Folate-mediated 1-carbon metabolism is a network of interconnected metabolic pathways necessary for the synthesis of purine nucleotides, thymidylate and the remethylation of homocysteine to methionine. Disruptions in this pathway influence both DNA synthesis and stability and chromatin methylation, and result from nutritional deficiencies and common gene variants. The mechanisms underlying folate-associated pathologies and developmental anomalies have yet to be established. This review focuses on the relationships among folate-mediated 1-carbon metabolism, chromatin methylation and human disease, and the role of gene-nutrient interactions in modifying epigenetic processes.
Epigenetics; Folate; Genetic variation; Methylation; One-carbon metabolism
The role of metabolic compartmentation in spatially organizing metabolic enzymes into pathways, regulating flux through metabolic pathways, and controlling the partitioning of metabolic intermediates among pathways is appreciated, but our understanding of the mechanisms that establish metabolic architecture and mediate communication and regulation among interconnected metabolic pathways and networks is still incomplete. This review discusses recent advancements in our understanding of metabolic compartmentation within the pathways that constitute the folate-mediated one-carbon metabolic network and emerging evidence for a need to regulate the trafficking of folates among compartmentalized metabolic pathways.
This report is based on the Federation of American Societies for Experimental Biology’s symposium, “Engaging basic Scientists in Translational Research: Identifying Opportunities, Overcoming Obstacles,” held in Chevy Chase, MD, March 24–25, 2011. Meeting participants examined the benefits of engaging basic scientists in translational research, the challenges to their participation in translational research, and the roles that research institutions, funding organizations, professional societies, and scientific publishers can play to address these challenges.
Basic science; Translational research; Benefits; Challenges; Recommendations
Folate-mediated one-carbon metabolism is required for the de novo synthesis of purines, thymidylate, and S-adenosylmethionine, the primary cellular methyl donor. Impairments in folate metabolism diminish cellular methylation potential and genome stability, which are risk factors for colorectal cancer (CRC). Cytoplasmic serine hydroxymethyltransferase (SHMT1) regulates the partitioning of folate-activated one-carbons between thymidylate and S-adenosylmethionine biosynthesis. Therefore, changes in SHMT1 expression enable the determination of the specific contributions made by thymidylate and S-adenosylmethionine biosynthesis to CRC risk. Shmt1 hemizygosity was associated with a decreased capacity for thymidylate synthesis, due to down regulation of enzymes in its biosynthetic pathway, namely thymidylate synthase and cytoplasmic thymidine kinase. Significant Shmt1-dependent changes to methylation capacity, gene expression and purine synthesis were not observed. Shmt1 hemizygosity was associated with increased risk for intestinal cancer in Apcmin/+ mice through a gene-by-diet interaction, indicating that the capacity for thymidylate synthesis modifies susceptibility to intestinal cancer in Apcmin/+ mice.
Cytoplasmic serine hydroxymethyltransferase; thymidylate synthesis; folate; colon cancer; Apc
The causal metabolic pathways underlying associations between folate and risk for colorectal cancer (CRC) have yet to be established. Folate-mediated one-carbon metabolism is required for the de novo synthesis of purines, thymidylate and methionine. Methionine is converted to S-adenosylmethionine (AdoMet), the major one-carbon donor for cellular methylation reactions. Impairments in folate metabolism can modify DNA synthesis, genomic stability and gene expression, characteristics associated with tumorigenesis. The Mthfd1 gene product, C1-tetrahydrofolate synthase, is a trifunctional enzyme that generates one-carbon substituted tetrahydrofolate cofactors for one-carbon metabolism. In this study, we use Mthfd1gt/+ mice, which demonstrate a 50% reduction in C1-tetrahydrofolate synthase, to determine its influence on tumor development in two mouse models of intestinal cancer, crosses between Mthfd1gt/+ and Apcmin/+ mice and azoxymethane (AOM)-induced colon cancer in Mthfd1gt/+ mice. Mthfd1 hemizygosity did not affect colon tumor incidence, number or load in Apcmin/+ mice. However, Mthfd1 deficiency increased tumor incidence 2.5-fold, tumor number 3.5-fold and tumor load 2-fold in AOM-treated mice. DNA uracil content in the colon was lower in Mthfd1gt/+ mice, indicating that thymidylate biosynthesis capacity does not play a significant role in AOM-induced colon tumorigenesis. Mthfd1 deficiency-modified cellular methylation potential, as indicated by the AdoMet: S-adenosylhomocysteine ratio and gene expression profiles, suggesting that changes in the transcriptome and/or decreased de novo purine biosynthesis and associated mutability cause cellular transformation in the AOM CRC model. This study emphasizes the impact and complexity of gene–nutrient interactions with respect to the relationships among folate metabolism and colon cancer initiation and progression.
