Martha S. Field

Trafficking of Intracellular Folates

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.

Regulation of folate-mediated one-carbon metabolism by 10-formyltetrahydrofolate dehydrogenase

10-Formyltetrahydrofolate dehydrogenase (FDH) catalyzes the NADP+-dependent conversion of 10-formyltetrahydrofolate to CO2 and tetrahydrofolate (THF) and is an abundant high affinity folate-binding protein. Although several activities have been ascribed to FDH, its metabolic role in folate-mediated one-carbon metabolism is not well understood. FDH has been proposed to: 1) inhibit purine biosynthesis by depleting 10-formyl-THF pools, 2) maintain cellular folate concentrations by sequestering THF, 3) deplete the supply of folate-activated one-carbon units, and 4) stimulate the generation of THF-activated one-carbon unit synthesis by channeling folate cofactors to other folate-dependent enzymes. The metabolic functions of FDH were investigated in neuroblastoma, which do not contain detectable levels of FDH. Both low and high FDH expression reduced total cellular folate concentrations by 60%, elevated rates of folate catabolism, and depleted cellular 5-methyl-THF and S-adenosylmethionine levels. Low FDH expression increased the formyl-THF/THF ratio nearly 10-fold, whereas THF accounted for nearly 50% of total folate in neuroblastoma with high FDH expression. FDH expression did not affect the enrichment of exogenous formate into methionine, serine, or purines and did not suppress de novo purine nucleotide biosynthesis. We conclude that low FDH expression facilitates the incorporation of one-carbon units into the one-carbon pool, whereas high levels of FDH expression deplete the folate-activated one-carbon pool by catalyzing the conversion of 10-formyl-THF to THF. Furthermore, FDH does not increase cellular folate concentrations by sequestering THF in neuroblastoma nor does it inhibit or regulate de novo purine biosynthesis. FDH expression does deplete cellular 5-methyl-THF and S-adenosylmethionine levels indicating that FDH impairs the folate-dependent homocysteine remethylation cycle.

Regulation of de Novo Purine Biosynthesis by Methenyltetrahydrofolate Synthetase in Neuroblastoma

5-Formyltetrahydrofolate (5-formylTHF) is the only folate derivative that does not serve as a cofactor in folate-dependent one-carbon metabolism. Two metabolic roles have been ascribed to this folate derivative. It has been proposed to 1) serve as a storage form of folate because it is chemically stable and accumulates in seeds and spores and 2) regulate folate-dependent one-carbon metabolism by inhibiting folate-dependent enzymes, specifically targeting folate-dependent de novo purine biosynthesis. Methenyltetrahydrofolate synthetase (MTHFS) is the only enzyme that metabolizes 5-formylTHF and catalyzes its ATP-dependent conversion to 5,10-methenylTHF. This reaction determines intracellular 5-formylTHF concentrations and converts 5-formylTHF into an enzyme cofactor. The regulation and metabolic role of MTHFS in one-carbon metabolism was investigated in vitro and in human neuroblastoma cells. Steady-state kinetic studies revealed that 10-formylTHF, which exists in chemical equilibrium with 5,10-methenylTHF, acts as a tight binding inhibitor of mouse MTHFS. [6R]-10-formylTHF inhibited MTHFS with a Ki of 150 nm, and [6R,S]-10-formylTHF triglutamate inhibited MTHFS with a Ki of 30 nm. MTHFS is the first identified 10-formylTHF tight-binding protein. Isotope tracer studies in neuroblastoma demonstrate that MTHFS enhances de novo purine biosynthesis, indicating that MTHFS-bound 10-formylTHF facilitates de novo purine biosynthesis. Feedback metabolic regulation of MTHFS by 10-formylTHF indicates that 5-formylTHF can only accumulate in the presence of 10-formylTHF, providing the first evidence that 5-formylTHF is a storage form of excess formylated folates in mammalian cells. The sequestration of 10-formylTHF by MTHFS may explain why de novo purine biosynthesis is protected from common disruptions in the folate-dependent one-carbon network.

Nuclear enrichment of folate cofactors and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) protect de novo thymidylate biosynthesis during folate deficiency

