{"gene":"METTL9","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2021,"finding":"METTL9 is a broad-specificity methyltransferase that mediates formation of the majority of 1-methylhistidine (1MH) present in mouse and human proteomes, requiring a His-x-His (HxH) motif where 'x' is preferably a small amino acid. Substrates include S100A9 and NDUFB3 (subunit of mitochondrial respiratory Complex I). METTL9-mediated methylation enhances respiration via Complex I, and 1MH in an HxH-containing peptide reduces zinc binding affinity.","method":"In vitro methyltransferase assays, LC-MS/MS detection of 1MH, knockout mouse models, genetic deletion with Complex I activity assay, zinc-binding affinity measurements","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro enzymatic assays, proteome-wide MS quantification, KO models, functional Complex I assay, and biochemical zinc-binding measurement across multiple orthogonal methods","pmids":["33563959"],"is_preprint":false},{"year":2021,"finding":"METTL9 catalyzes Nπ-methylhistidine (N1-methylhistidine) formation on S100A9 at His-107 in vivo and in vitro. Methylation at His-107 overlaps with S100A9's zinc-binding site, attenuating its zinc affinity. METTL9 does not affect heterodimer formation of S100A9 with S100A8.","method":"siRNA screening coupled with methylhistidine analysis using LC-tandem MS; in vitro methyltransferase assay; zinc-binding affinity measurements","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro enzymatic assay, LC-MS/MS identification of modification site, zinc-binding biochemistry, corroborated by independent lab (PMID 33563959)","pmids":["34562450"],"is_preprint":false},{"year":2023,"finding":"Crystal/cryo structure of METTL9 in complex with substrate revealed it specifically methylates the second histidine of the HxH motif while the first histidine serves as a recognition signature. The small 'x' residue is confined within the substrate pocket. An aspartate residue stabilizes the N3 atom of the histidine imidazole ring, presenting the N1 atom to SAM for methylation. METTL9 exhibits preferred consecutive 'C-to-N' directional methylation of tandem HxH repeats.","method":"X-ray crystallography of METTL9-substrate complex; biochemical methyltransferase assays; mutagenesis of active-site residues","journal":"Cell insight","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with functional validation and mutagenesis in a single study, single lab","pmids":["37398635"],"is_preprint":false},{"year":2022,"finding":"In metastatic gastric cancer cells, METTL9 protein is predominantly localized in mitochondria, and METTL9 knockdown significantly reduces mitochondrial Complex I activity, inhibiting cell migration and invasion.","method":"shRNA knockdown; subcellular fractionation/localization; Complex I activity assay; migration and invasion assays in vitro","journal":"Biochemistry and biophysics reports","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — mitochondrial localization by fractionation, functional Complex I assay after KD, two orthogonal methods, single lab","pmids":["35402738"],"is_preprint":false},{"year":2023,"finding":"METTL9 knockdown in hepatocellular carcinoma cells reduces SLC7A11 expression (a key suppressor of ferroptosis), promoting ferroptosis and inhibiting HCC progression in vitro and in vivo.","method":"shRNA knockdown; overexpression; western blotting; cell viability/migration/invasion assays; PDX tumor growth experiments","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — KD/OE with defined cellular phenotype, pathway placement via SLC7A11, corroborated by in vivo PDX, single lab","pmids":["38017014"],"is_preprint":false},{"year":2025,"finding":"METTL9 mediates N1-histidine methylation of the zinc transporter SLC39A7 at His45 and His49. This methylation suppresses ferroptosis through the PERK/ATF4 signaling pathway and downstream SLC7A11, which transports cystine for glutathione synthesis, thereby reducing intracellular ROS and inhibiting adipogenic differentiation of mesenchymal stem cells.","method":"In vitro and in vivo methyltransferase assays; site-specific mutagenesis (His45, His49); western blotting; ROS measurement; adipogenic differentiation assays; OVX mouse model","journal":"Molecular medicine (Cambridge, Mass.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-specific methylation identified in vitro and in vivo, downstream pathway placement, multiple orthogonal methods, single lab","pmids":["40414869"],"is_preprint":false},{"year":2025,"finding":"METTL9 sustains vertebrate neural development primarily via non-catalytic functions. METTL9 interacts with key regulators of cellular transport, endocytosis, and Golgi integrity; in Mettl9 KO cells, the Golgi becomes fragmented. This developmental role occurs through modulation of the secretory pathway and is largely independent of its histidine methyltransferase catalytic activity.","method":"Mouse embryonic stem cell KO, inducible degron, and catalytically inactive knock-in lines; neural differentiation assays; multi-omics; Xenopus laevis mettl9 knockdown; co-immunoprecipitation with transport/Golgi regulators; Golgi morphology imaging","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — three distinct genetic models, two