The differential translation capabilities of the human DHFR2 gene indicates a developmental and tissue specific endogenous protein of low abundance.

A functional role has been ascribed to the human Dihydrofolate reductase 2 (DHFR2) gene based on the enzymatic activity of recombinant versions of the predicted translated protein. However, the in vivo function is still unclear. The high amino acid sequence identity (92%) between DHFR2 and its parental homologue, DHFR, makes analysis of the endogenous protein challenging. This paper describes a targeted mass spectrometry proteomics approach in several human cell lines and tissue types to identify DHFR2 specific peptides as evidence of its translation. We show definitive evidence that the dihydrofolate reductase activity in the mitochondria is in fact mediated by DHFR, and not DHFR2. Analysis of Ribo-seq data and an experimental assessment of ribosome association using a sucrose cushion, showed that the two main Ensembl annotated mRNA isoforms of DHFR2, 201 and 202, show differential association with the ribosome. This indicates a functional role at both the RNA and protein level. However, we were unable to detect DHFR2 protein at a detectable level in most cell types examined despite various RNA isoforms of DHFR2 being relatively abundant. We did detect a DHFR2 specific peptide in embryonic heart, indicating that the protein may have a specific role during embryogenesis. We propose that the main functionality of the DHFR2 gene in adult cells is likely to arise at the RNA level

The role of mitochondrial folate enzymes in cancer

Folic acid is an essential B vitamin, the metabolism of folic acid via one carbon metabolism results in the production of important components for the cell, such as DNA bases and methyl donor groups. The importance of mitochondrial one carbon metabolism has recently been highlighted with the discovery of the novel enzyme Dihydrofolate Reductase like 1 (DHFRL1) and the association of other mitochondrial enzymes, in particular Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like (MTHFD1L) with rapid proliferation and mortality in cancer. The novel DHFRL1 enzyme has been shown to be a much less active enzyme with a reduced affinity for dihydrofolate relative to DHFR. The MTHFD1L enzyme is responsible for the last step in the production of formate for cytoplasmic one carbon metabolism. Due to MTHFD1L’s associations with rapid proliferation rate in cancer and mortality, a biomarker for its expression is desirable. Formate analysis by Gas chromatography–mass spectrometry (GC-MS) in Human Embryonic Kidney (HEK) 293 cells with modulated MTHFD1L expression demonstrated that knocking down the gene resulted in reduced formate levels and reduced cell growth. Similarly overexpressing the MTHFD1L gene in HEK 293 cells resulted in an increased formate level and growth rate relative to the controls. Investigation was undertaken into the amino acid differences between DHFR and DHFRL1 to begin to understand their functional relevance. It was identified that the arginine at amino acid position 24 may result in DHFRL1 having an altered structure, which may account in part for DHFRL1’s reduced affinity for dihydrofolate. In addition, DHFRL1 and other mitochondrial folate enzymes, MTHFD1L, Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2 (MTHFD2), Serine Hydroxymethyltransferase (mitochondrial isoform) 2 (SHMT2) were found to be up-regulated in a metastatic cancer cell line but their cytosolic paralogues showed no such up-regulation. The results presented provide further evidence of the mitochondrial driven role in cancer progression and are supportive for the use of formate as a biomarker for mitochondrial gene up-regulation