Background: MTHFD1 is the primary source of one-carbon units for thymidylate synthesis. Results: MTHFD1 localizes to the nucleus in folate deficiency and S- and G2/M phases in mammalian cells to support de novo thymidylate biosynthesis. Conclusion: MTHFD1 nuclear localization explains the incorporation of formate into thymidylate during de novo thymidylate biosynthesis. Significance: Nuclear localization of MTHFD1 protects DNA by limiting uracil misincorporation into DNA. Folate-mediated one-carbon metabolism is a metabolic network of interconnected pathways that is required for the de novo synthesis of three of the four DNA bases and the remethylation of homocysteine to methionine. Previous studies have indicated that the thymidylate synthesis and homocysteine remethylation pathways compete for a limiting pool of methylenetetrahydrofolate cofactors and that thymidylate biosynthesis is preserved in folate deficiency at the expense of homocysteine remethylation, but the mechanisms are unknown. Recently, it was shown that thymidylate synthesis occurs in the nucleus, whereas homocysteine remethylation occurs in the cytosol. In this study we demonstrate that methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), an enzyme that generates methylenetetrahydrofolate from formate, ATP, and NADPH, functions in the nucleus to support de novo thymidylate biosynthesis. MTHFD1 translocates to the nucleus in S-phase MCF-7 and HeLa cells. During folate deficiency mouse liver MTHFD1 levels are enriched in the nucleus >2-fold at the expense of levels in the cytosol. Furthermore, nuclear folate levels are resistant to folate depletion when total cellular folate levels are reduced by >50% in mouse liver. The enrichment of folate cofactors and MTHFD1 protein in the nucleus during folate deficiency in mouse liver and human cell lines accounts for previous metabolic studies that indicated 5,10-methylenetetrahydrofolate is preferentially directed toward de novo thymidylate biosynthesis at the expense of homocysteine remethylation during folate deficiency.

Human mutations in methylenetetrahydrofolate dehydrogenase 1 impair nuclear de novo thymidylate biosynthesis

Significance These studies have identified that human genetic mutations, which impair the function of the folate-dependent enzyme methylene tetrahydrofolate dehydrogenase I (MTHFD1), depress rates of de novo thymidylate synthesis, elevate uracil levels in human DNA, and increase genome instability. These findings provide insights into the role of MTHFD1 and thymidylate biosynthesis in the etiology of SCID and megaloblastic anemia. An inborn error of metabolism associated with mutations in the human methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) gene has been identified. The proband presented with SCID, megaloblastic anemia, and neurologic abnormalities, but the causal metabolic impairment is unknown. SCID has been associated with impaired purine nucleotide metabolism, whereas megaloblastic anemia has been associated with impaired de novo thymidylate (dTMP) biosynthesis. MTHFD1 functions to condense formate with tetrahydrofolate and serves as the primary entry point of single carbons into folate-dependent one-carbon metabolism in the cytosol. In this study, we examined the impact of MTHFD1 loss of function on folate-dependent purine, dTMP, and methionine biosynthesis in fibroblasts from the proband with MTHFD1 deficiency. The flux of formate incorporation into methionine and dTMP was decreased by 90% and 50%, respectively, whereas formate flux through de novo purine biosynthesis was unaffected. Patient fibroblasts exhibited enriched MTHFD1 in the nucleus, elevated uracil in DNA, lower rates of de novo dTMP synthesis, and increased salvage pathway dTMP biosynthesis relative to control fibroblasts. These results provide evidence that impaired nuclear de novo dTMP biosynthesis can lead to both megaloblastic anemia and SCID in MTHFD1 deficiency.

Mthfs is an Essential Gene in Mice and a Component of the Purinosome

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.

Reduced MTHFD1 Activity in Male Mice Perturbs Folate- and Choline-Dependent One-Carbon Metabolism As Well As Transsulfuration

Impaired utilization of folate is caused by insufficient dietary intake and/or genetic variation and has been shown to prompt changes in related pathways, including choline and methionine metabolism. These pathways have been shown to be sensitive to variation within the Mthfd1 gene, which codes for a folate-metabolizing enzyme responsible for generating 1-carbon (1-C)-substituted folate derivatives. The Mthfd1(gt/+) mouse serves as a potential model of human Mthfd1 loss-of-function genetic variants that impair MTHFD1 function. This study investigated the effects of the Mthfd1(gt/+) genotype and folate intake on markers of choline, folate, methionine, and transsulfuration metabolism. Male Mthfd1(gt/+) and Mthfd1(+/+) mice were randomly assigned at weaning (3 wk of age) to either a control (2 mg/kg folic acid) or folate-deficient (0 mg/kg folic acid) diet for 5 wk. Mice were killed at 8 wk of age following 12 h of food deprivation; blood and liver samples were analyzed for choline, methionine, and transsulfuration biomarkers. Independent of folate intake, mice with the Mthfd1(gt/+) genotype had higher hepatic concentrations of choline (P = 0.005), betaine (P = 0.013), and dimethylglycine (P = 0.004) and lower hepatic concentrations of glycerophosphocholine (P = 0.002) relative to Mthfd1(+/+) mice. Mthfd1(gt/+) mice also had higher plasma concentrations of homocysteine (P = 0.0016) and cysteine (P < 0.001) as well as lower plasma concentrations of methionine (P = 0.0003) and cystathionine (P = 0.011). The metabolic alterations observed in Mthfd1(gt/+) mice indicate perturbed choline and folate-dependent 1-C metabolism and support the future use of Mthfd1(gt/+) mice as a tool to investigate the impact of impaired 1-C metabolism on disease outcomes.

