{"gene":"GNMT","run_date":"2026-06-10T01:55:21","timeline":{"discoveries":[{"year":1998,"finding":"Crystal structure of apo-GNMT determined at 2.5 Å resolution, revealing it is a homotetramer (monomer Mr ~32,423 Da, 292 amino acids) with a three-domain monomer structure and a central channel. The tetramer assembles with intertwined N-terminal domains. Comparison with the AdoMet-bound structure showed only localized changes in the binding pocket residues between apo and substrate-bound forms.","method":"X-ray crystallography (2.5 Å resolution, orthorhombic space group P2(1)2(1)2, R-factor 21.9%)","journal":"Protein science","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional validation by comparison to substrate-bound form, published in peer-reviewed journal","pmids":["9655336"],"is_preprint":false},{"year":1998,"finding":"GNMT catalyzes methyl group transfer from S-adenosylmethionine (SAM) to glycine, producing S-adenosylhomocysteine (SAH) and sarcosine (N-methylglycine). The enzyme is inhibited by 5-methyltetrahydrofolate pentaglutamate, functioning as a regulatory enzyme controlling the SAM:SAH ratio.","method":"Crystal structure and prior biochemical characterization referenced in structural paper","journal":"Protein science","confidence":"High","confidence_rationale":"Tier 1 / Strong — enzymatic activity established by structural and biochemical characterization, replicated across multiple labs and papers in corpus","pmids":["9655336"],"is_preprint":false},{"year":2011,"finding":"GNMT expression increases hepatic folate concentration and promotes folate-dependent homocysteine remethylation via methionine synthase. GNMT transgenic mice had significantly increased hepatic folate, while GNMT knockout mice had reduced folate and decreased methionine synthase expression. GNMT also reduced antifolate methotrexate cytotoxicity in cell lines.","method":"GNMT transgenic and knockout mouse models; stable isotopic tracers with GC/MS for remethylation flux; quantitative folate measurement; Western blot for methionine synthase","journal":"Molecular medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (isotopic tracers, transgenic/KO mouse models, protein expression), replicated across cell and animal models","pmids":["21210071"],"is_preprint":false},{"year":2013,"finding":"GNMT supports folate-dependent pyrimidine and purine nucleotide synthesis and reduces uracil misincorporation into DNA. During prolonged folate depletion, GNMT translocates from cytoplasm into the nucleus. Loss of GNMT impairs nucleotide biosynthesis in vitro and in vivo.","method":"Stable isotopic tracers and GC/MS; uracil content assay in cells and Gnmt wildtype, heterozygote, and knockout mice; fluorescence microscopy for nuclear translocation","journal":"International journal of cancer","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods including isotopic tracers, genetic mouse models, and direct localization imaging with functional consequence","pmids":["23922098"],"is_preprint":false},{"year":2014,"finding":"In Drosophila, Gnmt is transcriptionally upregulated by dFoxO in the fat body in response to Toll-pathway activation (sterile inflammation from necrosis) and during fasting. Gnmt upregulation leads to increased sarcosine and reduced SAM levels in hemolymph, functioning as a rheostat for SAM metabolism and energy homeostasis.","method":"Drosophila genetic models (apoptosis-deficient mutants); metabolomic analysis of hemolymph; genetic epistasis with dFoxO and Toll pathway components","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — metabolomics plus genetic epistasis in vivo, multiple mutant combinations tested","pmids":["24746817"],"is_preprint":false},{"year":2014,"finding":"GNMT deficiency in mice triggers NK cell activation and TRAIL-mediated liver injury and fibrogenesis. Genetic deletion of TRAIL in GNMT-/- mice (double TRAIL-/-/GNMT-/- mice) protected against chronic liver injury and fibrogenesis. In vivo silencing of DR5 (TRAIL receptor) also protected GNMT-/- mice from liver injury, establishing the TRAIL/DR5 axis as a key pathway downstream of GNMT deficiency.","method":"Double knockout mouse model (TRAIL-/-/GNMT-/-); bile duct ligation model; in vivo NK cell depletion; in vivo DR5 silencing","journal":"Laboratory investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with double KO mice and multiple intervention strategies in vivo","pmids":["25531568"],"is_preprint":false},{"year":2017,"finding":"GNMT interacts with PREX2 (a PTEN inhibitor) and promotes its degradation through an E3 ligase HectH9-mediated proteasomal ubiquitination pathway. Depletion of GNMT or HectH9 results in PREX2 accumulation, AKT activation, and enhanced cell proliferation. Elevated PREX2 protein with AKT activation was confirmed in Gnmt knockout mouse liver.","method":"Co-immunoprecipitation; proteasomal ubiquitination assay; siRNA depletion of GNMT/HectH9; Western blot for AKT activation; Gnmt knockout mouse liver analysis; human HCC sample immunohistochemistry","journal":"International journal of cancer","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP identifying interaction, ubiquitination assay, genetic KO mouse model, and human tissue validation","pmids":["28205209"],"is_preprint":false},{"year":2019,"finding":"GNMT functions as an essential regulator of mitochondrial Complex II (succinate dehydrogenase) activity in the electron transport chain. In NAFLD, GNMT is post-transcriptionally repressed by miR-873-5p in hepatocytes, leading to disrupted mitochondrial functionality and impaired fatty acid β-oxidation.","method":"miR-873-5p overexpression/knockdown in vitro and in vivo NAFLD murine models; Complex II activity assay; anti-miR-873-5p therapy; liver biopsies from NAFLD/NASH patients","journal":"Molecular metabolism","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Complex II activity measured in cell and mouse models by single lab; miR-873-5p/GNMT axis confirmed in human tissue, but full mechanistic reconstitution of GNMT-Complex II interaction not provided in abstract","pmids":["31668391"],"is_preprint":false},{"year":2019,"finding":"Benzo[a]pyrene (BaP) treatment induces phosphorylation of GNMT at serine 9, which is required for BaP-induced nuclear translocation of GNMT. A serine 9 mutant incapable of phosphorylation showed dramatically decreased nuclear translocation and increased CYP1A1 expression upon BaP treatment. PKC and JNK were identified as candidate kinases for this phosphorylation.","method":"LC-MS/MS phosphoproteomics; site-directed mutagenesis (S9A mutant); fluorescence microscopy for nuclear translocation; CYP1A1 expression by Western blot","journal":"Journal of food and drug analysis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphorylation site identified by MS and validated by mutagenesis with functional consequence; kinase assignment (PKC, JNK) is pharmacological without direct reconstitution","pmids":["30987732"],"is_preprint":false},{"year":2019,"finding":"MYC transcriptionally represses GNMT by binding to the GNMT promoter. ChIP assay showed MYC antibodies precipitated the human GNMT promoter. MYC overexpression inhibited GNMT promoter activity and endogenous