Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
The manuscript by Dr. Shinkai and colleagues is about the posttranslational modification of a highly important protein, MT3, also known as the growth inhibitory factor. Authors postulate that MT3, or generally all MT isoforms, are sulfane sulfur binding proteins. The presence of sulfane sulfur at each Cys residue has, according to the authors, a critical impact on redox protein properties and almost does not affect zinc binding. They show a model in which 20 Cys residues with sulfane sulfur atoms can still bind seven zinc ions in the same clusters as unmodified protein. They also show that recombinant MT3 (but also MT1 and MT2) protein can react with HPE-IAM, an efficient trapping reagent of persulfides/polysulfides. This reaction performed in a new approach (high temperature and high reagent concentration) resulted in the formation of bis-S-HPE-AM product, which was quantitatively analyzed using LC-MS/MS. This analysis indicated that all Cys residues of MT proteins are modified by sulfane sulfur atoms. The authors performed a series of experiments showing that such protein can bind zinc, which dissociates in the reaction with hydrogen peroxide or SNAP. They also show that oxidized MT3 is reduced by thioredoxin. It gives a story about a new redox-dependent switching mechanism of zinc/persulfide cluster involving the formation of cystine tetrasulfide bridge.
The whole story is hard to follow due to the lack of many essential explanations or full discussion. What needs to be clarified is the conclusion (or its lack) about MT3 modification proven by mass spectrometry. Figure 1B shows the FT-ICR-MALDI-TOF/MS spectrum of recombinant MT3. It clearly shows the presence of unmodified MT3 protein without zinc ions. Ions dissociate in acidic conditions used for MALDI sample preparation. If the protein contained all Cys residues modified, its molecular weight would be significantly higher. Then, they show the MS spectrum (low quality) of oxidized protein (Fig. 1C), in which new signals (besides reduced apo-MT3) are observed. They conclude that new signals come from protein oxidation and modification with one or two sulfur atoms. If the conclusion on Cys residue oxidation is reasonable, how this protein contains sulfur is unclear. What is the origin of the sulfur if apo-MT does not contain it? Oxidized protein was obtained by acidification of the protein, leading to zinc dissociation and subsequent neutralization and air oxidation. Authors should perform a detailed isotope analysis of the isotopic envelope to prove that sulfur is bound to the protein. They say that the +32 mass increase is not due to the appearance of two oxygen donors. They do not provide evidence. This protein is not a sulfane sulfur binding protein, or its minority is modified. Moreover, it is unacceptable to write that during MT3 oxidation are "released nine molecules of H2". How is hydrogen molecule produced? Moreover, zinc is not "released", it dissociates from protein in a chemical process.
Thank you for your comment. According to your suggestion, we have rewritten the corresponding sentences below, together with addition of new Fig.1D.
First, the sentence “which corresponded to the mass of zinc-free apo-GIF/MT3 and indicated that zinc was removed during MS analysis.” was changed to “which corresponded to the mass of zinc-free apo-GIF/MT3 and indicated that zinc dissociates from protein in acidic conditions used for MALDI sample preparation.” in the introduction section. Second, we have added the following sentence “However, FT-ICR-MALDI-TOF/MS analysis failed to detect sulfur modifications in GIF/MT-3 (Fig. 1B), suggesting that sulfur modifications in the protein were dissociated during laser desorption/ionization. Therefore, we postulate that the small amount of sulfur detected in oxidized apo-GIF/MT-3 is derived from the effect of laser desorption/ionization rather than any actual modification of the minority component.” in the discussion section. Third, we have added new Fig. 1D and the corresponding citation in the introduction. Fourth, the sentence “An increase in mass of 32 Da can also result from addition of two oxygen atoms, but we attributed it to one sulfur atom for reasons described later.” was changed to “Note that an increase in mass of 32 Da can also result from addition of two oxygen atoms.”.
