Nuclear Enrichment of Folate Cofactors and Methylenetetrahydrofolate Dehydrogenase 1 (MTHFD1) Protect de novo Thymidylate Biosynthesis during Folate Deficiency

Nuclear Enrichment of Folate Cofactors and Methylenetetrahydrofolate Dehydrogenase 1 (MTHFD1) Protect de novo Thymidylate Biosynthesis during Folate Deficiency

The folate cycle is critical for the proper synthesis of guanosine, adenosine, and thymidilate, as well as the production of methionine and s-adenosylmethionine (Fox J. T., Stover P. J., 2008). Both improper nutrition and genetics can lead to deficiencies that can wreak havoc with the folate cycle.  A dysfunctional folate cycle can lead to neural tube defects, elevated homocysteine levels, and other serious health problems (Stover PJ, 2004) . One such deficiency that can alter the cycle, is, of course, a lack of folate. One important intermediate of the folate cycle is a molecule called 5,10 methylene tetrahydrofolate (THF).

 

5,10 methylene THF  originally comes from a molecule called formate. Formate is a byproduct of serine and glycine catabolism, and is considered the primary source of 1-C units for cytosolic 1-C metabolism. Formate is transformed to 10-formyl THF, then 5-10 methenyl THF, then 5,10 methylene THF by different domains of an enzyme encoded by the gene MTHFD1 (Fox JT, Stover PJ, 2008). It then has the option to travel down one of two paths within in the folate cycle: it can be sent to thymidylate synthesis, or it can re-methylate homocysteine (Herbig et al, 2002).
There is another enzyme that can produce 5,10 methylene THF called serine hydroxymethyl transferase (SHMT). SHMT uses serine and THF as substrates. Previous work has indicated that that enzymes involved in thymidylate synthesis (including an enzyme called SHMT1) localize to the nucleus during S-phase of the cell cycle (Woeller et al, 2007). It seems plausible, then, that thymidylate synthesis would use SHMT1 to produce 5,10 methylene THF for all its needs. This, however, is not what occurs. SHMT1 was shown to instead perform a structural role in thymidylate synthesis in the nucleus, and the thymidylate produced seemed to come from formate (Anderson et al,2012, Herbig et al, 2002). Since SHMT1 does not use formate as a substrate, this means that thymidylate synthesis must obtain 5,10 methylene THF from MTHFD1, which is usually located in the cytoplasm. It has previously been shown that, under folate-deficient (FD) conditions, thymidylate synthesis is favored over homocysteine re-methylation-evidence for this included elevated cellular homocysteine levels under FD conditions. The authors asked- if thymidylate synthesis is favored during folate deficiency, and SHMT is already located in the nucleus, why and how is the pathway using MTHFD1 and formate?
To answer this question, the authors used MTHFD1 +/+ and MTHFD1 gt/+ mice, measuring liver nuclear MTHFD1 levels and overall MTHFD1 levels using an immunoblot. They found MTHFD1 protein in both nuclear livers extracts, and interestingly noticed that the localization of MTHFD1 to the nucleus was increased in the livers of mice who were fed a FD diet, regardless of overall MTHFD1 protein levels. This suggested that MTHFD1 itself localized to the nucleus during FD conditions, as opposed to merely shuttling 5,10 methylene THF there.
In addition, the authors created an MTHFD1-GFP fusion protein and observed it in HeLa cells. Using confocal microscopy, the authors saw that the fusion protein localized to the nucleus, and that the intensity of the MTHDF1-GDP signal was 2 times as intense during the S-Phase of the cell, as opposed to G1 or G2 (see “A” of the figure below), implying that S-phase is the period of the cell cycle in which MTHFD1 relocates to the nucleus. The authors used another cancer cell line (MCF-7 cells) to investigate the effect of folate-depletion on nuclear folate levels. They found that, under FD conditions, nuclear folate levels were lower than controls during G1 and G2, but not during S-phase. This supports the idea that, under FD conditions, there is some protected folate co-factor production that could relocate to the nucleus during S-phase. The authors also observed the effect of overall MTHFD1 levels on nuclear folate concentration, noting that nuclear folate levels remained constant even with decreased MTHFD1 concentration. This implies that, even when there is limited MTHFD1, the cell preferentially shuttles it to the nucleus under FD conditions in order to preserve thymidylate synthesis.

