{"gene":"MTHFD1","run_date":"2026-06-10T02:59:51","timeline":{"discoveries":[{"year":1991,"finding":"MTHFD1 (MTHFD) encodes a single trifunctional polypeptide with three sequential catalytic activities: N5,N10-methylenetetrahydrofolate dehydrogenase, N5,N10-methenyltetrahydrofolate cyclohydrolase, and N10-formyltetrahydrofolate synthetase, all required for interconversion of tetrahydrofolate derivatives for purine, thymidylate, and methionine synthesis. In a CHO Ade-E mutant, all three activities were lost together with reduced/absent protein despite normal mRNA, demonstrating the activities reside on a single polypeptide and suggesting a post-transcriptional regulatory mechanism.","method":"Enzymatic activity assays, immunoblotting, immunoprecipitation, Northern blot analysis in CHO Ade-E mutant cells","journal":"Somatic cell and molecular genetics","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct enzymatic assays plus protein-level and mRNA-level orthogonal experiments in a defined mutant cell line demonstrating co-loss of all three activities","pmids":["1887335"],"is_preprint":false},{"year":2004,"finding":"Knockout of the Mthfd1 gene in murine embryonic stem cells eliminates all three cytoplasmic MTHFD1 activities (dehydrogenase, cyclohydrolase, synthetase), causes purine auxotrophy, and reveals a separate mitochondrial monofunctional 10-formyltetrahydrofolate synthetase (encoded by a recently identified mitochondrial transcript). Absence of NADP-dependent dehydrogenase activity in these null cells confirmed the mitochondrial enzyme lacks dehydrogenase/cyclohydrolase activities.","method":"Gene knockout in embryonic stem cells, enzymatic activity assays, subcellular fractionation/localization, Northern blot","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — genetic knockout with multiple enzymatic assays and subcellular fractionation providing multiple orthogonal lines of evidence","pmids":["15611115"],"is_preprint":false},{"year":2008,"finding":"Disruption of the Mthfd1 gene (gene-trap insertion inactivating formyl-THF synthetase/FTHFS activity) causes embryonic lethality in homozygous mice. Heterozygous Mthfd1gt/+ mice show lower hepatic S-adenosylmethionine (indicating formate-derived one carbons contribute to methylation reactions), decreased uracil in nuclear DNA (indicating enhanced thymidylate synthesis when FTHFS is reduced), demonstrating that formate-derived carbons compete with serine-derived carbons for THF cofactors used in thymidylate vs. homocysteine remethylation.","method":"Gene-trap mouse model, metabolite measurements (AdoMet, uracil in DNA), genetic analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic model with multiple metabolic readouts demonstrating pathway-level competition for THF cofactors","pmids":["19033438"],"is_preprint":false},{"year":2009,"finding":"The common MTHFD1 p.Arg653Gln (R653Q) variant reduces enzyme thermostability (36% reduction in half-life at 42°C) without altering substrate affinity. Thermolability is rescued by folate pentaglutamate and Mg-ATP. In murine Mthfd1 knockout cells transfected with the Arg653Gln variant, formate incorporation into DNA (a proxy for de novo purine synthesis) is reduced by 26% compared to wild-type, indicating impaired de novo purine synthesis.","method":"In vitro enzyme activity and stability assays (purified recombinant protein), mammalian cell transfection with formate incorporation into DNA measurement","journal":"Human mutation","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted in vitro enzyme assays plus cellular metabolic flux assay in defined knockout cells, two orthogonal methods in single study","pmids":["18767138"],"is_preprint":false},{"year":2014,"finding":"MTHFD1 translocates to the nucleus during S-phase in MCF-7 and HeLa cells. During folate deficiency, MTHFD1 is enriched >2-fold in the nucleus at the expense of cytosolic levels in mouse liver, and nuclear folate cofactors are maintained when total cellular folate is reduced by >50%. This nuclear enrichment supports de novo thymidylate biosynthesis preferentially over cytosolic homocysteine remethylation during folate deficiency.","method":"Subcellular fractionation, Western blot, cell cycle synchronization (S-phase), mouse dietary folate depletion model","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct fractionation experiments in both cultured cells (S-phase) and mouse liver, replicated across two experimental systems","pmids":["25213861"],"is_preprint":false},{"year":2017,"finding":"Arsenic trioxide (As2O3) increases MTHFD1 SUMOylation (confirmed in cultured cells and in vitro SUMOylation reactions) and promotes MTHFD1 ubiquitination and proteolytic degradation (along with SHMT1). This leads to inhibition of de novo thymidylate biosynthesis, increased uracil misincorporation into nuclear DNA, and genome instability. MTHFD1 and SHMT1 form a multienzyme complex with TYMS and DHFR at the nuclear DNA replication machinery during S-phase.","method":"In vitro SUMOylation assay, immunoprecipitation (Co-IP), Western blot for ubiquitination and protein levels, uracil-in-DNA measurement, genome instability assays in cultured cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro SUMOylation reconstitution plus Co-IP plus functional metabolic readouts, multiple orthogonal methods in single study","pmids":["28265077"],"is_preprint":false},{"year":2017,"finding":"The natural product carolacton inhibits both bacterial FolD and the human orthologs MTHFD1 and MTHFD2 in the low nanomolar range. Crystal structure of the bacterial FolD-carolacton complex reveals the binding mode; carolacton occupies the active site and inhibits the dehydrogenase/cyclohydrolase activities.","method":"Biophysical binding assay, X-ray crystallography of FolD-carolacton complex, in vitro enzymatic inhibition assays for MTHFD1 and MTHFD2","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus in vitro enzymatic inhibition assays demonstrating direct inhibition of human MTHFD1 at its active site","pmids":["29142318"],"is_preprint":false},{"year":2019,"finding":"A fraction of MTHFD1 resides in the nucleus where it is recruited to specific genomic loci by direct physical interaction with BRD4 (bromodomain-containing protein 4). Genetic and pharmacological screens identified this MTHFD1-BRD4 interaction. Inhibition of either BRD4 or MTHFD1 produces similar changes in nuclear metabolite composition and gene expression, and the two inhibitors synergize to impair cancer cell viability in vitro and in vivo.","method":"Complementary genetic interaction screen and physical interaction screen (converging on MTHFD1-BRD4), nuclear localization by fractionation, co-immunoprecipitation, metabolomics, in vitro and in vivo pharmacological studies","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal screens plus Co-IP plus metabolomics plus in vivo validation, multiple orthogonal methods across genetic and biochemical approaches","pmids":["31133746"],"is_preprint":false},{"year":2022,"finding":"PRMT5 directly binds MTHFD1 and symmetrically dimethylates it at arginine R173. Under suspension conditions (anoikis stress), the MTHFD1-PRMT5 interaction is strengthened, elevating R173 symmetric dimethylation, which augments MTHFD1 metabolic activity (NADPH generation), promoting anoikis resistance and distant organ metastasis. Genetic depletion or pharmacological inhibition of PRMT5 reduced tumor metastasis.","method":"CRISPR-Cas9 metabolic enzyme screen, Co-immunoprecipitation, in vitro methylation assay, site-directed mutagenesis (R173), NADPH/NADP+ ratio measurement, xenograft/metastasis models","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Moderate — Co-IP, in vitro methylation, mutagenesis of methylation site, and in vivo functional validation with multiple orthogonal methods","pmids":["35798877"],"is_preprint":false},{"year":2023,"finding":"TH9619, a dual inhibitor of MTHFD1 and MTHFD2 dehydrogenase/cyclohydrolase activities, selectively targets nuclear MTHFD2 but not mitochondrial MTHFD2. Continued mitochondrial formate overflow accumulates 10-formyl-THF downstream of MTHFD1 inhibition, creating a 'folate trap.' This results in thymidylate depletion and selective death of MTHFD2-expressing cancer cells. The trapping is exacerbated by physiological hypoxanthine which blocks de novo purine synthesis and prevents 10-formyl-THF consumption.","method":"Metabolomics (10-formyl-THF accumulation), isotope tracing, selective MTHFD2 nuclear vs. mitochondrial inhibition analysis, cellular viability assays, genetic and pharmacological perturbations","journal":"Nature metabolism","confidence":"High","confidence_rationale":"Tier 1 / Strong — metabolite tracing plus compartment-specific inhibitor studies plus multiple genetic/pharmacological perturbations establishing mechanism","pmids":["37012496"],"is_preprint":false},{"year":2023,"finding":"MTHFD1 negatively regulates retinoic acid receptor γ (RARγ) transcription factor activity. IP-mass spectrometry identified MTHFD1 as a specific RARγ-interacting protein; co-immunoprecipitation and immunofluorescence confirmed the interaction. MTHFD1 knockdown de-repressed RARγ signaling; low MTHFD1 expression and activated RAR signaling were observed in human anencephaly and a retinoic acid-induced NTD mouse model.","method":"Immunoprecipitation-mass spectrometry, co-immunoprecipitation, immunofluorescence confocal microscopy, luciferase reporter assay, ChIP-qPCR, mouse NTD model","journal":"Brain : a journal of neurology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus reporter assay plus ChIP in single study; replication in vivo is indirect (mouse NTD model with correlation not rescue)","pmids":["36928982"],"is_preprint":false},{"year":2023,"finding":"The MTHFD1 G1958A (R653Q) SNP reduces protein stability through ubiquitination-mediated proteasomal degradation. The R653Q variant shows enhanced binding to the E3 ubiquitin ligase TRIM21 compared to wild-type; lysine K504 is the primary ubiquitination site. R653Q expression results in reduced serine-derived methyl flux into purine synthesis precursors (confirmed by metabolic flux analysis) and impaired tumor growth in xenograft models.","method":"Immunoprecipitation for ubiquitination, mass spectrometry identification of ubiquitination site and interacting proteins, metabolic flux analysis with serine isotope, xenograft tumor growth assay","journal":"Cellular oncology (Dordrecht, Netherlands)","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — Co-IP for E3 ligase interaction, mass spectrometry site identification, metabolic flux; single lab, moderate replication","pmids":["36913067"],"is_preprint":false},{"year":2021,"finding":"Genome-wide CRISPR and RNAi screens in bat cells identified MTHFD1 as required for viral replication (mumps virus, influenza A, SARS-CoV-2). The MTHFD1 inhibitor carolacton potently blocked replication of several RNA viruses including SARS-CoV-2, demonstrating that MTHFD1 enzymatic activity supports viral replication in both bat and human cells.","method":"Genome-wide CRISPR and RNAi library screens, carolacton pharmacological inhibition, viral replication assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — orthogonal genetic screens (CRISPR + RNAi) converging on same target, plus pharmacological validation in multiple viral contexts","pmids":["34544865"],"is_preprint":false},{"year":2020,"finding":"MTHFD1 protein expression is markedly reduced (to 4.8–14.3% of control values) and methylenetetrahydrofolate dehydrogenase specific activity is undetectable in fibroblasts from MTHFD1-deficient patients bearing compound heterozygous MTHFD1 mutations, confirming that patient mutations abolish enzymatic function.","method":"Western blot