A new mathematical model of folate homeostasis in E. coli highlights the potential importance of the folinic acid futile cycle in cell growth.

Folate (vitamin B9) plays a central role in one-carbon metabolism in prokaryotes and eukaryotes. This pathway mediates the transfer of one-carbon units, playing a crucial role in nucleotide synthesis, methylation, and amino acid homeostasis. The folinic acid futile cycle adds a layer of intrigue to this pathway, due to its associations with metabolism, cell growth, and dormancy. It also introduces additional complexity to folate metabolism. A logical way to deal with such complexity is to examine it by using mathematical modelling. This work describes the construction and analysis of a model of folate metabolism, which includes the folinic acid futile cycle. This model was tested under three in silico growth conditions. Model simulations revealed: 1) the folate cycle behaved as a stable biochemical system in three growth states (slow, standard, and rapid); 2) the initial concentration of serine had the greatest impact on metabolite concentrations; 3) 5-formyltetrahydrofolate cyclo-ligase (5-FCL) activity had a significant impact on the levels of the 7 products that carry the one-carbon donated from folates, and the redox couple NADP/NADPH; this was particularly evident in the rapid growth state; 4) 5-FCL may be vital to the survival of the cells by maintaining low levels of homocysteine, as high levels can induce toxicity; and 5) the antifolate therapeutic trimethoprim had a greater impact on folate metabolism with higher nutrient availability. These results highlight the important role of 5-FCL in intracellular folate homeostasis and mass generation under different metabolic scenarios.

5-Formyltetrahydrofolate promotes conformational remodeling in a methylenetetrahydrofolate reductase active site and inhibits its activity.

The flavoprotein methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of N5, N10-methylenetetrahydrofolate (CH2-H4folate) to N5-methyltetrahydrofolate (CH3-H4folate), committing a methyl group from the folate cycle to the methionine one. This committed step is the sum of multiple ping-pong electron transfers involving multiple substrates, intermediates, and products all sharing the same active site. Insight into folate substrate binding is needed to better understand this multifunctional active site. Here, we performed activity assays with Thermus thermophilus MTHFR (tMTHFR), which showed pH-dependent inhibition by the substrate analog, N5-formyltetrahydrofolate (CHO-H4folate). Our crystal structure of a tMTHFR•CHO-H4folate complex revealed a unique folate-binding mode; tMTHFR subtly rearranges its active site to form a distinct folate-binding environment. Formation of a novel binding pocket for the CHO-H4folate p-aminobenzoic acid moiety directly affects how bent the folate ligand is and its accommodation in the active site. Comparative analysis of the available active (FAD- and folate-bound) MTHFR complex structures reveals that CHO-H4folate is accommodated in the active site in a conformation that would not support hydride transfer, but rather in a conformation that potentially reports on a different step in the reaction mechanism after this committed step, such as CH2-H4folate ring-opening. This active site remodeling provides insights into the functional relevance of the differential folate-binding modes and their potential roles in the catalytic cycle. The conformational flexibility displayed by tMTHFR demonstrates how a shared active site can use a few amino acid residues in lieu of extra domains to accommodate chemically distinct moieties and functionalities.

Immunodeficiency associated with a novel functionally defective variant of SLC19A1 benefits from folinic acid treatment.

Insufficient dietary folate intake, hereditary malabsorption, or defects in folate transport may lead to combined immunodeficiency (CID). Although loss of function mutations in the major intestinal folate transporter PCFT/SLC46A1 was shown to be associated with CID, the evidence for pathogenic variants of RFC/SLC19A1 resulting in immunodeficiency was lacking. We report two cousins carrying a homozygous pathogenic variant c.1042 G > A, resulting in p.G348R substitution who showed symptoms of immunodeficiency associated with defects of folate transport. SLC19A1 expression by peripheral blood mononuclear cells (PBMC) was quantified by real-time qPCR and immunostaining. T cell proliferation, methotrexate resistance, NK cell cytotoxicity, Treg cells and cytokine production by T cells were examined by flow cytometric assays. Patients were treated with and benefited from folinic acid. Studies revealed normal NK cell cytotoxicity, Treg cell counts, and naive-memory T cell percentages. Although SLC19A1 mRNA and protein expression were unaltered, remarkably, mitogen induced-T cell proliferation was significantly reduced at suboptimal folic acid and supraoptimal folinic acid concentrations. In addition, patients' PBMCs were resistant to methotrexate-induced apoptosis supporting a functionally defective SLC19A1. This study presents the second pathogenic SLC19A1 variant in the literature, providing the first experimental evidence that functionally defective variants of SLC19A1 may present with symptoms of immunodeficiency.