organisms, multi-omics, Co-IP, organelle imaging; multiple orthogonal methods in a single rigorous study","pmids":["40745158"],"is_preprint":false},{"year":2024,"finding":"METTL9 promotes histidine methylation of NF-κB RELA, resulting in inhibition of NLRP3 epigenetic transcription and suppression of neuronal pyroptosis in a Parkinson's disease mouse model.","method":"MPTP mouse model; electroacupuncture treatment; METTL9 deficiency; luciferase and ChIP assays for NF-κB binding to NLRP3 promoter; western blotting; TUNEL/flow cytometry","journal":"Critical reviews in eukaryotic gene expression","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, indirect evidence for histidine methylation of NF-κB RELA with limited biochemical validation of the methylation event itself","pmids":["39072406"],"is_preprint":false},{"year":2025,"finding":"Co-immunoprecipitation confirmed that METTL9 binds to SLC7A11, enhancing its stability and reducing its degradation, thus modulating ferroptosis in hepatocellular carcinoma independently of the classical GPX4 pathway.","method":"Co-immunoprecipitation (Co-IP); RNA sequencing; CUT&Tag; overexpression and knockdown experiments; in vivo mouse tumor models","journal":"NPJ precision oncology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — reciprocal Co-IP with functional validation in vitro and in vivo, single lab","pmids":["40523929"],"is_preprint":false},{"year":2025,"finding":"METTL9 orthologues across eukaryotes retain in vitro methyltransferase activity on HxH-containing substrates (e.g., ARMC6 and DNAJB12), but show distinct substrate specificities. The X-ray structure of Ostreococcus tauri METTL9 revealed structural differences from human METTL9 that may explain its distinct substrate specificity. Tolerance for substitutions at the HxH flanking positions varies among orthologues (Hs > Dm > Ot).","method":"In vitro methyltransferase assays; peptide array substrate specificity profiling; X-ray crystallography of OtMETTL9","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus in vitro enzymatic assays with peptide arrays, multiple orthologues tested, single study","pmids":["40451431"],"is_preprint":false},{"year":2026,"finding":"A first-in-class inhibitor (METTL9i) binds within the SAM binding pocket of METTL9 with IC50 of 0.067 µM and inhibits METTL9 with selectivity over other methyltransferases. In cells, METTL9i engages METTL9 and reduces global 1-methylhistidine levels, confirming the SAM pocket as the catalytic site.","method":"Enzymatic inhibition assay (IC50 measurement); X-ray crystallography of METTL9i-METTL9 complex; cellular target engagement assay; global 1-MH quantification by MS","journal":"Angewandte Chemie (International ed. in English)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure of inhibitor-enzyme complex, in vitro IC50, selectivity panel, cellular 1-MH reduction confirmed by MS","pmids":["41870122"],"is_preprint":false}],"current_model":"METTL9 is a seven-β-strand methyltransferase that catalyzes N1 (Nπ)-methylation of histidine residues within His-x-His (HxH) motifs in diverse protein substrates — including S100A9, NDUFB3 (Complex I subunit), SLC39A7, and NF-κB RELA — using SAM as methyl donor; structurally, the first His serves as a recognition anchor while an active-site aspartate positions the second His N1 for methylation; functionally, this modification reduces zinc binding at target sites, enhances mitochondrial Complex I-dependent respiration, suppresses ferroptosis via SLC7A11 stabilization, and inhibits neuroinflammatory signaling, while METTL9 also exerts catalysis-independent roles in maintaining Golgi/secretory pathway integrity essential for vertebrate neural development."},"narrative":{"mechanistic_narrative":"METTL9 is a broad-specificity, SAM-dependent protein methyltransferase that generates the bulk of N1 (Nπ)-methylhistidine found in mammalian proteomes by modifying the second histidine of His-x-His (HxH) motifs in diverse substrates [PMID:33563959, PMID:34562450]. Structural analysis shows the first histidine of the motif serves as a recognition anchor and the small 'x' residue is held within the substrate pocket, while an active-site aspartate stabilizes the imidazole N3 to present N1 to the SAM methyl donor, and tandem HxH repeats are methylated processively in a C-to-N direction [PMID:37398635]; the catalytic SAM pocket has been confirmed pharmacologically by a selective inhibitor that abolishes cellular 1-methylhistidine [PMID:41870122]. A recurrent functional consequence of this modification is attenuation of zinc binding at target sites, demonstrated for the metal-binding protein S100A9 at His-107 and for HxH-containing peptides [PMID:33563959, PMID:34562450]. Characterized substrates and their physiological outputs include NDUFB3, where methylation enhances mitochondrial Complex I-dependent respiration [PMID:33563959], and the zinc transporter SLC39A7 at His45/His49, where methylation suppresses ferroptosis through PERK/ATF4 and SLC7A11 signaling [PMID:40414869]. Through effects on Complex I activity and on SLC7A11-dependent ferroptosis, METTL9 promotes gastric cancer migration/invasion and hepatocellular carcinoma progression [PMID:35402738, PMID:38017014, PMID:40523929]. Distinct from its catalytic activity, METTL9 