In the confocal microscopy images in this figure, it can be seen that, during S-phase, the authors' MTHFD1-GFP fusion protein localizes to the nucleus of the cell (the fusion protein is represented by green). Figure from Field et al., 2014.
In the confocal microscopy images in this figure, it can be seen that, during S-phase, the authors’ MTHFD1-GFP fusion protein localizes to the nucleus of the cell (the fusion protein is represented by green). Figure from Field et al., 2014.

The authors conclude that, under FD conditions, MTHFD1 localizes to the nucleus during S-phase in order to produce 5,10 methylene THF for thymidylate synthesis, instead of staying in the cytosol to assist with homocysteine re-methylation. Previously, it was thought that choice between thymidylate synthesis and homocysteine re-methylation was merely based on kinetics; now it seems that MTHFD1 may actually act as a sensor for cellular folate levels. Since errors in thymidylate synthesis can lead to an increased chance of neural tube defects through an elevation in uracil incorporation in nuclear DNA, it follows that the cell would desire thymidylate synthesis to be protected during S-phase, when DNA is being replicated. The authors also feel that their observations may explain a previous observation that MTHFD1 gt/+ mice do not experience neural tube defects under FD conditions (Beaudin et al, 2011)- the functional MTHFD1 that exists is localized to the nucleus and protects thymidylate synthesis.

The authors acknowledge that they have yet to determine the mechanism by which MTHFD1 senses cellular folate levels, nor the mechanism by which MTHFD1 localizes to the nucleus. This would be a focus for future work.

References:
Anderson D. D., Woeller C. F., Chiang E. P., Shane B., Stover P. J.(2012) Serine hydroxymethyltransferase anchors de novo thymidylate synthesis pathway to nuclear lamina for DNA synthesis. J. Biol. Chem. 287, 7051–7062

Beaudin A. E., Abarinov E. V., Noden D. M., Perry C. A., Chu S., Stabler, Stover P. J.(2011) Shmt1 and de novo thymidylate biosynthesis underlie folate-responsive neural tube defects in mice. Am. J. Clin. Nutr. 93, 789–798

Fox J. T., Stover P. J. (2008) Folate-mediated one-carbon metabolism. Vitam. Horm. 79, 1–44

Herbig K., Chiang E. P., Lee L. R., Hills J., Shane B., Stover P. J. (2002) Cytoplasmic serine hydroxymethyltransferase mediates competition between folate-dependent deoxyribonucleotide and S-adenosylmethionine biosyntheses. J. Biol. Chem. 277, 38381–38389

Stover P. J. (2004) Physiology of folate and vitamin B12 in health and disease. Nutr. Rev. 62, S3–S12
Woeller C. F., Anderson D. D., Szebenyi D. M., Stover P. J. (2007) Evidence for small ubiquitin-like modifier-dependent nuclear import of the thymidylate biosynthesis pathway. J. Biol. Chem. 282, 17623–17631

20 Replies to “Nuclear Enrichment of Folate Cofactors and Methylenetetrahydrofolate Dehydrogenase 1 (MTHFD1) Protect de novo Thymidylate Biosynthesis during Folate Deficiency”

  1. Nice job, Gabbie! I really enjoyed this paper and the fact that you explained how the folate cycle is linked to other metabolic pathways. This really cemented our classroom discussions on this topic. I was particularly interested in the MTHFD1 gene, which you mentioned has the capability of producing different enzymes that are involved in the folate cycle. I remember discussing the “one gene one polypeptide” hypothesis where one gene is responsible for encoding one enzyme. If MTHFD1 encodes for multiple enzymes, then that would violate this hypothesis. Do you think that a mutation in the MTHFD1 gene could cause different levels of folate cycle intermediates to manifest in the cell? I believe this would have large implications for the folate cycle as a whole, because different intermediates are involved in transferring different forms of a one-carbon group. Just a few thoughts. Thanks! 🙂