The 5-formyl-tetrahydrofolate proteome links folates with C/N metabolism and reveals feedback regulation of folate biosynthesis.

Folates are indispensable for plant development, but their molecular mode of action remains elusive. We synthesized a probe, "5-F-THF-Dayne," comprising 5-formyl-tetrahydrofolate (THF) coupled to a photoaffinity tag. Exploiting this probe in an affinity proteomics study in Arabidopsis thaliana, we retrieved 51 hits. Thirty interactions were independently validated with in vitro expressed proteins to bind 5-F-THF with high or low affinity. Interestingly, the interactors reveal associations beyond one-carbon metabolism, covering also connections to nitrogen (N) metabolism, carbohydrate metabolism/photosynthesis, and proteostasis. Two of the interactions, one with the folate biosynthetic enzyme DIHYDROFOLATE REDUCTASE-THYMIDYLATE SYNTHASE 1 (AtDHFR-TS1) and another with N metabolism-associated glutamine synthetase 1;4 (AtGLN1;4), were further characterized. In silico and experimental analyses revealed G35/K36 and E330 as key residues for the binding of 5-F-THF in AtDHFR-TS1 and AtGLN1;4, respectively. Site-directed mutagenesis of AtGLN1;4 E330, which co-localizes with the ATP-binding pocket, abolished 5-F-THF binding as well as AtGLN1;4 activity. Furthermore, 5-F-THF was noted to competitively inhibit the activities of AtDHFR-TS1 and AtGLN1;4. In summary, we demonstrated a regulatory role for 5-F-THF in N metabolism, revealed 5-F-THF-mediated feedback regulation of folate biosynthesis, and identified a total of 14 previously unknown high-affinity binding cellular targets of 5-F-THF. Together, this sets a landmark toward understanding the role of folates in plant development.

Peptidomimetic-based identification of FDA-approved compounds inhibiting IRE1 activity.

Inositol-requiring enzyme 1 (IRE1) is a bifunctional serine/threonine kinase and endoribonuclease that is a major mediator of the unfolded protein response (UPR) during endoplasmic reticulum (ER) stress. Tumour cells experience ER stress due to adverse environmental cues such as hypoxia or nutrient shortage and high metabolic/protein-folding demand. To cope with those stresses, cancer cells utilise IRE1 signalling as an adaptive mechanism. Here, we report the discovery of the FDA-approved compounds methotrexate, cefoperazone, folinic acid and fludarabine phosphate as IRE1 inhibitors. These were identified through a structural exploration of the IRE1 kinase domain using IRE1 peptide fragment docking and further optimisation and pharmacophore development. The inhibitors were verified to have an impact on IRE1 activity in vitro and were tested for their ability to sensitise human cell models of glioblastoma multiforme (GBM) to chemotherapy. We show that all molecules identified sensitise glioblastoma cells to the standard-of-care chemotherapy temozolomide (TMZ).

Aldh1l2 knockout mouse metabolomics links the loss of the mitochondrial folate enzyme to deregulation of a lipid metabolism observed in rare human disorder.

