Nuclear Folate Metabolism

Abstract

Despite unequivocal evidence that folate deficiency increases risk for human pathologies, and that folic acid intake among women of childbearing age markedly decreases risk for birth defects, definitive evidence for a causal biochemical pathway linking folate to disease and birth defect etiology remains elusive. The de novo and salvage pathways for thymidylate synthesis translocate to the nucleus of mammalian cells during S- and G2/M-phases of the cell cycle and associate with the DNA replication and repair machinery, which limits uracil misincorporation into DNA and genome instability. There is increasing evidence that impairments in nuclear de novo thymidylate synthesis occur in many pathologies resulting from impairments in one-carbon metabolism. Understanding the roles and regulation of nuclear de novo thymidylate synthesis and its relationship to genome stability will increase our understanding of the fundamental mechanisms underlying folate- and vitamin B₁₂-associated pathologies.

Keywords: DNA synthesis; folate; neural tube defects; replitase; thymidylate.

Figure 1

FOCM is compartmentalized within the cell. FOCM compartmentalization enables efficient metabolic flux of THF-activated one-carbon units to biosynthetic reactions and cycling of THF. One-carbon metabolism in the cytosol includes the remethylation of homocysteine to methionine and the de novo synthesis of purines and thymidylate. Nuclear de novo dTMP biosynthesis requires TYMS, DHFR, MTHFD1, SHMT1, and SHMT2α. These enzymes are SUMOylated and imported into the nucleus at the onset of S-phase. The Shmt2 gene is expressed as two transcripts: SHMT2, for mitochondrial one-carbon metabolism, and SHMT2α, which functions in the cytosol and nucleus. Formate and serine are the primary one-carbon sources for cytosolic and nuclear FOCM. The hydroxymethyl group of serine enters the pool of activated one-carbon units through the SHMT-catalyzed reaction in the cytosol and mitochondria. In mitochondria, serine and glycine are converted to formate, which traverses to the cytosol and nucleus, where it is condensed with THF by MTHFD1. MTHFD1 is a trifunctional enzyme possessing formylTHF synthetase (S), methenylTHF cyclohydrolase (C), and methyleneTHF dehydrogenase (D) activities. 5,10-MethyleneTHF is synthesized by SHMT or MTHFD1 and used for de novo dTMP synthesis. Thymidylate is also produced through the salvage of dT nucleoside by the action of TK1 in the cytosol and nucleus and of TK2 in mitochondria. Abbreviations: AdoHcy, S-adenosylhomocysteine; AdoMet, S-adenosylmethionine; AICAR_Tfase, aminoimidazolecarboxamide ribonucleotide transformylase; DHFR, dihydrofolate reductase; dT, deoxythymidine; dTMP, deoxythymidine monophosphate; dUMP, deoxyuridine monophosphate; FOCM, folate-mediated one-carbon metabolism; GAR_Tfase, glycinamide ribonucleotide tranformylase; MTHFD1, methylenetetrahydrofolate dehydrogenase 1; MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; mtDNA, mitochondrial DNA; SHMT, serine hydroxymethyltransferase; SUMO, small ubiquitin-like modifier; THF, tetrahydrofolate; TK, thymidine kinase; TYMS, thymidylate synthase.

Figure 2

Folate and pathology. Impaired folate-mediated one-carbon metabolism is associated with biomarkers including decreased Met and AdoMet, which leads to chromatin hypomethylation, as well as decreased nucleotide synthesis, which leads to decreased cell division and increased uracil misincorporation into DNA. Impaired folate-mediated one-carbon metabolism is also associated with pathologies including cancer, neurodegenerative disease, and neural tube defects. All of these pathologies are complex traits, making it difficult to understand causal mechanisms that link impaired folate-mediated one-carbon metabolism to pathology. Abbreviations: AdoMet, S-adenosylmethionine; Met, methionine.

Figure 3

The de novo thymidylate synthesis pathway as a nuclear multienzyme complex at sites of DNA replication. SHMT, TYMS, and DHFR are SUMOylated covalently attached to the SUMO protein (red circles) at S-phase after which they translocate to the nucleus and form a multienzyme complex. This multienzyme complex is anchored to nuclear lamin proteins and DNA by SHMT (SHMT1 and/or SHMT2α isozymes). Both serine (through catalytic activity of SHMT1 and/or SHMT2α) and formate (through catalytic activity of MTHFD1) serve as one-carbon sources for the generation of CH₂THF. TYMS catalyzes conversion of dUMP to dTMP, which is converted to dTTP and incorporated into DNA as dT (the T base in DNA). Alternatively, dUMP can be converted to dUTP, which is then incorporated into DNA as deoxyuridine (known as uracil misincorporation). The dUTPase enzyme converts dUTP to dUMP, which both limits dUTP accumulation and provides dUMP substrate for TYMS. Abbreviations: ATP, adenosine triphosphate; DHF, dihydrofolate; DHFR, dihydrofolate reductase; dT, deoxythymidine; dTMP, deoxythymidine monophosphate; dTTP, deoxythymidine triphosphate; dUMP, deoxyuridine monophosphate; dUTP, deoxyuridine triphosphate; MTHFD1, methylenetetrahydrofolate dehydrogenase 1; NADPH, nicotinamide adenine dinucleotide phosphate; SHMT1, cytoplasmic serine hydroxymethyltransferase 1; SUMO, small ubiquitin-like modifier; THF, tetrahydrofolate; TYMS, thymidylate synthase.

Figure 4

Causes and consequences of uracil in DNA. Either dTTP or dUTP can be incorporated into DNA. dUTPase dephosphorylates dUTP into dUMP. TYMS transfers a one-carbon unit onto dUMP, generating dTMP. dTYMK and NDPK subsequently phosphorylate dTMP and dTDP, respectively, generating dTTP. Impaired de novo thymidylate biosynthesis can lead to dUTP incorporation into DNA. Uracil in DNA is excised primarily by the base excision repair pathway, initiated primarily by UNG1 in the mitochondria and UNG2 in the nucleus. Base excision repair generates strand breaks as a repair intermediate. However, if dTTP pools are relatively limited, uracil will be incorporated into DNA again. This constant generation of strand breaks leads to continuous activation of double-strand break repair machinery, triggering apoptosis. Abbreviations: dTDP, deoxythymidine diphosphate; dTMP, deoxythymidine monophosphate; dTTP, deoxythymidine triphosphate; dTYMK, deoxythymidylate kinase; dUMP, deoxyuracil monophosphate; dUTP, deoxyuracil triphosphate; dUTPase, dUTP phosphorylase; NDPK, nucleoside diphosphate kinase; TYMS, thymidylate synthase; UNG1/2, uracil DNA N-glycosylase 1/2.