GNMT protein; shRNA knockdown or pharmacological inhibition of MYC induced GNMT promoter activity and mRNA expression in hepatoma cells. GNMT and MYC expression were negatively correlated in human HCC samples.","method":"Chromatin immunoprecipitation (ChIP); luciferase reporter assay; shRNA knockdown of MYC; MYC overexpression; qRT-PCR; human HCC sample correlation","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct ChIP evidence for MYC binding GNMT promoter combined with loss- and gain-of-function experiments and human tissue validation","pmids":["30760754"],"is_preprint":false},{"year":2020,"finding":"GNMT is a direct molecular target of carnosine in renal tubular epithelial cells, identified by cellular thermal shift assay (CETSA) and molecular docking. Increased GNMT expression mimicked carnosine's protective effects (reducing inflammation and fibrosis), and inhibition of GNMT abolished carnosine's protective effects, placing GNMT downstream of carnosine in this pathway.","method":"CETSA; molecular docking; transient transfection (GNMT overexpression); siRNA knockdown of GNMT; in vivo DN mouse models","journal":"Clinical science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CETSA identifies direct binding; functional epistasis confirmed by overexpression and knockdown, single lab","pmids":["33241846"],"is_preprint":false},{"year":2024,"finding":"In Drosophila fat body, Gnmt protein is degraded via the nuclear ubiquitin-proteasome system (UPS) under conditions of SAM shortage (nutrient deprivation or inhibition of SAM synthesis). This degradation maintains SAM levels. Inhibition of nuclear UPS-mediated Gnmt degradation causes starvation tolerance, demonstrating that Gnmt turnover is a mechanism for buffering SAM consumption.","method":"Drosophila genetic models; metabolomics (SAM measurement); nuclear UPS inhibition; starvation assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and metabolomic evidence in Drosophila model, preprint not yet peer-reviewed","pmids":["bio_10.1101_2024.08.21.609067"],"is_preprint":true},{"year":2024,"finding":"Aurora kinase A (AurA) promotes nuclear localization of FOXO3, which induces GNMT expression, thereby consuming SAM. AurA inhibition increases GNMT expression and reduces cellular SAM levels, leading to decreased H3K4me3 and H3K36me3 on Il6 and Tnf gene regions in trained macrophages, dampening trained immunity. This places GNMT in the mTOR-FOXO3-GNMT axis regulating SAM-dependent histone methylation.","method":"ATAC-seq; RNA-seq; metabolomics; ChIP for histone methylation marks; AurA inhibitor treatment; β-glucan trained immunity model in mouse macrophages","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multi-omic approach with metabolomics and ChIP, preprint not yet peer-reviewed, single lab","pmids":["bio_10.1101_2024.11.11.622956"],"is_preprint":true},{"year":2026,"finding":"The GNMT N-terminal tail (specifically phosphorylation at serine 9, S9ph) is required for both catalytic turnover of SAM and for 5-methyltetrahydrofolate (5mTHF) binding/feedback inhibition. Distal N-terminal truncation (residues 1-8) or phosphomimetic substitution at S9 abolished 5mTHF binding while maintaining catalytic activity, uncoupling folate-feedback regulation from enzymatic function. S9ph is abundant in mouse liver and further enriched in aged mice. Constitutively active GNMT mutants in hepatocyte cell lines depleted SAM, increased SAH, disrupted protein methylation, impaired growth, and induced methyl-donor stress transcriptional responses.","method":"Structural analysis; biochemical binding assays; molecular dynamics simulations; phosphoproteomics in mouse liver; site-directed mutagenesis; lentiviral overexpression in hepatocyte cell lines; metabolomics (SAM/SAH); protein methylation assays; transcriptomic analysis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — structural, biochemical, and cell-based mutagenesis data, preprint not yet peer-reviewed but multiple orthogonal approaches in single study","pmids":["42244597"],"is_preprint":true},{"year":2025,"finding":"Choline upregulates GNMT expression (mRNA and protein) in hepatocytes in a concentration-dependent manner. GNMT knockdown reversed the protective effects of choline on lipid synthesis genes (FAS, ACC), fatty acid oxidation (CPT1), lipoprotein assembly (ApoB100, MTTP), and bile acid metabolism (CYP7A1, CYP27A1, BSEP). AMPK inhibition reduced GNMT protein expression and elevated MYC, suggesting choline regulates GNMT through the AMPK/MYC axis.","method":"siRNA knockdown of GNMT; AMPK inhibitor treatment; transcriptomic profiling; Western blot; RT-PCR in primary calf and human LO2 hepatocytes","journal":"Stress biology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, pharmacological inhibition for AMPK without direct reconstitution, AMPK/MYC/GNMT axis inferred rather than directly demonstrated","pmids":["41233636"],"is_preprint":false}],"current_model":"GNMT is a homotetrameric SAM-dependent methyltransferase that transfers a methyl group from SAM to glycine to produce sarcosine and SAH, thereby regulating the cellular SAM:SAH ratio and methyl-donor homeostasis; its activity is subject to feedback inhibition by 5-methyltetrahydrofolate through binding to its N-terminal tail (phosphorylatable at S9), and it supports hepatic folate retention, nucleotide biosynthesis, and DNA integrity; GNMT also interacts with PREX2 to promote its HectH9-mediated proteasomal degradation (suppressing AKT signaling), regulates mitochondrial Complex II activity, and undergoes nuclear translocation upon BaP-induced S9 phosphorylation, while its expression is transcriptionally repressed by MYC and post-transcriptionally by miR-873-5p, and its protein levels are buffered by nuclear ubiquitin-proteasome-mediated degradation under SAM-shortage conditions."},"narrative":{"mechanistic_narrative":"GNMT is a homotetrameric S-adenosylmethionine (SAM)-dependent methyltransferase that transfers a methyl group from SAM to glycine, generating sarcosine and S-adenosylhomocysteine (SAH) and thereby acting as a regulatory node controlling the cellular SAM:SAH ratio and methyl-donor homeostasis [PMID:9655336]. Its enzymatic output is held in check by feedback inhibition from 5-methyltetrahydrofolate, and structural and biochemical dissection shows this regulation is mediated through the N-terminal tail: truncation of residues 1-8 or a phosphomimetic at serine 9 abolishes 5-methyltetrahydrofolate binding while preserving catalytic turnover, uncoupling folate feedback from activity and—when constitutively active—depleting SAM, raising SAH, and inducing methyl-donor stress [PMID:42244597]. Through this control of one-carbon flux, GNMT increases hepatic folate retention and folate-dependent homocysteine remethylation via methionine synthase [PMID:21210071] and supports folate-dependent purine/pyrimidine synthesis, limiting uracil misincorporation into DNA, with the enzyme translocating from cytoplasm to nucleus under prolonged folate depletion [PMID:23922098]. Beyond catalysis, GNMT binds PREX2 and promotes its HectH9-mediated proteasomal degradation, restraining AKT signaling and proliferation [PMID:28205209], and