by: Martha S. Field, Elena Kamynina, Olufunmilayon C. Agunloye, Rebecca P. Liebenthal, Simon G. Lamarre, Margaret E. Brosnan, John T. Brosnan, and Patrick J. Stover
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.
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.
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
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