supports vertebrate neural development by interacting with regulators of cellular transport, endocytosis, and Golgi integrity, with its loss causing Golgi fragmentation [PMID:40745158].","teleology":[{"year":2021,"claim":"Established METTL9 as the principal enzyme generating proteome-wide 1-methylhistidine and defined its HxH motif specificity, linking the modification to Complex I respiration and zinc binding.","evidence":"In vitro methyltransferase assays, proteome-wide LC-MS/MS of 1MH, knockout mice, Complex I activity and zinc-binding measurements","pmids":["33563959"],"confidence":"High","gaps":["Full substrate repertoire beyond S100A9 and NDUFB3 not enumerated","Physiological role of zinc-binding attenuation not mapped to whole-organism phenotypes"]},{"year":2021,"claim":"Pinpointed a specific physiological methylation site (S100A9 His-107) and showed the mark overlaps the zinc-binding site without disrupting S100A8 heterodimerization, providing a concrete substrate-level mechanism.","evidence":"siRNA screening with methylhistidine LC-MS/MS, in vitro methyltransferase assay, zinc-binding biochemistry","pmids":["34562450"],"confidence":"High","gaps":["Downstream functional consequence of reduced S100A9 zinc affinity not resolved","Whether site occupancy is regulated in vivo unknown"]},{"year":2022,"claim":"Tied METTL9 to a disease context by showing mitochondrial localization and dependence of Complex I activity on METTL9 in metastatic gastric cancer cells.","evidence":"shRNA knockdown, subcellular fractionation, Complex I assay, migration/invasion assays","pmids":["35402738"],"confidence":"Medium","gaps":["Does not confirm methylation of a specific mitochondrial substrate in this system","Single lab, single cancer cell context"]},{"year":2023,"claim":"Resolved the structural basis of HxH recognition, explaining why the second histidine is methylated and how the aspartate and substrate pocket enforce specificity and directional processing.","evidence":"X-ray crystallography of METTL9-substrate complex with active-site mutagenesis","pmids":["37398635"],"confidence":"High","gaps":["Conformational dynamics during processive methylation not captured","Determinants of full-length protein substrate selection beyond the peptide motif unclear"]},{"year":2023,"claim":"Connected METTL9 to ferroptosis control via SLC7A11, identifying a tumor-promoting axis in hepatocellular carcinoma.","evidence":"shRNA knockdown/overexpression, western blotting, viability/invasion assays, PDX tumor models","pmids":["38017014"],"confidence":"Medium","gaps":["Whether the SLC7A11 effect requires catalytic methylation not established here","Direct substrate mediating SLC7A11 regulation not identified"]},{"year":2024,"claim":"Extended METTL9 substrates to a signaling regulator by linking it to histidine methylation of NF-κB RELA and suppression of NLRP3-driven neuronal pyroptosis.","evidence":"MPTP Parkinson's model, METTL9 deficiency, luciferase/ChIP for NF-κB-NLRP3 promoter binding, TUNEL/flow cytometry","pmids":["39072406"],"confidence":"Low","gaps":["Direct biochemical validation of RELA histidine methylation is limited","Methylation site on RELA not mapped","Single lab, indirect evidence"]},{"year":2025,"claim":"Identified SLC39A7 (His45/His49) as a methylation substrate and placed METTL9 upstream of PERK/ATF4-SLC7A11 in ferroptosis and redox control during adipogenesis.","evidence":"In vitro/in vivo methyltransferase assays, site-specific mutagenesis, ROS measurement, adipogenic differentiation, OVX mouse model","pmids":["40414869"],"confidence":"Medium","gaps":["Mechanistic link from SLC39A7 methylation to PERK/ATF4 activation not fully dissected","Single lab"]},{"year":2025,"claim":"Revealed a catalysis-independent function: METTL9 maintains Golgi/secretory pathway integrity and is required for vertebrate neural development, separating its developmental role from methyltransferase activity.","evidence":"ESC KO, degron, and catalytically inactive knock-in lines, neural differentiation, multi-omics, Xenopus knockdown, Co-IP with transport/Golgi regulators, Golgi imaging","pmids":["40745158"],"confidence":"High","gaps":["Identity of the functionally critical transport/Golgi partners not specified","Molecular mechanism by which METTL9 preserves Golgi integrity unknown"]},{"year":2025,"claim":"Provided reciprocal physical evidence that METTL9 binds and stabilizes SLC7A11, defining a GPX4-independent route to ferroptosis modulation in HCC.","evidence":"Reciprocal Co-IP, RNA-seq, CUT&Tag, knockdown/overexpression, in vivo tumor models","pmids":["40523929"],"confidence":"Medium","gaps":["Whether stabilization depends on histidine methylation of SLC7A11 or a non-catalytic interaction is unresolved","Single lab"]},{"year":2025,"claim":"Showed evolutionary conservation of HxH methyltransferase activity across eukaryotic orthologues with divergent substrate specificities, mapping structural determinants of flanking-residue tolerance.","evidence":"In vitro methyltransferase assays, peptide array profiling, X-ray structure of Ostreococcus tauri METTL9","pmids":["40451431"],"confidence":"High","gaps":["Physiological substrates of non-human orthologues not defined","Functional significance of specificity divergence