PREreview of One-carbon metabolism enzyme Ahcy is a redox sensor that modulates gene expression to protect against light stress-induced retinal degeneration

The manuscript by Stanhope et al. investigates the role of the one-carbon metabolism enzyme S-Adenosylhomocysteinase (Ahcy) in the alteration of neurodegeneration gene expression in Drosophila. The authors found that oxidation of C195 of Ahcy inhibits its activity, leading to neuroprotective changes in gene expression through an unknown mechanism. The findings highlight the importance of C195 oxidation in Ahcy, potentially limiting enzyme activity in a metabolism-dependent manner. We agree with the authors’ conclusions and we provide major and minor comments below to assist in the clarity and ease of interpretation of the manuscript. 

Major comments 

Please provide more information on Ahcy and AhcyL1, e.g. how many cysteines does Ahcy contain? Does AhcyL1 respond to blue light stress? 

Line 55, please include a brief explanation of the redox proteomics approach and whether it was concluded that C195 is involved in a disulfide bond. We suggest discussing early on from structural prediction of Ahcy whether the two cysteines (C195 and presumably C228) are in close proximity for a disulfide bond to form as otherwise it is unclear what is meant by oxidation of C195 (it could be e.g. a sulfenic acid) and what the ‘second’ cysteine is. 

Fig1E, include samples with reductant and oxidant +/- labeling to show where the fully reduced or oxidized protein migrates. There appears to be a slight band present in the “second labeled cys” in the C195S, which would contradict the statement in Line 99. Could you comment on it. Although not required for the conclusions, including protein samples from flies that are blue light-treated would inform on whether Ahcy-C195 becomes oxidized. 

We found it quite striking that the RNAi lines for AhcyL1 were also displaying a low level of Ahcy. Could it be an issue of sequence identity between Ahcy and AhcyL1 leading to off-target effects? Or could AhcyL1 positively regulate Ahcy gene expression? Also, could you explain the modest changes in the expression of AhcyL1 in the AhcyL1 line?

Fig2D, E, how do you explain the variation in AhcyL1 and Gnmt between the three Ahcy RNAi lines? Consider revising line 126-127: “without any corresponding changes in AhcyL1” as there are changes. 

Line 255, it would be of interest to provide more discussion of the discrepancy between this work and the previous work which showed Ahcy-dependent enhancement of H3K4me3. Line 273, consider revising this sentence as it was not shown here that Ahcy promotes methylation, which data indicate this point?

Minor comments  

Figures, figure panels are mostly small, with large white space between them. Increasing panel size and image fidelity would help to fully visualize data (e.g. Fig. 2A, 4E). Figure 1E, increasing the size and brightness/contrast would be good too.

Line 43, consider revising 'higher' to another term (e.g. multicellular organisms) because 'higher’ assumes that complexity increased with the evolution of eukaryotes, which is not always the case. 

Line 70, briefly describe S2 cells and why they are being used here.  

Line 77, define TMT. 

Lines 81-84, repeated information from introduction that could be removed or condensed. 

Fig1A, we found it unusual to include previously published data, is that because it wasn’t displayed as a graph in the previous study? 

Fig1B, add arrowheads and acronyms by the molecules (SAM, SAH) and an arrow indicating inhibition of methyltransferases. 

Fig 1E, define “IGMR”. 

Line 108, cite also Fig1C. 

Line 113, refer to Fig2A instead of Fig1A. 

Line 115, add Fig2A for the Ahcy interaction with itself. 

Line 140, refer to Supp Fig2B instead of 2A. Could you describe the data presented in Supp Fig2B in the legend.  

Lines 146-147, refer to Fig 3A only instead of 3A & 3B, as you are describing catalytic efficiency (kcat/km).

Line 158, could you discuss further how else C. elegans Ahcy differs from Drosophila? Maybe provide an alignment of the full protein sequences in supplemental. Are there other key differences between Drosophila and C. elegans Ahcy in structure or function? The rate shown in Fig. 3B appears different at lower SAH rates specifically, is this relevant? 

Line 170, refer to Supp Fig2A instead of 2B.

Figure 3E, add adenosine label. Could adenosine come from cleavage of NAD+ in these conditions? Or is the product of SAH hydrolysis (adenosine) still complexed with the enzyme Ahcy upon purification?

Line 180, refer to Fig3A instead of 3B.  

Line 181, consider including the structure of the C195S mutant (does it accumulate to wild type level or does the mutation destabilize the protein?) 

Lines 191-192, do you have other evidence than structural ones that suggests that C195 is completely protected from solvent (i.e., H2O2) when NAD+ is bound?

Line 207, add the word cells after "photoreceptor".

Figure 2, could you include in the legend what is meant by 3% input? In “(A) Ahcy, AhcyL1, and AhcyL1 in S2 cells” should read Ahcy, AhcyL1, and AhcyL2 in S2 cells. 

Fig2A, there’s a lower band for AhcyL1-V5 in the input, is that a N-term truncation product (if V5 is in C-term)? 

Fig 2C-E, the style of displaying the statistics on these graphs are initially a bit misleading as the straight lines across multiple bars seem to indicate significance across all the bars under the line, but on further inspection it might be representing a statistical comparison between the bars at the end of each line. It might be helpful to use bracketed lines instead of straight lines that point directly to the bars been compared.

Fig2C-F, the RNAi lines used in these panels are referenced in the Supplementary Table 1, but this table is missing.

Line 250, when discussing redox sensing, and the lack of Set1 oxidation in your previous analysis, clarify whether only cysteines were analyzed. It could be that methionine for example are oxidized in their sulfoxide form and possess a regulatory role. 

Lines 263-265, maybe rephrase “when compared both with untreated Ahcy RNAi flies and with mCherry RNAi flies exposed to blue light” to “when compared with mCherry RNAi flies exposed to blue light” because the untreated Ahcy RNAi flies had lower levels than the treated ones, and the original wording suggests that the treated is lower than the untreated.

Lines 296-297, missing space between using and anti. 

Line 332, include the specific type of qPCR kit used. 

Figure 4B, could you explain in the legend what the dashed arrows represent or are showing? 

Figure 4E, could you orient the non-initiated reader as to how these data are compared, how can one tell the differences observed e.g. between Ahcy RNAi blue light vs. control blue light is not significant?

Participants of a course on Peer Review (Indiana University Bloomington) - not prompted by a journal; this review was written within a Peer Review in Life Sciences graduate course led by Alizée Malnoë with input from Sally Abulaila, Kim Kissoon, Michael Kwakye, Madaline McPherson, Madison McReynolds, Mandkhai Molomjamts, Habib Ogunyemi, Octavio Origel, and Warren Wilson.

Competing interests

The authors declare that they have no competing interests.