From the journal RSC Chemical Biology Peer review history

30-Nov-2020

Dear Dr McCulla:

Manuscript ID: CB-ART-11-2020-000200
TITLE: Identifying Cysteine Residues Susceptible to Oxidation by Photoactivatable Atomic Oxygen Precursors using a Proteome-wide Analysis

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Associate Editor, RSC Chemical Biology

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

Reviewer 1

Isor et al synthesized a few DBTO derivatives that can generate O3P upon photoactivation and combined it with chemical proteomics to profile cysteines that are sensitive to oxidation by this specific ROS species. They analyzed sequence conservations around the identified cysteines as well as their solvent accessibility. Overall, the chemical synthesis and isoTOP-ABPP profiling were conducted with solid and convincing data and the manuscript is easy to follow, however, the reviewer did not find much biological insights that can be inferred from the current study. For example, how are the O3P sensitive cysteines different from other ROS-sensitive cysteines? In addition, are there any endogenous O3P in cells and it would boost the impact of this work if the authors can demonstrate a more biological source (e.g., an enzyme-mediated source) can lead to similar cysteine oxidation in the living cells. Technically, would DBTO treatment alter global proteome abundance? And did the authors find the protein/cysteine by mass spec that is with the increased labeling intensity as shown in Figure 7B?

Reviewer 2

This manuscript describes the use of novel photochemical precursors to atomic oxygen to target protein cysteine oxidation in live cells. The effects of atomic oxygen have been relatively unexplored and this study represents the first investigation of protein modification in a cellular environment. Publication in RSC Chemical Biology is recommended after the following issues are addressed.

1. The S-oxide functionality should be shown consistently as S(+)-O(-) as it is in Figures 1-3, not as S=O as it is in Schemes 1-3.

2. Why are the results shown in Table 1 significantly different in the current work vs. that reported in Ref. 19 for both DBTO and precursor 1?

3. What are the competing photodeoxygenation reactions of precursor 6 mentioned on page 7, middle of second column?

4. It seems that precursors 2 and 3 were developed as an analogue to precursor 1 to provide water solubility. But then precursor 1 was the main compound studied in cells. Is this compound really soluble at 10 uM in aqueous solution?

5. What is know about the diffusability of atomic oxygen? Is it so reactive that it is not expected to diffuse very far?

6. Is atomic oxygen expected to be hydrophilic or hydrophobic. I might anticipate that it is hydrophobic and could partition to hydrophobic regions (e.g., membranes). But this does not seem to be consistent with the experimental results. Could the authors comment on this?

7. On page 13, first column, it is stated that "membrane proteins are more likely to undergo cystine reduction when treated with 1 and UV." Are both 1 and UV needed? What is the effect of UV alone?

8. In the Supporting Information, the Cartesian Coordinates should be given along with the the figures of the optimized geometries shown on pages 44-46.

REVIEWER REPORT(S):
Referee: 1

Comments to the Author
Isor et al synthesized a few DBTO derivatives that can generate O3P upon photoactivation and combined it with chemical proteomics to profile cysteines that are sensitive to oxidation by this specific ROS species. They analyzed sequence conservations around the identified cysteines as well as their solvent accessibility. Overall, the chemical synthesis and isoTOP-ABPP profiling were conducted with solid and convincing data and the manuscript is easy to follow, however, the reviewer did not find much biological insights that can be inferred from the current study.

"We thank the reviewer for their favorable comments regarding the data in our manuscript. Regarding biological insights, we would like to emphasize that this study represents the first time that living cells were exposed to the oxidant O(3P). The ultimate goal of this manuscript is therefore to demonstrate that O(3P) is able to act as an oxidant within a native biological system. This has never been demonstrated before, and we think the novelty of the study lies in demonstrating the physiological compatibility of this unique oxidant. This study lays the foundation to address many of the biological insights that the reviewer alludes to below, and we are excited to focus on these questions in future studies with this oxidant."

