Evidence for Small Ubiquitin-like Modifier-dependent Nuclear Import of the Thymidylate Biosynthesis Pathway

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.

A UV-responsive Internal Ribosome Entry Site Enhances Serine Hydroxymethyltransferase 1 Expression for DNA Damage Repair

“…1). Nuclear thymidylate biosynthesis is enabled by the SUMO-dependent nuclear import of SHMT1 and TS (12,13). An increase in SUMOylation in response to UV treatment has previously been reported for several proteins involved in cellular stress response including TIP60 (41), xeroderma pigmentosum, complementation group C (42), and DJ-1 (43).…”

“…The concentration of free folate in the cell is negligible, and therefore, TS and methylene-THFR compete for limiting pools of the 5,10-methylene-THF cofactor (5)(6)(7)(8). Several studies have demonstrated that whereas the majority of 5,10-methylene-THF derived from the reduction of 10-formyl-THF is directed toward the synthesis of methionine (9, 10), SHMT1-derived 5,10-methylene-THF is partitioned to TS (11) through the cell cycle-dependent and small ubiquitin-like modifier (SUMO)-mediated nuclear localization of the thymidylate biosynthesis pathway (12,13) that enables the nuclear de novo synthesis of thymidylate (14) (Fig. 1).…”

Cytoplasmic Serine Hydroxymethyltransferase Regulates the Metabolic Partitioning of Methylenetetrahydrofolate but Is Not Essential in Mice

“…1) (1,2). Folate metabolism is compartmentalized in the cytoplasm, mitochondria, and the nucleus (2)(3)(4)(5). In the cytoplasm, folate-activated carbons are incorporated into the 2nd and 8th positions of the purine ring and are required for the conversion of uridylate to thymidylate and for the methylation of homocysteine to methionine.…”

“…Tetrahydrofolates (THF) 3 are present in cells as a family of metabolic cofactors that carry and chemically activate single carbons for a network of biosynthetic pathways referred to as folate-mediated one-carbon metabolism ( Fig. 1) (1,2).…”

“…Formate effluxes to the cytoplasm where it is incorporated into the folate-activated one-carbon pool through the activity of 10-formyl-THF synthetase. Nuclear folate metabolism occurs through the small ubiquitin-related modifier-dependent translocation of the de novo thymidylate synthase pathway from the cytoplasm to the nucleus during S-phase (3,4). SHMT isozymes are present in each of these three compartments.…”