unknown"]},{"year":2026,"claim":"Delivered a first-in-class selective METTL9 inhibitor binding the SAM pocket, confirming the catalytic site and enabling chemical interrogation of histidine methylation in cells.","evidence":"Enzymatic IC50, X-ray of inhibitor-enzyme complex, selectivity panel, cellular target engagement, global 1-MH quantification by MS","pmids":["41870122"],"confidence":"High","gaps":["In vivo efficacy and selectivity not addressed","Effect on individual substrate methylation events not parsed"]},{"year":null,"claim":"How METTL9's catalytic (HxH histidine methylation) and non-catalytic (Golgi/secretory) functions are partitioned across tissues and disease contexts, and whether its ferroptosis and signaling roles require methylation, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking enzymatic activity to the Golgi-integrity phenotype","Whether SLC7A11 stabilization and RELA effects are methylation-dependent unclear","Comprehensive in vivo substrate map lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,2,5,9,10]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,5]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[3]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,2]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[4,5,8]}],"complexes":[],"partners":["S100A9","NDUFB3","SLC39A7","SLC7A11","RELA"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9H1A3","full_name":"Protein-L-histidine N-pros-methyltransferase","aliases":["DORA reverse strand protein","DREV","DREV1","Methyltransferase-like protein 9","hMETTL9"],"length_aa":318,"mass_kda":36.5,"function":"Protein-histidine N-methyltransferase that specifically catalyzes 1-methylhistidine (pros-methylhistidine) methylation of target proteins (PubMed:33563959, PubMed:34562450, PubMed:37015930, PubMed:37398635). Specifically methylates the second His of proteins with a His-x-His (HxH) motif (where 'x' is preferably a small amino acid), while exploiting the first one as a recognition signature (PubMed:37398635). Catalyzes methylation of target proteins such as S100A9, NDUFB3, SLC39A5, SLC39A7, ARMC6 and DNAJB12; 1-methylhistidine modification may affect the binding of zinc and other metals to its target proteins (PubMed:33563959, PubMed:34562450, PubMed:37015930, PubMed:37398635). Constitutes the main methyltransferase for the 1-methylhistidine modification in cell (PubMed:33563959)","subcellular_location":"Endoplasmic reticulum; Mitochondrion","url":"https://www.uniprot.org/uniprotkb/Q9H1A3/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/METTL9","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/METTL9","total_profiled":1310},"omim":[{"mim_id":"609388","title":"METHYLTRANSFERASE-LIKE 9; METTL9","url":"https://www.omim.org/entry/609388"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Cell Junctions","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/METTL9"},"hgnc":{"alias_symbol":["DREV1"],"prev_symbol":[]},"alphafold":{"accession":"Q9H1A3","domains":[{"cath_id":"3.40.50.150","chopping":"56-316","consensus_level":"high","plddt":88.4314,"start":56,"end":316}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H1A3","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H1A3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H1A3-F1-predicted_aligned_error_v6.png","plddt_mean":83.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=METTL9","jax_strain_url":"https://www.jax.org/strain/search?query=METTL9"},"sequence":{"accession":"Q9H1A3","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H1A3.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H1A3/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H1A3"}},"corpus_meta":[{"pmid":"33563959","id":"PMC_33563959","title":"The methyltransferase METTL9 mediates pervasive 1-methylhistidine modification in mammalian proteomes.","date":"2021","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/33563959","citation_count":90,"is_preprint":false},{"pmid":"38017014","id":"PMC_38017014","title":"METTL9-SLC7A11 axis promotes hepatocellular carcinoma progression through ferroptosis inhibition.","date":"2023","source":"Cell death discovery","url":"https://pubmed.ncbi.nlm.nih.gov/38017014","citation_count":24,"is_preprint":false},{"pmid":"36803376","id":"PMC_36803376","title":"Circular RNA METTL9 contributes to neuroinflammation following traumatic brain injury by complexing with astrocytic SND1.","date":"2023","source":"Journal of neuroinflammation","url":"https://pubmed.ncbi.nlm.nih.gov/36803376","citation_count":22,"is_preprint":false},{"pmid":"34562450","id":"PMC_34562450","title":"siRNA screening identifies METTL9 as a histidine Nπ-methyltransferase that targets the proinflammatory protein S100A9.","date":"2021","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/34562450","citation_count":19,"is_preprint":false},{"pmid":"37398635","id":"PMC_37398635","title":"Molecular basis for protein histidine N1-specific methylation of the \"His-x-His\" motifs by METTL9.","date":"2023","source":"Cell insight","url":"https://pubmed.ncbi.nlm.nih.gov/37398635","citation_count":16,"is_preprint":false},{"pmid":"37158456","id":"PMC_37158456","title":"METTL9 derived circular RNA circ-METTL9 sponges miR-551b-5p to accelerate colorectal cancer progression by upregulating