{"gene":"NMT1","run_date":"2026-04-29T11:37:57","timeline":{"discoveries":[{"year":1985,"finding":"The transforming protein p60src of Rous sarcoma virus is N-terminally myristoylated at its amino-terminal glycine residue, establishing that myristoylation of N-terminal glycine is a posttranslational modification catalyzed by NMT on signaling kinases and retroviral transforming proteins.","method":"Isolation and sequencing of tryptic peptide from p60src; metabolic labeling with myristic acid","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 — direct biochemical identification of myristoylated peptide, foundational paper replicated broadly","pmids":["3917576"],"is_preprint":false},{"year":1989,"finding":"Myristoylation of the N-terminal glycine of HIV-1 Gag precursor Pr55gag, catalyzed by cellular NMT, is essential for virus particle assembly and budding; G2A mutation abolishing myristoylation prevents virus particle release.","method":"Site-directed mutagenesis of Gag glycine-2; metabolic labeling; virion morphogenesis assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis with functional virological readout, replicated across multiple HIV studies","pmids":["2788277"],"is_preprint":false},{"year":1990,"finding":"Myristoylation of G-protein alpha subunits (Gi1, Gi2, Gi3, Go, Gt, Gz but not Gs) by cellular NMT is required for their stable membrane association; mutation of the N-terminal glycine to alanine in Gαo abolishes both myristoylation and membrane binding.","method":"Metabolic [3H]myristate labeling of COS cell transfectants; site-directed mutagenesis; subcellular fractionation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 — mutagenesis plus subcellular fractionation in mammalian cells, multiple substrates tested","pmids":["2153964"],"is_preprint":false},{"year":1990,"finding":"Myristoylation of HIV-1 Pr55gag by cellular NMT is required for stable membrane association and virus assembly; non-myristoylated Gag accumulates in cells but is not incorporated into virions, though it can still be cleaved by HIV protease in vitro, demonstrating myristate is not required for protease recognition.","method":"G-to-A mutagenesis of Gag; metabolic labeling; complementation in trans; in vitro protease cleavage assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis, in vitro assay, and complementation","pmids":["2405382"],"is_preprint":false},{"year":1992,"finding":"The minimal catalytic domain of S. cerevisiae Nmt1p lies between residues Ile59–Phe96 (N-terminal boundary) and Gly451–Leu455 (C-terminal boundary); the N-terminal 59 residues play a non-catalytic targeting role enabling access to myristoyl-CoA pools from exogenous fatty acid. Peptide substrate specificity differences between yeast Nmt1p and human NMT involve elements distributed across both halves of the enzyme.","method":"Deletion mutagenesis; co-expression with substrates in E. coli; in vitro enzyme assay; yeast complementation of nmt1 null allele; human/yeast chimeric enzymes","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro assay plus mutagenesis and genetic rescue, ortholog of human NMT1","pmids":["1429724"],"is_preprint":false},{"year":1994,"finding":"The membrane-targeting domain of HIV-1 Gag consists of a bipartite signal: the myristoylated N-terminal 14 amino acids (added by NMT) and an adjacent highly basic region that binds acidic phospholipids. Both myristate and the basic region are required for efficient membrane binding and virion formation.","method":"Gag-Src chimera constructs; in vitro membrane binding assay; mutagenesis of basic residues; virion formation assay in vivo","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 1-2 — reconstituted membrane binding with chimeras and mutagenesis, in vivo confirmation","pmids":["8139035"],"is_preprint":false},{"year":1994,"finding":"Membrane binding of pp60c-src requires both NMT-mediated N-terminal myristoylation and six basic residues that interact electrostatically with acidic phospholipids; removal of either element drastically reduces membrane affinity (Kd shifts from ~0.6 µM to ~2 mM for neutral bilayers).","method":"In vitro lipid vesicle binding assay; mutagenesis of basic residues; metabolic labeling; transformation assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — quantitative in vitro reconstitution with mutagenesis and in vivo