Sequence variants in genes functioning in folate-mediated one-carbon metabolism are hypothesized to lead to changes in levels of homocysteine and DNA methylation, which, in turn, are associated with risk of cardiovascular disease.
330 SNPs in 52 genes were studied in relation to plasma homocysteine and global genomic DNA methylation. SNPs were selected based on functional effects and gene coverage, and assays were completed on the Illumina Goldengate platform. Age-, smoking-, and nutrient-adjusted genotype--phenotype associations were estimated in regression models.
Using a nominal P ≤ 0.005 threshold for statistical significance, 20 SNPs were associated with plasma homocysteine, 8 with Alu methylation, and 1 with LINE-1 methylation. Using a more stringent false discovery rate threshold, SNPs in FTCD, SLC19A1, and SLC19A3 genes remained associated with plasma homocysteine. Gene by vitamin B-6 interactions were identified for both Alu and LINE-1 methylation, and epistatic interactions with the MTHFR rs1801133 SNP were identified for the plasma homocysteine phenotype. Pleiotropy involving the MTHFD1L and SARDH genes for both plasma homocysteine and Alu methylation phenotypes was identified.
No single gene was associated with all three phenotypes, and the set of the most statistically significant SNPs predictive of homocysteine or Alu or LINE-1 methylation was unique to each phenotype. Genetic variation in folate-mediated one-carbon metabolism, other than the well-known effects of the MTHFR c.665C>T (known as c.677 C>T, rs1801133, p.Ala222Val), is predictive of cardiovascular disease biomarkers.
A roundtable to discuss the measurement of folate status biomarkers in NHANES took place in July 2010. NHANES has measured serum folate since 1974 and red blood cell (RBC) folate since 1978 with the use of several different measurement procedures. Data on serum 5-methyltetrahydrofolate (5MTHF) and folic acid (FA) concentrations in persons aged ≥60 y are available in NHANES 1999–2002. The roundtable reviewed data that showed that folate concentrations from the Bio-Rad Quantaphase II procedure (Bio-Rad Laboratories, Hercules, CA; used in NHANES 1991–1994 and NHANES 1999–2006) were, on average, 29% lower for serum and 45% lower for RBC than were those from the microbiological assay (MA), which was used in NHANES 2007–2010. Roundtable experts agreed that these differences required a data adjustment for time-trend analyses. The roundtable reviewed the possible use of an isotope-dilution liquid chromatography–tandem mass spectrometry (LC-MS/MS) measurement procedure for future NHANES and agreed that the close agreement between the MA and LC-MS/MS results for serum folate supported conversion to the LC-MS/MS procedure. However, for RBC folate, the MA gave 25% higher concentrations than did the LC-MS/MS procedure. The roundtable agreed that the use of the LC-MS/MS procedure to measure RBC folate is premature at this time. The roundtable reviewed the reference materials available or under development at the National Institute of Standards and Technology and recognized the challenges related to, and the scientific need for, these materials. They noted the need for a commutability study for the available reference materials for serum 5MTHF and FA.