acts as a regulator of mitochondrial Complex II activity [PMID:31668391]. GNMT loss in mouse liver triggers NK-cell activation and TRAIL/DR5-mediated injury and fibrogenesis [PMID:25531568]. Its expression is transcriptionally repressed by MYC at the GNMT promoter [PMID:30760754] and post-transcriptionally repressed by miR-873-5p [PMID:31668391]. Benzo[a]pyrene induces serine-9 phosphorylation that drives nuclear translocation and modulates CYP1A1 expression [PMID:30987732].","teleology":[{"year":1998,"claim":"Established the architecture and catalytic identity of GNMT, defining it as a homotetrameric SAM-dependent methyltransferase that converts glycine to sarcosine and is feedback-inhibited by methyltetrahydrofolate, framing it as a regulator of the SAM:SAH ratio rather than a simple metabolic enzyme.","evidence":"X-ray crystallography of apo- and SAM-bound forms with biochemical characterization","pmids":["9655336"],"confidence":"High","gaps":["Structural basis of folate feedback inhibition not resolved at this stage","No in vivo physiological role demonstrated"]},{"year":2011,"claim":"Connected GNMT activity to organismal one-carbon physiology, showing it controls hepatic folate retention and folate-dependent homocysteine remethylation.","evidence":"GNMT transgenic and knockout mice with isotopic remethylation tracers and methionine synthase Western blot","pmids":["21210071"],"confidence":"High","gaps":["Mechanism linking GNMT activity to folate retention not fully resolved","Does not address nucleotide-level consequences"]},{"year":2013,"claim":"Extended GNMT's metabolic role to genome integrity by showing it supports nucleotide synthesis and limits uracil misincorporation, and revealed regulated nuclear translocation during folate depletion.","evidence":"Isotopic tracers, uracil assays in Gnmt genotype-series mice, fluorescence microscopy","pmids":["23922098"],"confidence":"High","gaps":["Signal driving nuclear translocation not identified here","Nuclear function of GNMT not defined"]},{"year":2014,"claim":"Demonstrated GNMT acts as a tunable rheostat for SAM metabolism via dFoxO-driven transcriptional induction during inflammation and fasting, linking it to energy homeostasis.","evidence":"Drosophila genetic epistasis with dFoxO/Toll pathway and hemolymph metabolomics","pmids":["24746817"],"confidence":"High","gaps":["Conservation of dFoxO regulation in mammals not tested","Downstream consumers of altered SAM not mapped"]},{"year":2014,"claim":"Defined the pathological consequence of GNMT loss in liver as NK-cell activation and TRAIL/DR5-mediated injury and fibrogenesis, identifying an actionable downstream axis.","evidence":"TRAIL-/-/GNMT-/- double knockout, NK depletion, and in vivo DR5 silencing","pmids":["25531568"],"confidence":"High","gaps":["Link between SAM/SAH imbalance and NK activation not mechanistically resolved","Does not establish whether catalytic activity is required"]},{"year":2017,"claim":"Revealed a non-catalytic scaffolding function: GNMT promotes HectH9-mediated ubiquitination and degradation of PREX2, restraining AKT signaling and proliferation.","evidence":"Reciprocal Co-IP, ubiquitination assay, siRNA depletion, Gnmt KO liver, and human HCC tissue","pmids":["28205209"],"confidence":"High","gaps":["Whether GNMT enzymatic activity is required for PREX2 degradation unknown","Structural basis of GNMT-PREX2-HectH9 assembly not defined"]},{"year":2019,"claim":"Implicated GNMT in mitochondrial bioenergetics by linking its miR-873-5p-mediated repression to impaired Complex II activity and fatty acid oxidation in NAFLD.","evidence":"miR-873-5p gain/loss in vitro and in NAFLD mouse models, Complex II assays, human NASH biopsies","pmids":["31668391"],"confidence":"Medium","gaps":["Direct GNMT-Complex II molecular interaction not reconstituted","Whether effect is catalytic or independent of SAM metabolism unclear"]},{"year":2019,"claim":"Identified serine-9 phosphorylation as the trigger for xenobiotic-induced nuclear translocation of GNMT, coupling its localization to BaP exposure and CYP1A1 regulation.","evidence":"Phosphoproteomics, S9A mutagenesis, fluorescence microscopy, CYP1A1 Western blot","pmids":["30987732"],"confidence":"Medium","gaps":["Kinase assignment (PKC/JNK) pharmacological without direct reconstitution","Nuclear function downstream of translocation not defined"]},{"year":2019,"claim":"Established direct transcriptional repression of GNMT by MYC, providing a mechanism for GNMT loss in hepatocellular carcinoma.","evidence":"ChIP, luciferase reporter, MYC gain/loss, and human HCC correlation","pmids":["30760754"],"confidence":"High","gaps":["Cofactors mediating MYC repression not identified","Does not address combined transcriptional and post-transcriptional control"]},{"year":2020,"claim":"Placed GNMT downstream of carnosine as a direct binding target mediating renal anti-inflammatory and anti-fibrotic protection.","evidence":"CETSA, molecular docking, GNMT overexpression/knockdown, diabetic nephropathy mouse models","pmids":["33241846"],"confidence":"Medium","gaps":["Binding site and functional consequence of carnosine binding not defined","Single-lab finding without independent confirmation"]},{"year":2024,"claim":"Showed GNMT protein levels are buffered by nuclear UPS-mediated degradation under SAM shortage, defining protein turnover as a homeostatic mechanism for SAM conservation.","evidence":"Drosophila genetics, SAM metabolomics, nuclear UPS inhibition, starvation assays (preprint)","pmids":["bio_10.1101_2024.08.21.609067"],"confidence":"Medium","gaps":["E3 ligase mediating nuclear Gnmt degradation not identified","Conservation in mammals not tested","Preprint, not peer-reviewed"]},{"year":2024,"claim":"Positioned GNMT in an mTOR-FOXO3-GNMT axis that consumes SAM to limit SAM-dependent histone methylation and dampen trained immunity.","evidence":"ATAC-seq, RNA-seq, metabolomics, histone-mark ChIP, AurA inhibition in trained macrophage model (preprint)","pmids":["bio_10.1101_2024.11.11.622956"],"confidence":"Medium","gaps":["Direct demonstration that GNMT-driven SAM depletion causes the histone changes not isolated","Preprint, not peer-reviewed"]},{"year":2026,"claim":"Resolved how the N-terminal tail and serine-9 phosphorylation uncouple folate feedback from catalysis, showing constitutive GNMT activity depletes SAM and triggers methyl-donor stress.","evidence":"Structural analysis, MD simulations, binding assays, S9 mutagenesis, liver phosphoproteomics, hepatocyte overexpression with metabolomics (preprint)","pmids":["42244597"],"confidence":"Medium","gaps":["In vivo kinase responsible for S9 phosphorylation not established","Physiological trigger for loss of feedback control in aging not defined","Preprint, not peer-reviewed"]},{"year":2025,"claim":"Proposed that choline regulates GNMT through an AMPK/MYC axis to coordinate hepatic lipid and bile acid metabolism.","evidence":"GNMT knockdown, AMPK inhibition, transcriptomics in calf and human hepatocytes","pmids":["41233636"],"confidence":"Low","gaps":["AMPK/MYC/GNMT axis inferred from pharmacology, not directly reconstituted","Single-lab, low-confidence finding","Mechanism linking GNMT to