DHFR

Summary: Recombinant human DHFR binds its own mRNA in gel-shift assays and represses its translation in reticulocyte lysate; binding is relieved by substrates or methotrexate. This autoregulatory activity is supported but is secondary to the catalytic role.

Reason: In vitro evidence for translational autoregulation; a regulatory moonlighting activity rather than the gene's core function.

Summary: DHFR binds its own mRNA (and that of DHFRL1/DHFR2) for translational autoregulation.

Reason: Supported RNA-binding activity tied to autoregulation; non-core. The more informative term GO:0000900 is also annotated.

Summary: Human DHFR activity assayed as the target enzyme in an inhibitor/structure study. Core catalytic activity: NADPH-dependent reduction of 7,8-dihydrofolate to tetrahydrofolate.

Reason: Direct assay of human DHFR catalysis supports the core molecular function.

Summary: Human DHFR activity assayed as the target enzyme in an inhibitor/structure study. Core catalytic activity: NADPH-dependent reduction of 7,8-dihydrofolate to tetrahydrofolate.

Reason: Direct assay of human DHFR catalysis supports the core molecular function.

Summary: Human DHFR activity assayed as the target enzyme in an inhibitor/structure study. Core catalytic activity: NADPH-dependent reduction of 7,8-dihydrofolate to tetrahydrofolate.

Reason: Direct assay of human DHFR catalysis supports the core molecular function.

Summary: Human DHFR activity assayed as the target enzyme in an inhibitor/structure study. Core catalytic activity: NADPH-dependent reduction of 7,8-dihydrofolate to tetrahydrofolate.

Reason: Direct assay of human DHFR catalysis supports the core molecular function.

Summary: Human DHFR activity assayed as the target enzyme in an inhibitor/structure study. Core catalytic activity: NADPH-dependent reduction of 7,8-dihydrofolate to tetrahydrofolate.

Reason: Direct assay of human DHFR catalysis supports the core molecular function.

Summary: Phylogenetic inference of dihydrofolate reductase activity; DHFR is the canonical member of the family and has abundant direct human evidence. Core catalytic activity: NADPH-dependent reduction of 7,8-dihydrofolate to tetrahydrofolate.

Reason: Consistent with experimental data on the human enzyme.

Summary: Atomic-resolution ternary structures of human DHFR with NADPH and antifolates define the catalytic site. Core catalytic activity: NADPH-dependent reduction of 7,8-dihydrofolate to tetrahydrofolate.

Reason: Supports the core molecular function.

Summary: Human DHFR assayed alongside DHFRL1 (DHFR2), with Km values for dihydrofolate and NADPH. Core catalytic activity: NADPH-dependent reduction of 7,8-dihydrofolate to tetrahydrofolate.

Reason: Direct kinetic characterization of the human enzyme.

Summary: Full transient and steady-state kinetic scheme for recombinant human DHFR. Core catalytic activity: NADPH-dependent reduction of 7,8-dihydrofolate to tetrahydrofolate.

Reason: Definitive enzymological evidence for the core activity.

Summary: Activity assays with human DHFR, including its weak activity on 7,8-dihydrobiopterin. Core catalytic activity: NADPH-dependent reduction of 7,8-dihydrofolate to tetrahydrofolate.

Reason: Direct human activity assays.

Summary: Automated assignment of dihydrofolate reductase activity, consistent with direct evidence.

Reason: Correct core function.

Summary: Ortholog transfer of dihydrofolate reductase activity, consistent with direct human evidence.

Reason: Correct core function.

Summary: Traceable author statement from the human DHFR cDNA sequencing paper; the activity is independently established by direct assays.

Reason: Correct core function; supported here by direct evidence from other references.

Summary: Bare protein binding. PMID:22235121 reports DHFR in an SHMT1-scaffolded thymidylate-synthesis complex at the nuclear lamina; the other sources are proteome-scale interaction maps.

Reason: Bare protein binding carries no information about DHFR activity. The SHMT1/TYMS complex is better captured through the nuclear location and dTMP biosynthesis annotations. Removal concerns informativeness and does not assert that any interaction is false.

Summary: Bare protein binding. PMID:22235121 reports DHFR in an SHMT1-scaffolded thymidylate-synthesis complex at the nuclear lamina; the other sources are proteome-scale interaction maps.

Reason: Bare protein binding carries no information about DHFR activity. The SHMT1/TYMS complex is better captured through the nuclear location and dTMP biosynthesis annotations. Removal concerns informativeness and does not assert that any interaction is false.

Summary: Bare protein binding. PMID:22235121 reports DHFR in an SHMT1-scaffolded thymidylate-synthesis complex at the nuclear lamina; the other sources are proteome-scale interaction maps.

Reason: Bare protein binding carries no information about DHFR activity. The SHMT1/TYMS complex is better captured through the nuclear location and dTMP biosynthesis annotations. Removal concerns informativeness and does not assert that any interaction is false.

Summary: Folate binding is intrinsic to catalysis; DHFR also slowly reduces folic acid to dihydrofolate (Reactome R-HSA-9709098). The cited kinetic study concerns dihydrofolate and tetrahydrofolate binding rather than folic acid specifically.