for MTHFD1 protein expression in patient fibroblasts, enzymatic activity assay for dehydrogenase activity","journal":"Molecular genetics and metabolism","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — direct enzymatic measurement and protein quantification in patient-derived cells, single study with multiple patients","pmids":["32414565"],"is_preprint":false},{"year":2015,"finding":"Patient fibroblasts with MTHFD1 deficiency show severely reduced methionine formation from [14C]-formate (a direct measure of formyl-THF synthetase activity feeding homocysteine remethylation), which did not improve with cobalamin supplementation but was responsive to folic and folinic acid treatment, indicating that MTHFD1 is required for formate entry into folate metabolism for methionine synthesis.","method":"Radiolabeled formate ([14C]-formate) metabolic flux assay in patient fibroblasts, supplementation experiments","journal":"Journal of inherited metabolic disease","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — direct isotope tracing in patient-derived cells with pharmacological rescue, limited to single lab","pmids":["25633902"],"is_preprint":false},{"year":2024,"finding":"MTHFD1 maintains NADPH/NADP+ and GSH/GSSG redox homeostasis in MYCN-amplified neuroblastoma. MYCN directly activates MTHFD1 transcription (confirmed by ChIP-qPCR and dual-luciferase reporter assay). Knockdown of MTHFD1 reduces NADPH/NADP+ and GSH/GSSG ratios, increases ROS, and triggers apoptosis.","method":"ChIP-qPCR, dual-luciferase reporter assay, MTHFD1 knockdown, NADPH/NADP+ and GSH/GSSG ratio measurements, ROS measurement, mouse xenograft model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and reporter assay for transcriptional regulation plus metabolic readouts; single lab","pmids":["38336749"],"is_preprint":false},{"year":2024,"finding":"MTHFD1 regulates autophagy via the PI3K-AKT-mTOR signaling pathway in colorectal cancer cells. MTHFD1 overexpression suppresses autophagy and activates PI3K-AKT-mTOR, promoting proliferation and metastasis, while MTHFD1 knockdown increases autophagy and suppresses these phenotypes. Validated in xenograft models.","method":"Western blot for PI3K-AKT-mTOR pathway and autophagy markers, Transwell invasion assay, xenograft tumor models","journal":"Cancer medicine","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, Western blot pathway readout with overexpression/knockdown; no direct biochemical mechanism linking MTHFD1 enzymatic activity to PI3K-AKT-mTOR established","pmids":["39571599"],"is_preprint":false},{"year":2024,"finding":"METTL3-mediated m6A modification controls MTHFD1 mRNA stability in erythroid cells. Mettl3 deletion reduces Mthfd1 expression, causing nucleotide (dTMP and IMP) shortage, DNA damage, and apoptosis in erythroid progenitors. Re-introduction or rescue of MTHFD1 activity reverses the genome instability phenotype.","method":"m6A-seq and RNA-seq integration, Western blot, nucleotide measurement (dTMP and IMP), DNA damage assays, erythroid-specific Mettl3 knockout mouse (EpoR-Cre)","journal":"The Journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — integrated multi-omic approach plus genetic model with direct metabolic measurements; single lab","pmids":["41805631"],"is_preprint":false},{"year":2024,"finding":"MTHFD1 protein interacts with MTHFR (methylenetetrahydrofolate reductase) as identified by affinity purification-mass spectrometry and confirmed by immunoprecipitation-immunoblotting. An AlphaFold3 model places the MTHFD1 dehydrogenase/cyclohydrolase domain in direct contact with the MTHFR catalytic domain, suggesting substrate (methylenetetrahydrofolate) channeling between the two enzymes.","method":"Affinity purification-mass spectrometry (AP-MS), immunoprecipitation-immunoblotting, AlphaFold3 structural modeling","journal":"Biochimie","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP confirmed by single lab; structural model is computational prediction; no direct channeling experiment performed","pmids":["39571719"],"is_preprint":false},{"year":2025,"finding":"Genetic knockout and chemical degradation of NUDT5 revealed that NUDT5 interacts with PPAT (the rate-limiting enzyme of de novo purine synthesis) to repress the pathway in response to elevated purine levels. This NUDT5-PPAT scaffolding role (non-enzymatic) mediates sensitivity to adenosine in MTHFD1 deficiency, placing NUDT5 downstream of MTHFD1 in regulating purine de novo synthesis.","method":"CRISPR knockout, selective NUDT5 chemical degrader (PROTAC-type), genetic epistasis in MTHFD1 mutant background, interaction studies (NUDT5-PPAT)","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 2 / Weak — preprint; genetic epistasis with MTHFD1 deficiency establishes pathway placement but is a preprint and the direct MTHFD1 mechanism is secondary to NUDT5 findings","pmids":[],"is_preprint":true},{"year":2024,"finding":"RIPK4 (driven by TP53 mutations) phosphorylates MTHFD1, boosting NADPH production, reducing ROS, and promoting resistance to PANoptosis and metastasis in colorectal cancer.","method":"Phosphorylation assay (RIPK4-MTHFD1), NADPH measurement, ROS measurement, metastasis models in CRC","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 2 / Weak — preprint; mechanistic claim based on phosphorylation assay and NADPH/ROS readouts but not yet peer-reviewed","pmids":[],"is_preprint":true}],"current_model":"MTHFD1 encodes a cytoplasmic trifunctional enzyme (methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase, and formyltetrahydrofolate synthetase) that generates and interconverts folate-activated one-carbon units for de novo purine and thymidylate synthesis and homocysteine remethylation; a fraction of the protein translocates to the nucleus during S-phase (enriched further under folate deficiency) to support a nuclear thymidylate synthesis complex with SHMT, TYMS, and DHFR, and MTHFD1 is subject to multiple post-translational controls including PRMT5-mediated R173 symmetric dimethylation (enhancing NADPH output and anoikis resistance), TRIM21-mediated ubiquitination at K504 (accelerated by the R653Q variant), arsenic trioxide-induced SUMOylation and degradation, and RIPK4-mediated phosphorylation; it also interacts directly with BRD4 in the nucleus to couple folate metabolism to transcriptional regulation, and with MTHFR to potentially channel methylenetetrahydrofolate, while being transcriptionally activated by MYCN and post-transcriptionally stabilized by METTL3-dependent m6A modification."},"narrative":{"mechanistic_narrative":"MTHFD1 encodes a single cytoplasmic trifunctional polypeptide carrying N5,N10-methylenetetrahydrofolate dehydrogenase, N5,N10-methenyltetrahydrofolate cyclohydrolase, and N10-formyltetrahydrofolate synthetase activities that interconvert tetrahydrofolate-activated one-carbon units for de novo purine, thymidylate, and methionine synthesis [PMID:1887335, PMID:15611115]. Genetic disruption causes purine auxotrophy in stem cells and embryonic lethality in mice, with heterozygotes showing competition between formate-derived and serine-derived one-carbon units for THF cofactors used in thymidylate synthesis versus homocysteine remethylation [PMID:15611115, PMID:19033438]. A fraction of MTHFD1 translocates to the nucleus during S-phase—an enrichment intensified under folate deficiency—where it assembles with SHMT1, TYMS, and DHFR at the replication machinery to support de novo thymidylate biosynthesis and limit uracil misincorporation, and is also recruited to specific genomic loci through direct interaction with BRD4 to couple folate metabolism to transcriptional output [PMID:25213861, PMID:28265077, PMID:31133746]. The enzyme is governed by multiple post-translational controls: PRMT5-mediated symmetric dimethylation at R173 augments NADPH output and anoikis resistance [PMID:35798877], and TRIM21-dependent ubiquitination at K504—accelerated by the common R653Q variant—drives proteasomal degradation and reduces serine-derived methyl flux into purines [PMID:36913067]. MTHFD1 sustains NADPH/NADP+ and GSH/GSSG redox balance and is a direct transcriptional target of MYCN in neuroblastoma [PMID:38336749], while its mRNA is stabilized by METTL3-dependent m6A modification to maintain nucleotide supply in erythroid progenitors [PMID:41805631]. Compound heterozygous MTHFD1 mutations that abolish enzyme protein and dehydrogenase activity cause a human inborn error of folate metabolism with defective methionine formation from formate [PMID:32414565, PMID:25633902]. MTHFD1 enzymatic activity is also exploited to support replication of multiple RNA viruses and is inhibited at its active site by the natural product carolacton [PMID:29142318, PMID:34544865].","teleology":[{"year":1991,"claim":"Established that the three sequential folate-interconverting activities reside on a single polypeptide rather than separate proteins, defining MTHFD1 as a trifunctional enzyme and hinting at post-transcriptional control.","evidence":"Enzymatic assays, immunoblotting and Northern blot in a CHO Ade-E mutant showing co-loss of all three activities with normal mRNA","pmids":["1887335"],"confidence":"High","gaps":["Did not resolve domain architecture or how the three active sites are spatially organized","Mechanism of the inferred post-transcriptional regulation not defined"]},{"year":2004,"claim":"Genetic ablation confirmed MTHFD1 carries all three cytoplasmic activities and caused purine auxotrophy, while distinguishing it from a separate monofunctional mitochondrial formyltetrahydrofolate synthetase.","evidence":"Mthfd1 knockout in murine embryonic stem cells with enzymatic assays and subcellular fractionation","pmids":["15611115"],"confidence":"High","gaps":["Did not address tissue-specific or developmental requirements","Compartmental division of labor between cytosol and mitochondria left to later work"]},{"year":2008,"claim":"Demonstrated at the organismal level that MTHFD1-generated one-carbon units are partitioned between thymidylate synthesis and methylation, with formate-derived carbons competing with serine-derived carbons for THF cofactors.","evidence":"Gene-trap Mthfd1 mouse model with hepatic AdoMet and nuclear DNA uracil measurements","pmids":["19033438"],"confidence":"High","gaps":["Did not identify the molecular determinants of flux partitioning","Mechanism of homozygous embryonic lethality not dissected"]},{"year":2014,"claim":"Revealed that MTHFD1 is not constitutively cytosolic but translocates to the nucleus during S-phase and is further enriched there under folate deficiency, prioritizing thymidylate synthesis over homocysteine remethylation.","evidence":"Subcellular fractionation and cell-cycle synchronization in MCF-7/HeLa plus a folate-depletion mouse liver model","pmids":["25213861"],"confidence":"High","gaps":["Nuclear import signal and translocation machinery not identified","How nuclear folate cofactor pools are maintained unresolved"]},{"year":2017,"claim":"Defined the nuclear thymidylate synthesis complex and showed it is a regulated, degradable node, with arsenic trioxide driving MTHFD1 SUMOylation and ubiquitin-dependent degradation that destabilizes the genome.","evidence":"In vitro SUMOylation, Co-IP for the MTHFD1/SHMT1/TYMS/DHFR complex, ubiquitination and uracil-in-DNA assays in cultured cells","pmids":["28265077"],"confidence":"High","gaps":["SUMO/ubiquitin sites and responsible E3 ligase not pinpointed in this study","Stoichiometry and architecture of the nuclear multienzyme complex undefined"]},{"year":2017,"claim":"Provided direct structural and biochemical evidence that MTHFD1's dehydrogenase/cyclohydrolase active site is druggable, via low-nanomolar inhibition by carolacton.","evidence":"X-ray crystallography of a