A roundtable to discuss the measurement of vitamin B-12 (cobalamin) status biomarkers in NHANES took place in July 2010. NHANES stopped measuring vitamin B-12–related biomarkers after 2006. The roundtable reviewed 3 biomarkers of vitamin B-12 status used in past NHANES—serum vitamin B-12, methylmalonic acid (MMA), and total homocysteine (tHcy)—and discussed the potential utility of measuring holotranscobalamin (holoTC) for future NHANES. The roundtable focused on public health considerations and the quality of the measurement procedures and reference methods and materials that past NHANES used or that are available for future NHANES. Roundtable members supported reinstating vitamin B-12 status measures in NHANES. They noted evolving concerns and uncertainties regarding whether subclinical (mild, asymptomatic) vitamin B-12 deficiency is a public health concern. They identified the need for evidence from clinical trials to address causal relations between subclinical vitamin B-12 deficiency and adverse health outcomes as well as appropriate cutoffs for interpreting vitamin B-12–related biomarkers. They agreed that problems with sensitivity and specificity of individual biomarkers underscore the need for including at least one biomarker of circulating vitamin B-12 (serum vitamin B-12 or holoTC) and one functional biomarker (MMA or tHcy) in NHANES. The inclusion of both serum vitamin B-12 and plasma MMA, which have been associated with cognitive dysfunction and anemia in NHANES and in other population-based studies, was preferable to provide continuity with past NHANES. Reliable measurement procedures are available, and National Institute of Standards and Technology reference materials are available or in development for serum vitamin B-12 and MMA.
Tetrahydrofolates (THF) are a family of cofactors that function as one-carbon donors in folate-dependent one-carbon metabolism, a metabolic network required for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine in the cytoplasm. 5-FormylTHF is not a cofactor in one-carbon metabolism, but serves as a storage form of THF cofactors. 5-formylTHF is mobilized back into the THF cofactor pool by methenyltetrahydrofolate synthetase (MTHFS), which catalyzes the irreversible and ATP-dependent conversion 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate. Mthfs is not an essential gene in Arabidopsis, but MTHFS expression is elevated in animal tumors, enhances de novo purine synthesis, confers partial resistance to antifolate purine synthesis inhibitors and increases rates of folate catabolism in mammalian cell cultures. However, the mechanisms underlying these effects of MTHFS expression have yet to be established. The purpose of this study was to investigate the role and essentiality of MTHFS in mice. Mthfs was disrupted through the insertion of a gene trap vector between exons 1 and 2. Mthfsgt/+ mice were fertile and viable. No Mthfsgt/gt embryos were recovered from Mthfsgt/+ intercrosses, indicating Mthfs is an essential gene in mice. Tissue MTHFS protein levels are decreased in both Mthfsgt/+ and Mthfs+/+ mice placed on a folate and choline deficient diet, and mouse embryonic fibroblasts from Mthfsgt/+ embryos exhibit decreased capacity for de novo purine synthesis without impairment in de novo thymidylate synthesis. MTHFS was shown to co-localize with two enzymes of the de novo purine synthesis pathway in HeLa cells in a cell cycle-dependent manner, and to be modified by the small ubiquitin-like modifier (SUMO) protein. Mutation of the consensus SUMO modification sites on MTHFS eliminated co-localization of MTHFS with the de novo purine biosynthesis pathway under purine-deficient conditions. The results from this study indicate that MTHFS enhances purine biosynthesis by delivering 10-formylTHF to the purinosome in a SUMO-dependent fashion.
folate; MTHFS; purinosome; SHMT; 5-formyltetrahydrofolate; purine biosynthesis; leucovorin; SUMO
Increased breast cancer risk has been observed with both low folate status and a functional polymorphism in methylenetetrahydrofolate reductase (MTHFR 677C→T). Cytoplasmic serine hydroxymethyltransferase (cSHMT) affects the flow of one-carbon units through the folate metabolic network, but there is little research on a role for genetic variation in cSHMT in determining breast cancer risk.