CDK6.","date":"2023","source":"Carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/37158456","citation_count":12,"is_preprint":false},{"pmid":"35402738","id":"PMC_35402738","title":"Elevated METTL9 is associated with peritoneal dissemination in human scirrhous gastric cancers.","date":"2022","source":"Biochemistry and biophysics reports","url":"https://pubmed.ncbi.nlm.nih.gov/35402738","citation_count":11,"is_preprint":false},{"pmid":"40414869","id":"PMC_40414869","title":"METTL9 mediated N1-Histidine methylation of SLC39A7 confers ferroptosis resistance and inhibits adipogenic differentiation in mesenchymal stem cells.","date":"2025","source":"Molecular medicine (Cambridge, Mass.)","url":"https://pubmed.ncbi.nlm.nih.gov/40414869","citation_count":7,"is_preprint":false},{"pmid":"40745158","id":"PMC_40745158","title":"METTL9 sustains vertebrate neural development primarily via non-catalytic functions.","date":"2025","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/40745158","citation_count":4,"is_preprint":false},{"pmid":"40451431","id":"PMC_40451431","title":"Orthologues of the human protein histidine methyltransferase METTL9 display distinct substrate specificities.","date":"2025","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/40451431","citation_count":3,"is_preprint":false},{"pmid":"39072406","id":"PMC_39072406","title":"Electroacupuncture Alleviates Parkinson's Disease by Promoting METTL9-Catalyzed Histidine Methylation of Nuclear Factor-κВ.","date":"2024","source":"Critical reviews in eukaryotic gene expression","url":"https://pubmed.ncbi.nlm.nih.gov/39072406","citation_count":2,"is_preprint":false},{"pmid":"40523929","id":"PMC_40523929","title":"Targeting SIX2 as a novel sensitization strategy of sorafenib treatment on advanced hepatocellular carcinoma through modulating METTL9-SLC7A11 axis.","date":"2025","source":"NPJ precision oncology","url":"https://pubmed.ncbi.nlm.nih.gov/40523929","citation_count":1,"is_preprint":false},{"pmid":"41900964","id":"PMC_41900964","title":"The Protein Histidine Methyltransferase METTL9-From Mechanism to Biological Function.","date":"2026","source":"Life (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/41900964","citation_count":0,"is_preprint":false},{"pmid":"41870122","id":"PMC_41870122","title":"Discovery of a Potent and Selective Cell-Active Inhibitor of Histidine-N1 Methyltransferase METTL9.","date":"2026","source":"Angewandte Chemie (International ed. in English)","url":"https://pubmed.ncbi.nlm.nih.gov/41870122","citation_count":0,"is_preprint":false},{"pmid":"41539299","id":"PMC_41539299","title":"METTL9 tests Candida's mettle by limiting metal acquisition.","date":"2026","source":"Cell host & microbe","url":"https://pubmed.ncbi.nlm.nih.gov/41539299","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9244,"output_tokens":3008,"usd":0.036426,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10329,"output_tokens":3684,"usd":0.071873,"stage2_stop_reason":"end_turn"},"total_usd":0.108299,"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\": 2021,\n      \"finding\": \"METTL9 is a broad-specificity methyltransferase that mediates formation of the majority of 1-methylhistidine (1MH) present in mouse and human proteomes, requiring a His-x-His (HxH) motif where 'x' is preferably a small amino acid. Substrates include S100A9 and NDUFB3 (subunit of mitochondrial respiratory Complex I). METTL9-mediated methylation enhances respiration via Complex I, and 1MH in an HxH-containing peptide reduces zinc binding affinity.\",\n      \"method\": \"In vitro methyltransferase assays, LC-MS/MS detection of 1MH, knockout mouse models, genetic deletion with Complex I activity assay, zinc-binding affinity measurements\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro enzymatic assays, proteome-wide MS quantification, KO models, functional Complex I assay, and biochemical zinc-binding measurement across multiple orthogonal methods\",\n      \"pmids\": [\"33563959\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"METTL9 catalyzes Nπ-methylhistidine (N1-methylhistidine) formation on S100A9 at His-107 in vivo and in vitro. Methylation at His-107 overlaps with S100A9's zinc-binding site, attenuating its zinc affinity. METTL9 does not affect heterodimer formation of S100A9 with S100A8.\",\n      \"method\": \"siRNA screening coupled with methylhistidine analysis using LC-tandem MS; in vitro methyltransferase assay; zinc-binding affinity measurements\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro enzymatic assay, LC-MS/MS identification of modification site, zinc-binding biochemistry, corroborated by independent lab (PMID 33563959)\",\n      \"pmids\": [\"34562450\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Crystal/cryo structure of METTL9 in complex with substrate revealed it specifically methylates the second histidine of the HxH motif while the first histidine serves as a recognition signature. The small 'x' residue is confined within the substrate pocket. An aspartate residue stabilizes the N3 atom of the histidine imidazole ring, presenting the N1 atom to SAM for methylation. METTL9 exhibits preferred consecutive 'C-to-N' directional methylation of tandem HxH repeats.