bacterial FolD-carolacton complex with in vitro enzymatic inhibition of human MTHFD1 and MTHFD2","pmids":["29142318"],"confidence":"High","gaps":["No crystal structure of human MTHFD1 with the inhibitor","Selectivity between MTHFD1 and MTHFD2 not resolved"]},{"year":2019,"claim":"Connected MTHFD1's nuclear pool to transcriptional control by identifying a direct BRD4 interaction that recruits the enzyme to chromatin and couples folate metabolism to gene expression.","evidence":"Converging genetic and physical interaction screens, Co-IP, nuclear metabolomics and in vivo synergy of BRD4/MTHFD1 inhibitors","pmids":["31133746"],"confidence":"High","gaps":["Interaction interface and chromatin-recruitment mechanism not mapped","Whether enzymatic activity or scaffolding drives transcriptional effects unclear"]},{"year":2022,"claim":"Showed MTHFD1 is functionally tuned by arginine methylation, with PRMT5-mediated R173 symmetric dimethylation boosting NADPH output to confer anoikis resistance and metastasis.","evidence":"CRISPR screen, Co-IP, in vitro methylation, R173 mutagenesis, NADPH ratio measurement and metastasis xenografts","pmids":["35798877"],"confidence":"High","gaps":["How R173 methylation structurally alters catalytic output not defined","Demethylase counter-regulation unknown"]},{"year":2023,"claim":"Identified TRIM21 as the E3 ligase ubiquitinating MTHFD1 at K504 and explained how the common R653Q variant destabilizes the protein and reduces purine biosynthetic flux.","evidence":"Co-IP, mass spectrometry site mapping, serine isotope flux analysis and xenograft growth assays","pmids":["36913067"],"confidence":"Medium","gaps":["Single-lab finding without reciprocal validation of the TRIM21 interaction","Signals controlling TRIM21-MTHFD1 engagement not defined"]},{"year":2023,"claim":"Established a noncanonical role for MTHFD1 in repressing RARγ transcriptional activity, linking folate enzyme loss to neural tube defect pathology.","evidence":"IP-mass spectrometry, Co-IP, luciferase reporter and ChIP-qPCR with a retinoic acid-induced NTD mouse model","pmids":["36928982"],"confidence":"Medium","gaps":["In vivo link is correlative rather than rescue-based","Whether repression depends on enzymatic activity or physical binding unresolved"]},{"year":2023,"claim":"Demonstrated that pharmacological MTHFD1/MTHFD2 dehydrogenase inhibition creates a nuclear '10-formyl-THF folate trap' that selectively kills MTHFD2-expressing cancer cells through thymidylate depletion.","evidence":"Metabolomics, isotope tracing and compartment-specific inhibitor (TH9619) studies","pmids":["37012496"],"confidence":"High","gaps":["Determinants of nuclear versus mitochondrial inhibitor selectivity incompletely defined","Generality across tumor types not established"]},{"year":2024,"claim":"Placed MTHFD1 in redox homeostasis and oncogenic transcriptional circuitry, showing MYCN directly drives MTHFD1 expression to maintain NADPH and GSH pools and prevent apoptosis.","evidence":"ChIP-qPCR, dual-luciferase reporter, knockdown with NADPH/GSH/ROS measurements and xenografts","pmids":["38336749"],"confidence":"Medium","gaps":["Direct biochemical link between MTHFD1 catalysis and the measured redox ratios not isolated","Single-lab finding"]},{"year":2024,"claim":"Showed MTHFD1 abundance is set post-transcriptionally by METTL3-dependent m6A modification, gating nucleotide supply and genome stability in erythroid progenitors.","evidence":"m6A-seq/RNA-seq integration, erythroid-specific Mettl3 knockout mouse, nucleotide measurements and rescue","pmids":["41805631"],"confidence":"Medium","gaps":["m6A reader mediating MTHFD1 mRNA stabilization not identified","Tissue specificity of this regulation untested"]},{"year":2020,"claim":"Confirmed in patient-derived cells that disease-causing MTHFD1 mutations abolish protein and dehydrogenase activity, establishing MTHFD1 deficiency as a human inborn error of folate metabolism.","evidence":"Western blot and dehydrogenase activity assays in fibroblasts from compound heterozygous patients; complemented by [14C]-formate methionine-formation flux assays","pmids":["32414565","25633902"],"confidence":"Medium","gaps":["Genotype-phenotype relationships across mutation classes incomplete","Tissue-level pathophysiology beyond fibroblasts not addressed"]},{"year":2021,"claim":"Defined MTHFD1 as a host dependency factor whose enzymatic activity supports replication of multiple RNA viruses, nominating it as an antiviral target.","evidence":"Genome-wide CRISPR and RNAi screens in bat cells with carolacton inhibition across mumps, influenza A and SARS-CoV-2","pmids":["34544865"],"confidence":"Medium","gaps":["Which one-carbon outputs the viruses require not dissected","Direct dependence on individual MTHFD1 activities not separated"]},{"year":null,"claim":"How MTHFD1's enzymatic and scaffolding functions are integrated with its partner enzymes and signaling inputs—including direct substrate channeling to MTHFR and the regulatory consequences of RIPK4 phosphorylation—remains unresolved.","evidence":"MTHFD1-MTHFR Co-IP with AlphaFold3 modeling and RIPK4 phosphorylation/NADPH readouts are preliminary and not reconstituted","pmids":[],"confidence":"Low","gaps":["No direct experimental demonstration of methylenetetrahydrofolate channeling between MTHFD1 and MTHFR","RIPK4 phosphorylation site and mechanism in MTHFD1 not mapped (preprint)","Integration of nuclear scaffolding, redox, and metabolic roles into a unified regulatory logic absent"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[0,1,3,13]},{"term_id":"GO:0016829","term_label":"lyase activity","supporting_discovery_ids":[0]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1,14]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,1,4]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[4,5,7]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,1,2]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[7,10,15]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[17]}],"complexes":["nuclear thymidylate synthesis complex (MTHFD1-SHMT1-TYMS-DHFR)"],"partners":["BRD4","PRMT5","TRIM21","MTHFR","SHMT1","TYMS","DHFR","RARG"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P11586","full_name":"C-1-tetrahydrofolate synthase, cytoplasmic","aliases":["Epididymis secretory sperm binding protein"],"length_aa":935,"mass_kda":101.5,"function":"Trifunctional enzyme that catalyzes the interconversion of three forms of one-carbon-substituted tetrahydrofolate: (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate, 5,10-methenyltetrahydrofolate and (6S)-10-formyltetrahydrofolate (PubMed:10828945, PubMed:18767138, PubMed:1881876). These derivatives of tetrahydrofolate are differentially required in nucleotide and amino acid biosynthesis, (6S)-10-formyltetrahydrofolate being required for purine biosynthesis while (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate is used for serine and methionine biosynthesis for instance (PubMed:18767138, PubMed:25633902)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/P11586/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MTHFD1","classification":"Not Classified","n_dependent_lines":379,"n_total_lines":1208,"dependency_fraction":0.31374172185430466},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"ACTR2","stoichiometry":0.2},{"gene":"PSPC1","stoichiometry":0.2},{"gene":"RPS16","stoichiometry":0.2},{"gene":"SAR1B","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/MTHFD1","total_profiled":1310},"omim":[{"mim_id":"617780","title":"COMBINED IMMUNODEFICIENCY AND MEGALOBLASTIC ANEMIA WITH OR WITHOUT HYPERHOMOCYSTEINEMIA; CIMAH","url":"https://www.omim.org/entry/617780"},{"mim_id":"611427","title":"METHYLENETETRAHYDROFOLATE DEHYDROGENASE 1-LIKE, NADP(+)-DEPENDENT; MTHFD1L","url":"https://www.omim.org/entry/611427"},{"mim_id":"608749","title":"BROMODOMAIN-CONTAINING PROTEIN 4; BRD4","url":"https://www.omim.org/entry/608749"},{"mim_id":"604887","title":"METHYLENETETRAHYDROFOLATE DEHYDROGENASE 2; MTHFD2","url":"https://www.omim.org/entry/604887"},{"mim_id":"601634","title":"NEURAL TUBE DEFECTS, FOLATE-SENSITIVE; NTDFS","url":"https://www.omim.org/entry/601634"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Cytosol","reliability":"Enhanced"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"liver","ntpm":318.5}],"url":"https://www.proteinatlas.org/search/MTHFD1"},"hgnc":{"alias_symbol":[],"prev_symbol":["MTHFC","MTHFD"]},"alphafold":{"accession":"P11586","domains":[{"cath_id":"3.40.50.300","chopping":"320-444_618-808","consensus_level":"medium","plddt":94.9084,"start":320,"end":808},{"cath_id":"3.30.1510.10","chopping":"447-616","consensus_level":"medium","plddt":95.005,"start":447,"end":616},{"cath_id":"3.10.410.10","chopping":"823-905","consensus_level":"high","plddt":95.7933,"start":823,"end":905}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P11586","model_url":"https://alphafold.ebi.ac.uk/files/AF-P11586-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P11586-F1-predicted_aligned_error_v6.png","plddt_mean":94.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MTHFD1","jax_strain_url":"https://www.jax.org/strain/search?query=MTHFD1"},"sequence":{"accession":"P11586","fasta_url":"https://rest.uniprot.org/uniprotkb/P11586.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P11586/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P11586"}},"corpus_meta":[{"pmid":"14647408","id":"PMC_14647408","title":"Role of polymorphisms in MTHFR and MTHFD1 genes in the outcome of childhood acute lymphoblastic leukemia.","date":"2004","source":"The pharmacogenomics journal","url":"https://pubmed.ncbi.nlm.nih.gov/14647408","citation_count":136,"is_preprint":false},{"pmid":"9611072","id":"PMC_9611072","title":"Molecular genetic analysis of the gene encoding the trifunctional enzyme MTHFD (methylenetetrahydrofolate-dehydrogenase, methenyltetrahydrofolate-cyclohydrolase, formyltetrahydrofolate synthetase) in patients with neural tube defects.","date":"1998","source":"Clinical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/9611072","citation_count":124,"is_preprint":false},{"pmid":"18767138","id":"PMC_18767138","title":"The MTHFD1 p.Arg653Gln variant alters enzyme function and increases risk for congenital heart defects.","date":"2009","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/18767138","citation_count":88,"is_preprint":false},{"pmid":"19033438","id":"PMC_19033438","title":"Mthfd1 is an essential gene in mice and alters biomarkers of impaired one-carbon metabolism.","date":"2008","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19033438","citation_count":74,"is_preprint":false},{"pmid":"31133746","id":"PMC_31133746","title":"MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation.","date":"2019","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/31133746","citation_count":71,"is_preprint":false},{"pmid":"29142318","id":"PMC_29142318","title":"The natural product carolacton inhibits folate-dependent C1 metabolism by targeting FolD/MTHFD.","date":"2017","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/29142318","citation_count":67,"is_preprint":false},{"pmid":"25213861","id":"PMC_25213861","title":"Nuclear enrichment of folate cofactors and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) protect de novo thymidylate biosynthesis during folate deficiency.","date":"2014","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25213861","citation_count":59,"is_preprint":false},{"pmid":"14632302","id":"PMC_14632302","title":"Genotype frequencies and linkage disequilibrium in the CEPH human diversity panel for variants in folate pathway genes MTHFR, MTHFD, MTRR, RFC1, and GCP2.","date":"2003","source":"Birth defects research. Part A, Clinical and molecular