Dithiothreitol causes toxicity in C. elegans by modulating the methionine–homocysteine cycle

Essential revisions:

To support the conclusions made in the paper, the reviewers agreed on a number of essential revisions:

1. SAM levels should be quantified to support the idea that they are in fact depleted after DTT exposure.

We thank the reviewers for suggesting this critical experiment. We have quantified the SAM levels in the N2 and rips-1(jsn11) animals with and without exposure to 10 mM DTT. We observed that DTT indeed reduces SAM levels in N2, but not rips-1(jsn11) animals. The revised manuscript reads (lines 274-283): “The genetic and methionine supplementation experiments suggested that SAM depletion could be the primary cause of DTT toxicity. To establish that DTT indeed resulted in the depletion of SAM, we quantified SAM levels in animals with and without exposure to DTT. The exposure of the wild-type N2 animals to DTT resulted in a significant reduction in its SAM levels (Figure 5F). To study whether SAM depletion by DTT depended on the gene rips-1, we quantified SAM levels in rips-1(jsn11) animals. Surprisingly, rips-1(jsn11) animals had significantly increased SAM levels upon exposure to DTT (Figure 5G). It is unclear why DTT exposure resulted in the increased SAM levels in rips-1(jsn11) animals. Nevertheless, these results establish that DTT exposure depletes SAM levels in N2, but not rips-1(jsn11) animals.”

2. A genetic characterization of drm-1. It would be important to test if a genetic KO phenocopies the point mutants and if overexpression sensitize to DTT.

We would like to highlight that four of the rips-1 alleles isolated in our genetic screen are likely loss-of-function mutations (3 premature stop codons and one splice acceptor mutations). We have emphasized this information in the manuscript (lines 181-183): “Three of the isolated alleles of rips-1, jsn4, jsn11, and jsn12 had premature stop codon mutations and one allele (jsn8) had a splice acceptor mutation (Figure 3A). All of these mutations are expected to result in a loss-of-function of the gene.” The loss-of-function nature of rips-1 mutations is further confirmed by a complete rescue of the resistance of rips-1(jsn11) animals to DTT toxicity upon transgenic expression of wild-type copies of rips-1 (Figure 3F).

In addition, as suggested by the reviewers, we have now characterized the effects of rips-1 knockdown by RNAi on the toxic effects of DTT. Knockdown of rips-1 in the N2 animals resulted in a drastic improvement in their development in the presence of DTT (Figure 3—figure supplement 3A-B). Moreover, we have also characterized the impact of overexpression of rips-1 on the sensitivity to DTT toxicity. Animals overexpressing rips-1 exhibited enhanced sensitivity to DTT toxicity (Figure 3—figure supplement 3C-D). Taken together, these studies establish that the loss-of-function of rips-1 imparts DTT resistance while its overexpression sensitizes the animals to DTT toxicity.

3. Published work needs to be cited more accurately and comprehensively. Particularly the experiments with B12/DTT were published before and should be cited in the figure legend.

We have now cited the earlier work on acdh-1p::GFP and bacterial diets both in the figure legend and the text that describes the results on acdh-1p::GFP.

4. Alternative plausible hypotheses explaining the DTT effects need to be considered.

We have added a new paragraph in the discussion to provide a hypothesis for explaining the effects of DTT. The revised manuscript reads (lines 377-392): “One question that arises from the current study concerns the substrate for the SAM-methyltransferase RIPS-1. Because DTT results in the induction of rips-1 expression, one possibility is that DTT is the substrate for RIPS-1, and rips-1 expression is induced in response to DTT as a defense mechanism. This hypothesis is supported by studies that showed that multiple thiol reagents, including DTT and β-ME, could be methylated by SAM and SAM-dependent methyltransferases (Bremer and Greenberg, 1961; Coiner et al., 2006; Maldonato et al., 2021). The methylation of external thiols could be a defense response for some organisms. For example, the dithiol metabolite gliotoxin produced by Aspergillus fumigatus inhibits the growth of other fungi, including A. niger, and triggers a defense response in A. niger resulting in upregulation of SAM-dependent methyltransferases (Dolan et al., 2014; Manzanares-miralles et al., 2016; Owens et al., 2015). Methylation of gliotoxin by SAMdependent methyltransferases confers A. niger protection against the dithiol metabolite. Incidentally, the same methyltransferases that are required for the defense mechanism against gliotoxin are induced by DTT (MacKenzie et al., 2005; Manzanares-miralles et al., 2016). Therefore, upregulation of methyltransferases might be a general defense response against multiple thiol reagents.”