5,10-Methenyltetrahydrofolate synthetase activity is increased in tumors and modifies the efficacy of antipurine LY309887.

Methenyltetrahydrofolate synthetase (MTHFS) expression enhances folate-dependent de novo purine biosynthesis. In this study, the effect of increased MTHFS expression on the efficacy of the glycinamide ribonucleotide formyltransferase (GARFT) inhibitor LY309887 was investigated in SH-SY5Y neuroblastoma. GARFT catalyzes the incorporation of formate, in the form of 10-formyltetrahydrofolate, into the C8 position of the purine ring during de novo purine biosynthesis. SH-SY5Y neuroblastoma with increased MTHFS expression displayed a 4-fold resistance to the GARFT inhibitor LY309887, but did not exhibit resistance to the thymidylate synthase inhibitor Pemetrexed. This finding supports a mechanism whereby MTHFS increases the availability of 10-formyltetrahydrofolate for GARFT. MTHFS expression is elevated in animal tumor tissues compared to surrounding normal tissue, consistent with the dependence of transformed cells on de novo purine biosynthesis. The level of MTHFS expression in tumors may predict the efficacy of antipurine agents that target GARFT.

MTHFD1 regulates nuclear de novo thymidylate biosynthesis and genome stability.

Disruptions in folate-mediated one-carbon metabolism (FOCM) are associated with risk for several pathologies including developmental anomalies such as neural tube defects and congenital heart defects, diseases of aging including cognitive decline, neurodegeneration and epithelial cancers, and hematopoietic disorders including megaloblastic anemia. However, the causal pathways and mechanisms that underlie these pathologies remain unresolved. Because folate-dependent anabolic pathways are tightly interconnected and best described as a metabolic network, the identification of causal pathways and associated mechanisms of pathophysiology remains a major challenge in identifying the contribution of individual pathways to disease phenotypes. Investigations of genetic mouse models and human inborn errors of metabolism enable a more precise dissection of the pathways that constitute the FOCM network and enable elucidation of causal pathways associated with NTDs. In this overview, we summarize recent evidence that the enzyme MTHFD1 plays an essential role in FOCM in humans and in mice, and that it determines the partitioning of folate-activated one carbon units between the folate-dependent de novo thymidylate and homocysteine remethylation pathways through its regulated nuclear localization. We demonstrate that impairments in MTHFD1 activity compromise both homocysteine remethylation and de novo thymidylate biosynthesis, and provide evidence that MTHFD1-associated disruptions in de novo thymidylate biosynthesis lead to genome instability that may underlie folate-associated immunodeficiency and birth defects.

Maternal dietary uridine causes, and deoxyuridine prevents, neural tube closure defects in a mouse model of folate-responsive neural tube defects

BACKGROUND Folic acid prevents neural tube closure defects (NTDs), but the causal metabolic pathways have not been established. Serine hydroxymethyltransferase 1 (SHMT1) is an essential scaffold protein in folate-dependent de novo thymidylate synthesis in the nucleus. SHMT1-deficient mice provide a model to investigate folic acid-responsive NTDs wherein disruption of de novo thymidylate synthesis impairs neural tube closure. OBJECTIVE We examined the effects of maternal supplementation with the pyrimidine nucleosides uridine, thymidine, or deoxyuridine with and without folate deficiency on NTD incidence in the Shmt1 mouse model. DESIGN Shmt1(+/+) and Shmt1(-/-) female mice fed folate-replete or folate-deficient diets and supplemented with uridine, thymidine, or deoxyuridine were bred, and litters (n = 10-23 per group) were examined for the presence of NTDs. Biomarkers of impaired folate status and metabolism were measured, including plasma nucleosides, hepatic uracil content, maternal plasma folate concentrations, and incorporation of nucleoside precursors into DNA. RESULTS Shmt1(+/-) and Shmt1(-/-) embryos from dams fed the folate-deficient diet were susceptible to NTDs. No NTDs were observed in litters from dams fed the folate-deficient diet supplemented with deoxyuridine. Surprisingly, uridine supplementation increased NTD incidence, independent of embryo genotype and dietary folic acid. These dietary nucleosides did not affect maternal hepatic uracil accumulation in DNA but did affect plasma folate concentrations. CONCLUSIONS Maternal deoxyuridine supplementation prevented NTDs in dams fed the folate-deficient diet, whereas maternal uridine supplementation increased NTD incidence, independent of folate and embryo genotype. These findings provide new insights into the metabolic impairments and mechanisms of folate-responsive NTDs resulting from decreased Shmt1 expression.