A nested case-control study within the Nurses’ Health Study was used to investigate an association between cSHMT (1420C→T) and breast cancer risk.
No evidence for an association of cSHMT genotype and breast cancer was 10 observed. There was also no evidence of a gene-gene interaction between cSHMT and MTHFR.
There was no evidence of an association between cSHMT genotype and breast cancer occurrence. Further research in populations with differing average folate intake may be needed to fully understand the interactions of folate nutrition, sequence variation in folate genes, and breast cancer risk.
breast cancer; cSHMT; MTHFR; folate
Forward genetic screens in mice provide an unbiased means to identify genes and other functional genetic elements in the genome. Previously, a large scale ENU mutagenesis screen was conducted to query the functional content of a ~50 Mb region of the mouse genome on proximal Chr 5. The majority of phenotypic mutants recovered were embryonic lethals.
We report the high resolution genetic mapping, complementation analyses, and positional cloning of mutations in the target region. The collection of identified alleles include several with known or presumed functions for which no mutant models have been reported (Tbc1d14, Nol14, Tyms, Cad, Fbxl5, Haus3), and mutations in genes we or others previously reported (Tapt1, Rest, Ugdh, Paxip1, Hmx1, Otoe, Nsun7). We also confirmed the causative nature of a homeotic mutation with a targeted allele, mapped a lethal mutation to a large gene desert, and localized a spermiogenesis mutation to a region in which no annotated genes have coding mutations. The mutation in Tbc1d14 provides the first implication of a critical developmental role for RAB-GAP-mediated protein transport in early embryogenesis.
This collection of alleles contributes to the goal of assigning biological functions to all known genes, as well as identifying novel functional elements that would be missed by reverse genetic approaches.
The three enzymes that constitute the de novo thymidylate synthesis pathway in mammals, cytoplasmic serine hydroxymethyltransferase (SHMT1), thymidylate synthase (TYMS) and dihydrofolate reductase (DHFR) undergo sumoylation and nuclear import during S-phase. In this study, we demonstrate that purified intact mouse liver nuclei convert dUMP to dTMP in the presence of NADPH and serine. Neither nuclear extracts nor intact nuclei exposed to aminomethylphosphonate, a SHMT inhibitor, exhibit thymidylate synthesis activity. Nuclei isolated from Shmt1−/− mouse livers retained 25% of thymidylate synthesis activity exhibited by nuclei isolated from wild type mice. This residual activity was due to the presence of a cytoplasmic/nuclear isozyme of SHMT encoded by Shmt2. Shmt2 is shown to encode two transcripts, one which encodes a protein that localizes exclusively to the mitochondria (SHMT2), and a second transcript that lacks exon 1 and encodes a protein that localizes to the cytoplasm and nucleus during S-phase (SHMT2α). The ability of Shmt2 to encode a cytoplasmic isozyme of SHMT may account for the viability of Shmt1−/− mice and provide redundancy that permitted the expansion of the human SHMT1 L474F polymorphism that impairs SHMT1 sumoylation and nuclear translocation.
