions with the final concentration of 0‒25 μM were added to each sample respectively and incubated at 37 °C for another 24 h. We have completed the details of these experiments. 

 

C4: At the end of the introduction the authors state that to their knowledge MnPM is the first inorganic compound capable of both modulating the Abeta peptide aggregation activity and of antioxidant activity; in section 2.4 (line 288) they state that "most of mono-functional modulators .... do not possess antioxidant activity". Given they say "most" and not "all", the doubt remains in the reader whether MnPM is really the first modulator to possess antioxidant activity or not.

A4: Thanks a lot for your question. I apologize for the misunderstanding of the words used here.  A more accurate word here would be chelator. It is obvious that chelator are different from manganese-substituted polyoxometalates in terms of structure and effect. Therefore, we revised this sentence as follows to avoid misunderstanding: “In previous literature reports, most of the mono-functional chelators only prevent the formation of β-sheet-rich metal-Aβ species from chelating metal ions, but do not possess antioxidant activity.”

 

C5: - page 9, line 273-274: in discussing Fig. 8 data, the authors state that "the peaks of Abeta40 with MnPM are much more simpler than that without MnPm, which may indicate that MnPM can suppress the aggregation of advanced misfolding of Abeta". Leaving aside the unclear meaning of "aggregation of advances misfolding", the authors should explain why peaks with a simpler structure should imply a suppression of the misfolding/aggregation pathway.

A5: Thanks a lot for your question. It is well known that β-sheet is one of the main type secondary structure of protein, which was first suggested by Pauling et al. [1]. It refers to highly regular local sub-structures on the actual polypeptide backbone chain, which are defined by patterns of hydrogen bonds between the main-chain peptide groups. Therefore, the hydrogen environment on their skeleton may reflect their structural information. It is reported that the change of signal of ¹H-NMR can indicate the change of hydrogen bond structure in those protein [2]. Coordination effect is a stronger intermolecular interaction than hydrogen bond. And thus, if some residue on proteins form coordination bonds, the environment of hydrogen atoms will change dramatically [3]. As shown in Figure 8, the proton signals assignable to the imidazole of His residues were changed, which may be concluded that MnPM affect the chemical environment of protons in Aβ40 by coordinated with His residues. Herein, thank you again for your reminder. Since the coordination structure formed by the protein and MnPM may also be more regular than the β-sheet structure, we revised and deleted the expression about “aggregation of advances misfolding”.

1. Pauling, L.; Corey, R.B.; Branson, H.R. The structure of proteins; two hydrogen-bonded helical configurations of the polypeptide chain. Natl. Acad. Sci. USA1951, 37, 205–211.
2. Del Bene, J.E.; Jordan, M.J.T. Vibrational spectroscopic and NMR properties of hydrogen-bonded complexes: Do they tell us the same thing? Am. Chem. Soc.2000, 122, 4794–4797.
3. Hou, L.M.; Zagorski, M.G. NMR reveals anomalous copper(II) binding to the amyloid Aβ peptide of Alzheimer’s disease. Am. Chem. Soc.2006, 128, 9260–9261.

 

C6: - page 2, line 68: is polyphospolybdate a typo for polyposphomolybdate?

A6: Thanks a lot. This is a typo and we have revised it.

 

C7: - page 3, line 97: B as defined in the equation for bond valence sum is a length, so setting B=0.37 should also include the info about the length unit measure.

A7: Thanks a lot. The value of B was set to 0.37 Å. It was also added to the manuscript.

 

C8: - there are several acronyms which are never defined throughout the text: en ligands;  TGA; cal. (in the steps for weight loss); SOD;

A8: Thanks a lot. en represents for ethanediamine. TGA represents thermogravimetric analysis. Cal. represents for calculation value. SOD represents for superoxide dismutase. All of them have been revised.

 

C9: - page 5, line 166: I do not get what "dove derivatives" are

A9: It has been revised as polyphenols and their derivatives.

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have made all the changes as suggested. Overall, the manuscript looks much better in all aspects!

Reviewer 2 Report

I am happy with the revised version as is.

However, for the record, I wish to stress again the point about the competition between soluble and insoluble beta-sheet rich species. What the authors state in their rebuttal is, I think, incorrect. Luckily for them, the sentences reported in the main text can still be considered correct. The main point, again, is that the result from CD spectrum on soluble species allow the authors to conclude that the beta-sheet content of soluble oligomeric species is decreasing upon addition of POM.

Yet, by themselves, the ThT fluorescence data do not allow to conclude the above (yet they are consistent with it, as the authors correctly state), because oligomeric soluble species are not in equilibrium with insoluble species. Oligomers are transient species that eventually disappears in the aggregation process. So having a drug dissolving fibrils may result in the increase of oligomer concentration. Even if oligomers were in equilibrium with fibrils, a drug could influence such equilibrium by shifting it in favor of oligomeric soluble species, resulting again in the effect of less fibrils and more oligomers. Again, the authors are lucky that the CD data show that this not the case.