lipid/bile gene expression undefined"]},{"year":null,"claim":"Whether GNMT's catalytic methyltransferase activity is required for its non-enzymatic functions (PREX2 degradation, Complex II regulation, nuclear signaling) remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No experiment separates catalytic-dead GNMT from scaffolding roles","Nuclear function of translocated GNMT undefined","Mammalian relevance of Drosophila UPS-buffering not tested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,13]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[1,13]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3,8]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[3,8,11]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[1,2,4]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[6,13]}],"complexes":[],"partners":["PREX2","HECTH9"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q14749","full_name":"Glycine N-methyltransferase","aliases":[],"length_aa":295,"mass_kda":32.7,"function":"Catalyzes the methylation of glycine by using S-adenosylmethionine (AdoMet) to form N-methylglycine (sarcosine) with the concomitant production of S-adenosylhomocysteine (AdoHcy), a reaction regulated by the binding of 5-methyltetrahydrofolate. Plays an important role in the regulation of methyl group metabolism by regulating the ratio between S-adenosyl-L-methionine and S-adenosyl-L-homocysteine","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q14749/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/GNMT","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/GNMT","total_profiled":1310},"omim":[{"mim_id":"606664","title":"GLYCINE N-METHYLTRANSFERASE DEFICIENCY","url":"https://www.omim.org/entry/606664"},{"mim_id":"606628","title":"GLYCINE N-METHYLTRANSFERASE; GNMT","url":"https://www.omim.org/entry/606628"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"liver","ntpm":278.9},{"tissue":"pancreas","ntpm":563.6}],"url":"https://www.proteinatlas.org/search/GNMT"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"Q14749","domains":[{"cath_id":"3.40.50.150","chopping":"25-179_248-292","consensus_level":"high","plddt":93.5772,"start":25,"end":292}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14749","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q14749-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q14749-F1-predicted_aligned_error_v6.png","plddt_mean":88.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=GNMT","jax_strain_url":"https://www.jax.org/strain/search?query=GNMT"},"sequence":{"accession":"Q14749","fasta_url":"https://rest.uniprot.org/uniprotkb/Q14749.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q14749/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14749"}},"corpus_meta":[{"pmid":"24746817","id":"PMC_24746817","title":"Necrosis-driven systemic immune response alters SAM metabolism through the FOXO-GNMT axis.","date":"2014","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/24746817","citation_count":68,"is_preprint":false},{"pmid":"12566309","id":"PMC_12566309","title":"Genotypic and phenotypic characterization of a putative tumor susceptibility gene, GNMT, in liver cancer.","date":"2003","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/12566309","citation_count":63,"is_preprint":false},{"pmid":"33241846","id":"PMC_33241846","title":"Carnosine alleviates diabetic nephropathy by targeting GNMT, a key enzyme mediating renal inflammation and fibrosis.","date":"2020","source":"Clinical science (London, England : 1979)","url":"https://pubmed.ncbi.nlm.nih.gov/33241846","citation_count":45,"is_preprint":false},{"pmid":"31668391","id":"PMC_31668391","title":"miR-873-5p targets mitochondrial GNMT-Complex II interface contributing to non-alcoholic fatty liver disease.","date":"2019","source":"Molecular metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/31668391","citation_count":43,"is_preprint":false},{"pmid":"21210071","id":"PMC_21210071","title":"GNMT expression increases hepatic folate contents and folate-dependent methionine synthase-mediated homocysteine remethylation.","date":"2011","source":"Molecular medicine (Cambridge, Mass.)","url":"https://pubmed.ncbi.nlm.nih.gov/21210071","citation_count":38,"is_preprint":false},{"pmid":"23147572","id":"PMC_23147572","title":"The multi-functional roles of GNMT in toxicology and cancer.","date":"2012","source":"Toxicology and applied pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/23147572","citation_count":34,"is_preprint":false},{"pmid":"28205209","id":"PMC_28205209","title":"Characterization of the GNMT-HectH9-PREX2 tripartite relationship in the pathogenesis of hepatocellular carcinoma.","date":"2017","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/28205209","citation_count":28,"is_preprint":false},{"pmid":"23922098","id":"PMC_23922098","title":"A novel role of the tumor suppressor GNMT in cellular defense against DNA damage.","date":"2013","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/23922098","citation_count":25,"is_preprint":false},{"pmid":"25531568","id":"PMC_25531568","title":"TRAIL-producing NK cells contribute to liver injury and related fibrogenesis in the context of GNMT deficiency.","date":"2014","source":"Laboratory investigation; a journal of technical methods and pathology","url":"https://pubmed.ncbi.nlm.nih.gov/25531568","citation_count":24,"is_preprint":false},{"pmid":"30217986","id":"PMC_30217986","title":"AAV serotype 8-mediated liver specific GNMT expression delays progression of hepatocellular carcinoma and prevents carbon tetrachloride-induced liver damage.","date":"2018","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/30217986","citation_count":22,"is_preprint":false},{"pmid":"9655336","id":"PMC_9655336","title":"Crystal structure of apo-glycine N-methyltransferase (GNMT).","date":"1998","source":"Protein science : a publication of the Protein Society","url":"https://pubmed.ncbi.nlm.nih.gov/9655336","citation_count":22,"is_preprint":false},{"pmid":"33714108","id":"PMC_33714108","title":"Polymorphisms in GNMT and DNMT3b are associated with methotrexate treatment outcome in plaque psoriasis.","date":"2021","source":"Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie","url":"https://pubmed.ncbi.nlm.nih.gov/33714108","citation_count":20,"is_preprint":false},{"pmid":"22807109","id":"PMC_22807109","title":"Human liver methionine cycle: MAT1A and GNMT gene resequencing, functional genomics, and hepatic genotype-phenotype correlation.","date":"2012","source":"Drug metabolism and disposition: the biological fate of chemicals","url":"https://pubmed.ncbi.nlm.nih.gov/22807109","citation_count":17,"is_preprint":false},{"pmid":"30760754","id":"PMC_30760754","title":"Induction of GNMT by 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranoside through proteasome-independent MYC downregulation in hepatocellular carcinoma.","date":"2019","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/30760754","citation_count":16,"is_preprint":false},{"pmid":"26598833","id":"PMC_26598833","title":"Regulation of Folate-Mediated One-Carbon Metabolism by Glycine N-Methyltransferase (GNMT) and Methylenetetrahydrofolate Reductase (MTHFR).","date":"2015","source":"Journal