teratology","url":"https://pubmed.ncbi.nlm.nih.gov/14632302","citation_count":56,"is_preprint":false},{"pmid":"17691219","id":"PMC_17691219","title":"Oxidative DNA damage and level of thiols as related to polymorphisms of MTHFR, MTR, MTHFD1 in Alzheimer's and Parkinson's diseases.","date":"2007","source":"Acta neurobiologiae experimentalis","url":"https://pubmed.ncbi.nlm.nih.gov/17691219","citation_count":55,"is_preprint":false},{"pmid":"21813566","id":"PMC_21813566","title":"Novel inborn error of folate metabolism: identification by exome capture and sequencing of mutations in the MTHFD1 gene in a single proband.","date":"2011","source":"Journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/21813566","citation_count":51,"is_preprint":false},{"pmid":"17438114","id":"PMC_17438114","title":"Polymorphisms of MTHFD, plasma homocysteine levels, and risk of gastric cancer in a high-risk Chinese population.","date":"2007","source":"Clinical cancer research : an official journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/17438114","citation_count":47,"is_preprint":false},{"pmid":"37012496","id":"PMC_37012496","title":"Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells.","date":"2023","source":"Nature metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/37012496","citation_count":46,"is_preprint":false},{"pmid":"16123074","id":"PMC_16123074","title":"A polymorphism in the MTHFD1 gene increases a mother's risk of having an unexplained second trimester pregnancy loss.","date":"2005","source":"Molecular human reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/16123074","citation_count":46,"is_preprint":false},{"pmid":"15611115","id":"PMC_15611115","title":"Disruption of the mthfd1 gene reveals a monofunctional 10-formyltetrahydrofolate synthetase in mammalian mitochondria.","date":"2004","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15611115","citation_count":44,"is_preprint":false},{"pmid":"23296427","id":"PMC_23296427","title":"Severe combined immunodeficiency resulting from mutations in MTHFD1.","date":"2013","source":"Pediatrics","url":"https://pubmed.ncbi.nlm.nih.gov/23296427","citation_count":42,"is_preprint":false},{"pmid":"24532985","id":"PMC_24532985","title":"Homocysteine Level and Mechanisms of Injury in Parkinson's Disease as Related to MTHFR, MTR, and MTHFD1 Genes Polymorphisms and L-Dopa Treatment.","date":"2013","source":"Current genomics","url":"https://pubmed.ncbi.nlm.nih.gov/24532985","citation_count":35,"is_preprint":false},{"pmid":"25633902","id":"PMC_25633902","title":"Characterization and review of MTHFD1 deficiency: four new patients, cellular delineation and response to folic and folinic acid treatment.","date":"2015","source":"Journal of inherited metabolic disease","url":"https://pubmed.ncbi.nlm.nih.gov/25633902","citation_count":33,"is_preprint":false},{"pmid":"28265077","id":"PMC_28265077","title":"Arsenic trioxide targets MTHFD1 and SUMO-dependent nuclear de novo thymidylate biosynthesis.","date":"2017","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/28265077","citation_count":32,"is_preprint":false},{"pmid":"34544865","id":"PMC_34544865","title":"Orthogonal genome-wide screens of bat cells identify MTHFD1 as a target of broad antiviral therapy.","date":"2021","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/34544865","citation_count":32,"is_preprint":false},{"pmid":"35798877","id":"PMC_35798877","title":"Arginine methylation of MTHFD1 by PRMT5 enhances anoikis resistance and cancer metastasis.","date":"2022","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/35798877","citation_count":31,"is_preprint":false},{"pmid":"26853819","id":"PMC_26853819","title":"MTHFD1 regulates nuclear de novo thymidylate biosynthesis and genome stability.","date":"2016","source":"Biochimie","url":"https://pubmed.ncbi.nlm.nih.gov/26853819","citation_count":31,"is_preprint":false},{"pmid":"23685927","id":"PMC_23685927","title":"Polymorphisms in MTHFR, MTHFD, and PAI-1 and recurrent miscarriage among North Indian women.","date":"2013","source":"Archives of gynecology and obstetrics","url":"https://pubmed.ncbi.nlm.nih.gov/23685927","citation_count":30,"is_preprint":false},{"pmid":"22378735","id":"PMC_22378735","title":"Maternal Mthfd1 disruption impairs fetal growth but does not cause neural tube defects in mice.","date":"2012","source":"The American journal of clinical nutrition","url":"https://pubmed.ncbi.nlm.nih.gov/22378735","citation_count":27,"is_preprint":false},{"pmid":"19130090","id":"PMC_19130090","title":"Analysis of the MTHFD1 promoter and risk of neural tube defects.","date":"2009","source":"Human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/19130090","citation_count":26,"is_preprint":false},{"pmid":"21156972","id":"PMC_21156972","title":"Mthfd1 is a modifier of chemically induced intestinal carcinogenesis.","date":"2010","source":"Carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/21156972","citation_count":26,"is_preprint":false},{"pmid":"21543238","id":"PMC_21543238","title":"MTHFR, MTR, and MTHFD1 gene polymorphisms compared to homocysteine and asymmetric dimethylarginine concentrations and their metabolites in epileptic patients treated with antiepileptic drugs.","date":"2011","source":"Seizure","url":"https://pubmed.ncbi.nlm.nih.gov/21543238","citation_count":25,"is_preprint":false},{"pmid":"31377316","id":"PMC_31377316","title":"Mitochondrial MTHFD isozymes display distinct expression, regulation, and association with cancer.","date":"2019","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/31377316","citation_count":24,"is_preprint":false},{"pmid":"17417062","id":"PMC_17417062","title":"MTHFD 1958G>A and MTR 2756A>G polymorphisms are associated with bipolar disorder and schizophrenia.","date":"2007","source":"Psychiatric genetics","url":"https://pubmed.ncbi.nlm.nih.gov/17417062","citation_count":24,"is_preprint":false},{"pmid":"27707701","id":"PMC_27707701","title":"Moderate folic acid supplementation and MTHFD1-synthetase deficiency in mice, a model for the R653Q variant, result in embryonic defects and abnormal placental development.","date":"2016","source":"The American journal of clinical nutrition","url":"https://pubmed.ncbi.nlm.nih.gov/27707701","citation_count":23,"is_preprint":false},{"pmid":"24977710","id":"PMC_24977710","title":"Association between MTHFD1 G1958A polymorphism and neural tube defects susceptibility: a meta-analysis.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24977710","citation_count":20,"is_preprint":false},{"pmid":"23190757","id":"PMC_23190757","title":"Reduced MTHFD1 activity in male mice perturbs folate- and choline-dependent one-carbon metabolism as well as transsulfuration.","date":"2012","source":"The Journal of nutrition","url":"https://pubmed.ncbi.nlm.nih.gov/23190757","citation_count":19,"is_preprint":false},{"pmid":"28968444","id":"PMC_28968444","title":"One-carbon genetic variants and the role of MTHFD1 1958G>A in liver and colon cancer risk according to global DNA methylation.","date":"2017","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/28968444","citation_count":18,"is_preprint":false},{"pmid":"21630102","id":"PMC_21630102","title":"MTHFD1 G1958A, BHMT G742A, TC2 C776G and TC2 A67G polymorphisms and head and neck squamous cell carcinoma risk.","date":"2011","source":"Molecular biology reports","url":"https://pubmed.ncbi.nlm.nih.gov/21630102","citation_count":16,"is_preprint":false},{"pmid":"27707659","id":"PMC_27707659","title":"Precision Molecular Diagnosis Defines Specific Therapy in Combined Immunodeficiency with Megaloblastic Anemia Secondary to MTHFD1 Deficiency.","date":"2016","source":"The journal of allergy and clinical immunology. In practice","url":"https://pubmed.ncbi.nlm.nih.gov/27707659","citation_count":16,"is_preprint":false},{"pmid":"18261183","id":"PMC_18261183","title":"The MTHFD1 gene is not involved in cleft lip with or without palate onset among the Italian population.","date":"2008","source":"Annals of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/18261183","citation_count":14,"is_preprint":false},{"pmid":"28865601","id":"PMC_28865601","title":"Relationship of the MTHFD1 (rs2236225), eNOS (rs1799983), CBS (rs2850144) and ACE (rs4343) gene polymorphisms in a population of Iranian pediatric patients with congenital heart defects.","date":"2017","source":"The Kaohsiung journal of medical sciences","url":"https://pubmed.ncbi.nlm.nih.gov/28865601","citation_count":14,"is_preprint":false},{"pmid":"26408344","id":"PMC_26408344","title":"MTHFD1 formyltetrahydrofolate synthetase deficiency, a model for the MTHFD1 R653Q variant, leads to congenital heart defects in mice.","date":"2015","source":"Birth defects research. Part A, Clinical and molecular teratology","url":"https://pubmed.ncbi.nlm.nih.gov/26408344","citation_count":14,"is_preprint":false},{"pmid":"37630697","id":"PMC_37630697","title":"Association of Maternal Folate Intake and Offspring MTHFD1 and MTHFD2 Genes with Congenital Heart Disease.","date":"2023","source":"Nutrients","url":"https://pubmed.ncbi.nlm.nih.gov/37630697","citation_count":13,"is_preprint":false},{"pmid":"35693982","id":"PMC_35693982","title":"The Emerging Role of MTHFD Family Genes in Regulating the Tumor Immunity of Oral Squamous Cell Carcinoma.","date":"2022","source":"Journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/35693982","citation_count":13,"is_preprint":false},{"pmid":"25524527","id":"PMC_25524527","title":"Association between MTHFD1 polymorphisms and neural tube defect susceptibility.","date":"2014","source":"Journal of the neurological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/25524527","citation_count":12,"is_preprint":false},{"pmid":"35100977","id":"PMC_35100977","title":"Association of MTHFD1 gene polymorphisms and maternal smoking with risk of congenital heart disease: a hospital-based case-control study.","date":"2022","source":"BMC pregnancy and childbirth","url":"https://pubmed.ncbi.nlm.nih.gov/35100977","citation_count":12,"is_preprint":false},{"pmid":"21254748","id":"PMC_21254748","title":"Methylenetetrahydrofolate dehydrogenase (MTHFD) enzyme polymorphism as a maternal risk factor for trisomy 21: a clinical study.","date":"2010","source":"Journal of medicine and life","url":"https://pubmed.ncbi.nlm.nih.gov/21254748","citation_count":12,"is_preprint":false},{"pmid":"38336749","id":"PMC_38336749","title":"MTHFD1 regulates the NADPH redox homeostasis in MYCN-amplified neuroblastoma.","date":"2024","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/38336749","citation_count":11,"is_preprint":false},{"pmid":"28734179","id":"PMC_28734179","title":"Computational analysis for the determination of deleterious nsSNPs in human MTHFD1 gene.","date":"2017","source":"Computational biology and chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28734179","citation_count":11,"is_preprint":false},{"pmid":"29659962","id":"PMC_29659962","title":"Low Dietary Folate Interacts with MTHFD1 Synthetase Deficiency in Mice, a Model for the R653Q Variant, to Increase Incidence of Developmental Delays and Defects.","date":"2018","source":"The Journal of nutrition","url":"https://pubmed.ncbi.nlm.nih.gov/29659962","citation_count":11,"is_preprint":false},{"pmid":"32238907","id":"PMC_32238907","title":"CpG-SNP site methylation regulates allele-specific expression of MTHFD1 gene in type 2 diabetes.","date":"2020","source":"Laboratory investigation; a journal of technical methods and pathology","url":"https://pubmed.ncbi.nlm.nih.gov/32238907","citation_count":10,"is_preprint":false},{"pmid":"25039261","id":"PMC_25039261","title":"Association study of MTHFD1 coding polymorphisms R134K and R653Q with migraine