For example, how are the O3P sensitive cysteines different from other ROS-sensitive cysteines?

"As mentioned previously, this study was designed to demonstrate the physiological compatibility of oxidants generated from photodeoxygenation of O(3P)-precursors. Our data from this study demonstrates that O(3P) can be applied as a physiologically compatible oxidant, and in future studies we plant to investigate some more specific hypotheses, for example, how is the effect of O(3P) different than other ROS."

In addition, are there any endogenous O3P in cells and it would boost the impact of this work if the authors can demonstrate a more biological source (e.g., an enzyme-mediated source) can lead to similar cysteine oxidation in the living cells.

"Endogenous O(3P) has not been observed in cells, and we hope our work will help answer if it is potentially an endogenous ROS. That said, many enzymes, such as cytochrome-P450’s, can be considered as metal-stabilized oxygen atoms. Thus, we also plan to investigate how close the reactivity of freely diffusing O(3P) mimics these types of enzymes. In the revised manuscript, we have included a sentence that discusses this potential biological relevance.

Additionally, the photodeoxygenation enables the use of DBTO as a ‘caged oxidant’ that can be selectively appended to specific biological targets to enable targeted oxidation. Therefore, even if O(3P) is not an endogenous oxidant, we envision many potential applications of this chemotype in redox biology applications."

Technically, would DBTO treatment alter global proteome abundance?

"In the live-cell labeling experiments, cells were exposed to DBTO for a total time of 30 mins, after which the cells were lysed for downstream processing. Given the short incubation time, we do not think that there are substantial changes in protein abundance occurring in response to DBTO. "

And did the authors find the protein/cysteine by mass spec that is with the increased labeling intensity as shown in Figure 7B?


'We have included an excel file with our supporting information that lists all our isoTOP-ABPP data. As can be seen in the data, of the 20 proteins with the largest increase in cysteine reactivity (lowest ratio values), there are several that lie within the general molecular weight range (~37-50kD), as the band the reviewer is indicating in Figure 7b. These putative proteins are PSMC5, PDHA1, and HNRNPA2B1. However, considerable work needs to be undertaken to determine which of these proteins are represented by the gel band, and why these particular cysteines show an increase in reactivity. Given that the focus of this study is to identify the sites of oxidation (increased ratios), we feel that identification and characterization of this protein is beyond the scope of this current study."


Referee: 2

Comments to the Author
This manuscript describes the use of novel photochemical precursors to atomic oxygen to target protein cysteine oxidation in live cells. The effects of atomic oxygen have been relatively unexplored and this study represents the first investigation of protein modification in a cellular environment. Publication in RSC Chemical Biology is recommended after the following issues are addressed.

We thank this reviewer for their favorable comments on our manuscript.

1. The S-oxide functionality should be shown consistently as S(+)-O(-) as it is in Figures 1-3, not as S=O as it is in Schemes 1-3.

Thank you for pointing this out. This has been addressed.


2. Why are the results shown in Table 1 significantly different in the current work vs. that reported in Ref. 19 for both DBTO and precursor 1?

In ref 19 (Zheng et al., 2016),1 the solutions were made where the concentrations ranged from 1-6 mM. The solutions were then degassed using argon gas and placed in a photoreactor with 14 LZC UVA bulbs. The solutions were irradiated such that the corresponding sulphide conversion was less than 30%.
In contrast, the solutions made in this work had concentrations ranging from 8-10mM. The solutions were degassed and then placed in the photoreactor with 8 LZC-UVA bulbs. The photoirradiation times ranged from 1h 15 min – 1h 37 min such that the corresponding sulphide after photodeoxygenation did not exceed 20%. Difference in experimental conditions, particularly how the solution was degassed are known to have significant effects on the observed yields.2 Thus, we believe that the difference in experimental procedures and the influence of experimental conditions on the oxidation yields2 result in differing yields reported in ref 19 and our work.