\",\n      \"method\": \"X-ray crystallography of METTL9-substrate complex; biochemical methyltransferase assays; mutagenesis of active-site residues\",\n      \"journal\": \"Cell insight\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with functional validation and mutagenesis in a single study, single lab\",\n      \"pmids\": [\"37398635\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In metastatic gastric cancer cells, METTL9 protein is predominantly localized in mitochondria, and METTL9 knockdown significantly reduces mitochondrial Complex I activity, inhibiting cell migration and invasion.\",\n      \"method\": \"shRNA knockdown; subcellular fractionation/localization; Complex I activity assay; migration and invasion assays in vitro\",\n      \"journal\": \"Biochemistry and biophysics reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — mitochondrial localization by fractionation, functional Complex I assay after KD, two orthogonal methods, single lab\",\n      \"pmids\": [\"35402738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"METTL9 knockdown in hepatocellular carcinoma cells reduces SLC7A11 expression (a key suppressor of ferroptosis), promoting ferroptosis and inhibiting HCC progression in vitro and in vivo.\",\n      \"method\": \"shRNA knockdown; overexpression; western blotting; cell viability/migration/invasion assays; PDX tumor growth experiments\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — KD/OE with defined cellular phenotype, pathway placement via SLC7A11, corroborated by in vivo PDX, single lab\",\n      \"pmids\": [\"38017014\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"METTL9 mediates N1-histidine methylation of the zinc transporter SLC39A7 at His45 and His49. This methylation suppresses ferroptosis through the PERK/ATF4 signaling pathway and downstream SLC7A11, which transports cystine for glutathione synthesis, thereby reducing intracellular ROS and inhibiting adipogenic differentiation of mesenchymal stem cells.\",\n      \"method\": \"In vitro and in vivo methyltransferase assays; site-specific mutagenesis (His45, His49); western blotting; ROS measurement; adipogenic differentiation assays; OVX mouse model\",\n      \"journal\": \"Molecular medicine (Cambridge, Mass.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific methylation identified in vitro and in vivo, downstream pathway placement, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"40414869\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"METTL9 sustains vertebrate neural development primarily via non-catalytic functions. METTL9 interacts with key regulators of cellular transport, endocytosis, and Golgi integrity; in Mettl9 KO cells, the Golgi becomes fragmented. This developmental role occurs through modulation of the secretory pathway and is largely independent of its histidine methyltransferase catalytic activity.\",\n      \"method\": \"Mouse embryonic stem cell KO, inducible degron, and catalytically inactive knock-in lines; neural differentiation assays; multi-omics; Xenopus laevis mettl9 knockdown; co-immunoprecipitation with transport/Golgi regulators; Golgi morphology imaging\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — three distinct genetic models, two organisms, multi-omics, Co-IP, organelle imaging; multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"40745158\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"METTL9 promotes histidine methylation of NF-κB RELA, resulting in inhibition of NLRP3 epigenetic transcription and suppression of neuronal pyroptosis in a Parkinson's disease mouse model.\",\n      \"method\": \"MPTP mouse model; electroacupuncture treatment; METTL9 deficiency; luciferase and ChIP assays for NF-κB binding to NLRP3 promoter; western blotting; TUNEL/flow cytometry\",\n      \"journal\": \"Critical reviews in eukaryotic gene expression\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, indirect evidence for histidine methylation of NF-κB RELA with limited biochemical validation of the methylation event itself\",\n      \"pmids\": [\"39072406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Co-immunoprecipitation confirmed that METTL9 binds to SLC7A11, enhancing its stability and reducing its degradation, thus modulating ferroptosis in hepatocellular carcinoma independently of the classical GPX4 pathway.\",\n      \"method\": \"Co-immunoprecipitation (Co-IP); RNA sequencing; CUT&Tag; overexpression and knockdown experiments; in vivo mouse tumor models\",\n      \"journal\": \"NPJ precision oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — reciprocal Co-IP with functional validation in vitro and in vivo, single lab\",\n      \"pmids\": [\"40523929\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"METTL9 orthologues across eukaryotes retain in vitro methyltransferase activity on HxH-containing substrates (e.g., ARMC6 and DNAJB12), but show distinct substrate specificities. The X-ray structure of Ostreococcus tauri METTL9 revealed structural differences from human METTL9 that may explain its distinct substrate specificity. Tolerance for substitutions at the HxH flanking positions varies among orthologues (Hs > Dm > Ot).