susceptibility.","date":"2014","source":"Headache","url":"https://pubmed.ncbi.nlm.nih.gov/25039261","citation_count":10,"is_preprint":false},{"pmid":"32414565","id":"PMC_32414565","title":"Biochemical analysis of patients with mutations in MTHFD1 and a diagnosis of methylenetetrahydrofolate dehydrogenase 1 deficiency.","date":"2020","source":"Molecular genetics and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/32414565","citation_count":10,"is_preprint":false},{"pmid":"20217437","id":"PMC_20217437","title":"Association analysis of CbetaS 844ins68 and MTHFD1 G1958A polymorphisms with Alzheimer's disease in Chinese.","date":"2010","source":"Journal of neural transmission (Vienna, Austria : 1996)","url":"https://pubmed.ncbi.nlm.nih.gov/20217437","citation_count":10,"is_preprint":false},{"pmid":"24668664","id":"PMC_24668664","title":"Combined folate gene MTHFD and TC polymorphisms as maternal risk factors for Down syndrome in China.","date":"2014","source":"Genetics and molecular research : GMR","url":"https://pubmed.ncbi.nlm.nih.gov/24668664","citation_count":9,"is_preprint":false},{"pmid":"26394717","id":"PMC_26394717","title":"Paternal transmission of MTHFD1 G1958A variant predisposes to neural tube defects in the offspring.","date":"2015","source":"Developmental medicine and child neurology","url":"https://pubmed.ncbi.nlm.nih.gov/26394717","citation_count":9,"is_preprint":false},{"pmid":"27597531","id":"PMC_27597531","title":"Murine MTHFD1-synthetase deficiency, a model for the human MTHFD1 R653Q polymorphism, decreases growth of colorectal tumors.","date":"2016","source":"Molecular carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/27597531","citation_count":8,"is_preprint":false},{"pmid":"1916813","id":"PMC_1916813","title":"A pseudogene on the X chromosome for the human trifunctional enzyme MTHFD (methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase).","date":"1991","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/1916813","citation_count":8,"is_preprint":false},{"pmid":"28559181","id":"PMC_28559181","title":"Deletion of one allele of Mthfd1 (methylenetetrahydrofolate dehydrogenase 1) impairs learning in mice.","date":"2017","source":"Behavioural brain research","url":"https://pubmed.ncbi.nlm.nih.gov/28559181","citation_count":8,"is_preprint":false},{"pmid":"28398708","id":"PMC_28398708","title":"Evidence of gene-gene interactions between MTHFD1 and MTHFR in relation to anterior encephalocele susceptibility in Northeast India.","date":"2017","source":"Birth defects research","url":"https://pubmed.ncbi.nlm.nih.gov/28398708","citation_count":7,"is_preprint":false},{"pmid":"35735795","id":"PMC_35735795","title":"A Common Polymorphism in the MTHFD1 Gene Is a Modulator of Risk of Congenital Heart Disease.","date":"2022","source":"Journal of cardiovascular development and disease","url":"https://pubmed.ncbi.nlm.nih.gov/35735795","citation_count":7,"is_preprint":false},{"pmid":"19946345","id":"PMC_19946345","title":"Association between HIC1 and RASSF1A promoter hypermethylation with MTHFD1 G1958A polymorphism and clinicopathological features of breast cancer in Iranian patients.","date":"2009","source":"Iranian biomedical journal","url":"https://pubmed.ncbi.nlm.nih.gov/19946345","citation_count":7,"is_preprint":false},{"pmid":"24368157","id":"PMC_24368157","title":"The MTHFD1 1958G>A variant is associated with elevated C-reactive protein and body mass index in Canadian women from a premature birth cohort.","date":"2013","source":"Molecular genetics and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/24368157","citation_count":7,"is_preprint":false},{"pmid":"25129243","id":"PMC_25129243","title":"Significant association of MTHFD1 1958G>A single nucleotide polymorphism with nonsyndromic cleft lip and palate in Indian population.","date":"2014","source":"Medicina oral, patologia oral y cirugia bucal","url":"https://pubmed.ncbi.nlm.nih.gov/25129243","citation_count":7,"is_preprint":false},{"pmid":"39857769","id":"PMC_39857769","title":"Identifying MTHFD1 and LGALS4 as Potential Therapeutic Targets in Prostate Cancer Through Multi-Omics Mendelian Randomization Analysis.","date":"2025","source":"Biomedicines","url":"https://pubmed.ncbi.nlm.nih.gov/39857769","citation_count":6,"is_preprint":false},{"pmid":"36928982","id":"PMC_36928982","title":"MTHFD1 is critical for the negative regulation of retinoic acid receptor signalling in anencephaly.","date":"2023","source":"Brain : a journal of neurology","url":"https://pubmed.ncbi.nlm.nih.gov/36928982","citation_count":6,"is_preprint":false},{"pmid":"39571599","id":"PMC_39571599","title":"MTHFD1 Regulates Autophagy to Promote Growth and Metastasis in Colorectal Cancer via the PI3K-AKT-mTOR Signaling Pathway.","date":"2024","source":"Cancer medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39571599","citation_count":6,"is_preprint":false},{"pmid":"26343515","id":"PMC_26343515","title":"Polymorphisms in MTHFD1 Gene and Susceptibility to Neural Tube Defects: A Case-Control Study in a Chinese Han Population with Relatively Low Folate Levels.","date":"2015","source":"Medical science monitor : international medical journal of experimental and clinical research","url":"https://pubmed.ncbi.nlm.nih.gov/26343515","citation_count":6,"is_preprint":false},{"pmid":"32443475","id":"PMC_32443475","title":"Independent and Interactive Influences of Environmental UVR, Vitamin D Levels, and Folate Variant MTHFD1-rs2236225 on Homocysteine Levels.","date":"2020","source":"Nutrients","url":"https://pubmed.ncbi.nlm.nih.gov/32443475","citation_count":6,"is_preprint":false},{"pmid":"30459299","id":"PMC_30459299","title":"Methylenetetrahydrofolate Dehydrogenase 1 (MTHFD1) is Underexpressed in Clear Cell Renal Cell Carcinoma Tissue and Transfection and Overexpression in Caki-1 Cells Inhibits Cell Proliferation and Increases Apoptosis.","date":"2018","source":"Medical science monitor : international medical journal of experimental and clinical research","url":"https://pubmed.ncbi.nlm.nih.gov/30459299","citation_count":6,"is_preprint":false},{"pmid":"33934113","id":"PMC_33934113","title":"A redox probe screens MTHFD1 as a determinant of gemcitabine chemoresistance in cholangiocarcinoma.","date":"2021","source":"Cell death discovery","url":"https://pubmed.ncbi.nlm.nih.gov/33934113","citation_count":6,"is_preprint":false},{"pmid":"35186819","id":"PMC_35186819","title":"Association of Maternal Dietary Habits and MTHFD1 Gene Polymorphisms With Ventricular Septal Defects in Offspring: A Case-Control Study.","date":"2022","source":"Frontiers in pediatrics","url":"https://pubmed.ncbi.nlm.nih.gov/35186819","citation_count":4,"is_preprint":false},{"pmid":"39871280","id":"PMC_39871280","title":"Association of MTHFD1 G1958A (rs2236225) gene polymorphism with the risk of congenital heart disease: a systematic review and meta-analysis.","date":"2025","source":"BMC medical genomics","url":"https://pubmed.ncbi.nlm.nih.gov/39871280","citation_count":4,"is_preprint":false},{"pmid":"30882176","id":"PMC_30882176","title":"Detection of Polymorphisms in MTHFD1 G1958A and Its Possible Association with Idiopathic Male Infertility.","date":"2019","source":"Urology journal","url":"https://pubmed.ncbi.nlm.nih.gov/30882176","citation_count":4,"is_preprint":false},{"pmid":"39442756","id":"PMC_39442756","title":"Betaine and B12 Intake, Glutathione Concentration, and MTHFR, PEMT, and MTHFD1 Genotypes Are Associated with Diabetes-Related Parameters in Polish Adults.","date":"2024","source":"The Journal of nutrition","url":"https://pubmed.ncbi.nlm.nih.gov/39442756","citation_count":4,"is_preprint":false},{"pmid":"20334533","id":"PMC_20334533","title":"The MTHFD1 c.1958 G>A polymorphism and recurrent spontaneous abortions.","date":"2010","source":"The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstetricians","url":"https://pubmed.ncbi.nlm.nih.gov/20334533","citation_count":4,"is_preprint":false},{"pmid":"26834978","id":"PMC_26834978","title":"Is MTHFD1 polymorphism rs 2236225 (c.1958G>A) associated with the susceptibility of NSCL/P? A systematic review and meta-analysis.","date":"2015","source":"F1000Research","url":"https://pubmed.ncbi.nlm.nih.gov/26834978","citation_count":4,"is_preprint":false},{"pmid":"39074897","id":"PMC_39074897","title":"Specific association of MTHFD1 expressions with small cell lung cancer development and chemoradiotherapy outcome.","date":"2024","source":"Saudi medical journal","url":"https://pubmed.ncbi.nlm.nih.gov/39074897","citation_count":3,"is_preprint":false},{"pmid":"39256448","id":"PMC_39256448","title":"Selectivity analysis of diaminopyrimidine-based inhibitors of MTHFD1, MTHFD2 and MTHFD2L.","date":"2024","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/39256448","citation_count":3,"is_preprint":false},{"pmid":"37628752","id":"PMC_37628752","title":"Common Variants in One-Carbon Metabolism Genes (MTHFR, MTR, MTHFD1) and Depression in Gynecologic Cancers.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/37628752","citation_count":3,"is_preprint":false},{"pmid":"25304051","id":"PMC_25304051","title":"[G894T (NOS3) and G1958A (MTHFD1) gene polymorphisms and risk of ischemic heart disease in Yucatan, Mexico].","date":"2014","source":"Clinica e investigacion en arteriosclerosis : publicacion oficial de la Sociedad Espanola de Arteriosclerosis","url":"https://pubmed.ncbi.nlm.nih.gov/25304051","citation_count":3,"is_preprint":false},{"pmid":"36913067","id":"PMC_36913067","title":"The negative effect of G1958A polymorphism on MTHFD1 protein stability and HCC growth.","date":"2023","source":"Cellular oncology (Dordrecht, Netherlands)","url":"https://pubmed.ncbi.nlm.nih.gov/36913067","citation_count":2,"is_preprint":false},{"pmid":"38435941","id":"PMC_38435941","title":"Association of MTHFD1 G1958A Polymorphism with Gestational Diabetes Mellitus.","date":"2024","source":"Cureus","url":"https://pubmed.ncbi.nlm.nih.gov/38435941","citation_count":2,"is_preprint":false},{"pmid":"1887335","id":"PMC_1887335","title":"Deficient synthesis of MTHFD, a trifunctional folate-dependent enzyme, in the CHO Ade E mutant.","date":"1991","source":"Somatic cell and molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/1887335","citation_count":2,"is_preprint":false},{"pmid":"36685872","id":"PMC_36685872","title":"Doubly bi-allelic variants of MTHFR and MTHFD1 in a Chinese patient with hyperhomocysteinemia and failure of folic acid therapy.","date":"2023","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/36685872","citation_count":2,"is_preprint":false},{"pmid":"22202336","id":"PMC_22202336","title":"[Prediction of the efficacy of modified FOLFOX6 therapy according to the mRNA levels of thymidylate synthase (TS), excision repair cross-complementing-1 and -2( ERCC-1 and ERCC-2) and methylenetetrahydrofolate dehydrogenase( MTHFD) in the primary lesion of colorectal cancer].","date":"2011","source":"Gan to kagaku ryoho. Cancer & chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/22202336","citation_count":2,"is_preprint":false},{"pmid":"30388610","id":"PMC_30388610","title":"Impact of RFC1, MTHFR, and MTHFD1 polymorphism on unexplained pregnancy loss (UPL): comparative analysis of maternal and fetal components using mother-abortus paired samples.","date":"2018","source":"European journal of obstetrics, gynecology, and reproductive biology","url":"https://pubmed.ncbi.nlm.nih.gov/30388610","citation_count":2,"is_preprint":false},{"pmid":"39571719","id":"PMC_39571719","title":"Evidence for interaction of 5,10-methylenetetrahydrofolate reductase (MTHFR) with methylenetetrahydrofolate dehydrogenase (MTHFD1) and general control nonderepressible 1 (GCN1).","date":"2024","source":"Biochimie","url":"https://pubmed.ncbi.nlm.nih.gov/39571719","citation_count":2,"is_preprint":false},{"pmid":"25118499","id":"PMC_25118499","title":"The