Reviewer #1 (Recommendations for the authors):

To better support the conclusions, I would like to suggest the following key experiments.

1. To put the effect of DTT on SAM in context and to make comparisons in other systems possible, it would be important to measure the absolute SAM concentration in worms with and without DTT treatment, and after methionine supplementation.

We thank the reviewer for suggesting this important experiment. We have quantified the SAM levels in the N2 and rips-1(jsn11) animals with and without exposure to 10 mM DTT. We observed that DTT indeed lowers SAM levels in N2, but not rips-1(jsn11) animals. The revised manuscript reads (lines 274-283): “The genetic and methionine supplementation experiments suggested that SAM depletion could be the primary cause of DTT toxicity. To establish that DTT indeed resulted in the depletion of SAM, we quantified SAM levels in animals with and without exposure to DTT. The exposure of the wild-type N2 animals to DTT resulted in a significant reduction in its SAM levels (Figure 5F). To study whether SAM depletion by DTT depended on the gene rips-1, we quantified SAM levels in rips-1(jsn11) animals. Surprisingly, rips-1(jsn11) animals had significantly increased SAM levels upon exposure to DTT (Figure 5G). It is unclear why DTT exposure resulted in the increased SAM levels in rips-1(jsn11) animals. Nevertheless, these results establish that DTT exposure depletes SAM levels in N2, but not rips-1(jsn11) animals.”

2. To address a broad biological significance of the finding that DTT treatment first leads to toxicity through SAM depletion and only at higher concentrations to ER stress it would be important to perform the key experiments in cultured mammalian cells.

We thank the reviewer for this important suggestion. We believe that studying the impact of DTT on SAM and the methionine-homocysteine cycle in mammalian cells merits an in-depth investigation and is beyond the scope of the current manuscript.

3. What is the effect of 5mM DTT on hsp-4::GFP expression/UPR signaling?

We have added data on the effect of 5 mM DTT on hsp-4p::GFP expression. The revised manuscript reads (lines 314-318): “Next, we explored the role of ER stress in DTTmediated development retardation. First, we tested whether a lower concentration of DTT, which results in development retardation, would lead to ER stress. Exposure to 5 mM DTT resulted in the upregulation of hsp-4p::GFP to an intermediate level between the control and 10 mM DTT exposure levels (Figure 6—figure supplement 1A-B), indicating that lower concentrations of DTT also cause ER stress.”

Reviewer #2 (Recommendations for the authors):

1. drm-1 should be fully characterized. It is a point mutation, therefore could be a loss, partial or gain of function allele. In addition, identification of the methylation pathway the drm-1 gene works in critical to bring together aspects of this story.

We would like to highlight that four of the rips-1 alleles isolated in our genetic screen are likely loss-of-function mutations (3 premature stop codons and one splice acceptor mutations). We have emphasized this information in the manuscript (lines 181-183): “Three of the isolated alleles of rips-1, jsn4, jsn11, and jsn12 had premature stop codon mutations and one allele (jsn8) had a splice acceptor mutation (Figure 3A). All of these mutations are expected to result in a loss-of-function of the gene.” The loss-of-function nature of rips-1 mutations is further confirmed by a complete rescue of the resistance of rips-1(jsn11) animals to DTT toxicity upon transgenic expression of wild-type copies of rips-1 (Figure 3F).

In addition, we have now characterized the effects of rips-1 knockdown by RNAi on the toxic effects of DTT. Knockdown of rips-1 in the N2 animals resulted in a drastic improvement in their development in the presence of DTT (Figure 3—figure supplement 3AB). Moreover, we have also characterized the impact of overexpression of rips-1 on sensitivity to DTT toxicity. Animals overexpressing rips-1 exhibited enhanced sensitivity to DTT toxicity (Figure 3—figure supplement 3C-D). Taken together, these studies establish that the loss-of-function of rips-1 imparts DTT resistance while its overexpression sensitizes the animals to DTT toxicity.

