METTL9

METTL9 is a seven-β-strand methyltransferase that catalyzes N1 (Nπ) methylation of histidine residues within His-x-His (HxH) motifs across a broad substrate repertoire, generating 1-methylhistidine (1MH) on proteins including S100A9, NDUFB3 (Complex I subunit), SLC39A7, and ARMC6 (PMID:33563959, PMID:34562450, PMID:40451431). Crystal structures show that an active-site aspartate orients the target imidazole ring by engaging N3, presenting N1 to the SAM methyl donor, with the first histidine of the HxH dipeptide serving as a recognition element and methylation proceeding C-to-N through tandem repeats (PMID:37398635). Functionally, METTL9-mediated methylation reduces zinc-binding affinity of target HxH sites, enhances mitochondrial Complex I respiration, and suppresses ferroptosis via SLC39A7/PERK/ATF4-dependent upregulation of SLC7A11 (PMID:33563959, PMID:40414869, PMID:38017014). Beyond its catalytic role, METTL9 sustains vertebrate neurogenesis through a catalysis-independent function in maintaining Golgi and secretory pathway integrity (PMID:40745158).

1. 2021 High

   The enzymatic function of METTL9 was unknown; proteomic and biochemical analyses established it as the major mammalian histidine methyltransferase generating 1-methylhistidine at HxH motifs, with substrates including S100A9 and NDUFB3, linking it to Complex I respiration and zinc-binding regulation.

   Evidence METTL9 KO mice, proteomic MS, in vitro methyltransferase assays, Complex I respiration and zinc-binding measurements

   PMID:33563959 PMID:34562450

   Open questions at the time

   • Identity of the full in vivo substrate repertoire beyond identified targets
   • Mechanism by which 1MH modification enhances Complex I activity not resolved at structural level
   • Selectivity rules explaining why some HxH-containing proteins (e.g. MYLK2) are not methylated in vivo
2. 2022 Medium

   Where METTL9 acts within cells was unclear; subcellular fractionation showed predominant mitochondrial localization in gastric cancer cells, directly coupling METTL9 presence to Complex I activity.

   Evidence Subcellular fractionation, immunofluorescence, and Complex I activity assay in shRNA-knockdown gastric cancer cells

   PMID:35402738

   Open questions at the time

   • Localization studied only in one cancer cell type
   • Whether mitochondrial targeting requires a signal peptide or import machinery not determined
   • Relative distribution across other compartments not systematically quantified
3. 2023 High

   How METTL9 achieves N1-specific methylation was structurally unknown; the crystal structure of METTL9 bound to substrate revealed that an active-site aspartate stabilizes N3 of the target histidine imidazole, presenting N1 to SAM, with the first histidine serving as a recognition element and a confined pocket selecting for small intervening residues.

   Evidence X-ray crystallography of METTL9–substrate complex, site-directed mutagenesis, in vitro assays on tandem HxH repeats

   PMID:37398635

   Open questions at the time

   • Full-length protein substrate complexes not crystallized
   • Structural basis for C-to-N processivity on tandem repeats not fully defined
4. 2023 Medium

   Whether METTL9 influenced cell death pathways was unexplored; knockdown in HCC cells reduced SLC7A11 expression and promoted ferroptosis, linking METTL9 to ferroptosis resistance.

   Evidence shRNA knockdown, cell viability/migration assays, PDX xenograft models

   PMID:38017014

   Open questions at the time

   • Whether SLC7A11 regulation depends on METTL9 catalytic activity not established
   • No direct methylation of SLC7A11 or its transcriptional regulators demonstrated
5. 2025 Medium

   The molecular mechanism connecting METTL9 to ferroptosis was clarified: METTL9 methylates SLC39A7 (ZIP7) at His45/49, suppressing ferroptosis through PERK/ATF4-mediated SLC7A11 induction and glutathione synthesis.

   Evidence In vitro methyltransferase assay on SLC39A7 peptides, site-directed mutagenesis, OVX mouse model, adipogenic differentiation assays

   PMID:40414869

   Open questions at the time

   • How histidine methylation of SLC39A7 triggers PERK/ATF4 signaling is mechanistically unclear
   • Single-lab finding awaiting independent replication
6. 2025 High

   Whether METTL9 functions solely through its catalytic activity was untested; genetic dissection in mouse ESCs and Xenopus revealed a conserved catalysis-independent role in neurogenesis, with METTL9 maintaining Golgi integrity and secretory pathway function through interaction with transport and endocytic regulators.

   Evidence METTL9 KO, catalytically dead knock-in, Degron mouse ESC lines; Xenopus knockdown; multi-omics; Co-IP; Golgi morphology imaging

   PMID:40745158

   Open questions at the time

   • Identity of the specific Golgi/transport interactors mediating the non-catalytic function not fully characterized
   • Whether the neurogenesis role extends to adult neurogenesis not tested
7. 2025 High

   Evolutionary conservation and divergence of METTL9 substrate specificity was uncharted; cross-species analysis showed conserved HxH methylation activity but distinct substrate preferences between human, Drosophila, and algal orthologues, explained by structural differences in the substrate pocket.

   Evidence In vitro methyltransferase assays with recombinant orthologues, peptide arrays, X-ray crystallography of O. tauri METTL9

   PMID:40451431

   Open questions at the time

   • In vivo substrates of non-mammalian METTL9 orthologues not determined
   • Functional consequences of species-specific substrate selectivity unknown
8. 2025 Medium

   METTL9 was shown to physically bind SLC7A11 and stabilize it against degradation, providing a direct protein-level mechanism for METTL9-dependent ferroptosis suppression in HCC independent of GPX4.

   Evidence Reciprocal Co-IP, RNA-seq, overexpression/knockdown functional assays in HCC cells

   PMID:40523929

   Open questions at the time

   • Whether the METTL9–SLC7A11 interaction depends on methyltransferase activity not tested
   • Single-lab Co-IP without domain mapping or structural characterization of the interaction
9. 2026 High

   Pharmacological targeting of METTL9 was previously impossible; a first-in-class small-molecule inhibitor (IC50 = 67 nM) was developed that occupies the SAM-binding pocket and selectively reduces global cellular 1MH levels, providing a chemical tool to dissect METTL9 biology.

   Evidence X-ray crystallography of inhibitor–METTL9 complex, in vitro IC50 determination, selectivity profiling, cellular 1MH proteomics

   PMID:41870122

   Open questions at the time

   • In vivo pharmacokinetics and efficacy not reported
   • Effects of inhibitor on non-catalytic METTL9 functions not assessed

• Key unresolved questions include: whether METTL9's ferroptosis-related effects operate through its catalytic activity or non-catalytic protein stabilization of SLC7A11; the structural basis and in vivo significance of the catalysis-independent Golgi/neurogenesis role; and the full physiological substrate repertoire in different tissues.
• Catalytic vs. non-catalytic contributions to ferroptosis not genetically separated
• Golgi-maintaining mechanism and relevant interactors not biochemically reconstituted
• Tissue-specific in vivo substrate profiles not mapped