role of 401a>G polymorphism of methylenetetrahydrofolate dehydrogenase gene (MTHFD1) in fetal hypotrophy.","date":"2014","source":"Ginekologia polska","url":"https://pubmed.ncbi.nlm.nih.gov/25118499","citation_count":2,"is_preprint":false},{"pmid":"34904448","id":"PMC_34904448","title":"Association Between MTHFD1 1958G > A Variant and non-Syndromic Cleft lip and Palate: An Updated Meta-Analysis.","date":"2021","source":"The Cleft palate-craniofacial journal : official publication of the American Cleft Palate-Craniofacial Association","url":"https://pubmed.ncbi.nlm.nih.gov/34904448","citation_count":1,"is_preprint":false},{"pmid":"35894196","id":"PMC_35894196","title":"[Association of maternal MTHFD1 and MTHFD2 gene polymorphisms with congenital heart disease in offspring].","date":"2022","source":"Zhongguo dang dai er ke za zhi = Chinese journal of contemporary pediatrics","url":"https://pubmed.ncbi.nlm.nih.gov/35894196","citation_count":1,"is_preprint":false},{"pmid":"34168100","id":"PMC_34168100","title":"Triglyceride regulate ACE2 level through MTHFD1.","date":"2021","source":"Journal of biosciences","url":"https://pubmed.ncbi.nlm.nih.gov/34168100","citation_count":1,"is_preprint":false},{"pmid":"28299500","id":"PMC_28299500","title":"MTHFR and MTHFD1 gene polymorphisms are not associated with pseudoexfoliation syndrome in South Indian population.","date":"2017","source":"International ophthalmology","url":"https://pubmed.ncbi.nlm.nih.gov/28299500","citation_count":1,"is_preprint":false},{"pmid":"26221324","id":"PMC_26221324","title":"MTHFD1 gene polymorphisms as risk factors involved in orofacial cleft: an independent case-control study and a meta-analysis.","date":"2015","source":"International journal of clinical and experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/26221324","citation_count":1,"is_preprint":false},{"pmid":"41668133","id":"PMC_41668133","title":"CRISPR screen of human pancreatic cancer xenografts identifies a KLF5 proliferation vulnerability through epigenetic modifiers NCAPD2 and MTHFD1.","date":"2026","source":"Molecular cancer","url":"https://pubmed.ncbi.nlm.nih.gov/41668133","citation_count":1,"is_preprint":false},{"pmid":"40714174","id":"PMC_40714174","title":"Contrasting Effects of Phosphatidylcholine and Betaine Supplementation on Embryonic Development in a Mouse Model of the MTHFD1 R653Q Variant.","date":"2025","source":"The Journal of nutrition","url":"https://pubmed.ncbi.nlm.nih.gov/40714174","citation_count":0,"is_preprint":false},{"pmid":"41805631","id":"PMC_41805631","title":"Methyltransferase complex subunit METTL3 maintains genome stability of erythroid cells via MTHFD1-mediated nucleotide biosynthesis.","date":"2026","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/41805631","citation_count":0,"is_preprint":false},{"pmid":"41859783","id":"PMC_41859783","title":"Discovery of 1,2,3,4-Tetrahydrochromeno[3,4-c]pyridin-5-one Derivatives as Novel MTHFD Inhibitors for the Treatment of Acute Myeloid Leukemia.","date":"2026","source":"Journal of medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/41859783","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.03.16.643506","title":"Functional profiling reveals a non-enzymatic role of NUDT5 in repressing purine  <i>de novo</i>  synthesis","date":"2025-03-16","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.16.643506","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.05.15.654252","title":"DNA methylation marks associated with body composition in children from India and the Gambia - findings from the EMPHASIS study","date":"2025-05-19","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.15.654252","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.08.06.606759","title":"RIPK4 driven by TP53 mutations promotes resistance to redox stress of CRC by phosphorylating MTHFD1","date":"2024-08-06","source":"bioRxiv","url":"https://doi.org/10.1101/2024.08.06.606759","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":54995,"output_tokens":6033,"usd":0.12774,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":14799,"output_tokens":5150,"usd":0.101373,"stage2_stop_reason":"end_turn"},"total_usd":0.229113,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1991,\n      \"finding\": \"MTHFD1 (MTHFD) encodes a single trifunctional polypeptide with three sequential catalytic activities: N5,N10-methylenetetrahydrofolate dehydrogenase, N5,N10-methenyltetrahydrofolate cyclohydrolase, and N10-formyltetrahydrofolate synthetase, all required for interconversion of tetrahydrofolate derivatives for purine, thymidylate, and methionine synthesis. In a CHO Ade-E mutant, all three activities were lost together with reduced/absent protein despite normal mRNA, demonstrating the activities reside on a single polypeptide and suggesting a post-transcriptional regulatory mechanism.\",\n      \"method\": \"Enzymatic activity assays, immunoblotting, immunoprecipitation, Northern blot analysis in CHO Ade-E mutant cells\",\n      \"journal\": \"Somatic cell and molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct enzymatic assays plus protein-level and mRNA-level orthogonal experiments in a defined mutant cell line demonstrating co-loss of all three activities\",\n      \"pmids\": [\"1887335\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Knockout of the Mthfd1 gene in murine embryonic stem cells eliminates all three cytoplasmic MTHFD1 activities (dehydrogenase, cyclohydrolase, synthetase), causes purine auxotrophy, and reveals a separate mitochondrial monofunctional 10-formyltetrahydrofolate synthetase (encoded by a recently identified mitochondrial transcript). Absence of NADP-dependent dehydrogenase activity in these null cells confirmed the mitochondrial enzyme lacks dehydrogenase/cyclohydrolase activities.\",\n      \"method\": \"Gene knockout in embryonic stem cells, enzymatic activity assays, subcellular fractionation/localization, Northern blot\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — genetic knockout with multiple enzymatic assays and subcellular fractionation providing multiple orthogonal lines of evidence\",\n      \"pmids\": [\"15611115\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Disruption of the Mthfd1 gene (gene-trap insertion inactivating formyl-THF synthetase/FTHFS activity) causes embryonic lethality in homozygous mice. Heterozygous Mthfd1gt/+ mice show lower hepatic S-adenosylmethionine (indicating formate-derived one carbons contribute to methylation reactions), decreased uracil in nuclear DNA (indicating enhanced thymidylate synthesis when FTHFS is reduced), demonstrating that formate-derived carbons compete with serine-derived carbons for THF cofactors used in thymidylate vs. homocysteine remethylation.\",\n      \"method\": \"Gene-trap mouse model, metabolite measurements (AdoMet, uracil in DNA), genetic analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic model with multiple metabolic readouts demonstrating pathway-level competition for THF cofactors\",\n      \"pmids\": [\"19033438\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The common MTHFD1 p.Arg653Gln (R653Q) variant reduces enzyme thermostability (36% reduction in half-life at 42°C) without altering substrate affinity. Thermolability is rescued by folate pentaglutamate and Mg-ATP. In murine Mthfd1 knockout cells transfected with the Arg653Gln variant, formate incorporation into DNA (a proxy for de novo purine synthesis) is reduced by 26% compared to wild-type, indicating impaired de novo purine synthesis.\",\n      \"method\": \"In vitro enzyme activity and stability assays (purified recombinant protein), mammalian cell transfection with formate incorporation into DNA measurement\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted in vitro enzyme assays plus cellular metabolic flux assay in defined knockout cells, two orthogonal methods in single study\",\n      \"pmids\": [\"18767138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"MTHFD1 translocates to the nucleus during S-phase in MCF-7 and HeLa cells. During folate deficiency, MTHFD1 is enriched >2-fold in the nucleus at the expense of cytosolic levels in mouse liver, and nuclear folate cofactors are maintained when total cellular folate is reduced by >50%. This nuclear enrichment supports de novo thymidylate biosynthesis preferentially over cytosolic homocysteine remethylation during folate deficiency.\",\n      \"method\": \"Subcellular fractionation, Western blot, cell cycle synchronization (S-phase), mouse dietary folate depletion model\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct fractionation experiments in both cultured cells (S-phase) and mouse liver, replicated across two experimental systems\",\n      \"pmids\": [\"25213861\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Arsenic trioxide (As2O3) increases MTHFD1 SUMOylation (confirmed in cultured cells and in vitro SUMOylation reactions) and promotes MTHFD1 ubiquitination and proteolytic degradation (along with SHMT1). This leads to inhibition of de novo thymidylate biosynthesis, increased uracil misincorporation into nuclear DNA, and genome instability. MTHFD1 and SHMT1 form a multienzyme complex with TYMS and DHFR at the nuclear DNA replication machinery during S-phase.\",\n      \"method\": \"In vitro SUMOylation assay, immunoprecipitation (Co-IP), Western blot for ubiquitination and protein levels, uracil-in-DNA measurement, genome instability assays in cultured cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro SUMOylation reconstitution plus Co-IP plus functional metabolic readouts, multiple orthogonal methods in single study\",\n      \"pmids\": [\"28265077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The natural product carolacton inhibits both bacterial FolD and the human orthologs MTHFD1 and MTHFD2 in the low nanomolar range. Crystal structure of the bacterial FolD-carolacton complex reveals the binding mode; carolacton occupies the active site and inhibits the dehydrogenase/cyclohydrolase activities.\",\n      \"method\": \"Biophysical binding assay, X-ray crystallography of FolD-carolacton complex, in vitro enzymatic inhibition assays for MTHFD1 and MTHFD2\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus in vitro enzymatic inhibition assays demonstrating direct inhibition of human MTHFD1 at its active site\",\n      \"pmids\": [\"29142318\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"A fraction of MTHFD1 resides in the nucleus where it is recruited to specific genomic loci by direct physical interaction with BRD4 (bromodomain-containing protein 4). Genetic and pharmacological screens identified this MTHFD1-BRD4 interaction. Inhibition of either BRD4 or MTHFD1 produces similar changes in nuclear metabolite composition and gene expression, and the two inhibitors synergize to impair cancer cell viability in vitro and in vivo.\",\n      \"method\": \"Complementary genetic interaction screen and physical interaction screen (converging on MTHFD1-BRD4), nuclear localization by fractionation, co-immunoprecipitation, metabolomics, in vitro and in vivo pharmacological studies\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal screens plus Co-IP plus metabolomics plus in vivo validation, multiple orthogonal methods across genetic and biochemical approaches\",\n      \"pmids\": [\"31133746\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PRMT5 directly binds MTHFD1 and symmetrically dimethylates it at arginine R173. Under suspension conditions (anoikis stress), the MTHFD1-PRMT5 interaction is strengthened, elevating R173 symmetric dimethylation, which augments MTHFD1 metabolic activity (NADPH generation), promoting anoikis resistance and distant organ metastasis. Genetic depletion or pharmacological inhibition of PRMT5 reduced tumor metastasis.