Another important point is a new approach to the HPE-IAM application. Zinc-binding MT3 was incubated with 5 mM reagent at 60°C for 36 h. Authors claim that high concentration was required because apoMT3 has stable conformation. Figure 2B shows that product concentration increases with higher temperature, but it is unclear why such a high temperature was used. Figure 1D shows that at 37°C, there is almost no reaction at 5 mM reagent. Changing parameters sounds reasonable only when the reaction is monitored by mass spectrometry. In conclusion, about 20 sulfane sulfur atoms present in MT3 would be clearly visible. Such evidence was not provided. Increased temperature and reagent concentration could cause modification of cysteinyl thiol/thiolates as well, not only persulfides/polysulfides. Therefore, it is highly possible that non-modified MT3 protein could react with HPE-IAM, giving false results. Besides mass spectrometry, which would clearly prove modifications of 20 Cys, authors should use very important control, which could be chemically synthesized beta- or alfa-domain of MT3 reconstituted with zinc (many protocols are present in the literature). Such models are commonly used to test any kind of chemistry of MTs. If a non-modified chemically obtained domain would undergo a reaction with HPE-IAM under such rigorous conditions, then my expectation would be right.
Thank you for your comments. Although we have already confirmed that no false-positive results were observed using this method in Fig. 5 (previously Fig. 4), we have conducted additional experiments by preparing chemically synthesized α- and β-domains of GIF/MT-3, as well as recombinant α- and β-domains of GIF/MT-3. As shown in the new Fig. S2A, the chemically synthesized α- and β-domains of GIF/MT-3 detected almost no sulfane sulfur (less than 1 molecule per protein), whereas the recombinant α- and β-domains detected several molecules of sulfane sulfur (more than 5 molecules per protein) (Fig. S2A). Therefore, I would like to emphasize here that the cysteine residue itself cannot be the source of the bis-S-HPE-AM product (sulfane sulfur derivative).
Accordingly, we have added the following sentence in the results section: “Because this assay was performed at relatively high temperatures (60°C), we also examined the sulfane sulfur levels of several mutant proteins using chemically synthesized α- and β-domains of GIF/MT-3 to eliminate false-positive results. As shown in Fig. S2A, sulfane sulfur (less than 1 molecule per protein) was undetectable in chemically synthesized α- and β-domains of GIF/MT-3, whereas several molecules of sulfane sulfur per protein were detected in recombinant α- and β-domains exhibited (Fig. S2B, left panel). These findings indicated that the sulfane sulfur detected in our assay was derived from biological processes executed during the production of GIF/MT-3 protein. We further analyzed mutant proteins with β-Cys-to-Ala and α-Cys-to-Ala substitutions and found that their sulfane sulfur levels were comparable with those of the α- and β-domains of GIF/MT-3, respectively (Fig. S2B, left panel). Additionally, Ser-to-Ala mutation did not affect the sulfane sulfur levels of GIF/MT-3. The zinc content of each mutant protein was also determined under these conditions (Fig. S2B, right panel).”
- The remaining experiments provided in the manuscript can also be applied for non-modified protein (without sulfane sulfur modification) and do not provide worthwhile evidence. For instance, hydrogen peroxide or SNAP may interact with non-modified MTs. Zinc ions dissociate due to cysteine residue modification, and TCEP may reduce oxidized residue to rescue zinc binding. Again, mass spectrometry would provide nice evidence.
Thank you for your comment. We understand that such experiments can also be applied to non-modified proteins (without sulfane sulfur modification). However, the experiments shown in Fig. 4 and Fig. 6 were conducted to investigate the role of sulfane sulfur under oxidative stress conditions, rather than to examine sulfur modification in the protein itself. As mentioned previously, it is difficult to detect sulfur modifications directly in the protein using MALDI-TOF/MS (Fig. 1), as sulfur modifications appear to dissociate during the laser desorption/ionization process.
- The same is thioredoxin (Fig. 7) and its reaction with oxidized MT3. Nonmodified and oxidized MT3 would react as well.