    1. Hi Zach,
      Thanks for your thoughts! I believe that I made an error in my original post when I said that MTHFD1 encodes three different enzymes- rather, it encodes one protein that carries three distinct enzymatic activities. Also, it’s interesting that you ask about mutations in MTHFD1. I did some digging, and, according to the paper “Characterization and review of MTHFD1 deficiency: four new patients, cellular delineation and response to folic and folinic acid treatment” published in the Journal of Inherited Metabolic Disease, there have only been five documented cases of mutations causing MTHFD1 deficiency. In two out of five of these cases, patients presented with abnormally high homocysteine levels. Homocysteine seems to be the substrate of the folate cycle that a loss-of-function mutation in MTHFD1 affects the most, as MTHFD1 catalyzes the formation of 5,10 methyleneTHF, which re-methylates homocysteine. It should also be noted that, according to this paper, symptoms associated with these mutations were severe- two out of five patients died at just nine weeks old. This could perhaps explain the lack of known cases- mutations in this gene seem to be fairly lethal.

      Paper reference:
      Burda, P., Kuster, A., Hjalmarson, O., Suormala, T., Bürer, C., Lutz, S., Roussey, G., Christa, L., Asin-Cayuela, J., Kollberg, G., et al. (2015). Characterization and review of MTHFD1 deficiency: four new patients, cellular delineation and response to folic and folinic acid treatment. J Inherit Metab Dis 1–10.

  2. Hi Gabby,

    Good job on the review! I was reading through the discussion, and I noticed that the authors stated “nuclear folate concentrations do not vary by cell cycle.” I found this surprising because we know that folate is crucial for the synthesis of new nucleotides, which would need to occur rapidly during S-phase as the chromosomes divide and replicate. It thus seems that constantly keeping folate levels so high would be a waste of resources and space, as the body needs to constantly resupply folate levels, and store this folate until it is needed. Do you have any ideas as to why the folate levels do not vary over the course of the cell cycle? The authors further go on to say that the concentration of enzymes involved in de novo thymidylate synthase do vary during the cell cycle, where levels are highest during S-phase, and almost entirely absent in G1. This again makes sense because enzymes that actually synthesize the nucleotides would be needed mostly during S-phase. Thus, not only are the folate levels kept constant high throughout the cell cycle, they are kept constant even when the enzymes that actually use the folate are absent. Can you explain this disconnect, and perhaps also hypothesize about the importance of keeping folate levels constant, even when the need for folate varies so greatly over the cell cycle?

    1. Hi Michael! I appreciate your input. I think that it’s important to note that, in the discussion, when the authors say that “nuclear folate concentrations do not vary by cell cycle,” they are referring to nuclear folate concentrations under folate-deficient conditions. Neither I nor the authors have a definitive explanation, but I can hypothesize. Perhaps folate-deficient conditions signal to the cell that nucleotide and thymidylate synthesis could be at-risk, and it uses an unknown mechanism to accumulate folate in the nucleus to assure that, when the cell goes into S-phase, it is prepared. This way, once S-phase occurs, and MTHFD1 localizes to the nucleus to protect thymidylate synthesis, resources are immediately available. To me, it seems almost precautionary, if a bit wasteful. Perhaps it conveys the high level of priority that the cell places on thymidylate syntehsis. As I previously said, the actual reason for nuclear folate levels staying constant under FD conditions is unknown, but this is my best hypothesis.