BACKGROUND: Mitochondrial folate enzyme ALDH1L2 (aldehyde dehydrogenase 1 family member L2) converts 10-formyltetrahydrofolate to tetrahydrofolate and CO2 simultaneously producing NADPH. We have recently reported that the lack of the enzyme due to compound heterozygous mutations was associated with neuro-ichthyotic syndrome in a male patient. Here, we address the role of ALDH1L2 in cellular metabolism and highlight the mechanism by which the enzyme regulates lipid oxidation. METHODS: We generated Aldh1l2 knockout (KO) mouse model, characterized its phenotype, tissue histology, and levels of reduced folate pools and applied untargeted metabolomics to determine metabolic changes in the liver, pancreas, and plasma caused by the enzyme loss. We have also used NanoString Mouse Inflammation V2 Code Set to analyze inflammatory gene expression and evaluate the role of ALDH1L2 in the regulation of inflammatory pathways. RESULTS: Both male and female Aldh1l2 KO mice were viable and did not show an apparent phenotype. However, H&E and Oil Red O staining revealed the accumulation of lipid vesicles localized between the central veins and portal triads in the liver of Aldh1l2-/- male mice indicating abnormal lipid metabolism. The metabolomic analysis showed vastly changed metabotypes in the liver and plasma in these mice suggesting channeling of fatty acids away from ß-oxidation. Specifically, drastically increased plasma acylcarnitine and acylglycine conjugates were indicative of impaired ß-oxidation in the liver. Our metabolomics data further showed that mechanistically, the regulation of lipid metabolism by ALDH1L2 is linked to coenzyme A biosynthesis through the following steps. ALDH1L2 enables sufficient NADPH production in mitochondria to maintain high levels of glutathione, which in turn is required to support high levels of cysteine, the coenzyme A precursor. As the final outcome, the deregulation of lipid metabolism due to ALDH1L2 loss led to decreased ATP levels in mitochondria. CONCLUSIONS: The ALDH1L2 function is important for CoA-dependent pathways including ß-oxidation, TCA cycle, and bile acid biosynthesis. The role of ALDH1L2 in the lipid metabolism explains why the loss of this enzyme is associated with neuro-cutaneous diseases. On a broader scale, our study links folate metabolism to the regulation of lipid homeostasis and the energy balance in the cell.

Cellular Pharmacodynamics of a Novel Pyrrolo[3,2-d]pyrimidine Inhibitor Targeting Mitochondrial and Cytosolic One-Carbon Metabolism.

Folate-dependent one-carbon (C1) metabolism is compartmentalized in the mitochondria and cytosol and is a source of critical metabolites for proliferating tumors. Mitochondrial C1 metabolism including serine hydroxymethyltransferase 2 (SHMT2) generates glycine for de novo purine nucleotide and glutathione biosynthesis and is an important source of NADPH, ATP, and formate, which affords C1 units as 10-formyl-tetrahydrofolate and 5,10-methylene-tetrahydrofolate for nucleotide biosynthesis in the cytosol. We previously discovered novel first-in-class multitargeted pyrrolo[3,2-d]pyrimidine inhibitors of SHMT2 and de novo purine biosynthesis at glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase with potent in vitro and in vivo antitumor efficacy toward pancreatic adenocarcinoma cells. In this report, we extend our findings to an expanded panel of pancreatic cancer models. We used our lead analog AGF347 [(4-(4-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)butyl)-2-fluorobenzoyl)-l-glutamic acid] to characterize pharmacodynamic determinants of antitumor efficacy for this series and demonstrated plasma membrane transport into the cytosol, uptake from cytosol into mitochondria, and metabolism to AGF347 polyglutamates in both cytosol and mitochondria. Antitumor effects of AGF347 downstream of SHMT2 and purine biosynthesis included suppression of mammalian target of rapamycin signaling, and glutathione depletion with increased levels of reactive oxygen species. Our results provide important insights into the cellular pharmacology of novel pyrrolo[3,2-d]pyrimidine inhibitors as antitumor compounds and establish AGF347 as a unique agent for potential clinical application for pancreatic cancer, as well as other malignancies. SIGNIFICANCE STATEMENT: This study establishes the antitumor efficacies of novel inhibitors of serine hydroxymethyltransferase 2 and of cytosolic targets toward a panel of clinically relevant pancreatic cancer cells and demonstrates the important roles of plasma membrane transport, mitochondrial accumulation, and metabolism to polyglutamates of the lead compound AGF347 to drug activity. We also establish that loss of serine catabolism and purine biosynthesis resulting from AGF347 treatment impacts mammalian target of rapamycin signaling, glutathione pools, and reactive oxygen species, contributing to antitumor efficacy.

A novel miniature transposon-like element discovered in the coding sequence of a gene that encodes for 5-formyltetrahydrofolate in wheat.