Thank you for your comment. We understand that such experiments can also be applied to non-modified MT-3 protein. However, to the best of our knowledge, this is the first report demonstrating that apo-MT-3 can serve as a good substrate for the Trx system. In fact, this experiment is not intended to prove that MT-3 is sulfane sulfur-binding protein. Rather, it demonstrates the novel finding that apo-MT3 serves as an excellent substrate for Trx and that the sulfane sulfur (persulfide structure) remains intact throughout the reduction process.
- If HPE-IAM reacts with Cys residues with unmodified MT3, which is more likely the case under used conditions, the protein product of such reaction will not bind zinc. It could be an explanation of the cyanolysis experiment (Fig. 6).
Thank you for your comment. As you pointed out, HPE-IAM reacts with cysteine residues in unmodified MT-3, thereby preventing zinc from binding to the protein. However, we did not use HPE-IAM prior to measuring zinc binding. Instead, HPE-IAM was used solely for determining the sulfane sulfur content in the protein, and thus it cannot explain the results of the cyanolysis experiment.
- Figure 4 shows the reactivity of (pol)sulfides with TCEP and HPE-IAM. What are redox potentials? Do they correlate with the obtained results?
Thank you for your comment. However, we must apologize as we do not fully understand the rationale behind determining redox potentials in this experiment. We believe the data itself to be very clear and presenting convincing results.
- Raman spectroscopy experiments would illustrate the presence of sulfane sulfur in MT3 only if all Cys were modified.
Yes, that is correct. Since approximately 20 sulfane sulfur atoms are detected in the protein with 20 cysteine residues, we believe that nearly all cysteine residues are modified by sulfane sulfur. Therefore, Raman spectroscopy is considered applicable to our current study.
- The modeling presented in this study is very interesting and confirms the flexibility of metallothioneins. MT domains are known to bind various metal ions of different diameters. They adopt in this way to larger size the ions. The same mechanism could be present from the protein site. The presence of 9 or 11 sulfur atoms in the beta or alfa domain would increase the size of the domains without changing the cluster structure.
We truly appreciate your positive evaluation of this work.
- Comment to authors. Apo-MT is not present in the cell. It exists as a partially metallated species. The term "apo-MT" was introduced to explain that MTs are not fully saturated by metals and function as a metal buffer system. Apo-MT comes from old ages when MT was considered to be present only in two forms: apo-form and fully saturated forms.
Thank you for your insightful comments. We find it reasonable to understand that apo-MT exists as a partially metallated species within the cell.
Reviewer #2 (Public Review):
Summary:
In this manuscript, the authors reveal that GIF/MT-3 regulates zinc homeostasis depending on the cellular redox status. The manuscript technically sounds, and their data concretely suggest that the recombinant MTs, not only GIF/MT-3 but also canonical MTs such as MT-1 and MT-2, contain sulfane sulfur atoms for the Zn-binding. The scenario proposed by the authors seems to be reasonable to explain the Zn homeostasis by the cellular redox balance.
Strengths:
The data presented in the manuscript solidly reveal that recombinant GIF/MT-3 contains sulfane sulfur.
Weaknesses:
It is still unclear whether native MTs, in particular, induced MTs in vivo contain sulfane sulfur or not.
Thank you for pointing out the strengths and weaknesses of this manuscript. Based on your suggestions, we have determined the sulfane sulfur content in the native GIF/MT-3 protein, as explained in our response to "Recommendations for the Authors #2."
Reviewer #3 (Public Review):
Summary:
The authors were trying to show that a novel neuronal metallothionein of poorly defined function, GIF/MT3, is actually heavily persulfidated in both the Zn-bound and apo (metal-free) forms of the molecule as purified from a heterologous or native host. Evidence in support of this conclusion is compelling, with both spectroscopic and mass spectrometry evidence strongly consistent with this general conclusion. The authors would appear to have achieved their aims.
Strengths:
The analytical data are compelling in support of the author's primary conclusions are strong. The authors also provide some modeling evidence that strongly supports the contention that MT3 (and other MTs) can readily accommodate sulfane sulfur on each of the 20 cysteines in the Zn-bound structure, with little perturbation of the structure. This is not the case with Cys trisulfides, which suggests that the persulfide-metallated state is clearly positioned at lower energy relative to the immediately adjacent thiolate- or trisulfidated metal coordination complexes.
