Abstract
Perturbations in folate-mediated one-carbon metabolism increase rates of uracil misincorporation into DNA during replication, impair cellular methylation reactions, and increase risk for neural tube defects and cancer. One-carbon metabolism is compromised by folate deficiency and common genetic polymorphisms. In this study, the mechanism for the preferential partitioning of cytoplasmic serine hydroxymethyltransferase (cSHMT)-derived methylenetetrahydrofolate to de novo thymidylate biosynthesis was investigated. The cSHMT enzyme was shown to interact with UBC9 and was a substrate for UBC9-catalyzed small ubiquitin-like modifier (SUMO) modification in vitro. SUMOylated cSHMT was detected in extracts from S phase MCF-7 cells, and cSHMT was shown to localize to the nucleus and nuclear periphery during the S and G(2)/M phases of the cell cycle. A common single nucleotide polymorphism (L474F-cSHMT) impaired the UBC9-cSHMT interaction and inhibited cSHMT SUMOylation in vitro. The three folate-dependent enzymes that constitute the de novo thymidylate biosynthesis pathway, cSHMT, thymidylate synthase, and dihydrofolate reductase, all contain SUMO modification consensus sequences. Compartmentation of the folate-dependent de novo thymidylate biosynthesis pathway in the nucleus accounts for the preferential partitioning of cSHMT-derived folate-activated one-carbon units into thymidylate biosynthesis; the efficiency of nuclear folate metabolism is likely to be modified by the cSHMT L474F polymorphism.
Highlights
The term folate refers to a family of enzyme cofactors that carry and chemically activate single carbons at three oxidation states [1, 2]
This competition is most pronounced for the two reactions that utilize methylene-THF: thymidylate synthesis catalyzed by thymidylate synthase (TS) and 5-methyl-THF synthesis catalyzed by methylenetetrahydrofolate reductase (MTHFR) (Fig. 1)
The concept of nuclear folate metabolism was first described by Prem veer Reddy and Pardee [49], who proposed the existence of a nuclear “replitase,” which is a putative multienzyme complex that synthesizes nucleotides de novo at the replication fork during S phase
Summary
MCF-7 mammary adenocarcinoma cells (HTB22) and HeLa cells (CCL2) were obtained from ATCC. Cells were cultured in ␣-minimal essential medium (Hyclone Laboratories) supplemented with 11% fetal calf serum (Hyclone Laboratories) and maintained at 37 °C in a 5% CO2 atmosphere
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