The revised manuscript now has a sentence that discusses the sensitivity to experimental conditions.


3. What are the competing photodeoxygenation reactions of precursor 6 mentioned on page 7, middle of second column?

According to a study published in 20161 that explored the photodeoxygenation of benzannulated DBTO derivatives, a competing photodeoxygenation mechanism was noted to be similar to the triplet sensitization of DBTO where the S-O bond cleavage proceeds through the dissociative T1 state instead of the T2 state. Since evidence indicates that O(3P) generation proceeds through the dissociative T2 state,3 a S-O bond cleavage resulting from relaxation from the T1 state would not produce O(3P). The difference in T1 energies of 1-3 and 6 as compared to DBTO suggest that the intersystem crossing of S1 to T1 and non-radiative relaxation of T2 to T1 are more favored leading to a decrease in O(3P) yields.

The revised manuscript includes text on this page that mentions the alternate photodeoxygenation mechanism.

4. It seems that precursors 2 and 3 were developed as an analogue to precursor 1 to provide water solubility. But then precursor 1 was the main compound studied in cells. Is this compound really soluble at 10 uM in aqueous solution?

Prior to cell treatment, a 1mM stock of 1 was made in DMSO to aid in its solubility into the aqueous media. The treatment media was made by diluting this 1mM stock 1:100 into complete media resulting in a final concentration of 10µM 1 and 1% DMSO. The oxidation profiles that we observe upon treatment of cells with compound 1 support that this compound is in fact soluble under the conditions used.


5. What is know about the diffusability of atomic oxygen? Is it so reactive that it is not expected to diffuse very far?

The rate constants in the following publications suggest O(3P)’s high reactivity:
Gas phase:
1. D. L. Singleton and R. J. Cvetanović, J. Phys. Chem. Ref. Data, 1988, 17, 1377–1437.
2. R. J. Cvetanović, J. Phys. Chem. Ref. Data, 1987, 16, 261–326.

Liquid phase:
1. G. Bucher and J. C. Scaiano, J. Phys. Chem., 1994, 98, 12411–12413.
2. S. M. Omlid, M. Zhang, A. Isor and R. D. McCulla, J. Org. Chem., 2017, 82, 13333–13341.
3. E. Lucien and A. Greer, J. Org. Chem., 2001, 66, 4576–4579.
Evidence of diffusing O(3P) was presented in a study by Omlid et al., 2019.4 The study indicated a freely diffusing O(3P) being generated as a result of photodeoxyengation of a water soluble DBTO derivative. In the experimental setup outlined within that study, the O(3P) pre-cursor was separated from the O(3P) accepting molecule though a polymer nanocapsule barrier with a < 1nm pore size. The concentration of O(3P) pre-cursor loading within nanocapsule ranged from 1-8mM and the O(3P)-accepting molecule at 20±2 mM in acetonitrile. After 5h of irradiation with 14 LZC-UVA bulbs (fwhm, 325-375 nm), the oxidized product of O(3P)-accepting molecule was found to range from 8.4µM-10.4µM depending on the degassing technique used pre-photoirradiation of the reaction solution. The diffusing distance for O(3P) in the system was assumed to be 65 nm. The photoreaction was carried out with 14 LZC-UVA bulbs. Based on results in this work, ~1.5h of irradiation of DBTO with 8 UV-A bulbs produced a toluene oxidation yield of 57% and the thiophene conversion from photodeoxygenation of DBTO remained <20%. This suggests that the concentration of oxidized product of O(3P)-accepting molecule in the study by Omlid et al., 2019 is quite low. This comparative insight suggest that a diffusing distance of 65 nm can significantly alter the oxidation yield further proposing that O(3P) may not diffuse very far.

We hope to do further studies in the future to help estimate the diffusion distance of O(3P) within a cell.