We have also added a discussion on the potential substrate of rips-1. The revised manuscript reads (lines 377-392): “One question that arises from the current study concerns the substrate for the SAM-methyltransferase RIPS-1. Because DTT results in the induction of rips-1 expression, one possibility is that DTT is the substrate for RIPS-1, and rips-1 expression is induced in response to DTT as a defense mechanism. This hypothesis is supported by studies that showed that multiple thiol reagents, including DTT and β-ME, could be methylated by SAM and SAM-dependent methyltransferases (Bremer and Greenberg, 1961; Coiner et al., 2006; Maldonato et al., 2021). The methylation of external thiols could be a defense response for some organisms. For example, the dithiol metabolite gliotoxin produced by Aspergillus fumigatus inhibits the growth of other fungi, including A. niger, and triggers a defense response in A. niger resulting in upregulation of SAMdependent methyltransferases (Dolan et al., 2014; Manzanares-miralles et al., 2016; Owens et al., 2015). Methylation of gliotoxin by SAM-dependent methyltransferases confers A. niger protection against the dithiol metabolite. Incidentally, the same methyltransferases that are required for the defense mechanism against gliotoxin are induced by DTT (MacKenzie et al., 2005; Manzanares-miralles et al., 2016). Therefore, upregulation of methyltransferases might be a general defense response against multiple thiol reagents.”

2. The data from this paper is largely self reported (C. elegans larval stage) or low magnification images. More quantitive assays should be used (for example, expression of adult collagens such as col-19), or higher magnification images of vulval development shown.

We thank the reviewer for this suggestion. However, most of the comparative development data reported in the manuscript have a great contrast in the development stages (for example, L1/L2 stages vs. L4/adult stages) which is readily observable without the need for development stage-specific reporters. We believe that the current resolution of data is adequate for the observed phenotypes.

3. GFP images should be quantitated.

We have quantified the GFP images. The quantification data is shown in Figure 3I, Figure 3—figure supplement 4B, Figure 4D, Figure 6B, and Figure 6—figure supplement 1B.

4. Data from the Walhout lab should be directly cited in the figure legend and in the same paragraph that discuss the results.

We have now cited the earlier work on acdh-1p::GFP and bacterial diets both in the figure legend and the text that describes the results on acdh-1p::GFP.

5. Direct mechanistic links from the action of DTT to the Met/SAM cycle are needed for the conclusions of the paper to be supportable. There are multiple types of experiments that might be acceptable here.

To provide a direct link between DTT and Met/SAM cycle, we have quantified the SAM levels in the N2 and rips-1(jsn11) animals with and without exposure to 10 mM DTT. We observed that DTT indeed reduces SAM levels in N2, but not rips-1(jsn11) animals. The revised manuscript reads (lines 274-283): “The genetic and methionine supplementation experiments suggested that SAM depletion could be the primary cause of DTT toxicity. To establish that DTT indeed resulted in the depletion of SAM, we quantified SAM levels in animals with and without exposure to DTT. The exposure of the wild-type N2 animals to DTT resulted in a significant reduction in its SAM levels (Figure 5F). To study whether SAM depletion by DTT depended on the gene rips-1, we quantified SAM levels in rips-1(jsn11) animals. Surprisingly, rips-1(jsn11) animals had significantly increased SAM levels upon exposure to DTT (Figure 5G). It is unclear why DTT exposure resulted in the increased SAM levels in rips-1(jsn11) animals. Nevertheless, these results establish that DTT exposure depletes SAM levels in N2, but not rips-1(jsn11) animals.”

Reviewer #3 (Recommendations for the authors):

1. The authors conclude on L305 that "By upregulating the expression of SAM-dependent methyltransferase gene drm-1, DTT leads to the depletion of SAM that results in toxicity". No experiment has shown that drm-1 activation causes lower SAM – this is merely implied from the supplementation data. The authors should provide support of that model and measure SAM levels with and without DTT and with and without B12, as well as in the drm-1 mutant. Such molecular evidence of actual SAM depletion is critical to the main conclusion of the paper – that SAM levels and their depletion are a major driver of DTT toxicity.

We thank the reviewer for suggesting this important experiment. We have quantified the SAM levels in the N2 and rips-1(jsn11) animals with and without exposure to 10 mM DTT. We observed that DTT indeed reduces SAM levels in N2, but not rips-1(jsn11) animals. The revised manuscript reads (lines 274-283): “The genetic and methionine supplementation experiments suggested that SAM depletion could be the primary cause of DTT toxicity. To establish that DTT indeed resulted in the depletion of SAM, we quantified SAM levels in animals with and without exposure to DTT. The exposure of the wild-type N2 animals to DTT resulted in a significant reduction in its SAM levels (Figure 5F). To study whether SAM depletion by DTT depended on the gene rips-1, we quantified SAM levels in rips-1(jsn11) animals. Surprisingly, rips^-1(jsn11) animals had significantly increased SAM levels upon exposure to DTT (Figure 5G). It is unclear why DTT exposure resulted in the increased SAM levels in rips-1(jsn11) animals. Nevertheless, these results establish that DTT exposure depletes SAM levels in N2, but not rips-1(jsn11) animals.”