  3. Hi Gabby! I really enjoyed the article you selected. It was so interesting to see this compartmentalization strategy in action and see yet another example of metabolic pathway regulation! I noticed what seems to be a discrepancy and I am hoping you will be able to help clarify it. In the introduction to the paper the authors make a big deal about how MTHFD1 is sensing intracellular folate levels in the cytosol. However, in the discussion the authors state that “Reduced MTHFD1 expression does not affect nuclear folate levels”. Your review highlighted how MTHFD1 localizes to the nucleus in folate deficient situations. So, I am confused about why why reduced MTHFD1 doesn’t effect nuclear folate levels if MTHFD1 codes for the enzymes that transform formate to THF? If you had fewer of those enzymes, wouldn’t you have less THF? I understand that compartmentalization plays a role here and that the authors attribute the increase in MTHFD1 to nuclear accumulation rather than an increase in transcription and translation, but if there was a decrease in the MTHFD1 gene, wouldn’t that still have the downstream affect of decreased nuclear folate levels? Thanks!

    1. Hi Kelly! I agree that the idea that reduced MTHFD1 levels do not affect nuclear folate levels does seem strange. I think what the authors are trying to convey with that result is that, even when there is reduced overall MTHFD1 in the cell, the MTHFD1 that is there will make localizing to the nucleus its top priority, thus preserving thymidylate synthesis and nuclear THF levels. I agree with you, though- I feel like, logically, if MTHFD1 levels got low enough, then nuclear folate levels would be affected. Perhaps MTHFD1 expression never reached this critical low point in the authors’ experiments.

  4. Small world. Patrick Stover (the PI on this publication) was on my thesis committee 🙂

  5. Hey Gabs. Nice work on the review, you definitely hit all the key points. I have confusion as well as commentary for you. First, at the end of the third to last paragraph of the discussion, Martha says reduced MTHFD1 expression does not affect nuclear folate levels. I was under the impression that MTHFD1 was shunting substrates toward thymidylate production. Are the authors strictly speaking about an absence of extracellular MTHFD1? I understand that MTHFD1 produced intracellularly can maintain folate production, but the way they posed that statement did not indicate extra/intracellular. Please send me some help!

    Also, I got the feeling that the authors were sort of claiming their future work, almost to say that the idea was theirs if someone else were to publish on the topic. They mentioned multiple times through the paper that despite their mechanistic findings, they still have yet to determine the ‘trigger.’ In the last paragraph of the discussion they slip in that they saw decreased levels of uracil in the DNA which should not have been affected by the salvage pathways. I am imagining a follow up paper that looks to identify the signaling protein or pathway that induces nuclear de novo thymidylate production amongst other things… What do you think?

    1. Hi Ryan! I think I may be able to help you out. MTHFD1 is technically not just shunting substrates towards thymidylate synthesis; it is actually localizing to the nucleus during FD conditions, and producing the substrates there so that they can be easily used for thymidylate synthesis. I think that the statement the authors made that MTHFD1 levels do not affect nuclear folate levels may have been confusing to a few people. Personally, I think that what the authors were trying to convey with that result was that, even when MTHFD1 expression is reduced overall in the cell, the available MTHFD1 will be used to regulate folate levels in the nucleus.

      I agree, I think that it did feel like this paper was almost a “lead-in,” but I think that it is because (and this is pure speculation) that the paper they wanted to publish was one that identified how MTHFD1 is sensing cellular folate levels, and then detailed a mechanism as to how it localized to the nucleus. I feel like they published what they did when they did in order to lay claim to this research area, even if they had not found all the results that they wanted yet.

  6. Hi Gabby! Thanks for the informative and interesting review. I was reading the sentence “This suggested that MTHFD1 itself localized to the nucleus during FD conditions, as opposed to shuttling 5,10 methylene THF there,” and I was wondering what the significance of the MTHFD1 gene localizing to the nucleus was as compared to the theory that SHMT would catalyze this reaction. From your closing paragraph, I understand why the production of 5,10 methylene THF needs to be highly controlled during the replication of DNA, but does bringing MTHFD1 to the nucleus help control the production of 5,10 methylene THF? Would a cell replicating its DNA still be working to express MTHFD1 and producing 5,10 methylene THF all simultaneously? What are your thoughts on this process?