BACKGROUND: Transposable elements (TEs) comprise over 80% of the wheat genome and usually possess unique features for specific super-families and families. However, the role of TEs in wheat evolution and reshaping the wheat genome remains largely unclear. RESULTS: In this study, we discovered a miniature (307 bp in length) TE-like sequence in exon 6 of a gene that encodes for 5-formyltetrahydrofolate, in two accessions of wild emmer wheat (T. turgidum ssp. dicoccoides) and has interfered with the gene translation by creating a shorter reading frame as a result of a stop codon. The sequence that was termed Mariam, does not show any structural similarity to known TEs. It does not possess terminal inverted repeats (TIRs) that would allow us to assign this element to one of the TIR DNA super-families, and it does not possess characteristic features of SINE, such as a Pol-III promotor or a poly-A tail. In-silico analysis of five publicly available genome drafts of Triticum and Aegilops species revealed that Mariam element appears in a very low copy number (1-3 insertions) in diploid wheat species and ~ 12 insertions in tetraploid and hexaploidy wheat species. In addition, Mariam element was found to be unique to wheat, as it was not found in other plant genomes. The dynamic nature of Mariam in the wheat genome was assessed by site-specific PCR analysis and revealed that it retained activity in wild emmer populations in a population-specific manner. CONCLUSIONS: This study provides additional insight into the evolutionary impact of TEs in wheat.

Cytosolic 10-formyltetrahydrofolate dehydrogenase regulates glycine metabolism in mouse liver.

ALDH1L1 (10-formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism highly expressed in liver, metabolizes 10-formyltetrahydrofolate to produce tetrahydrofolate (THF). This reaction might have a regulatory function towards reduced folate pools, de novo purine biosynthesis, and the flux of folate-bound methyl groups. To understand the role of the enzyme in cellular metabolism, Aldh1l1-/- mice were generated using an ES cell clone (C57BL/6N background) from KOMP repository. Though Aldh1l1-/- mice were viable and did not have an apparent phenotype, metabolomic analysis indicated that they had metabolic signs of folate deficiency. Specifically, the intermediate of the histidine degradation pathway and a marker of folate deficiency, formiminoglutamate, was increased more than 15-fold in livers of Aldh1l1-/- mice. At the same time, blood folate levels were not changed and the total folate pool in the liver was decreased by only 20%. A two-fold decrease in glycine and a strong drop in glycine conjugates, a likely result of glycine shortage, were also observed in Aldh1l1-/- mice. Our study indicates that in the absence of ALDH1L1 enzyme, 10-formyl-THF cannot be efficiently metabolized in the liver. This leads to the decrease in THF causing reduced generation of glycine from serine and impaired histidine degradation, two pathways strictly dependent on THF.

Investigations of Amino Acids in the 5-Formyltetrahydrofolate Binding Site of 5,10-Methenyltetrahydrofolate Synthetase from Mycoplasma pneumonia.

5,10-Methenyltetrahydrofolate synthetase plays a significant role in folate metabolism by catalyzing the conversion of 5-formyltetrahydrofolate into 5,10-methenyltetrahydrofolate. The enzyme is important in some forms of chemotherapy, and it has been implicated in resistance to antifolate antibiotics. A co-crystal structure of the enzyme (1U3G) and primary sequence analysis were used to select highly conserved amino acids in close proximity to bound 5-formyltetrahydrofolate. The amino acids were then investigated using site directed mutagenesis and kinetics. Y123, E55, and F118 were concluded to be important for binding 5-formyltetrahydrofolate in the active site and/or for substrate turnover of the enzyme. Replacement of E55 or Y123 with alanine resulted in no detectable activity. The more subtle replacement of E55 with glutamine was also inactive suggesting an ionic interaction with 5-formyltetrahydrofolate. Mutations to F118 resulted in substantial increases in apparent Km for both 5-formyltetrahydrofolate and ATP, but did not substantially affect catalytic turnover. Outside the active site, the replacement of Q144 with alanine yielded an enzyme that bound the substrates of ATP and 5-formyltetrahydrofolate with higher apparent Km values than the wild-type enzyme, but demonstrated a 3.1 fold increase in kcat.

Responsive self-assembly of tectoRNAs with loop-receptor interactions from the tetrahydrofolate (THF) riboswitch.

Naturally occurring RNAs are known to exhibit a high degree of modularity, whereby specific structural modules (or motifs) can be mixed and matched to create new molecular architectures. The modular nature of RNA also affords researchers the ability to characterize individual structural elements in controlled synthetic contexts in order to gain new and critical insights into their particular structural features and overall performance. Here, we characterized the binding affinity of a unique loop-receptor interaction found in the tetrahydrofolate (THF) riboswitch using rationally designed self-assembling tectoRNAs. Our work suggests that the THF loop-receptor interaction has been fine-tuned for its particular role as a riboswitch component. We also demonstrate that the thermodynamic stability of this interaction can be modulated by the presence of folinic acid, which induces a local structural change at the level of the loop-receptor. This corroborates the existence of a THF binding site within this tertiary module and paves the way for its potential use as a THF responsive module for RNA nanotechnology and synthetic biology.