6. Is atomic oxygen expected to be hydrophilic or hydrophobic. I might anticipate that it is hydrophobic and could partition to hydrophobic regions (e.g., membranes). But this does not seem to be consistent with the experimental results. Could the authors comment on this?

Atomic oxygen is a small electrophilic and transient oxidant.5,6 Its reactivity with membranes have shown to be dependent on the lipophilicity of the O(3P)-precursor. In the study by Petroff et al., 2020,7 the extent of oxidation of the lipoprotein by O(3P) was revealed to increase with using DBTO derivatives that localized in plasma membrane. Because of the highly reactive nature of O(3P),6 it is predicted that it would react with an O(3P)-accepting molecule or functional group in the immediate environment of its generation as opposed to partitioning within membranes and then oxidizing cysteines in membrane proteins.

7. On page 13, first column, it is stated that "membrane proteins are more likely to undergo cystine reduction when treated with 1 and UV." Are both 1 and UV needed? What is the effect of UV alone?

In Figure 7A, we show that UV treatment alone does not affect cysteine reactivity (see lanes 1 and 2; DMSO +/- UV). Additionally, treatment with compound 1 alone does not affect cysteine reactivity (see lanes 1 and 3; DMSO versus 1 with no UV). The only observed changes in cysteine reactivity occur upon addition of both 1 and UV (lane 4). Therefore, we are confident that both 1 and UV irradiation are necessary to induce cysteine oxidation.

8. In the Supporting Information, the Cartesian Coordinates should be given along with the figures of the optimized geometries shown on pages 44-46. –

This is now included in the supporting information.

References
1 X. Zheng, S. M. Baumann, S. M. Chintala, K. D. Galloway, J. B. Slaughter and R. D. McCulla, Photochem. Photobiol. Sci., 2016, 15, 791–800.
2 J. T. Petroff, S. M. Omlid, S. M. Chintala and R. D. McCulla, J. Photochem. Photobiol. A Chem., 2018, 358, 130–137.
3 S. A. Stoffregen, S. Y. Lee, P. Dickerson and W. S. Jenks, Photochem. Photobiol. Sci., 2014, 13, 431–8.
4 S. M. Omlid, S. A. Dergunov, A. Isor, K. L. Sulkowski, J. T. Petroff, E. Pinkhassik and R. D. Mcculla, Chem. Commun., 2019, 55.
5 E. Lucien and A. Greer, J. Org. Chem., 2001, 66, 4576–4579.
6 G. Bucher and J. C. Scaiano, J. Phys. Chem., 1994, 98, 12411–12413.
7 J. T. Petroff, A. Isor, S. M. Chintala, C. J. Albert, J. D. Franke, D. Weinstein, S. M. Omlid, C. K. Arnatt, D. A. Ford and R. D. McCulla, RSC Adv., 2020, 10, 26553–26565.

03-Jan-2021

Dear Dr McCulla:

Manuscript ID: CB-ART-11-2020-000200.R1
TITLE: Identifying Cysteine Residues Susceptible to Oxidation by Photoactivatable Atomic Oxygen Precursors using a Proteome-wide Analysis

Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below. One of referees raised a serious concern about your manuscript, but I decided to accept this manuscript on the basis of my own evaluation.

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With best wishes,

Prof Seung Bum Park

Associate Editor, RSC Chemical Biology
Professor, Chemistry Department, Seoul National University, Korea

Reviewer 2

The authors have addressed all of the previous review's concerns and publication is recommended.

Reviewer 1

I still don’t get the argument that how O3P oxidant is biologically or physiologically relevant in this case if no endogenous counterpart is found. I would like to point out that because it is the first time for such a profiling to be done, it does not mean it is novel or fits the scope of the journal. With the current data, I will leave the editor to decide whether it deserves a chance to be published in this specific journal.

Reviewer 3

Authors have addressed reviewers' all review comments. Therefore, I herein recommend the revised manuscript to accept for publication in this journal.