\",\n      \"method\": \"CRISPR-Cas9 metabolic enzyme screen, Co-immunoprecipitation, in vitro methylation assay, site-directed mutagenesis (R173), NADPH/NADP+ ratio measurement, xenograft/metastasis models\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — Co-IP, in vitro methylation, mutagenesis of methylation site, and in vivo functional validation with multiple orthogonal methods\",\n      \"pmids\": [\"35798877\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TH9619, a dual inhibitor of MTHFD1 and MTHFD2 dehydrogenase/cyclohydrolase activities, selectively targets nuclear MTHFD2 but not mitochondrial MTHFD2. Continued mitochondrial formate overflow accumulates 10-formyl-THF downstream of MTHFD1 inhibition, creating a 'folate trap.' This results in thymidylate depletion and selective death of MTHFD2-expressing cancer cells. The trapping is exacerbated by physiological hypoxanthine which blocks de novo purine synthesis and prevents 10-formyl-THF consumption.\",\n      \"method\": \"Metabolomics (10-formyl-THF accumulation), isotope tracing, selective MTHFD2 nuclear vs. mitochondrial inhibition analysis, cellular viability assays, genetic and pharmacological perturbations\",\n      \"journal\": \"Nature metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — metabolite tracing plus compartment-specific inhibitor studies plus multiple genetic/pharmacological perturbations establishing mechanism\",\n      \"pmids\": [\"37012496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MTHFD1 negatively regulates retinoic acid receptor γ (RARγ) transcription factor activity. IP-mass spectrometry identified MTHFD1 as a specific RARγ-interacting protein; co-immunoprecipitation and immunofluorescence confirmed the interaction. MTHFD1 knockdown de-repressed RARγ signaling; low MTHFD1 expression and activated RAR signaling were observed in human anencephaly and a retinoic acid-induced NTD mouse model.\",\n      \"method\": \"Immunoprecipitation-mass spectrometry, co-immunoprecipitation, immunofluorescence confocal microscopy, luciferase reporter assay, ChIP-qPCR, mouse NTD model\",\n      \"journal\": \"Brain : a journal of neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus reporter assay plus ChIP in single study; replication in vivo is indirect (mouse NTD model with correlation not rescue)\",\n      \"pmids\": [\"36928982\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The MTHFD1 G1958A (R653Q) SNP reduces protein stability through ubiquitination-mediated proteasomal degradation. The R653Q variant shows enhanced binding to the E3 ubiquitin ligase TRIM21 compared to wild-type; lysine K504 is the primary ubiquitination site. R653Q expression results in reduced serine-derived methyl flux into purine synthesis precursors (confirmed by metabolic flux analysis) and impaired tumor growth in xenograft models.\",\n      \"method\": \"Immunoprecipitation for ubiquitination, mass spectrometry identification of ubiquitination site and interacting proteins, metabolic flux analysis with serine isotope, xenograft tumor growth assay\",\n      \"journal\": \"Cellular oncology (Dordrecht, Netherlands)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — Co-IP for E3 ligase interaction, mass spectrometry site identification, metabolic flux; single lab, moderate replication\",\n      \"pmids\": [\"36913067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Genome-wide CRISPR and RNAi screens in bat cells identified MTHFD1 as required for viral replication (mumps virus, influenza A, SARS-CoV-2). The MTHFD1 inhibitor carolacton potently blocked replication of several RNA viruses including SARS-CoV-2, demonstrating that MTHFD1 enzymatic activity supports viral replication in both bat and human cells.\",\n      \"method\": \"Genome-wide CRISPR and RNAi library screens, carolacton pharmacological inhibition, viral replication assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — orthogonal genetic screens (CRISPR + RNAi) converging on same target, plus pharmacological validation in multiple viral contexts\",\n      \"pmids\": [\"34544865\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MTHFD1 protein expression is markedly reduced (to 4.8–14.3% of control values) and methylenetetrahydrofolate dehydrogenase specific activity is undetectable in fibroblasts from MTHFD1-deficient patients bearing compound heterozygous MTHFD1 mutations, confirming that patient mutations abolish enzymatic function.\",\n      \"method\": \"Western blot for MTHFD1 protein expression in patient fibroblasts, enzymatic activity assay for dehydrogenase activity\",\n      \"journal\": \"Molecular genetics and metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct enzymatic measurement and protein quantification in patient-derived cells, single study with multiple patients\",\n      \"pmids\": [\"32414565\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Patient fibroblasts with MTHFD1 deficiency show severely reduced methionine formation from [14C]-formate (a direct measure of formyl-THF synthetase activity feeding homocysteine remethylation), which did not improve with cobalamin supplementation but was responsive to folic and folinic acid treatment, indicating that MTHFD1 is required for formate entry into folate metabolism for methionine synthesis.\",\n      \"method\": \"Radiolabeled formate ([14C]-formate) metabolic flux assay in patient fibroblasts, supplementation experiments\",\n      \"journal\": \"Journal of inherited metabolic disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct isotope tracing in patient-derived cells with pharmacological rescue, limited to single lab\",\n      \"pmids\": [\"25633902\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MTHFD1 maintains NADPH/NADP+ and GSH/GSSG redox homeostasis in MYCN-amplified neuroblastoma. MYCN directly activates MTHFD1 transcription (confirmed by ChIP-qPCR and dual-luciferase reporter assay). Knockdown of MTHFD1 reduces NADPH/NADP+ and GSH/GSSG ratios, increases ROS, and triggers apoptosis.\",\n      \"method\": \"ChIP-qPCR, dual-luciferase reporter assay, MTHFD1 knockdown, NADPH/NADP+ and GSH/GSSG ratio measurements, ROS measurement, mouse xenograft model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and reporter assay for transcriptional regulation plus metabolic readouts; single lab\",\n      \"pmids\": [\"38336749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MTHFD1 regulates autophagy via the PI3K-AKT-mTOR signaling pathway in colorectal cancer cells. MTHFD1 overexpression suppresses autophagy and activates PI3K-AKT-mTOR, promoting proliferation and metastasis, while MTHFD1 knockdown increases autophagy and suppresses these phenotypes. Validated in xenograft models.\",\n      \"method\": \"Western blot for PI3K-AKT-mTOR pathway and autophagy markers, Transwell invasion assay, xenograft tumor models\",\n      \"journal\": \"Cancer medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, Western blot pathway readout with overexpression/knockdown; no direct biochemical mechanism linking MTHFD1 enzymatic activity to PI3K-AKT-mTOR established\",\n      \"pmids\": [\"39571599\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"METTL3-mediated m6A modification controls MTHFD1 mRNA stability in erythroid cells. Mettl3 deletion reduces Mthfd1 expression, causing nucleotide (dTMP and IMP) shortage, DNA damage, and apoptosis in erythroid progenitors. Re-introduction or rescue of MTHFD1 activity reverses the genome instability phenotype.\",\n      \"method\": \"m6A-seq and RNA-seq integration, Western blot, nucleotide measurement (dTMP and IMP), DNA damage assays, erythroid-specific Mettl3 knockout mouse (EpoR-Cre)\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — integrated multi-omic approach plus genetic model with direct metabolic measurements; single lab\",\n      \"pmids\": [\"41805631\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MTHFD1 protein interacts with MTHFR (methylenetetrahydrofolate reductase) as identified by affinity purification-mass spectrometry and confirmed by immunoprecipitation-immunoblotting. An AlphaFold3 model places the MTHFD1 dehydrogenase/cyclohydrolase domain in direct contact with the MTHFR catalytic domain, suggesting substrate (methylenetetrahydrofolate) channeling between the two enzymes.\",\n      \"method\": \"Affinity purification-mass spectrometry (AP-MS), immunoprecipitation-immunoblotting, AlphaFold3 structural modeling\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP confirmed by single lab; structural model is computational prediction; no direct channeling experiment performed\",\n      \"pmids\": [\"39571719\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Genetic knockout and chemical degradation of NUDT5 revealed that NUDT5 interacts with PPAT (the rate-limiting enzyme of de novo purine synthesis) to repress the pathway in response to elevated purine levels. This NUDT5-PPAT scaffolding role (non-enzymatic) mediates sensitivity to adenosine in MTHFD1 deficiency, placing NUDT5 downstream of MTHFD1 in regulating purine de novo synthesis.\",\n      \"method\": \"CRISPR knockout, selective NUDT5 chemical degrader (PROTAC-type), genetic epistasis in MTHFD1 mutant background, interaction studies (NUDT5-PPAT)\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 2 / Weak — preprint; genetic epistasis with MTHFD1 deficiency establishes pathway placement but is a preprint and the direct MTHFD1 mechanism is secondary to NUDT5 findings\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RIPK4 (driven by TP53 mutations) phosphorylates MTHFD1, boosting NADPH production, reducing ROS, and promoting resistance to PANoptosis and metastasis in colorectal cancer.\",\n      \"method\": \"Phosphorylation assay (RIPK4-MTHFD1), NADPH measurement, ROS measurement, metastasis models in CRC\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 2 / Weak — preprint; mechanistic claim based on phosphorylation assay and NADPH/ROS readouts but not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"MTHFD1 encodes a cytoplasmic trifunctional enzyme (methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase, and formyltetrahydrofolate synthetase) that generates and interconverts folate-activated one-carbon units for de novo purine and thymidylate synthesis and homocysteine remethylation; a fraction of the protein translocates to the nucleus during S-phase (enriched further under folate deficiency) to support a nuclear thymidylate synthesis complex with SHMT, TYMS, and DHFR, and MTHFD1 is subject to multiple post-translational controls including PRMT5-mediated R173 symmetric dimethylation (enhancing NADPH output and anoikis resistance), TRIM21-mediated ubiquitination at K504 (accelerated by the R653Q variant), arsenic trioxide-induced SUMOylation and degradation, and RIPK4-mediated phosphorylation; it also interacts directly with BRD4 in the nucleus to couple folate metabolism to transcriptional regulation, and with MTHFR to potentially channel methylenetetrahydrofolate, while being transcriptionally activated by MYCN and post-transcriptionally stabilized by METTL3-dependent m6A modification.