Reason: Binding of the folate substrate is part of the catalytic activity rather than an independent function; kept as non-core.

Summary: DHFR translocates to the nucleus with SHMT1 and TYMS during S and G2/M, where de novo thymidylate synthesis supports DNA replication and repair.

Reason: Experimental nuclear localization relevant to the dTMP-synthesis role.

Summary: UniProt-derived nuclear localization, based on PMID:22235121.

Reason: Consistent with experimental data.

Summary: As part of the nuclear thymidylate-synthesis complex, DHFR is enriched at sites of DNA replication.

Reason: Supported, specialized sub-nuclear location; non-core.

Summary: DHFR is a soluble cytoplasmic enzyme (cytosol, with a cell-cycle-regulated nuclear pool).

Reason: Correct general location; cytosol is the more specific term.

Summary: DHFR is a soluble cytoplasmic enzyme (cytosol, with a cell-cycle-regulated nuclear pool).

Reason: Correct general location; cytosol is the more specific term.

Summary: A mitochondrial pool of canonical DHFR is supported: a 2024 proteomic study of purified HEK293 mitochondria (PMID:38224738) measured reductase activity and found peptides unique to DHFR but none unique to the paralog DHFR2, revising the earlier attribution of mitochondrial reductase activity to DHFR2/DHFRL1 (PMID:21876184). The mitochondrial pool supports local THF regeneration, including mitochondrial dTMP synthesis.

Reason: Consistent with the high-throughput mitochondrial proteome (PMID:34800366), UniProt (by similarity to mouse) and the newer human proteomic evidence. The cytosol remains the principal site; the tissue generality of the mitochondrial pool is not established.

Summary: A mitochondrial pool of canonical DHFR is supported: a 2024 proteomic study of purified HEK293 mitochondria (PMID:38224738) measured reductase activity and found peptides unique to DHFR but none unique to the paralog DHFR2, revising the earlier attribution of mitochondrial reductase activity to DHFR2/DHFRL1 (PMID:21876184). The mitochondrial pool supports local THF regeneration, including mitochondrial dTMP synthesis.

Reason: Consistent with the high-throughput mitochondrial proteome (PMID:34800366), UniProt (by similarity to mouse) and the newer human proteomic evidence. The cytosol remains the principal site; the tissue generality of the mitochondrial pool is not established. Examined as a synthesis row (ORGANELLES, 2026-10-20): the PAINT IBD at PTN000556119 (PTHR48069-paint.tsv, Eukaryota) is seeded by mouse Dhfr (MGI:94890), rat Dhfr (RGD:2500) and human DHFR2 (Q86XF0). The DHFR2 seed is a paralog, and on its own would be a paralog transfer of the kind seen for SHMT1, but the two rodent seeds are canonical DHFR orthologues with their own IDA for mitochondrion (PMID:25980602, which found mitochondrial reductase activity in rat and mouse and no second enzyme to carry it), and the 2024 human study assigns the mitochondrial activity to canonical DHFR. So the organelle claim holds for the target. No sub-compartment is added: DHFR has no presequence or transmembrane segment, and the only sub-mitochondrial data (matrix and inner membrane, PMID:21876188) were reported for DHFRL1/DHFR2, an attribution the 2024 study revised; that leaves fewer than two agreeing lines for DHFR itself.

Summary: A mitochondrial pool of canonical DHFR is supported: a 2024 proteomic study of purified HEK293 mitochondria (PMID:38224738) measured reductase activity and found peptides unique to DHFR but none unique to the paralog DHFR2, revising the earlier attribution of mitochondrial reductase activity to DHFR2/DHFRL1 (PMID:21876184). The mitochondrial pool supports local THF regeneration, including mitochondrial dTMP synthesis.

Reason: Consistent with the high-throughput mitochondrial proteome (PMID:34800366), UniProt (by similarity to mouse) and the newer human proteomic evidence. The cytosol remains the principal site; the tissue generality of the mitochondrial pool is not established. Examined as a synthesis row (ORGANELLES, 2026-10-20): the mapping is UniProt SubCell SL-0173 (Mitochondrion), and the source line is itself by similarity (ECO:0000250) to mouse P00375, whose mitochondrial location rests on direct evidence (PMID:25980602). The feature the mapping assumes, a mitochondrial pool, is supported for human DHFR by PMID:38224738, so the root term is correct. No sub-compartment is added (see the IBA row).

Summary: A mitochondrial pool of canonical DHFR is supported: a 2024 proteomic study of purified HEK293 mitochondria (PMID:38224738) measured reductase activity and found peptides unique to DHFR but none unique to the paralog DHFR2, revising the earlier attribution of mitochondrial reductase activity to DHFR2/DHFRL1 (PMID:21876184). The mitochondrial pool supports local THF regeneration, including mitochondrial dTMP synthesis.