confirmation","pmids":["7527558"],"is_preprint":false},{"year":1998,"finding":"Cryptococcus neoformans NMT is an essential enzyme whose inhibition causes under-myristoylation of ADP ribosylation factor (Arf) and cell death; this fungicidal effect of a depeptidized NMT inhibitor is NMT-dependent, as demonstrated using isogenic strains expressing either fungal or human NMT.","method":"Homologous recombination to introduce conditional lethal nmt allele; strain-specific inhibitor sensitivity assay; kinetic studies of purified fungal vs. human NMT; protein synthesis inhibition assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — genetic and biochemical evidence with isogenic strains and purified enzyme kinetics","pmids":["9575206"],"is_preprint":false},{"year":1999,"finding":"NMT is targeted to membranes in mammary epithelial cells via interaction with ribosomal proteins, and its subcellular localization is an important variable in the regulation of cellular proliferation; NMT expression and membrane/cytosol distribution are differentially regulated during normal mammary gland development and in breast cancer transformation.","method":"Subcellular fractionation; expression analysis during pregnancy/lactation and malignant transformation; comparison of normal vs. cancerous human breast tissue","journal":"Advances in enzyme regulation","confidence":"Medium","confidence_rationale":"Tier 3 — fractionation without functional manipulation; single lab, partial mechanistic follow-up","pmids":["10470373"],"is_preprint":false},{"year":2000,"finding":"BID undergoes posttranslational (non-cotranslational) N-myristoylation by NMT after caspase-8 cleavage exposes a glycine residue; this myristoylation enables BID fragments to target mitochondria and enhances cytochrome c release and apoptosis, functioning as a molecular activating switch.","method":"In vitro myristoylation assay; liposome binding assay with defined lipid compositions; mitochondria targeting assay; cytochrome c release assay; mutagenesis of glycine acceptor","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution, mutagenesis, and functional apoptosis assay; highly cited","pmids":["11099414"],"is_preprint":false},{"year":2000,"finding":"CaBP1 and CaBP2, novel Ca²⁺-binding proteins expressed in the retina, contain consensus sequences for N-terminal myristoylation and are fatty acid acylated in vitro by NMT; their subcellular localization patterns depend on whether they are myristoylated.","method":"In vitro myristoylation assay; heterologous expression with subcellular localization analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro NMT assay plus localization, single lab","pmids":["10625670"],"is_preprint":false},{"year":2001,"finding":"HIV-1 Nef is N-terminally myristoylated by cellular NMT; this myristoylation is required for membrane association of Nef and is critical for Nef-mediated CD4 downregulation and viral pathogenesis.","method":"Structure-function analysis; mutagenesis of N-terminal glycine; metabolic labeling; CD4 downregulation assays","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1-2 — mutagenesis with functional assays, extensively reviewed and replicated","pmids":["11463741"],"is_preprint":false},{"year":2003,"finding":"The HIV-1 matrix protein (myr-MA) adopts myristate-exposed and myristate-sequestered states that are in equilibrium; myristate exposure is coupled to trimerization (not mechanically induced conformational change), constituting an entropic myristyl switch. This switch regulates Gag targeting to the plasma membrane during assembly.","method":"NMR structural studies of myr-MA in both states; sedimentation equilibrium analytical ultracentrifugation; equilibrium constant measurements for monomer-trimer transition","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — NMR structure plus biophysical quantification of equilibrium, mechanistically novel finding","pmids":["14699046"],"is_preprint":false},{"year":2018,"finding":"Genetic inhibition of NMT1 in breast cancer cells promotes ROS accumulation and ER stress, which cross-talk to produce more ROS. Both oxidative stress and ER stress activate the JNK pathway, leading to autophagy that suppresses breast cancer progression. ROS negatively regulate NMT1 expression, forming a feedback loop.","method":"shRNA knockdown of NMT1; ROS measurement; ER stress markers; JNK pathway analysis; autophagy