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MTHFD1 encodes a single cytoplasmic trifunctional polypeptide carrying N5,N10-methylenetetrahydrofolate dehydrogenase, N5,N10-methenyltetrahydrofolate cyclohydrolase, and N10-formyltetrahydrofolate synthetase activities that interconvert tetrahydrofolate-activated one-carbon units for de novo purine, thymidylate, and methionine synthesis [#0, #1]. Genetic disruption causes purine auxotrophy in stem cells and embryonic lethality in mice, with heterozygotes showing competition between formate-derived and serine-derived one-carbon units for THF cofactors used in thymidylate synthesis versus homocysteine remethylation [#1, #2]. A fraction of MTHFD1 translocates to the nucleus during S-phase—an enrichment intensified under folate deficiency—where it assembles with SHMT1, TYMS, and DHFR at the replication machinery to support de novo thymidylate biosynthesis and limit uracil misincorporation, and is also recruited to specific genomic loci through direct interaction with BRD4 to couple folate metabolism to transcriptional output [#4, #5, #7]. The enzyme is governed by multiple post-translational controls: PRMT5-mediated symmetric dimethylation at R173 augments NADPH output and anoikis resistance [#8], and TRIM21-dependent ubiquitination at K504—accelerated by the common R653Q variant—drives proteasomal degradation and reduces serine-derived methyl flux into purines [#11]. MTHFD1 sustains NADPH/NADP+ and GSH/GSSG redox balance and is a direct transcriptional target of MYCN in neuroblastoma [#15], while its mRNA is stabilized by METTL3-dependent m6A modification to maintain nucleotide supply in erythroid progenitors [#17]. Compound heterozygous MTHFD1 mutations that abolish enzyme protein and dehydrogenase activity cause a human inborn error of folate metabolism with defective methionine formation from formate [#13, #14]. MTHFD1 enzymatic activity is also exploited to support replication of multiple RNA viruses and is inhibited at its active site by the natural product carolacton [#6, #12].\",\n  \"teleology\": [\n    {\n      \"year\": 1991,\n      \"claim\": \"Established that the three sequential folate-interconverting activities reside on a single polypeptide rather than separate proteins, defining MTHFD1 as a trifunctional enzyme and hinting at post-transcriptional control.\",\n      \"evidence\": \"Enzymatic assays, immunoblotting and Northern blot in a CHO Ade-E mutant showing co-loss of all three activities with normal mRNA\",\n      \"pmids\": [\"1887335\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve domain architecture or how the three active sites are spatially organized\", \"Mechanism of the inferred post-transcriptional regulation not defined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Genetic ablation confirmed MTHFD1 carries all three cytoplasmic activities and caused purine auxotrophy, while distinguishing it from a separate monofunctional mitochondrial formyltetrahydrofolate synthetase.\",\n      \"evidence\": \"Mthfd1 knockout in murine embryonic stem cells with enzymatic assays and subcellular fractionation\",\n      \"pmids\": [\"15611115\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address tissue-specific or developmental requirements\", \"Compartmental division of labor between cytosol and mitochondria left to later work\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrated at the organismal level that MTHFD1-generated one-carbon units are partitioned between thymidylate synthesis and methylation, with formate-derived carbons competing with serine-derived carbons for THF cofactors.\",\n      \"evidence\": \"Gene-trap Mthfd1 mouse model with hepatic AdoMet and nuclear DNA uracil measurements\",\n      \"pmids\": [\"19033438\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the molecular determinants of flux partitioning\", \"Mechanism of homozygous embryonic lethality not dissected\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Revealed that MTHFD1 is not constitutively cytosolic but translocates to the nucleus during S-phase and is further enriched there under folate deficiency, prioritizing thymidylate synthesis over homocysteine remethylation.\",\n      \"evidence\": \"Subcellular fractionation and cell-cycle synchronization in MCF-7/HeLa plus a folate-depletion mouse liver model\",\n      \"pmids\": [\"25213861\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nuclear import signal and translocation machinery not identified\", \"How nuclear folate cofactor pools are maintained unresolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defined the nuclear thymidylate synthesis complex and showed it is a regulated, degradable node, with arsenic trioxide driving MTHFD1 SUMOylation and ubiquitin-dependent degradation that destabilizes the genome.\",\n      \"evidence\": \"In vitro SUMOylation, Co-IP for the MTHFD1/SHMT1/TYMS/DHFR complex, ubiquitination and uracil-in-DNA assays in cultured cells\",\n      \"pmids\": [\"28265077\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"SUMO/ubiquitin sites and responsible E3 ligase not pinpointed in this study\", \"Stoichiometry and architecture of the nuclear multienzyme complex undefined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Provided direct structural and biochemical evidence that MTHFD1's dehydrogenase/cyclohydrolase active site is druggable, via low-nanomolar inhibition by carolacton.\",\n      \"evidence\": \"X-ray crystallography of a bacterial FolD-carolacton complex with in vitro enzymatic inhibition of human MTHFD1 and MTHFD2\",\n      \"pmids\": [\"29142318\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No crystal structure of human MTHFD1 with the inhibitor\", \"Selectivity between MTHFD1 and MTHFD2 not resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected MTHFD1's nuclear pool to transcriptional control by identifying a direct BRD4 interaction that recruits the enzyme to chromatin and couples folate metabolism to gene expression.\",\n      \"evidence\": \"Converging genetic and physical interaction screens, Co-IP, nuclear metabolomics and in vivo synergy of BRD4/MTHFD1 inhibitors\",\n      \"pmids\": [\"31133746\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Interaction interface and chromatin-recruitment mechanism not mapped\", \"Whether enzymatic activity or scaffolding drives transcriptional effects unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed MTHFD1 is functionally tuned by arginine methylation, with PRMT5-mediated R173 symmetric dimethylation boosting NADPH output to confer anoikis resistance and metastasis.\",\n      \"evidence\": \"CRISPR screen, Co-IP, in vitro methylation, R173 mutagenesis, NADPH ratio measurement and metastasis xenografts\",\n      \"pmids\": [\"35798877\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How R173 methylation structurally alters catalytic output not defined\", \"Demethylase counter-regulation unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified TRIM21 as the E3 ligase ubiquitinating MTHFD1 at K504 and explained how the common R653Q variant destabilizes the protein and reduces purine biosynthetic flux.\",\n      \"evidence\": \"Co-IP, mass spectrometry site mapping, serine isotope flux analysis and xenograft growth assays\",\n      \"pmids\": [\"36913067\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab finding without reciprocal validation of the TRIM21 interaction\", \"Signals controlling TRIM21-MTHFD1 engagement not defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Established a noncanonical role for MTHFD1 in repressing RARγ transcriptional activity, linking folate enzyme loss to neural tube defect pathology.\",\n      \"evidence\": \"IP-mass spectrometry, Co-IP, luciferase reporter and ChIP-qPCR with a retinoic acid-induced NTD mouse model\",\n      \"pmids\": [\"36928982\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo link is correlative rather than rescue-based\", \"Whether repression depends on enzymatic activity or physical binding unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated that pharmacological MTHFD1/MTHFD2 dehydrogenase inhibition creates a nuclear '10-formyl-THF folate trap' that selectively kills MTHFD2-expressing cancer cells through thymidylate depletion.\",\n      \"evidence\": \"Metabolomics, isotope tracing and compartment-specific inhibitor (TH9619) studies\",\n      \"pmids\": [\"37012496\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Determinants of nuclear versus mitochondrial inhibitor selectivity incompletely defined\", \"Generality across tumor types not established\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Placed MTHFD1 in redox homeostasis and oncogenic transcriptional circuitry, showing MYCN directly drives MTHFD1 expression to maintain NADPH and GSH pools and prevent apoptosis.\",\n      \"evidence\": \"ChIP-qPCR, dual-luciferase reporter, knockdown with NADPH/GSH/ROS measurements and xenografts\",\n      \"pmids\": [\"38336749\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical link between MTHFD1 catalysis and the measured redox ratios not isolated\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed MTHFD1 abundance is set post-transcriptionally by METTL3-dependent m6A modification, gating nucleotide supply and genome stability in erythroid progenitors.\",\n      \"evidence\": \"m6A-seq/RNA-seq integration, erythroid-specific Mettl3 knockout mouse, nucleotide measurements and rescue\",\n      \"pmids\": [\"41805631\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"m6A reader mediating MTHFD1 mRNA stabilization not identified\", \"Tissue specificity of this regulation untested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Confirmed in patient-derived cells that disease-causing MTHFD1 mutations abolish protein and dehydrogenase activity, establishing MTHFD1 deficiency as a human inborn error of folate metabolism.\",\n      \"evidence\": \"Western blot and dehydrogenase activity assays in fibroblasts from compound heterozygous patients; complemented by [14C]-formate methionine-formation flux assays\",\n      \"pmids\": [\"32414565\", \"25633902\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Genotype-phenotype relationships across mutation classes incomplete\", \"Tissue-level pathophysiology beyond fibroblasts not addressed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined MTHFD1 as a host dependency factor whose enzymatic activity supports replication of multiple RNA viruses, nominating it as an antiviral target.\",\n      \"evidence\": \"Genome-wide CRISPR and RNAi screens in bat cells with carolacton inhibition across mumps, influenza A and SARS-CoV-2\",\n      \"pmids\": [\"34544865\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which one-carbon outputs the viruses require not dissected\", \"Direct dependence on individual MTHFD1 activities not separated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How MTHFD1's enzymatic and scaffolding functions are integrated with its partner enzymes and signaling inputs—including direct substrate channeling to MTHFR and the regulatory consequences of RIPK4 phosphorylation—remains unresolved.\",\n      \"evidence\": \"MTHFD1-MTHFR Co-IP with AlphaFold3 modeling and RIPK4 phosphorylation/NADPH readouts are preliminary and not reconstituted\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct experimental demonstration of methylenetetrahydrofolate channeling between MTHFD1 and MTHFR\", \"RIPK4 phosphorylation site and mechanism in MTHFD1 not mapped (preprint)\", \"Integration of nuclear scaffolding, redox, and metabolic roles into a unified regulatory logic absent\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [0, 1, 3, 13]},\n      {\"term_id\": \"GO:0016829\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1, 14]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 1, 4]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [4, 5, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [7, 10, 15]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [17]}\n    ],\n    \"complexes\": [\"nuclear thymidylate synthesis complex (MTHFD1-SHMT1-TYMS-DHFR)\"],\n    \"partners\": [\"BRD4\", \"PRMT5\", \"TRIM21\", \"MTHFR\", \"SHMT1\", \"TYMS\", \"DHFR\", \"RARG\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}