2. If the authors' model is correct that DTT-driven drm-1 induction is the source of developmental delay due to SAM depletion, they should cross their overexpression transgene into the WT background; the prediction would be that this should cause developmental delay on its own. Alternatively, they could use a stronger intestinal promoter to overexpress drm-1. Again, is in the above point, the authors should also quantify SAM in these worms.

We thank the reviewer for another very important suggestion. We tested the sensitivity of rips-1 overexpressing animals to DTT and indeed found them to be more sensitive to DTT. The revised manuscript reads (lines 216-218): “To establish that the toxic effects of DTT are indeed due to the upregulation of rips-1, we tested the effects of overexpression of rips-1 on DTT sensitivity. Animals overexpressing rips-1 exhibited enhanced sensitivity to DTT toxicity (Figure 3—figure supplement 3C-D).” We could not quantify SAM levels in the rips-1 overexpressing animals as the array is extrachromosomal (resulting in both transgenic and non-transgenic progeny), and several thousands of worms are required to quantify SAM levels.

3. One idea that emerges from the data provided here is that drm-1 may be a SAM-dependent enzyme that produces PC; this might be important in the condition of DTT-induced ER stress as it might allow synthesizing more ER membrane, allowing for accumulation of misfolded proteins. It would be great if the authors could test whether the drm-1 mutant has lower levels of PC.

We thank the reviewer for this interesting conjecture. However, we observed that choline supplementation, which enhances PC synthesis, rescues DTT toxicity. Because SAM levels are critical for maintaining PC levels (in the absence of choline supplementation), it is likely that DTT leads to reduced levels of PC (supported by the rescue of DTT toxicity by choline supplementation). Therefore, if the rips-1 mutants had lower levels of PC, they would have been more sensitive to DTT. However, because rips-1 mutants are resistant to DTT, it is unlikely that rips-1 is involved in PC synthesis.

4. The role of thiol agents should be expanded beyond DTT. The authors could readily test whether the mechanism described here also protects against toxic concentrations of other agents, e.g. test B12/methionine/choline and/or drm-1 rescue of toxic concentration of NAC.

We thank the reviewer for this important suggestion. We studied whether the thiol reagents, β-mercaptoethanol (β-ME) and NAC, caused toxicity via rips-1. Interestingly, we observed that β-ME, but not NAC, shares the toxicity pathway with DTT. These results are described in a new Results subsection and a new Figure (Figure 4). The revised manuscript reads (lines 263-272): “β-Mercaptoethanol, but not NAC, shares toxicity pathway with DTT We tested whether the other thiol reagents, β-mercaptoethanol (β-ME) and NAC, caused toxicity via rips-1. Interestingly, rips-1(jsn11) animals exhibited resistance to β-ME toxicity, but not NAC toxicity (Figure 4A-B). We studied the effect of these thiol reagents on the expression levels of rips-1p::GFP. While NAC exposure did not affect the expression of rips1p::GFP, β-ME resulted in its dramatic upregulation (Figure 4C-D). Finally, we studied the effects of vitamin B12 supplementation of the toxicities of β-ME and NAC. Vitamin B12 supplementation alleviated β-ME, but not NAC, toxicity (Figure 4E-F). These results suggested that β-ME shares the toxicity mechanism with DTT. On the other hand, NAC causes toxicity by a mechanism different from DTT.”

5.The authors showed that drm-1 is induced by DTT, and so were two of its orthologs, R08E5.1, R08F11.4 (but not K12D9.1). To examine whether protection against DTT is is specific to drm-1, the authors should use mutants in the other three genes to test whether they, too, block toxic effects of DTT (one might predict the former two should, the latter maybe not, because it is not induced by DTT). RNAi might work as well, there is now an option to do RNAi in OP50. The screen did not identify alleles in these genes, and while this doesn't rule out their importance, it is somewhat compelling and suggests that drm-1 is key here, so these experiments are not essential.