Weaknesses:
The biological significance of the findings is not entirely clear. On the one hand, the analytical data are clearly solid (albeit using a protein derived from a bacterial over-expression experiment), and yes, it's true that sulfane S can protect Cys from overoxidation, but everything shown in the summary figure (Fig. 8D) can be done with Zn release from a thiol by ROS, and subsequent reduction by the Trx/TR system. In addition, it's long been known that Zn itself can protect Cys from oxidation. I view this as a minor weakness that will motivate follow-up studies. Fig. 1 was incomplete in its discussion and only suggests that a few S atoms may be covalently bound to MT3 as isolated. This is in contrast to the sulfate S "release" experiment, which I find quite compelling.
Impact:
The impact will be high since the finding is potentially disruptive to the metals in the biology field in general and the MT field for sure. The sulfane sulfur counting experiment (the HPE-IAM electrophile trapping experiment) may well be widely adopted by the field. Those of us in the metals field always knew that this was a possibility, and it will interesting to see the extent to which metal-binding thiolates broadly incorporate sulfate sulfur into their first coordination shells.
Thank you for pointing out the strengths and weaknesses of this manuscript. As you noted, the explanations and discussions regarding Fig. 1 were missing. To address this, we have added the following sentences to the discission section: “However, FT-ICR-MALDI-TOF/MS analysis failed to detect sulfur modifications in GIF/MT-3 (Fig. 1B), suggesting that sulfur modifications in the protein were dissociated during laser desorption/ionization. Therefore, we postulate that the small amount of sulfur detected in oxidized apo-GIF/MT-3 is derived from the effect of laser desorption/ionization rather than any actual modification of the minority component.”
Reviewer #1 (Recommendations For The Authors):
Overall, the topic of the study is interesting, but the provided evidence is insufficient to claim that MT3 is a sulfane sulfur-binding protein. Indeed, some recent studies showed that natural and recombinant MT proteins can be modified, but only one or a few cysteine residues were modified. Authors should follow my suggestion and apply mass spectrometry to all performed reactions and, first of all, to freshly obtained protein. I strongly suggest using chemically synthesized and reconstituted domains to test whether the home-developed approach is appropriate. Moreover, native MS and ICP-MS analysis of MT3 would support their claims.
Thank you for your insightful comments. Following your suggestions, we have prepared chemically synthesized proteins of the α- and β-domains of GIF/MT-3 and conducted additional experiments, as explained in response comments to “Public Review #1”. Regarding the MS analysis, we have also added a discussion on the difficulty of detecting sulfur modifications in the protein.
Reviewer #2 (Recommendations For The Authors):
I have some minor points which should be considered by the authors.
(1) Table 1: In the simulation by MOE, the authors speculated 7 atoms of metal bound to GIF/MT-3. Although a total of 7 atoms of Zn or Cd are actually bound to MTs as a divalent ion, the number of Cu and Hg bound to MTs as a monovalent ion is scientifically controversial. Several ideas have been proposed in the literature, however, "7 atoms of Cu or Hg" could be inappropriate as far as I know. The authors should simulate again using a more appropriate number of Cu or Hg in MTs.
Thank you for providing this valuable information. We reviewed several papers by the Stillman group and found that the relative binding constants of Cu4-MT, Cu6-MT, and Cu10-MT were determined after the addition of Cu(I) to apo MT-1A, MT-2, and MT-3 (Melenbacher and Stillman, Metallomics, 2024). However, incorporating these copper numbers into our GIF/MT-3 simulation model proved challenging. Therefore, we decided to omit the score value for copper in Table 1.
On the other hand, some researchers have reported that mercury binds to MT as a divalent ion, and the formation of Hg7MT is possible (not just other forms). Therefore, we decided to continue using the score value for mercury shown in Table 1.