of nutritional science and vitaminology","url":"https://pubmed.ncbi.nlm.nih.gov/26598833","citation_count":15,"is_preprint":false},{"pmid":"16317120","id":"PMC_16317120","title":"The glycine N-methyltransferase (GNMT) 1289 C->T variant influences plasma total homocysteine concentrations in young women after restricting folate intake.","date":"2005","source":"The Journal of nutrition","url":"https://pubmed.ncbi.nlm.nih.gov/16317120","citation_count":15,"is_preprint":false},{"pmid":"25560641","id":"PMC_25560641","title":"Epigenetic Silencing of GNMT Gene in Pancreatic Adenocarcinoma.","date":"2015","source":"Cancer genomics & proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/25560641","citation_count":13,"is_preprint":false},{"pmid":"34132534","id":"PMC_34132534","title":"Discovery of an Orally Efficacious MYC Inhibitor for Liver Cancer Using a GNMT-Based High-Throughput Screening System and Structure-Activity Relationship Analysis.","date":"2021","source":"Journal of medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/34132534","citation_count":11,"is_preprint":false},{"pmid":"30987732","id":"PMC_30987732","title":"Utilizing proteomic approach to identify nuclear translocation related serine kinase phosphorylation site of GNMT as downstream effector for benzo[a]pyrene.","date":"2019","source":"Journal of food and drug analysis","url":"https://pubmed.ncbi.nlm.nih.gov/30987732","citation_count":6,"is_preprint":false},{"pmid":"38250306","id":"PMC_38250306","title":"The Role of GNMT and MMP12 Expression in Determining TACE Efficacy: Validation at Transcription and Protein Levels.","date":"2024","source":"Journal of hepatocellular carcinoma","url":"https://pubmed.ncbi.nlm.nih.gov/38250306","citation_count":5,"is_preprint":false},{"pmid":"30061177","id":"PMC_30061177","title":"Differential expression of NPM, GSTA3, and GNMT in mouse liver following long-term in vivo irradiation by means of uranium tailings.","date":"2018","source":"Bioscience reports","url":"https://pubmed.ncbi.nlm.nih.gov/30061177","citation_count":5,"is_preprint":false},{"pmid":"38003554","id":"PMC_38003554","title":"ERVK13-1/miR-873-5p/GNMT Axis Promotes Metastatic Potential in Human Bladder Cancer though Sarcosine Production.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/38003554","citation_count":4,"is_preprint":false},{"pmid":"33802396","id":"PMC_33802396","title":"The Case for GNMT as a Biomarker and a Therapeutic Target in Pancreatic Cancer.","date":"2021","source":"Pharmaceuticals (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/33802396","citation_count":3,"is_preprint":false},{"pmid":"41828318","id":"PMC_41828318","title":"GNMT and Its Regulatory MicroRNAs as Biomarkers and Therapeutic Targets for Metabolic Dysfunction-Associated Fatty Liver Disease and Hepatocellular Carcinoma.","date":"2026","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/41828318","citation_count":1,"is_preprint":false},{"pmid":"38877335","id":"PMC_38877335","title":"Brucine Suppresses Malignant Progression of Prostate Cancer by Decreasing Sarcosine Accumulation via Downregulation of GNMT in the Glycine/sarcosine Metabolic Pathway.","date":"2024","source":"Cell biochemistry and biophysics","url":"https://pubmed.ncbi.nlm.nih.gov/38877335","citation_count":1,"is_preprint":false},{"pmid":"41233636","id":"PMC_41233636","title":"Choline attenuates NEFA-induced hepatic steatosis via GNMT regulation in hepatocytes.","date":"2025","source":"Stress biology","url":"https://pubmed.ncbi.nlm.nih.gov/41233636","citation_count":0,"is_preprint":false},{"pmid":"42244597","id":"PMC_42244597","title":"The GNMT N-terminus Couples Folate Feedback to Methyl-donor Homeostasis.","date":"2026","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/42244597","citation_count":0,"is_preprint":false},{"pmid":"42252606","id":"PMC_42252606","title":"Computational Investigation of GNMT-Catalyzed Methyl Transfer Reaction: Integrating MD, QM, and ML Approaches.","date":"2026","source":"Journal of computational chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/42252606","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.08.21.609067","title":"S-adenosylmethionine metabolism buffering is regulated by glycine N-methyltransferase decrease via nuclear ubiquitin-proteasome system","date":"2024-08-22","source":"bioRxiv","url":"https://doi.org/10.1101/2024.08.21.609067","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.11.11.622956","title":"Aurora kinase A promotes trained immunity via regulation of endogenous S-adenosylmethionine metabolism","date":"2024-11-11","source":"bioRxiv","url":"https://doi.org/10.1101/2024.11.11.622956","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":17645,"output_tokens":4181,"usd":0.057825,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12229,"output_tokens":4323,"usd":0.08461,"stage2_stop_reason":"end_turn"},"total_usd":0.142435,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1998,\n      \"finding\": \"Crystal structure of apo-GNMT determined at 2.5 Å resolution, revealing it is a homotetramer (monomer Mr ~32,423 Da, 292 amino acids) with a three-domain monomer structure and a central channel. The tetramer assembles with intertwined N-terminal domains. Comparison with the AdoMet-bound structure showed only localized changes in the binding pocket residues between apo and substrate-bound forms.\",\n      \"method\": \"X-ray crystallography (2.5 Å resolution, orthorhombic space group P2(1)2(1)2, R-factor 21.9%)\",\n      \"journal\": \"Protein science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional validation by comparison to substrate-bound form, published in peer-reviewed journal\",\n      \"pmids\": [\"9655336\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"GNMT catalyzes methyl group transfer from S-adenosylmethionine (SAM) to glycine, producing S-adenosylhomocysteine (SAH) and sarcosine (N-methylglycine). The enzyme is inhibited by 5-methyltetrahydrofolate pentaglutamate, functioning as a regulatory enzyme controlling the SAM:SAH ratio.\",\n      \"method\": \"Crystal structure and prior biochemical characterization referenced in structural paper\",\n      \"journal\": \"Protein science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — enzymatic activity established by structural and biochemical characterization, replicated across multiple labs and papers in corpus\",\n      \"pmids\": [\"9655336\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"GNMT expression increases hepatic folate concentration and promotes folate-dependent homocysteine remethylation via methionine synthase. GNMT transgenic mice had significantly increased hepatic folate, while GNMT knockout mice had reduced folate and decreased methionine synthase expression. GNMT also reduced antifolate methotrexate cytotoxicity in cell lines.