assays; in vitro and in vivo xenograft models","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — KD with multiple pathway readouts, single lab","pmids":["30446635"],"is_preprint":false},{"year":2021,"finding":"NMT1-mediated N-myristoylation differentially regulates two protein classes in liver cancer: NDP proteins (LXN, RPL29, FAU) are destabilized by myristoylation via HIST1H4H-mediated ubiquitination facilitated by RPL7A, while NUP proteins (AHSG, ALB, TF) are stabilized by myristoylation because HBB prevents their ubiquitination by HIST1H4H. Both effects depend on NMT1 and POTEE.","method":"Conditional liver-specific NMT1 knockout mice; click chemistry N-myristoylation assay; iTRAQ proteomics; co-immunoprecipitation; ubiquitination assays","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2 — KO mouse model, proteomics, and mechanistic ubiquitination assays; single lab","pmids":["34136404"],"is_preprint":false},{"year":2022,"finding":"NMT catalyzes both G-myristoylation (N-terminal glycine α-amino group, efficient) and K-myristoylation (lysine ε-amino group, poor efficiency); G-myristoylation uses a water-mediated interaction with the NMT catalytic base while K-myristoylation uses direct interaction. Evolutionary pressure has depleted K-myristoylation motifs in humans. K-myristoylation likely only results from post-translational events.","method":"Mass spectrometry; enzyme kinetics; in silico analysis; X-ray crystallography of NMT complexes","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus kinetics and MS, multiple orthogonal methods in single study","pmids":["36181773"],"is_preprint":false},{"year":2023,"finding":"NMT1 myristoylates VILIP3 at its N-terminus; desloratadine binds Asn-246 in NMT1 and inhibits its enzymatic activity, blocking VILIP3 myristoylation and suppressing downstream NFκB/Bcl-2 signaling to reduce HCC tumor growth.","method":"DARTS and SPR assays to identify NMT1 as desloratadine target; metabolic labeling and mass spectrometry to identify VILIP3 as NMT1 substrate; NMT1 knockdown/overexpression; CDX/PDO/PDX models","journal":"Signal transduction and targeted therapy","confidence":"High","confidence_rationale":"Tier 1-2 — direct target identification by SPR, substrate identification by metabolic labeling/MS, multiple in vivo models","pmids":["36617552"],"is_preprint":false},{"year":2023,"finding":"NMT1 N-myristoylates the N-terminus of ICAM-1 and prevents its ubiquitination by inhibiting the Ub E3 ligase FBXO4 (F-box protein 4), thereby stabilizing ICAM-1 and sustaining cell adhesion while suppressing tumor cell migration.","method":"NMT1 knockdown; co-immunoprecipitation; ubiquitination assay; protein half-life measurement; wound healing and Transwell migration assay","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2-3 — co-IP and ubiquitination assays with functional migration readout; single lab","pmids":["37269961"],"is_preprint":false},{"year":2024,"finding":"TNF-α stimulation of osteoblasts increases NMT1 expression but paradoxically inhibits overall protein myristoylation; Sorbs2 was identified as a novel NMT1 binding protein by immunoprecipitation/mass spectrometry, and TNF-α-induced Sorbs2–NMT1 binding inhibits myristoylation activity.","method":"Click-it myristoylation assay; RNA interference; immunoprecipitation/mass spectrometry; immunocytochemistry; western blotting in MC3T3-E1 osteoblastic cells","journal":"In vivo","confidence":"Medium","confidence_rationale":"Tier 2-3 — biochemical identification of Sorbs2 as NMT1 interactor with functional myristoylation readout; single lab","pmids":["38148048"],"is_preprint":false},{"year":2024,"finding":"NMT1 and NMT2 myristoylate the mammarenavirus matrix Z protein and stable signal peptide (SSP) at their N-terminal glycines; pharmacological NMT inhibition with DDD85464 reduces Z-mediated budding, impairs GP2-mediated fusion activity, and promotes proteasomal degradation of Z protein, exerting potent antiviral activity against LCMV, JUNV, and LASV.","method":"NMT inhibitor DDD85464 treatment; Z budding assay; GP2 fusion assay; proteasome inhibition rescue; antiviral titer assays for multiple arenaviruses","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — validated on-target NMT inhibitor with multiple functional assays; preprint, single lab","pmids":[],"is_preprint":true},{"year":2025,"finding":"Hypoxia-inducible