(2) If possible, native MT samples isolated from an experimental animal should be evaluated for the sulfane sulfur content. Canonical MTs, MT-1 and MT-2, are highly inducible by not only heavy metals but also oxidative stress. Under the oxidative stress condition such as the exposure of hydrogen peroxide, it is questionable whether the induced Zn-MTs contain sulfane sulfur or not.
According to your suggestion, we evaluated the sulfane sulfur content in native GIF/MT-3 samples isolated from mouse brain cytosol (Fig. 10). The measured amount was 3.3 per protein. This suggests that sulfane sulfur in GIF/MT-3 could be consumed under oxidative conditions, as you anticipated. Another possible explanation for the discrepancy between the native form and recombinant protein is likely related to metal binding in the protein. It is generally understood that both zinc and copper bind to GIF/MT-3 in approximately equal proportions in vivo. When we prepared recombinant copper-binding GIF/MT-3 protein, the sulfane sulfur content in the protein was significantly different (approximately 4.0 per protein) compared to the Zn7GIF/MT-3 form. Further studies are needed to clarify the relationship between sulfane sulfur binding and the types of metals in the future.
(3) The biological significance of sulfane sulfur in MTs is still unclear to me.
Thank you for your comments. To address this question, we have added the following sentence to the discussion section: “The biological significance of sulfane sulfur in MTs lies in its ability to 1) contribute to metal binding affinity, 2) provide a sensing mechanism against oxidative stress, and 3) aid in the regeneration of the protein.”
(4) According to the widely accepted nomenclature of MT, "MT3" should be amended to "MT-3".
According to your suggestion, we have amended from MT3 to MT-3 throughout the manuscript.
Reviewer #3 (Recommendations For The Authors):
Most of my comments are editorial in nature, largely focused on what I perceive as overinterpretation or unnecessary speculation.
The authors state in the abstract that the intersection of sulfane sulfur and Zn enzymes "has been overlooked." This is not actually true - please tone down to "under investigated" or something like this.
Based on your suggestion, we have replaced the term “has been overlooked” with “has been under investigated” in the abstract.
Line 228: The discussion of Fig. 6C involved too much speculation. I cannot see a quantitative experiment that supports this.
Based on your suggestion, we have removed Fig. 6C (currently referred to as Fig. 7C). Additionally, we have revised the sentence from “implying that the sulfane sulfur is an essential zinc ligand in apo-GIF/MT3 and that an asymmetric SSH or SH ligand is insufficient for native zinc binding (Fig. 6C)” to “implying the contribution of sulfane sulfur to zinc binding in GIF/MT-3”.
Line 247 "persulfide in apo-GIF/MT3 seems.." I think the authors mean that the Zn form of the protein is resistant to Trx or TCEP.
Thank you for pointing this out. We realized that the term “persulfide in apo-GIF/MT3” might be confusing. Therefore, we have replaced it with “persulfide formation derived from apo-GIF/MT3” in the corresponding sentence.
Molecular modeling: We need more details- were these structures energy-minimized in any way? Can the authors comment on the plethora of S-S dihedral angles in these structures, and whether they are consistent with expectations of covalent geometry? Please add text to explain or even a table that compiles these data.
Thank you for your comment. Yes, energy minimization calculations for structural optimization were conducted during homology modeling in MOE. In fact, we have already stated in the Methods section that “Refinement of the model with the lowest generalized Born/volume integral (GBVI) score was achieved through energy minimization of outlier residues in Ramachandran plots generated within MOE.” In this model, covalent geometry, including the S-S dihedral angles, is also taken into consideration.
What is a thermostability score? Perhaps a bit more discussion here and what relationship this has to an apparent (or macroscopic) metal affinity constant.