\",\n      \"method\": \"GNMT transgenic and knockout mouse models; stable isotopic tracers with GC/MS for remethylation flux; quantitative folate measurement; Western blot for methionine synthase\",\n      \"journal\": \"Molecular medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (isotopic tracers, transgenic/KO mouse models, protein expression), replicated across cell and animal models\",\n      \"pmids\": [\"21210071\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"GNMT supports folate-dependent pyrimidine and purine nucleotide synthesis and reduces uracil misincorporation into DNA. During prolonged folate depletion, GNMT translocates from cytoplasm into the nucleus. Loss of GNMT impairs nucleotide biosynthesis in vitro and in vivo.\",\n      \"method\": \"Stable isotopic tracers and GC/MS; uracil content assay in cells and Gnmt wildtype, heterozygote, and knockout mice; fluorescence microscopy for nuclear translocation\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods including isotopic tracers, genetic mouse models, and direct localization imaging with functional consequence\",\n      \"pmids\": [\"23922098\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In Drosophila, Gnmt is transcriptionally upregulated by dFoxO in the fat body in response to Toll-pathway activation (sterile inflammation from necrosis) and during fasting. Gnmt upregulation leads to increased sarcosine and reduced SAM levels in hemolymph, functioning as a rheostat for SAM metabolism and energy homeostasis.\",\n      \"method\": \"Drosophila genetic models (apoptosis-deficient mutants); metabolomic analysis of hemolymph; genetic epistasis with dFoxO and Toll pathway components\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — metabolomics plus genetic epistasis in vivo, multiple mutant combinations tested\",\n      \"pmids\": [\"24746817\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"GNMT deficiency in mice triggers NK cell activation and TRAIL-mediated liver injury and fibrogenesis. Genetic deletion of TRAIL in GNMT-/- mice (double TRAIL-/-/GNMT-/- mice) protected against chronic liver injury and fibrogenesis. In vivo silencing of DR5 (TRAIL receptor) also protected GNMT-/- mice from liver injury, establishing the TRAIL/DR5 axis as a key pathway downstream of GNMT deficiency.\",\n      \"method\": \"Double knockout mouse model (TRAIL-/-/GNMT-/-); bile duct ligation model; in vivo NK cell depletion; in vivo DR5 silencing\",\n      \"journal\": \"Laboratory investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with double KO mice and multiple intervention strategies in vivo\",\n      \"pmids\": [\"25531568\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"GNMT interacts with PREX2 (a PTEN inhibitor) and promotes its degradation through an E3 ligase HectH9-mediated proteasomal ubiquitination pathway. Depletion of GNMT or HectH9 results in PREX2 accumulation, AKT activation, and enhanced cell proliferation. Elevated PREX2 protein with AKT activation was confirmed in Gnmt knockout mouse liver.\",\n      \"method\": \"Co-immunoprecipitation; proteasomal ubiquitination assay; siRNA depletion of GNMT/HectH9; Western blot for AKT activation; Gnmt knockout mouse liver analysis; human HCC sample immunohistochemistry\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP identifying interaction, ubiquitination assay, genetic KO mouse model, and human tissue validation\",\n      \"pmids\": [\"28205209\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"GNMT functions as an essential regulator of mitochondrial Complex II (succinate dehydrogenase) activity in the electron transport chain. In NAFLD, GNMT is post-transcriptionally repressed by miR-873-5p in hepatocytes, leading to disrupted mitochondrial functionality and impaired fatty acid β-oxidation.\",\n      \"method\": \"miR-873-5p overexpression/knockdown in vitro and in vivo NAFLD murine models; Complex II activity assay; anti-miR-873-5p therapy; liver biopsies from NAFLD/NASH patients\",\n      \"journal\": \"Molecular metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Complex II activity measured in cell and mouse models by single lab; miR-873-5p/GNMT axis confirmed in human tissue, but full mechanistic reconstitution of GNMT-Complex II interaction not provided in abstract\",\n      \"pmids\": [\"31668391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Benzo[a]pyrene (BaP) treatment induces phosphorylation of GNMT at serine 9, which is required for BaP-induced nuclear translocation of GNMT. A serine 9 mutant incapable of phosphorylation showed dramatically decreased nuclear translocation and increased CYP1A1 expression upon BaP treatment. PKC and JNK were identified as candidate kinases for this phosphorylation.\",\n      \"method\": \"LC-MS/MS phosphoproteomics; site-directed mutagenesis (S9A mutant); fluorescence microscopy for nuclear translocation; CYP1A1 expression by Western blot\",\n      \"journal\": \"Journal of food and drug analysis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphorylation site identified by MS and validated by mutagenesis with functional consequence; kinase assignment (PKC, JNK) is pharmacological without direct reconstitution\",\n      \"pmids\": [\"30987732\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MYC transcriptionally represses GNMT by binding to the GNMT promoter. ChIP assay showed MYC antibodies precipitated the human GNMT promoter. MYC overexpression inhibited GNMT promoter activity and endogenous GNMT protein; shRNA knockdown or pharmacological inhibition of MYC induced GNMT promoter activity and mRNA expression in hepatoma cells. GNMT and MYC expression were negatively correlated in human HCC samples.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP); luciferase reporter assay; shRNA knockdown of MYC; MYC overexpression; qRT-PCR; human HCC sample correlation\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct ChIP evidence for MYC binding GNMT promoter combined with loss- and gain-of-function experiments and human tissue validation\",\n      \"pmids\": [\"30760754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GNMT is a direct molecular target of carnosine in renal tubular epithelial cells, identified by cellular thermal shift assay (CETSA) and molecular docking. Increased GNMT expression mimicked carnosine's protective effects (reducing inflammation and fibrosis), and inhibition of GNMT abolished carnosine's protective effects, placing GNMT downstream of carnosine in this pathway.\",\n      \"method\": \"CETSA; molecular docking; transient transfection (GNMT overexpression); siRNA knockdown of GNMT; in vivo DN mouse models\",\n      \"journal\": \"Clinical science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CETSA identifies direct binding; functional epistasis confirmed by overexpression and knockdown, single lab\",\n      \"pmids\": [\"33241846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In Drosophila fat body, Gnmt protein is degraded via the nuclear ubiquitin-proteasome system (UPS) under conditions of SAM shortage (nutrient deprivation or inhibition of SAM synthesis). This degradation maintains SAM levels. Inhibition of nuclear UPS-mediated Gnmt degradation causes starvation tolerance, demonstrating that Gnmt turnover is a mechanism for buffering SAM consumption.