Mechanism of Action of Methotrexate Against Zika Virus.

Zika virus (ZIKV), which is associated with microcephaly in infants and Guillain-Barré syndrome, reemerged as a serious public health threat in Latin America in recent years. Previous high-throughput screening (HTS) campaigns have revealed several potential hit molecules against ZIKV, including methotrexate (MTX), which is clinically used as an anti-cancer chemotherapy and anti-rheumatoid agent. We studied the mechanism of action of MTX against ZIKV in relation to its inhibition of dihydrofolate reductase (DHFR) in vitro using Vero and human neural stem cells (hNSCs). As expected, an antiviral effect for MTX against ZIKV was observed, showing up to 10-fold decrease in virus titer during MTX treatment. We also observed that addition of leucovorin (a downstream metabolite of DHFR pathway) rescued the ZIKV replication impaired by MTX treatment in ZIKV-infected cells, explaining the antiviral effect of MTX through inhibition of DHFR. We also found that addition of adenosine to ZIKV-infected cells was able to rescue ZIKV replication inhibited by MTX, suggesting that restriction of de novo synthesis adenosine triphosphate (ATP) pools suppresses viral replication. These results confirm that the DHFR pathway can be targeted to inhibit replication of ZIKV, similar to other published results showing this effect in related flaviviruses.

The 5-formyltetrahydrofolate futile cycle reduces pathway stochasticity in an extended hybrid-stochastic model of folate-mediated one-carbon metabolism.

In folate-mediated one-carbon metabolism (FOCM), 5-formyltetrahydrofolate (5fTHF), a one-carbon substituted tetrahydrofolate (THF) vitamer, acts as an intracellular storage form of folate and as an inhibitor of the folate-dependent enzymes phosphoribosylaminoimidazolecarboxamide formyltransferase (AICARFT) and serine hydroxymethyltransferase (SHMT). Cellular levels of 5fTHF are regulated by a futile cycle comprising the enzymes SHMT and 5,10-methenyltetrahydrofolate synthetase (MTHFS). MTHFS is an essential gene in mice; however, the roles of both 5fTHF and MTHFS in mammalian FOCM remain to be fully elucidated. We present an extension of our previously published hybrid-stochastic model of FOCM by including the 5fTHF futile-cycle to explore its effect on the FOCM network. Model simulations indicate that MTHFS plays an essential role in preventing 5fTHF accumulation, which consequently averts inhibition of all other reactions in the metabolic network. Moreover, in silico experiments show that 10-formylTHF inhibition of MTHFS is critical for regulating purine synthesis. Model simulations also provide evidence that 5-methylTHF (and not 5fTHF) is the predominant physiological binder/inhibitor of SHMT. Finally, the model simulations indicate that the 5fTHF futile cycle dampens the stochastic noise in FOCM that results from both folate deficiency and a common variant in the methylenetetrahydrofolate reductase (MTHFR) gene.

Folinic acid improves verbal communication in children with autism and language impairment: a randomized double-blind placebo-controlled trial.

We sought to determine whether high-dose folinic acid improves verbal communication in children with non-syndromic autism spectrum disorder (ASD) and language impairment in a double-blind placebo control setting. Forty-eight children (mean age 7 years 4 months; 82% male) with ASD and language impairment were randomized to receive 12 weeks of high-dose folinic acid (2 mg kg-1 per day, maximum 50 mg per day; n=23) or placebo (n=25). Children were subtyped by glutathione and folate receptor-α autoantibody (FRAA) status. Improvement in verbal communication, as measured by a ability-appropriate standardized instrument, was significantly greater in participants receiving folinic acid as compared with those receiving placebo, resulting in an effect of 5.7 (1.0,10.4) standardized points with a medium-to-large effect size (Cohen's d=0.70). FRAA status was predictive of response to treatment. For FRAA-positive participants, improvement in verbal communication was significantly greater in those receiving folinic acid as compared with those receiving placebo, resulting in an effect of 7.3 (1.4,13.2) standardized points with a large effect size (Cohen's d=0.91), indicating that folinic acid treatment may be more efficacious in children with ASD who are FRAA positive. Improvements in subscales of the Vineland Adaptive Behavior Scale, the Aberrant Behavior Checklist, the Autism Symptom Questionnaire and the Behavioral Assessment System for Children were significantly greater in the folinic acid group as compared with the placebo group. There was no significant difference in adverse effects between treatment groups. Thus, in this small trial of children with non-syndromic ASD and language impairment, treatment with high-dose folinic acid for 12 weeks resulted in improvement in verbal communication as compared with placebo, particularly in those participants who were positive for FRAAs.