Reason: Consistent with the high-throughput mitochondrial proteome (PMID:34800366), UniProt (by similarity to mouse) and the newer human proteomic evidence. The cytosol remains the principal site; the tissue generality of the mitochondrial pool is not established. Examined as a synthesis row (ORGANELLES, 2026-10-20): the source is mouse DHFR P00375, the 1:1 orthologue (not the DHFR2 paralog), which holds mitochondrion by IDA from PMID:25980602; rodents have no active second reductase, so their mitochondrial activity is canonical DHFR, which matches the human finding in PMID:38224738. The source records only the organelle, so the root term is right. No sub-compartment is added (see the IBA row).

Summary: Reactome places DHFR reactions in the cytosol, the principal site of the folate cycle.

Reason: Correct principal location.

Summary: Reactome places DHFR reactions in the cytosol, the principal site of the folate cycle.

Reason: Correct principal location.

Summary: Reactome places DHFR reactions in the cytosol, the principal site of the folate cycle.

Reason: Correct principal location.

Summary: DHFR supports de novo thymidylate (dTMP) synthesis by regenerating tetrahydrofolate for the methylene-THF-dependent thymidylate synthase reaction. Thymidylate is a nucleotide; DHFR does not act in pyrimidine nucleobase synthesis.

Reason: The process studied is dTMP biosynthesis; replace with GO:0006231.

Proposed replacements: dTMP biosynthetic process

Summary: The thymidylate-synthesis complex localizes to replication forks and supplies dTMP for DNA synthesis, but DHFR does not perform a step of DNA replication itself.

Reason: DHFR provides precursor supply; DNA replication is an indirect consequence.

Summary: DHFR reduces 7,8-dihydrobiopterin back to tetrahydrobiopterin (a salvage reaction), albeit with low affinity; DHFR-deficient patients have cerebral BH4 deficiency.

Reason: Supported secondary pathway role (BH4 salvage rather than de novo biosynthesis); non-core.

Summary: Ortholog transfer of the BH4 salvage role, consistent with human data.

Reason: Supported secondary role; non-core.

Summary: DHFR protein inhibits translation of its own mRNA in vitro.

Reason: Supported autoregulatory role; non-core.

Summary: Ortholog transfer of a role in axon regeneration, presumably from folate-supplementation studies in rodents. Any requirement reflects general folate supply for nucleotide and methylation reactions rather than a specific role of DHFR in the regenerative process.

Reason: Indirect, pleiotropic consequence of folate metabolism.

Summary: DHFR consumes dihydrofolate in its core reaction.

Reason: Accurate process description of the core activity.

Summary: DHFR consumes dihydrofolate in its core reaction.

Reason: Accurate process description of the core activity.

Summary: DHFR consumes dihydrofolate in its core reaction.

Reason: Accurate process description of the core activity.

Summary: DHFR produces tetrahydrofolate, the central carrier of one-carbon units.

Reason: Accurate process; GO:0046654 is the more specific biosynthetic term.

Summary: DHFR produces tetrahydrofolate, the central carrier of one-carbon units.

Reason: Accurate process; GO:0046654 is the more specific biosynthetic term.

Summary: DHFR produces tetrahydrofolate, the central carrier of one-carbon units.

Reason: Accurate process; GO:0046654 is the more specific biosynthetic term.

Summary: DHFR catalyzes the only step that produces tetrahydrofolate from dihydrofolate, both for dietary folate and for DHF released by thymidylate synthase.

Reason: Core biological process.

Summary: DHFR catalyzes the only step that produces tetrahydrofolate from dihydrofolate, both for dietary folate and for DHF released by thymidylate synthase.

Reason: Core biological process.

Summary: DHFR catalyzes the only step that produces tetrahydrofolate from dihydrofolate, both for dietary folate and for DHF released by thymidylate synthase.

Reason: Core biological process.

Summary: DHFR reduces folic acid and dihydrofolate and so acts in folic acid metabolism.

Reason: Accurate, if general.

Summary: DHFR reduces folic acid and dihydrofolate and so acts in folic acid metabolism.

Reason: Accurate, if general.

Summary: DHFR binds NADPH as hydride donor and releases NADP+; nucleotide-cofactor binding is part of catalysis.

Reason: Correct cofactor binding; captured by the catalytic activity term.

Summary: DHFR binds NADPH as hydride donor and releases NADP+; nucleotide-cofactor binding is part of catalysis.

Reason: Correct cofactor binding; captured by the catalytic activity term.

Summary: Stopped-flow kinetics define NADPH binding to human DHFR.

Reason: Correct cofactor binding; part of the catalytic activity.

Summary: Broad ARBA term; DHFR supports dTMP synthesis indirectly via cofactor regeneration.

Reason: Overly general; dTMP biosynthetic process captures the specific contribution.

Summary: DHFR binds a specific region of its own mRNA.

Reason: Supported autoregulatory activity; non-core.

Summary: Transferred from rodent studies of BH4 recycling and endothelial NO synthase coupling. Effects on superoxide are an indirect consequence of BH4 supply, and human endothelial DHFR has low activity on 7,8-dihydrobiopterin.

Reason: Indirect downstream effect, not a function of DHFR itself.