\",\n      \"method\": \"Drosophila genetic models; metabolomics (SAM measurement); nuclear UPS inhibition; starvation assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and metabolomic evidence in Drosophila model, preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2024.08.21.609067\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Aurora kinase A (AurA) promotes nuclear localization of FOXO3, which induces GNMT expression, thereby consuming SAM. AurA inhibition increases GNMT expression and reduces cellular SAM levels, leading to decreased H3K4me3 and H3K36me3 on Il6 and Tnf gene regions in trained macrophages, dampening trained immunity. This places GNMT in the mTOR-FOXO3-GNMT axis regulating SAM-dependent histone methylation.\",\n      \"method\": \"ATAC-seq; RNA-seq; metabolomics; ChIP for histone methylation marks; AurA inhibitor treatment; β-glucan trained immunity model in mouse macrophages\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multi-omic approach with metabolomics and ChIP, preprint not yet peer-reviewed, single lab\",\n      \"pmids\": [\"bio_10.1101_2024.11.11.622956\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"The GNMT N-terminal tail (specifically phosphorylation at serine 9, S9ph) is required for both catalytic turnover of SAM and for 5-methyltetrahydrofolate (5mTHF) binding/feedback inhibition. Distal N-terminal truncation (residues 1-8) or phosphomimetic substitution at S9 abolished 5mTHF binding while maintaining catalytic activity, uncoupling folate-feedback regulation from enzymatic function. S9ph is abundant in mouse liver and further enriched in aged mice. Constitutively active GNMT mutants in hepatocyte cell lines depleted SAM, increased SAH, disrupted protein methylation, impaired growth, and induced methyl-donor stress transcriptional responses.\",\n      \"method\": \"Structural analysis; biochemical binding assays; molecular dynamics simulations; phosphoproteomics in mouse liver; site-directed mutagenesis; lentiviral overexpression in hepatocyte cell lines; metabolomics (SAM/SAH); protein methylation assays; transcriptomic analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — structural, biochemical, and cell-based mutagenesis data, preprint not yet peer-reviewed but multiple orthogonal approaches in single study\",\n      \"pmids\": [\"42244597\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Choline upregulates GNMT expression (mRNA and protein) in hepatocytes in a concentration-dependent manner. GNMT knockdown reversed the protective effects of choline on lipid synthesis genes (FAS, ACC), fatty acid oxidation (CPT1), lipoprotein assembly (ApoB100, MTTP), and bile acid metabolism (CYP7A1, CYP27A1, BSEP). AMPK inhibition reduced GNMT protein expression and elevated MYC, suggesting choline regulates GNMT through the AMPK/MYC axis.\",\n      \"method\": \"siRNA knockdown of GNMT; AMPK inhibitor treatment; transcriptomic profiling; Western blot; RT-PCR in primary calf and human LO2 hepatocytes\",\n      \"journal\": \"Stress biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, pharmacological inhibition for AMPK without direct reconstitution, AMPK/MYC/GNMT axis inferred rather than directly demonstrated\",\n      \"pmids\": [\"41233636\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"GNMT is a homotetrameric SAM-dependent methyltransferase that transfers a methyl group from SAM to glycine to produce sarcosine and SAH, thereby regulating the cellular SAM:SAH ratio and methyl-donor homeostasis; its activity is subject to feedback inhibition by 5-methyltetrahydrofolate through binding to its N-terminal tail (phosphorylatable at S9), and it supports hepatic folate retention, nucleotide biosynthesis, and DNA integrity; GNMT also interacts with PREX2 to promote its HectH9-mediated proteasomal degradation (suppressing AKT signaling), regulates mitochondrial Complex II activity, and undergoes nuclear translocation upon BaP-induced S9 phosphorylation, while its expression is transcriptionally repressed by MYC and post-transcriptionally by miR-873-5p, and its protein levels are buffered by nuclear ubiquitin-proteasome-mediated degradation under SAM-shortage conditions.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GNMT is a homotetrameric S-adenosylmethionine (SAM)-dependent methyltransferase that transfers a methyl group from SAM to glycine, generating sarcosine and S-adenosylhomocysteine (SAH) and thereby acting as a regulatory node controlling the cellular SAM:SAH ratio and methyl-donor homeostasis [#0, #1]. Its enzymatic output is held in check by feedback inhibition from 5-methyltetrahydrofolate, and structural and biochemical dissection shows this regulation is mediated through the N-terminal tail: truncation of residues 1-8 or a phosphomimetic at serine 9 abolishes 5-methyltetrahydrofolate binding while preserving catalytic turnover, uncoupling folate feedback from activity and—when constitutively active—depleting SAM, raising SAH, and inducing methyl-donor stress [#13]. Through this control of one-carbon flux, GNMT increases hepatic folate retention and folate-dependent homocysteine remethylation via methionine synthase [#2] and supports folate-dependent purine/pyrimidine synthesis, limiting uracil misincorporation into DNA, with the enzyme translocating from cytoplasm to nucleus under prolonged folate depletion [#3]. Beyond catalysis, GNMT binds PREX2 and promotes its HectH9-mediated proteasomal degradation, restraining AKT signaling and proliferation [#6], and acts as a regulator of mitochondrial Complex II activity [#7]. GNMT loss in mouse liver triggers NK-cell activation and TRAIL/DR5-mediated injury and fibrogenesis [#5]. Its expression is transcriptionally repressed by MYC at the GNMT promoter [#9] and post-transcriptionally repressed by miR-873-5p [#7]. Benzo[a]pyrene induces serine-9 phosphorylation that drives nuclear translocation and modulates CYP1A1 expression [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established the architecture and catalytic identity of GNMT, defining it as a homotetrameric SAM-dependent methyltransferase that converts glycine to sarcosine and is feedback-inhibited by methyltetrahydrofolate, framing it as a regulator of the SAM:SAH ratio rather than a simple metabolic enzyme.\",\n      \"evidence\": \"X-ray crystallography of apo- and SAM-bound forms with biochemical characterization\",\n      \"pmids\": [\"9655336\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of folate feedback inhibition not resolved at this stage\", \"No in vivo physiological role demonstrated\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Connected GNMT activity to organismal one-carbon physiology, showing it controls hepatic folate retention and folate-dependent homocysteine remethylation.\",\n      \"evidence\": \"GNMT transgenic and knockout mice with isotopic remethylation tracers and methionine synthase Western blot\",\n      \"pmids\": [\"21210071\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking GNMT activity to folate retention not fully resolved\", \"Does not address nucleotide-level consequences\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Extended GNMT's metabolic role to genome integrity by showing it supports nucleotide synthesis and limits uracil misincorporation, and revealed regulated nuclear translocation during folate depletion.