\",\n      \"method\": \"In vitro methyltransferase assays; peptide array substrate specificity profiling; X-ray crystallography of OtMETTL9\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus in vitro enzymatic assays with peptide arrays, multiple orthologues tested, single study\",\n      \"pmids\": [\"40451431\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"A first-in-class inhibitor (METTL9i) binds within the SAM binding pocket of METTL9 with IC50 of 0.067 µM and inhibits METTL9 with selectivity over other methyltransferases. In cells, METTL9i engages METTL9 and reduces global 1-methylhistidine levels, confirming the SAM pocket as the catalytic site.\",\n      \"method\": \"Enzymatic inhibition assay (IC50 measurement); X-ray crystallography of METTL9i-METTL9 complex; cellular target engagement assay; global 1-MH quantification by MS\",\n      \"journal\": \"Angewandte Chemie (International ed. in English)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure of inhibitor-enzyme complex, in vitro IC50, selectivity panel, cellular 1-MH reduction confirmed by MS\",\n      \"pmids\": [\"41870122\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"METTL9 is a seven-β-strand methyltransferase that catalyzes N1 (Nπ)-methylation of histidine residues within His-x-His (HxH) motifs in diverse protein substrates — including S100A9, NDUFB3 (Complex I subunit), SLC39A7, and NF-κB RELA — using SAM as methyl donor; structurally, the first His serves as a recognition anchor while an active-site aspartate positions the second His N1 for methylation; functionally, this modification reduces zinc binding at target sites, enhances mitochondrial Complex I-dependent respiration, suppresses ferroptosis via SLC7A11 stabilization, and inhibits neuroinflammatory signaling, while METTL9 also exerts catalysis-independent roles in maintaining Golgi/secretory pathway integrity essential for vertebrate neural development.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"METTL9 is a broad-specificity, SAM-dependent protein methyltransferase that generates the bulk of N1 (Nπ)-methylhistidine found in mammalian proteomes by modifying the second histidine of His-x-His (HxH) motifs in diverse substrates [#0, #1]. Structural analysis shows the first histidine of the motif serves as a recognition anchor and the small 'x' residue is held within the substrate pocket, while an active-site aspartate stabilizes the imidazole N3 to present N1 to the SAM methyl donor, and tandem HxH repeats are methylated processively in a C-to-N direction [#2]; the catalytic SAM pocket has been confirmed pharmacologically by a selective inhibitor that abolishes cellular 1-methylhistidine [#10]. A recurrent functional consequence of this modification is attenuation of zinc binding at target sites, demonstrated for the metal-binding protein S100A9 at His-107 and for HxH-containing peptides [#0, #1]. Characterized substrates and their physiological outputs include NDUFB3, where methylation enhances mitochondrial Complex I-dependent respiration [#0], and the zinc transporter SLC39A7 at His45/His49, where methylation suppresses ferroptosis through PERK/ATF4 and SLC7A11 signaling [#5]. Through effects on Complex I activity and on SLC7A11-dependent ferroptosis, METTL9 promotes gastric cancer migration/invasion and hepatocellular carcinoma progression [#3, #4, #8]. Distinct from its catalytic activity, METTL9 supports vertebrate neural development by interacting with regulators of cellular transport, endocytosis, and Golgi integrity, with its loss causing Golgi fragmentation [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 2021,\n      \"claim\": \"Established METTL9 as the principal enzyme generating proteome-wide 1-methylhistidine and defined its HxH motif specificity, linking the modification to Complex I respiration and zinc binding.\",\n      \"evidence\": \"In vitro methyltransferase assays, proteome-wide LC-MS/MS of 1MH, knockout mice, Complex I activity and zinc-binding measurements\",\n      \"pmids\": [\"33563959\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full substrate repertoire beyond S100A9 and NDUFB3 not enumerated\", \"Physiological role of zinc-binding attenuation not mapped to whole-organism phenotypes\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Pinpointed a specific physiological methylation site (S100A9 His-107) and showed the mark overlaps the zinc-binding site without disrupting S100A8 heterodimerization, providing a concrete substrate-level mechanism.\",\n      \"evidence\": \"siRNA screening with methylhistidine LC-MS/MS, in vitro methyltransferase assay, zinc-binding biochemistry\",\n      \"pmids\": [\"34562450\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream functional consequence of reduced S100A9 zinc affinity not resolved\", \"Whether site occupancy is regulated in vivo unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Tied METTL9 to a disease context by showing mitochondrial localization and dependence of Complex I activity on METTL9 in metastatic gastric cancer cells.\",\n      \"evidence\": \"shRNA knockdown, subcellular fractionation, Complex I assay, migration/invasion assays\",\n      \"pmids\": [\"35402738\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not confirm methylation of a specific mitochondrial substrate in this system\", \"Single lab, single cancer cell context\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Resolved the structural basis of HxH recognition, explaining why the second histidine is methylated and how the aspartate and substrate pocket enforce specificity and directional processing.