An LC-MS chemical derivatization method for the measurement of five different one-carbon states of cellular tetrahydrofolate.

The cofactor tetrahydrofolate (THF) is used to reduce, oxidize, and transfer one-carbon (1C) units required for the synthesis of nucleotides, glycine, and methionine. Measurement of intracellular THF species is complicated by their chemical instability, signal dilution caused by variable polyglutamation, and the potential for interconversion among these species. Here, we describe a method using negative mode liquid chromatography-mass spectrometry (LC-MS) to measure intracellular folate species from mammalian cells. Application of this method with isotope-labeled substrates revealed abiotic interconversion of THF and methylene-THF, which renders their separate quantitation particularly challenging. Chemical reduction of methylene-THF using deuterated sodium cyanoborohydride traps methylene-THF, which is unstable, as deuterated 5-methyl-THF, which is stable. Together with proper sample handling and LC-MS, this enables effective measurements of five active folate pools (THF, 5-methyl-THF, methylene-THF, methenyl-THF/10-formyl-THF, and 5-formyl-THF) representing the biologically important 1C oxidation states of THF in mammalian cells. Graphical abstract Chemical derivatization with deuterated cyanoborohydride traps unstable methylene-THF as isotope-labeled 5-methyl-THF, enabling accurate quantification by LC-MS.

Crystal Structure of the Emerging Cancer Target MTHFD2 in Complex with a Substrate-Based Inhibitor.

To sustain their proliferation, cancer cells become dependent on one-carbon metabolism to support purine and thymidylate synthesis. Indeed, one of the most highly upregulated enzymes during neoplastic transformation is MTHFD2, a mitochondrial methylenetetrahydrofolate dehydrogenase and cyclohydrolase involved in one-carbon metabolism. Because MTHFD2 is expressed normally only during embryonic development, it offers a disease-selective therapeutic target for eradicating cancer cells while sparing healthy cells. Here we report the synthesis and preclinical characterization of the first inhibitor of human MTHFD2. We also disclose the first crystal structure of MTHFD2 in complex with a substrate-based inhibitor and the enzyme cofactors NAD+ and inorganic phosphate. Our work provides a rationale for continued development of a structural framework for the generation of potent and selective MTHFD2 inhibitors for cancer treatment. Cancer Res; 77(4); 937-48. ©2017 AACR.

Reversal of Cytosolic One-Carbon Flux Compensates for Loss of the Mitochondrial Folate Pathway.

One-carbon (1C) units for purine and thymidine synthesis can be generated from serine by cytosolic or mitochondrial folate metabolism. The mitochondrial 1C pathway is consistently overexpressed in cancer. Here, we show that most but not all proliferating mammalian cell lines use the mitochondrial pathway as the default for making 1C units. Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated mitochondrial pathway knockout activates cytosolic 1C-unit production. This reversal in cytosolic flux is triggered by depletion of a single metabolite, 10-formyl-tetrahydrofolate (10-formyl-THF), and enables rapid cell growth in nutrient-replete conditions. Loss of the mitochondrial pathway, however, renders cells dependent on extracellular serine to make 1C units and on extracellular glycine to make glutathione. HCT-116 colon cancer xenografts lacking mitochondrial 1C pathway activity generate the 1C units required for growth by cytosolic serine catabolism. Loss of both pathways precludes xenograft formation. Thus, either mitochondrial or cytosolic 1C metabolism can support tumorigenesis, with the mitochondrial pathway required in nutrient-poor conditions.

Folate-Dependent Purine Nucleotide Biosynthesis in Humans.