\",\n      \"evidence\": \"Isotopic tracers, uracil assays in Gnmt genotype-series mice, fluorescence microscopy\",\n      \"pmids\": [\"23922098\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signal driving nuclear translocation not identified here\", \"Nuclear function of GNMT not defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrated GNMT acts as a tunable rheostat for SAM metabolism via dFoxO-driven transcriptional induction during inflammation and fasting, linking it to energy homeostasis.\",\n      \"evidence\": \"Drosophila genetic epistasis with dFoxO/Toll pathway and hemolymph metabolomics\",\n      \"pmids\": [\"24746817\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conservation of dFoxO regulation in mammals not tested\", \"Downstream consumers of altered SAM not mapped\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined the pathological consequence of GNMT loss in liver as NK-cell activation and TRAIL/DR5-mediated injury and fibrogenesis, identifying an actionable downstream axis.\",\n      \"evidence\": \"TRAIL-/-/GNMT-/- double knockout, NK depletion, and in vivo DR5 silencing\",\n      \"pmids\": [\"25531568\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Link between SAM/SAH imbalance and NK activation not mechanistically resolved\", \"Does not establish whether catalytic activity is required\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Revealed a non-catalytic scaffolding function: GNMT promotes HectH9-mediated ubiquitination and degradation of PREX2, restraining AKT signaling and proliferation.\",\n      \"evidence\": \"Reciprocal Co-IP, ubiquitination assay, siRNA depletion, Gnmt KO liver, and human HCC tissue\",\n      \"pmids\": [\"28205209\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether GNMT enzymatic activity is required for PREX2 degradation unknown\", \"Structural basis of GNMT-PREX2-HectH9 assembly not defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Implicated GNMT in mitochondrial bioenergetics by linking its miR-873-5p-mediated repression to impaired Complex II activity and fatty acid oxidation in NAFLD.\",\n      \"evidence\": \"miR-873-5p gain/loss in vitro and in NAFLD mouse models, Complex II assays, human NASH biopsies\",\n      \"pmids\": [\"31668391\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct GNMT-Complex II molecular interaction not reconstituted\", \"Whether effect is catalytic or independent of SAM metabolism unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified serine-9 phosphorylation as the trigger for xenobiotic-induced nuclear translocation of GNMT, coupling its localization to BaP exposure and CYP1A1 regulation.\",\n      \"evidence\": \"Phosphoproteomics, S9A mutagenesis, fluorescence microscopy, CYP1A1 Western blot\",\n      \"pmids\": [\"30987732\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Kinase assignment (PKC/JNK) pharmacological without direct reconstitution\", \"Nuclear function downstream of translocation not defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established direct transcriptional repression of GNMT by MYC, providing a mechanism for GNMT loss in hepatocellular carcinoma.\",\n      \"evidence\": \"ChIP, luciferase reporter, MYC gain/loss, and human HCC correlation\",\n      \"pmids\": [\"30760754\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cofactors mediating MYC repression not identified\", \"Does not address combined transcriptional and post-transcriptional control\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Placed GNMT downstream of carnosine as a direct binding target mediating renal anti-inflammatory and anti-fibrotic protection.\",\n      \"evidence\": \"CETSA, molecular docking, GNMT overexpression/knockdown, diabetic nephropathy mouse models\",\n      \"pmids\": [\"33241846\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding site and functional consequence of carnosine binding not defined\", \"Single-lab finding without independent confirmation\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed GNMT protein levels are buffered by nuclear UPS-mediated degradation under SAM shortage, defining protein turnover as a homeostatic mechanism for SAM conservation.\",\n      \"evidence\": \"Drosophila genetics, SAM metabolomics, nuclear UPS inhibition, starvation assays (preprint)\",\n      \"pmids\": [\"bio_10.1101_2024.08.21.609067\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ligase mediating nuclear Gnmt degradation not identified\", \"Conservation in mammals not tested\", \"Preprint, not peer-reviewed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Positioned GNMT in an mTOR-FOXO3-GNMT axis that consumes SAM to limit SAM-dependent histone methylation and dampen trained immunity.\",\n      \"evidence\": \"ATAC-seq, RNA-seq, metabolomics, histone-mark ChIP, AurA inhibition in trained macrophage model (preprint)\",\n      \"pmids\": [\"bio_10.1101_2024.11.11.622956\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct demonstration that GNMT-driven SAM depletion causes the histone changes not isolated\", \"Preprint, not peer-reviewed\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Resolved how the N-terminal tail and serine-9 phosphorylation uncouple folate feedback from catalysis, showing constitutive GNMT activity depletes SAM and triggers methyl-donor stress.\",\n      \"evidence\": \"Structural analysis, MD simulations, binding assays, S9 mutagenesis, liver phosphoproteomics, hepatocyte overexpression with metabolomics (preprint)\",\n      \"pmids\": [\"42244597\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo kinase responsible for S9 phosphorylation not established\", \"Physiological trigger for loss of feedback control in aging not defined\", \"Preprint, not peer-reviewed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Proposed that choline regulates GNMT through an AMPK/MYC axis to coordinate hepatic lipid and bile acid metabolism.\",\n      \"evidence\": \"GNMT knockdown, AMPK inhibition, transcriptomics in calf and human hepatocytes\",\n      \"pmids\": [\"41233636\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"AMPK/MYC/GNMT axis inferred from pharmacology, not directly reconstituted\", \"Single-lab, low-confidence finding\", \"Mechanism linking GNMT to lipid/bile gene expression undefined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether GNMT's catalytic methyltransferase activity is required for its non-enzymatic functions (PREX2 degradation, Complex II regulation, nuclear signaling) remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No experiment separates catalytic-dead GNMT from scaffolding roles\", \"Nuclear function of translocated GNMT undefined\", \"Mammalian relevance of Drosophila UPS-buffering not tested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 13]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [1, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3, 8]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3, 8, 11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [1, 2, 4]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [6, 13]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"PREX2\", \"HectH9\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}