Evidence for interaction of 5,10-methylenetetrahydrofolate reductase (MTHFR) with methylenetetrahydrofolate dehydrogenase (MTHFD1) and general control nonderepressible 1 (GCN1).

Linda R B��chler, Linnea K M Blomgren, C��line B��rer, Vito R T Zanotelli, D Sean Froese
Author Information
  1. Linda R B��chler: Division of Metabolism and Children's Research Center, University Children's Hospital Z��rich, University of Z��rich, Z��rich, Switzerland.
  2. Linnea K M Blomgren: Division of Metabolism and Children's Research Center, University Children's Hospital Z��rich, University of Z��rich, Z��rich, Switzerland.
  3. C��line B��rer: Division of Metabolism and Children's Research Center, University Children's Hospital Z��rich, University of Z��rich, Z��rich, Switzerland.
  4. Vito R T Zanotelli: Division of Metabolism and Children's Research Center, University Children's Hospital Z��rich, University of Z��rich, Z��rich, Switzerland.
  5. D Sean Froese: Division of Metabolism and Children's Research Center, University Children's Hospital Z��rich, University of Z��rich, Z��rich, Switzerland. Electronic address: Sean.Froese@kispi.uzh.ch.

Abstract

5,10-Methylenetetrahydrofolate reductase (MTHFR) is a folate cycle enzyme required for the intracellular synthesis of methionine. MTHFR was previously shown to be partially phosphorylated at 16 residues, which was abrogated by conversion of threonine 34 to alanine (T34A) or truncation of the first 37 amino acids (i.e. expression of amino acids 38-656), and promoted by methionine supplementation. Here, we over-expressed wild-type MTHFR (MTFHR), as well as the variants MTHFR and MTHFR in 293T cells to provide further insights into these mechanisms. We demonstrate that following incubation in high methionine conditions (100-1000 ��M) MTHFR is almost completely phosphorylated, but in methionine restricted conditions (0-10 ��M) phosphorylation is reduced, while MTHFR always remains unphosphorylated. Following affinity purification coupled mass spectrometry of an empty vector, MTHFR, MTHFR and MTHFR in three separate experiments, we identified 134 proteins consistently pulled-down by all three MTHFR protein variants, of which 5 were indicated to be likely true interactors (SAINT prediction threshold of 0.95 and 2 fold-change). Amongst these were the folate cycle enzyme methylenetetrahydrofolate dehydrogenase (MTHFD1) and the amino acid starvation sensor general control nonderepressible 1 (GCN1). Immunoprecipitation-immunoblotting of MTHFR replicated interaction with both proteins. An AlphaFold 3 generated model of the MTHFR-MTHFD1 interaction places the MTHFD1 dehydrogenase/cyclohydrolase domain in direct contact with the MTHFR catalytic domain, suggesting their interaction may facilitate direct delivery of methylenetetrahydrofolate. Overall, we confirm methionine availability increases MTHFR phosphorylation, and identified potential interaction of MTHFR with MTHFD1 and GCN1.

MeSH Term

Methylenetetrahydrofolate Reductase (NADPH2)
Humans
Methylenetetrahydrofolate Dehydrogenase (NADP)
Phosphorylation
HEK293 Cells
Minor Histocompatibility Antigens
Protein Binding
Methionine

Chemicals

Methylenetetrahydrofolate Reductase (NADPH2)
Methylenetetrahydrofolate Dehydrogenase (NADP)
MTHFR protein, human
MTHFD1 protein, human
Minor Histocompatibility Antigens
Methionine

Word Cloud

Created with Highcharts 10.0.0MTHFRmethionineinteractionMTHFD15aminomethylenetetrahydrofolateGCN1reductasefolatecycleenzymephosphorylatedacidsvariantsconditionsphosphorylationthreeidentifiedproteinsdehydrogenasegeneralcontrolnonderepressible1domaindirect10-Methylenetetrahydrofolaterequiredintracellularsynthesispreviouslyshownpartially16residuesabrogatedconversionthreonine34alanineT34Atruncationfirst37ieexpression38-656promotedsupplementationover-expressedwild-typeMTFHRwell293Tcellsprovideinsightsmechanismsdemonstratefollowingincubationhigh100-1000 ��Malmostcompletelyrestricted0-10 ��MreducedalwaysremainsunphosphorylatedFollowingaffinitypurificationcoupledmassspectrometryemptyvectorseparateexperiments134consistentlypulled-downproteinindicatedlikelytrueinteractorsSAINTpredictionthreshold0952fold-changeAmongstacidstarvationsensorImmunoprecipitation-immunoblottingreplicatedAlphaFold3generatedmodelMTHFR-MTHFD1placesdehydrogenase/cyclohydrolasecontactcatalyticsuggestingmayfacilitatedeliveryOverallconfirmavailabilityincreasespotentialEvidence10-methylenetetrahydrofolate

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