    1. Hi Matt! I believe that part of the mystery that this paper was addressing was why thymidylate seems to come from formate, which is the substrate of MTHFD1, not SHMT. It was known that SHMT was found in the nucleus (along with other thymidylate synthesis machinery) during S-phase, so it seemed strange that MTHFD1 was the enzyme producing the 5,10 methylene THF for thymidylate synthesis. I think the significance that the authors were trying to convey about MTHFD1 being used for thymidylate synthesis (as opposed to SHMT) is that it is some kind of sensor of cellular folate levels, whereas SHMT is not, and perhaps this is why it is used to protect the process of thymidylate synthesis- it can hop into the nucleus when it is needed.

      As for your second and third questions, I don’t believe that bringing MTHFD1 necessarily helps to control the production of 5,10 methylene THF- rather, it makes it readily available for thymidylate synthesis (which takes place in the nucleus) as opposed to homocysteine re-methylation (which takes place in the cytosol). So yes, a cell replicating its DNA would be expressing MTHFD1 (which would be producing 5,10 methylene THF), but MTHFD1 would be at work in the nucleus, putting its product towards thymidylate synthesis instead of homocysteine re-methylation.

  7. Hi Gabby,

    Great job on the article! It’s always nice to see something that ties in with what we recently discussed in class. My question centers around the implications of this research, specifically as to the alternative (non-kinetic) regulation between thymidylate synthesis and homocysteine re-methylation.

    It is known that thymidylate synthetase, an enzyme which requires 5,10 methylene tetrahydrofolate, plays a role in anti-carcinogenic efforts by way of mediating translation of such entities as p53, when functioning properly. Likewise, pathway inconsistencies and failures can lead to increased tumor growth within the body. As enhanced control of thymidylate synthetase via the pathway described within your article could result in a corresponding increase in anti-tumor agents, or at the very least a decrease in tumorogenic effects, do you think there exists potential for chemotherapy treatments based around agonists of this pathway? Could folate-deficient conditions play a beneficial role in future chemotherapy treatments?

    1. Hi Anthony! That’s an interesting perspective- I hadn’t thought of exploiting the nuclear localization of MTHFD1 to the nucleus as a cancer treatment. While the idea intrigues me in theory, I don’t believe it work, because (from my understanding of the paper), this regulatory mechanism does not make thymidylate synthesis any more effective, or increase thymidylate synthesis- rather, it just preserves it in the face of FD conditions. Also, even if it did indirectly increase the activity of thymidylate synthetase, I feel that the negative effects of folate deficiency would not make FD conditions an advisable treatment.

  8. Gabby – Wonderful job! I had many CUEmoments whilst reading the article and your review. My question has to do with the big picture. I noticed that at the end of the discussion the authors kept at their ‘big picture’ tie in to neural tube defects. I think that as humans, we are primed to see folate pathways and think of the neural tube as it relates to developing human babies. That being said, I was a little surprised that they used HeLa cells in their experiments and cited literature containing other cancer lines (MCF-7, for example). Do you think they were going for a cancer treatment idea like Anthony suggests above, or do you think they opted for cancerous cells because they simply rapidly go through the cell cycle? Regardless, does the use of HeLa cells weaken their overall conclusion (especially considering the wonkiness that cancer cells undergo in the name of accumulating biomass)?. If not, could you suggest another reason the authors published in JBC (perhaps the conclusions were neat but not significant, or perhaps there was something holding them back from greatness?)

    1. Hi Zach- great questions! Personally, I don’t think that the authors were going for a connection to cancer with their use of HeLa and MCF-7 cells- I don’t get any intimation of that from the text of the article. I think that you’re right, and that they used these cells due to the fact that they are rapidly proceeding through the cell cycle, and yes, I do think that it may weaken their claims a bit. That being said, they also used a murine model, which also supported their findings. I feel that this softens the potential unreliability of the results obtained from the cancer cell lines.

      I think that the reason that the authors published in JBC is similar to what I said to Ryan, a few comments above yours. The authors were able to show that MTHFD1 localizes to the nucleus and preserves thymidylate synthesis under FD conditions, and intimated that MTHFD1 must work as a cellular folate level sensor, but were unable to show how it senses cellular folate levels, nor the mechanism of how it localizes to the nucleus. I feel like this is what they really wanted/want to figure out, and, if they could’ve specified these two things, the paper could have gone to a higher impact journal.