The thermostability score is used to compare the thermal stability between the wild-type and mutant proteins. As shown in Equation (1) in the method section, it is calculated by subtracting the energy of the hypothetical unfolded state from the energy of the folded state. Since obtaining the structure of the unfolded state requires extensive computational effort, MOE employs an empirical formula based on two-dimensional structural features to estimate it. The ΔΔG values represent the difference between ΔGf(WT) and ΔGf(Mut). However, because it is difficult to directly determine ΔGf(Mut) and ΔGf(WT), MOE calculates ΔΔG using the thermodynamic cycle equivalence: ΔΔGs =ΔGsf (WT→Mut) - ΔGsu (WT→Mut), as expressed in Equation (1).
On the other hand, the affinity score represents the interaction energy between the target ligand and the protein. In this study, we calculated the affinity score by selecting metal atoms as the ligands. The interaction energy (E int) is defined as:
E int = E complex − E receptor − E ligand
where each term is as follows:
E complex : Potential energy of the complex.
E receptor : Potential energy of the receptor alone.
E ligand : Potential energy of the ligand alone.
Each potential energy term includes contributions from bonded interactions such as bond lengths and bond angles. However, since there is no structural difference among E receptor, and E ligand, the bonded energy components cancel out. Consequently, E int is determined as:
E int = ΔEele +ΔEvdW +ΔE sol
Here, a negative E int indicates that the complex is more stable, while a positive E int implies that the receptor and ligand are more stable in their dissociated states.
We have revised the sentence "The affinity score was also calculated using MOE software as the difference between the ΔΔGs values of the protein, free zinc, and metal–protein complex” to "The affinity score was also calculated using MOE software as the difference between the potential energy values of the protein, free zinc, and metal–protein complex” to correct the misdescription.
Lines 278-280: The authors state that they observe a "marked enhancement of metal binding affinity, and rearrangement of zinc ions." I don't see support for this rather provocative conclusion. This is the expectation of course. I would love to see actual experimental data on this point, direct binding titrations with metals performed before and after the release of the sulfate sulfur atoms.
Thank you for your comments. Although this statement is based on the 3D modeling simulation, we have also experimentally observed that the diminishment of sulfane sulfur in GIF/MT-3 resulted in a decrease in zinc binding levels, as shown in Fig. 7. However, conducting direct binding titration experiments was difficult for us due to the difficulty in preparing pure GIF/MT-3 protein with or without sulfane sulfur. Therefore, we have revised the sentence "marked enhancement of metal binding affinity, and rearrangement of zinc ions" to simply "enhancement of metal binding affinity" to avoid over-speculation.
Table I- quantitatively lower stability for the Cu complex- the stoichiometry is clearly wrong in this simulation- please redo this simulation with the right stoichiometry or Cu to MT3- consult a Stillman paper.
Thank you for providing this valuable information. We reviewed several papers by the Stillman group and found that the relative binding constants of Cu4-MT, Cu6-MT, and Cu10-MT were determined after the addition of Cu(I) to apo MT-1A, MT-2, and MT-3 (Melenbacher and Stillman, Metallomics, 2024). However, incorporating these copper numbers into our GIF/MT-3 simulation model proved challenging. Therefore, we decided to omit the score value for copper in Table 1.
I like the model for reversible metal release mediated by the thioredoxin system (Fig. 8D)- but you can also do this with thiols- nothing really novel here. Has it been generally established that tetraulfides are better substrates for the Trx/TR system? The data shown in Fig. 7B seems to suggest this, but is this broadly true, from the literature?
There are reports describing that persulfides and polysulfides are reduced by the thioredoxin system. However, it is not well-established that tetraulfides are better substrates for the Trx/TR system. To the best of our knowledge, this is the first report demonstrating that apo-MT-3 can serve as a good substrate for the Trx/TR system. Further research is required to compare the catalytic efficiency between proteins containing disulfide and those with tetraulfide moieties.
Line 380: Many groups have reported that many proteins are per- or polysulfidated in a whole host of cells using mass spectrometry workflows, and that terminal persulfides can be readily reduced by general or specific Trx/TR systems. This work could be better acknowledged in the context of the authors' demonstration of the reduction of the tetrasulfides, which itself would appear to be novel (and exciting!).
We truly appreciate your positive evaluation of this work.