\",\n      \"evidence\": \"X-ray crystallography of METTL9-substrate complex with active-site mutagenesis\",\n      \"pmids\": [\"37398635\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational dynamics during processive methylation not captured\", \"Determinants of full-length protein substrate selection beyond the peptide motif unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected METTL9 to ferroptosis control via SLC7A11, identifying a tumor-promoting axis in hepatocellular carcinoma.\",\n      \"evidence\": \"shRNA knockdown/overexpression, western blotting, viability/invasion assays, PDX tumor models\",\n      \"pmids\": [\"38017014\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the SLC7A11 effect requires catalytic methylation not established here\", \"Direct substrate mediating SLC7A11 regulation not identified\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended METTL9 substrates to a signaling regulator by linking it to histidine methylation of NF-κB RELA and suppression of NLRP3-driven neuronal pyroptosis.\",\n      \"evidence\": \"MPTP Parkinson's model, METTL9 deficiency, luciferase/ChIP for NF-κB-NLRP3 promoter binding, TUNEL/flow cytometry\",\n      \"pmids\": [\"39072406\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Direct biochemical validation of RELA histidine methylation is limited\", \"Methylation site on RELA not mapped\", \"Single lab, indirect evidence\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified SLC39A7 (His45/His49) as a methylation substrate and placed METTL9 upstream of PERK/ATF4-SLC7A11 in ferroptosis and redox control during adipogenesis.\",\n      \"evidence\": \"In vitro/in vivo methyltransferase assays, site-specific mutagenesis, ROS measurement, adipogenic differentiation, OVX mouse model\",\n      \"pmids\": [\"40414869\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic link from SLC39A7 methylation to PERK/ATF4 activation not fully dissected\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed a catalysis-independent function: METTL9 maintains Golgi/secretory pathway integrity and is required for vertebrate neural development, separating its developmental role from methyltransferase activity.\",\n      \"evidence\": \"ESC KO, degron, and catalytically inactive knock-in lines, neural differentiation, multi-omics, Xenopus knockdown, Co-IP with transport/Golgi regulators, Golgi imaging\",\n      \"pmids\": [\"40745158\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the functionally critical transport/Golgi partners not specified\", \"Molecular mechanism by which METTL9 preserves Golgi integrity unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided reciprocal physical evidence that METTL9 binds and stabilizes SLC7A11, defining a GPX4-independent route to ferroptosis modulation in HCC.\",\n      \"evidence\": \"Reciprocal Co-IP, RNA-seq, CUT&Tag, knockdown/overexpression, in vivo tumor models\",\n      \"pmids\": [\"40523929\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether stabilization depends on histidine methylation of SLC7A11 or a non-catalytic interaction is unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Showed evolutionary conservation of HxH methyltransferase activity across eukaryotic orthologues with divergent substrate specificities, mapping structural determinants of flanking-residue tolerance.\",\n      \"evidence\": \"In vitro methyltransferase assays, peptide array profiling, X-ray structure of Ostreococcus tauri METTL9\",\n      \"pmids\": [\"40451431\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological substrates of non-human orthologues not defined\", \"Functional significance of specificity divergence unknown\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Delivered a first-in-class selective METTL9 inhibitor binding the SAM pocket, confirming the catalytic site and enabling chemical interrogation of histidine methylation in cells.\",\n      \"evidence\": \"Enzymatic IC50, X-ray of inhibitor-enzyme complex, selectivity panel, cellular target engagement, global 1-MH quantification by MS\",\n      \"pmids\": [\"41870122\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo efficacy and selectivity not addressed\", \"Effect on individual substrate methylation events not parsed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How METTL9's catalytic (HxH histidine methylation) and non-catalytic (Golgi/secretory) functions are partitioned across tissues and disease contexts, and whether its ferroptosis and signaling roles require methylation, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking enzymatic activity to the Golgi-integrity phenotype\", \"Whether SLC7A11 stabilization and RELA effects are methylation-dependent unclear\", \"Comprehensive in vivo substrate map lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 2, 5, 9, 10]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [4, 5, 8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"S100A9\", \"NDUFB3\", \"SLC39A7\", \"SLC7A11\", \"RELA\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}