SUMMARY Mammalian folate metabolism is comprised of cytosolic and mitochondrial pathways with nearly identical core reactions, yet the functional advantages of such an organization are not well understood. Using genome-editing and biochemical approaches, we find that ablating folate metabolism in the mitochondria of mammalian cell lines results in folate degradation in the cytosol. Mechanistically, we show that QDPR, an enzyme in tetrahydrobiopterin metabolism, moonlights to repair oxidative damage to tetrahydrofolate (THF). This repair capacity is overwhelmed when cytosolic THF hyperaccumulates in the absence of mitochondrially produced formate, leading to THF degradation. Unexpectedly, we also find that the classic antifolate methotrexate, by inhibiting its well-known target DHFR, causes even more extensive folate degradation in nearly all tested cancer cell lines. These findings shed light on design features of folate metabolism, provide a biochemical basis for clinically observed folate deficiency in QDPR-deficient patients, and reveal a hitherto unknown and unexplored cellular effect of methotrexate.

Type 2 phosphatidylinositol-5-phosphate 4-kinase (PI5P4K) converts phosphatidylinositol-5-phosphate to phosphatidylinositol-4,5-bisphosphate. Mammals have three enzymes PI5P4Kα, PI5P4Kβ, and PI5P4Kγ, and these enzymes have been implicated in metabolic control, growth control, and a variety of stress responses. Here, we show that mice with germline deletion of type 2 phosphatidylinositol-5-phosphate 4-kinase gamma (Pip4k2c), the gene encoding PI5P4Kγ, appear normal in regard to growth and viability but have increased inflammation and T-cell activation as they age. Immune cell infiltrates increased in Pip4k2c −/− mouse tissues. Also, there was an increase in proinflammatory cytokines, including IFNγ, interleukin 12, and interleukin 2 in plasma of Pip4k2c −/− mice. Pip4k2c −/− mice had an increase in T-helper-cell populations and a decrease in regulatory T-cell populations with increased proliferation of T cells. Interestingly, mammalian target of rapamycin complex 1 (mTORC1) signaling was hyperactivated in several tissues from Pip4k2c −/− mice and treating Pip4k2c−/− mice with rapamycin reduced the inflammatory phenotype, resulting in a decrease in mTORC1 signaling in tissues and a decrease in proinflammatory cytokines in plasma. These results indicate that PI5P4Kγ plays a role in the regulation of the immune system via mTORC1 signaling.

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the irreversible conversion of 5,10-methylene-tetrahydrofolate (THF) to 5-methyl-THF, thereby committing one-carbon units to the methionine cycle. While MTHFR has long been known to be allosterically inhibited by S-adenosylmethionine (SAM), only relatively recently has N-terminal multisite phosphorylation been shown to provide an additional layer of regulation. In vitro, the multiply phosphorylated form of MTHFR is more sensitive to allosteric inhibition by SAM. Here we sought to investigate the kinases responsible for MTHFR multisite phosphorylation and the physiological function of MTHFR phosphorylation in cells. We identified DYRK1A/2 and GSK3A/B among the kinases that phosphorylate MTHFR. In addition, we found that MTHFR phosphorylation is maintained by adequate cellular SAM levels, which are sensed through the C-terminal SAM binding domain of MTHFR. To understand the function of MTHFR phosphorylation in cells, we generated MTHFR CRISPR knockin mutant lines that effectively abolished MTHFR phosphorylation and compared them with the parental cell lines. Whereas the parental cell lines showed increased 5-methyl-THF production in response to homocysteine treatment, the knockin cell lines had high basal levels of 5-methyl-THF and did not respond to homocysteine treatment. Overall, our results suggest that MTHFR multisite phosphorylation coordinates with SAM binding to inhibit MTHFR activity in cells.