We studied the role of rips-1 and rips-1-related methyltransferases in DTT toxicity by their RNAi-mediated knockdown. The revised manuscript reads (202-215): “Because DTT resulted in the upregulation of methyltransferases closely related to rips-1, we used RNAi knockdown to study the role of these methyltransferases in the toxic effects of DTT. As expected, knockdown of rips-1 in the N2 animals resulted in a drastic improvement in their development in the presence of DTT (Figure 3—figure supplement 3A-B). Interestingly, knockdown of R08E5.1 resulted in a phenotype similar to rips-1 knockdown. On the other hand, knockdown of R08F11.4 and K12D9.1 improved the development in the presence of DTT only marginally (Figure 3—figure supplement 3A-B). These results suggest that the rips-1-related methyltransferases might also be required for DTT toxicity. It is also possible that the knockdown of these methyltransferases results in the knockdown of rips-1 by cross RNAi. The level of sequence similarity between rips-1 and R08E5.1 (78 % identical) indicates that these genes would undergo complete cross RNAi (Rual et al., 2007). Because we did not recover mutants of any of the rips-1-related methyltransferases in our genetic screen in contrast to the twelve alleles of rips-1, the rips-1-related methyltransferases likely have only minor roles in DTT toxicity.”

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

1. SAM data should be displayed in absolute values and in the same graph so that it's possible to understand the effect of rips-1 mutation on SAM.

We thank the reviewer for these suggestions. We have displayed the SAM quantification data for both N2 and rips-1(jsn11) animals in the same graph (Figure 5F).

SAM is a very unstable molecule. The SAM standard that we purchased from Σ (#A7007) has this cautionary note: “This material is 80-90% pure when prepared, but is very unstable. As much as 10% purity loss per day at 25 °C has been noted.” Therefore, its absolute quantification may not be reliable. It is a standard practice to present SAM levels in relative values. Because for each experiment, all four of the samples (N2 and rips-1(jsn11) with and without 10 mM DTT) were prepared and processed simultaneously, the relative SAM quantification in these samples is reliable and sufficient to understand the effects of DTT on relative SAM levels.

2. For the choline rescue experiment, it was noted that the concentrations used were unusually high. It would be important to show rescue of a sams-1 mutant with choline.

We have shown the rescue of sams-1(ok2946) animals with choline in Figure 5GH. While the rescue with choline is complete in N2 and metr-1(ok521) animals, it is only partial in sams-1(ok2946) animals. This suggested that while phosphatidylcholine is one important downstream product of SAM involved in attenuating DTT toxicity, other SAMrelated functions may also be involved in rescuing DTT toxicity. We have added these points in the manuscript (lines 295-301): “Supplementation of choline rescued the developmental defects in N2 and metr-1(ok521) animals on an E. coli OP50 diet containing 10 mM DTT (Figure 5G-H). The rescue of DTT toxicity was only partial in sams-1(ok2946) animals (Figure 5G-H). These results suggested that phosphatidylcholine is a major SAM product responsible for combating DTT toxicity. However, the partial rescue of DTT toxicity by choline supplementation in sams-1(ok2946) animals suggested that other SAM-related functions may also be involved in attenuating DTT toxicity.”

3. From the data it appears that knockdown of all 4 methyltransferases results in some rescue, so perhaps they are all involved? This requires no experimentation, just a change in the wording of the interpretation.

We thank the reviewer for this suggestion. Indeed, the knockdown of all 4 methyltransferases results in some rescue of DTT toxicity. This could be because of the following two reasons: Either all the 4 methyltransferases are involved in mediating DTT toxicity, or because of high sequence similarity among these methyltransferases, cross-RNAi takes place. We have now discussed both of these possibilities. We have added a new sentence in the manuscript (lines 215-216): “However, our studies do not rule out the involvement of the rips-1-related methyltransferases in DTT toxicity.” The whole paragraph on methyltransferases RNAi reads: “Because DTT resulted in the upregulation of methyltransferases closely related to rips-1, we used RNAi knockdown to study the role of these methyltransferases in the toxic effects of DTT. As expected, knockdown of rips-1 in the N2 animals resulted in a drastic improvement in their development in the presence of DTT (Figure 3—figure supplement 3A-B). Interestingly, knockdown of R08E5.1 resulted in a phenotype similar to rips-1 knockdown. On the other hand, knockdown of R08F11.4 and K12D9.1 improved the development in the presence of DTT only marginally (Figure 3— figure supplement 3A-B). These results suggest that the rips-1-related methyltransferases might also be required for DTT toxicity. It is also possible that the knockdown of these methyltransferases results in the knockdown of rips-1 by cross RNAi. The level of sequence similarity between rips-1 and R08E5.1 (78 % identical) indicates that these genes would undergo complete cross RNAi (Rual et al., 2007). Because we did not recover mutants of any of the rips-1-related methyltransferases in our genetic screen in contrast to the twelve alleles of rips-1, the rips-1-related methyltransferases likely have only minor roles in DTT toxicity. However, our studies do not rule out the involvement of the rips-1-related methyltransferases in DTT toxicity.”