Folate-activated one-carbon units are derived from serine through the activity of the pyridoxal-phosphate (PLP)-dependent isozymes of serine hydroxymethyltransferase (SHMT). The effect of vitamin B6 availability on the activity and expression of the human mitochondrial and cytoplasmic SHMT isozymes was investigated in human MCF-7 cells. Cells were cultured for 6 months in vitamin B6 replete (4.9 μM pyridoxine) minimal essential medium (αMEM) or vitamin B6-deficient medium containing 49 nM, 4.9 nM or 0.49 nM pyridoxine. Total cellular PLP levels and SHMT activity were reduced 72% and 7% respectively when medium pyridoxine was decreased from 4.9 μM to 49 nM. Cells cultured in medium containing 4.9 nM pyridoxine exhibited 75%, 27% and 60% reduced levels of PLP, SHMT activity and S-adenosylmethionine, respectively compared to cells cultured in αMEM. Cytoplasmic SHMT activity and protein levels, but not mRNA levels, were decreased in cells cultured in vitamin B6 deficient medium, whereas mitochondrial SHMT activity and protein levels were less sensitive to vitamin B6 availability. PLP bound to cytoplasmic SHMT with a Kd = 850 nM, a value two orders of magnitude lower than previously reported for the bovine cytoplasmic SHMT isozyme. Collectively, these data indicate that vitamin B6 restriction decreases the activity and stability of SHMT, and that the cytoplasmic isozyme is more sensitive to vitamin B6 deficiency than the mitochondrial isozyme in MCF-7 cells.
serine hydroxymethyltransferase; vitamin B6; pyridoxal-phosphate; one-carbon metabolism; homocysteine; folate
The interaction of 5-formyltetrahydrofolate analogs with murine methenyltetrahydrofolate synthetase (MTHFS) was investigated using steady-state kinetics, molecular modeling, and site-directed mutagenesis. MTHFS catalyzes the irreversible cyclization of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate. Folate analogs that cannot undergo the rate-limiting step in catalysis were inhibitors of murine MTHFS. 5-formyltetrahydrohomofolate was an effective inhibitor of murine MTHFS (Ki = 0.7 μM), whereas 5-formyl, 10-methyltetrahydrofolate was a weak inhibitor (Ki = 10 μM). The former, but not the latter, was slowly phosphorylated by MTHFS. 5-formyltetrahydrohomofolate was not a substrate for murine MTHFS, but was metabolized when the MTHFS active site Y151 was mutated to Ala. MTHFS active site residues do not directly facilitate N10 attack on the on the N5-iminium phosphate intermediate, but rather restrict N10 motion around N5. Inhibitors specifically designed to block N10 attack appear to be less effective than the natural 10-formyltetrahydrofolate polyglutamate inhibitors.
Recombinant mouse 5,10-methenyltetrahydrofolate synthetase (MTHFS) was expressed in Escherichia coli and shown to co-purify with a chromophore that had a λmax at 320 nm. The chromophore remained bound to MTHFS during extensive dialysis, but dissociated from MTHFS when its substrate, 5-formyltetrahydrofolate, was bound. The chromophore was identified as an oxidized catecholamine by mass spectrometry and absorption spectroscopy. Purified recombinant mouse MTHFS and rabbit liver MTHFS proteins were shown to bind oxidized N-acetyldopamine (NADA) tightly. The addition of NADA to cell culture medium accelerated markedly folate turnover and decreased both folate accumulation and total cellular folate concentrations in MCF-7 cells. Expression of the MTHFS cDNA in MCF-7 cells increased the concentration of NADA required to deplete cellular folate. The results of this study are the first to identify a link between catecholamines and one-carbon metabolism and demonstrate that NADA accelerates folate turnover and impairs cellular folate accumulation in MCF-7 cells.
5; 10-methenyltetrahydrofolate synthetase; MTHFS; catecholamines; folate; 5-formyltetrahydrofolate; N-acetyldopamine; one-carbon metabolism; NADA; MTHFS - 5,10 - methenyltetrahydrofolate synthetase; DHF - dihydrofolate; THF - tetrahydrofolate; NADA - N-acetyldopamine; oxNADA - oxidized N-acetyldopamine; AdoMet - S-adenosylmethionine; pABG - para-aminobenzoyl(poly)glutamate; ESI-MS - electrospray ionization mass spectrometry; MALDI-TOF - matrix-assisted laser desorption ionization, time-of-flight; ICP-MS - inductively coupled plasma mass spectrometry; MS - mass spectrometry; E. coli - Escherichia coli; αMEM - α-minimal essential medium