Purine nucleotide biosynthesis de novo (PNB) requires 2 folate-dependent transformylases-5'-phosphoribosyl-glycinamide (GAR) and 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) transformylases-to introduce carbon 8 (C8) and carbon 2 (C2) into the purine ring. Both transformylases utilize 10-formyltetrahydrofolate (10-formyl-H4folate), where the formyl-carbon sources include ring-2-C of histidine, 3-C of serine, 2-C of glycine, and formate. Our findings in human studies indicate that glycine provides the carbon for GAR transformylase (exclusively C8), whereas histidine and formate are the predominant carbon sources for AICAR transformylase (C2). Contrary to the previous notion, these carbon sources may not supply a general 10-formyl-H4folate pool, which was believed to equally provide carbons to C8 and C2. To explain these phenomena, we postulate that GAR transformylase is in a complex with the trifunctional folate-metabolizing enzyme (TFM) and serine hydroxymethyltransferase to channel carbons of glycine and serine to C8. There is no evidence for channeling carbons of histidine and formate to AICAR transformylase (C2). GAR transformylase may require the TFM to furnish 10-formyl-H4folate immediately after its production from serine to protect its oxidation to 10-formyldihydrofolate (10-formyl-H2folate), whereas AICAR transformylase can utilize both 10-formyl-H2folate and 10-formyl-H4folate. Human liver may supply AICAR to AICAR transformylase in erythrocytes/erythroblasts. Incorporation of ring-2-C of histidine and formate into C2 of urinary uric acid presented a circadian rhythm with a peak in the morning, which corresponds to the maximum DNA synthesis in the bone marrow, and it may be useful in the timing of the administration of drugs that block PNB for the treatment of cancer and autoimmune disease.

Serine hydroxymethyltransferase: a key player connecting purine, folate and methionine metabolism in Saccharomyces cerevisiae.

Previous genetic analyses showed phenotypic interactions between 5-amino-4-imidazole carboxamide ribonucleotide 5'-phosphate (AICAR) produced from the purine and histidine pathways and methionine biosynthesis. Here, we revisited the effect of AICAR on methionine requirement due to AICAR accumulation in the presence of the fau1 mutation invalidating folinic acid remobilization. We found that this methionine auxotrophy could be suppressed by overexpression of the methionine synthase Met6 or by deletion of the serine hydroxymethyltransferase gene SHM2. We propose that in a fau1 background, AICAR, by stimulating the transcriptional expression of SHM2, leads to a folinic acid accumulation inhibiting methionine synthesis by Met6. In addition, we uncovered a new methionine auxotrophy for the ade3 bas1 double mutant that can be rescued by overexpressing the SHM2 gene. We propose that methionine auxotrophy in this mutant is the result of a competition for 5,10-methylenetetrahydrofolate between methionine and deoxythymidine monophosphate synthesis. Altogether, our data show intricate genetic interactions between one-carbon units, purine and methionine metabolism through fine-tuning of serine hydroxymethyltransferase by AICAR and the transcription factor Bas1.

Quantification of folate metabolites in serum using ultraperformance liquid chromatography tandem mass spectrometry.

Folate deficiency is considered a risk factor for many diseases such as cancer, congenital heart disease and neural tube defects (NTDs). There is a pressing need for more methods of detecting folate and its main metabolites in the human body. Here, we developed a simple, fast and sensitive ultraperformance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) method for the simultaneous quantifications of folate metabolites including folic acid, 5-methyltetrahydrofolate (5-MeTHF), 5-formyltetrahydrofolate (5-FoTHF), homocysteine (Hcy), S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). The method was validated by determining the linearity (r(2)>0.998), sensitivity (limit of detection ranged from 0.05 to 0.200ng/mL), intra- and inter-day precision (both CV<6%) and recovery (each analyte was >90%). The total analysis time was 7min. Serum samples of NTD-affected pregnancies and controls from a NTD high-risk area in China were analyzed by this method, the NTD serum samples showed lower concentrations of 5-MeTHF (P<0.05) and 5-FoTHF (P<0.05), and higher concentrations of Hcy (P<0.05) and SAH (P<0.05) compared with serum samples from controls, consistent with a previous study. These results showed that the method is sensitive and reliable for simultaneous determination of six metabolites, which might indicate potential risk factors for NTDs, aid early diagnosis and provide more insights into the pathogenesis of NTDs.