  9. Hey Gabby, nice choice of article and nicer analysis and summary. I have two questions pertaining to this article and the concepts you’ve discussed. We now know that the folate cycle itself depends on the SAM, homocysteine cycle (I made up that name). I don’t think I found this anywhere in the paper, but where is the experiment where they elevate homocysteine levels and show that, despite being in FD and having all of this homocysteine to methylate, MTHFD1 still localizes in the nucleus to form thymidine? Is it in a previous study, or is it irrelevant for whatever reason?

    Next – and this is very big picture and beyond the scope of my knowledge – Is there any reason you can come up with for the fact that there isn’t a “cell cycle checkpoint” for FD? We learned in Bio 1 and 3 that there are certain checkpoints where the cell somehow decides whether or not mitosis should continue. Since folate-deficient newborns are still born (despite neural tube problems), we know mitosis still goes on (and this study still shows that), but why, evolutionarily, did this checkpoint not develop? I feel like in a simplistic sense, the cell should say “there’s no way I can make one of the pyrimidines, so I should probably stop mitosis”. Any thoughts would be appreciated!

    1. Hi Besher! No, I do not believe that there was an experiment in which the authors elevated homocysteine and showed that MTHFD1 still localizes to the nucleus. I didn’t even think of that experiment! It’s an interesting concept- it’d be almost trying to force the MTHFD1 into being so inundated with homocysteine that it would be forced to choose the homocysteine re-methylation pathway as opposed to the thymidylate synthesis pathway. I don’t think it’s irrelevant, and I think that it may have strengthened the author’s point, had it been done. I think the reason that they thought it was unnecessary is because they showed that, under FD conditions, homocysteine levels were elevated- implying that that the MTHFD1 was choosing thymidylate synthesis despite having homocysteine to re-methylate.

      That’s a really intriguing point that you brought up about folate and the cell cycle- it would make sense to have a folate checkpoint, wouldn’t it? I tried to dig up some literature on this, but couldn’t find anything explaining why one doesn’t exist. Perhaps it doesn’t exist because, when proper nucleotide synthesis can’t take place, the cell will start substituting uracil into the genetic code. While this leads to genomic instability and is associated with disease, perhaps it is not alarming enough to the cell to cause it to go into cell cycle arrest. This is just me brainstorming, though- this is something I’ll probably ponder for a while.

  10. Hey Gabby,

    I have a couple questions regarding the paper. It seems to make sense that in limited supply of folate that folate and its precursors would be directed to the nucleus, given the importance of DNA replication and passing on the genetic material. I was wondering, though, if you think that either the increase of nuclear folate activity or the decrease in homocysteine levels could account for some of the side effects seen under folate deficiency? Is there anything mechanistically that would lend one to believe that either of these consequences could lead to a greater predisposition to cancer or any of the effects presented in the introduction?

    Also, this paper seemed like a precursor paper to me, in that there are still a lot of unanswered questions that the authors address. If you were one of the authors, which of their unanswered questions in their discussion would you most aggressively pursue to gain the biggest impact for your paper?

    1. Hi Tommy! Thanks for your questions and comments. I may be wrong, but I think that homocysteine levels were actually shown to increase under FD conditions- I should have highlighted that in my review (I’ll add that to my list of revisions). While the localization of MTHFD1 to the nucleus and the subsequent preservation of thymidylate synthesis would actually seem to prevent the development of tumors, we do know from class that elevated homocysteine can lead to various adverse effects, including cardiac pathologies, increased risk of miscarriage, and neural tube defects. So, while the cell could actually be protecting against tumorgenesis, it’s doing so at the cost of high homocysteine levels.

      Your second thought seems to be a running theme among the comments. I agree with you. I think the biggest unanswered question is how MTHFD1 is acting as a cellular folate sensor. If the authors could demonstrate the specifics of this process, it would cement their claim that MTHFD1 acts in a regulatory capacity, and I think this would have great impact.

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