factor HIF1α upregulates NMT1, which mediates myristoylation of calcineurin B homologous protein 1 (CHP1); myristoylated CHP1 binds PD-L1 and facilitates its rapid translocation to the cell membrane, enhancing immune evasion. NMT1 inhibitor PCLX-001 blocks CHP1 myristoylation and disrupts excessive PD-L1 membrane localization.","method":"HIF1α overexpression/knockdown; NMT1 knockdown; click chemistry myristoylation assay; co-immunoprecipitation of CHP1 and PD-L1; membrane fractionation; HNSCC xenograft mouse model with anti-PD-1 + PCLX-001 combination","journal":"Journal of experimental & clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP, fractionation, functional assays and in vivo model; single lab","pmids":["40605065"],"is_preprint":false},{"year":2025,"finding":"TMEM106B is myristoylated by NMT1/2 at both its Gly2 α-amino group (G-myristoylation) and Lys3 ε-amino group (K-myristoylation); myristoylation promotes lysosomal degradation of TMEM106B and regulates its trafficking, as non-myristoylated TMEM106B accumulates at the cell surface rather than lysosomes.","method":"Mutagenesis of Gly2 and Lys3; NMT1/2 overexpression and knockdown; subcellular fractionation; live-cell imaging; lysosomal inhibitor experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — mutagenesis, NMT1/2 manipulation, subcellular localization with functional consequence; multiple orthogonal methods","pmids":["40451428"],"is_preprint":false},{"year":2025,"finding":"NMT1-mediated myristoylation in the ischemic brain regulates VPS15 (PI3K regulatory subunit 4) expression: ischemia-induced downregulation of NMT1 increases myristic acid (MA) levels in the penumbra, and elevated MA reduces VPS15 expression to exacerbate ischemia injury. The NMT1/MA/VPS15 pathway operates in the ischemic cerebral cortex.","method":"NMT1 knockdown in rat cerebral ischemia-reperfusion model; intraperitoneal MA injection; BCtDCS intervention; VPS15 expression analysis; infarct volume measurement","journal":"Experimental neurology","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo rat model with NMT1 KD and substrate manipulation; single lab, pathway placement","pmids":["40221008"],"is_preprint":false}],"current_model":"NMT1 is a monomeric N-myristoyltransferase (EC 2.3.1.97) that cotranslationally and posttranslationally transfers myristate from myristoyl-CoA to N-terminal glycine residues (and, with lower efficiency, to lysine ε-amino groups via direct catalytic base contact) of substrate proteins including G-protein α-subunits, Src-family kinases, HIV Gag/Nef/matrix, BID, VILIP3, CHP1, ICAM-1, and TMEM106B, thereby regulating their membrane association, subcellular trafficking, stability (through modulation of ubiquitination), and downstream signaling (NFκB/Bcl-2, JNK, PI3K/VPS15); its activity is inhibited by Sorbs2 binding upon TNF-α stimulation and by small-molecule inhibitors (e.g., desloratadine binding Asn-246, PCLX-001), and its catalytic domain spans residues Ile59–Leu455 with the N-terminal 59 residues providing non-catalytic targeting to myristoyl-CoA pools."},"narrative":{"teleology":[{"year":1985,"claim":"The identification of N-terminal myristate on p60src established that NMT-catalyzed myristoylation is a defined covalent modification of signaling kinases and retroviral transforming proteins, opening the field of protein myristoylation.","evidence":"Tryptic peptide sequencing and metabolic [3H]myristate labeling of p60src from Rous sarcoma virus","pmids":["3917576"],"confidence":"High","gaps":["NMT enzyme not yet purified or cloned","substrate scope unknown"]},{"year":1990,"claim":"Mutagenesis of the N-terminal glycine of HIV-1 Gag and Gα subunits demonstrated that NMT-mediated myristoylation is required for stable membrane association, viral assembly, and G-protein signaling, establishing myristoylation as a functional membrane-targeting signal rather than an inert modification.","evidence":"G2A mutagenesis of HIV-1 Gag and Gαo; metabolic labeling; subcellular fractionation and virion assembly assays in mammalian cells","pmids":["2788277","2153964","2405382"],"confidence":"High","gaps":["Relative contribution of myristate vs. adjacent basic residues not yet dissected","no crystal structure of NMT–substrate complex"]},{"year":1992,"claim":"Deletion and chimera mapping of yeast Nmt1p defined the minimal catalytic domain (Ile59–Leu455) and revealed that the N-terminal 59 residues play a non-catalytic role in accessing myristoyl-CoA pools, establishing the domain architecture conserved in human NMT1.","evidence":"Deletion mutagenesis, E. coli reconstitution, yeast nmt1-null complementation, and human/yeast chimeric enzymes","pmids":["1429724"],"confidence":"High","gaps":["Human NMT1 domain boundaries not directly mapped","structural basis of substrate specificity determinants unresolved"]},{"year":1994,"claim":"Quantitative in vitro membrane-binding studies showed that NMT-added myristate cooperates with adjacent polybasic sequences to form a bipartite membrane-targeting signal on Src and HIV Gag, explaining how a single 14-carbon acyl chain achieves high-affinity membrane anchoring.","evidence":"Chimera constructs, mutagenesis of basic residues, and liposome-binding assays with quantified Kd values for p60c-src and HIV-1 Gag","pmids":["7527558","8139035"],"confidence":"High","gaps":["Myristyl-switch mechanism not yet characterized","contribution of palmitoylation or other secondary signals unclear"]},{"year":2000,"claim":"The discovery that caspase-8-cleaved BID undergoes posttranslational myristoylation by NMT, which redirects tBID to mitochondria and promotes cytochrome c release, demonstrated that NMT activity is not limited to cotranslational modification and directly controls apoptotic signaling.","evidence":"In vitro myristoylation, liposome binding, and mitochondrial cytochrome c release assays with glycine-mutant controls","pmids":["11099414"],"confidence":"High","gaps":["Relative contributions of NMT1 vs. NMT2 to posttranslational BID myristoylation not separated","in vivo apoptotic relevance not fully dissected"]},{"year":2003,"claim":"NMR studies of HIV-1 myristoylated matrix protein revealed an entropic myristyl switch coupled to protein trimerization, showing how NMT-installed myristate is regulated downstream of the transferase reaction to control membrane targeting in a conformationally gated manner.","evidence":"NMR structure determination and analytical ultracentrifugation of myr-MA monomer–trimer equilibrium","pmids":["14699046"],"confidence":"High","gaps":["Switch mechanism not demonstrated for non-viral NMT substrates","kinetic regulation of switch in the virion assembly context unclear"]},{"year":2018,"claim":"NMT1 knockdown in breast cancer cells established a feedback loop where reduced myristoylation triggers ROS accumulation and ER stress, activating JNK-mediated autophagy that suppresses tumor growth, placing NMT1 as a regulatory node integrating lipid modification with stress-response signaling.","evidence":"shRNA knockdown of NMT1; ROS, ER stress, and JNK pathway analysis; in vivo xenograft models","pmids":["30446635"],"confidence":"Medium","gaps":["Specific substrates mediating ROS/ER stress not identified","NMT2 compensation not assessed","single lab"]},{"year":2021,"claim":"Liver-specific NMT1 knockout mice revealed that myristoylation differentially controls protein stability: NDP-class substrates are destabilized via HIST1H4H-mediated ubiquitination, while NUP-class substrates are stabilized because myristoylation prevents their ubiquitination, establishing that NMT1 modulates the ubiquitin–proteasome system.","evidence":"Conditional liver-specific NMT1 KO; click chemistry; iTRAQ proteomics; co-IP and ubiquitination assays","pmids":["34136404"],"confidence":"Medium","gaps":["Mechanism by which myristoylation recruits or blocks ubiquitin ligases is unclear","relevance of POTEE as co-factor needs independent validation"]},{"year":2022,"claim":"Structural and kinetic analysis demonstrated that NMT catalyzes both efficient G-myristoylation (water-mediated catalytic base) and inefficient K-myristoylation (direct catalytic base contact), revealing a dual-mechanism catalytic architecture and explaining evolutionary depletion of K-myristoylation motifs in humans.","evidence":"X-ray crystallography of NMT–substrate complexes; mass spectrometry; enzyme kinetics; in silico motif analysis","pmids":["36181773"],"confidence":"High","gaps":["Physiological relevance of K-myristoylation in vivo remains limited to few substrates","structural basis of substrate selectivity between NMT1 and NMT2 not resolved"]},{"year":2023,"claim":"Identification of VILIP3 as an NMT1 substrate whose myristoylation drives NFκB/Bcl-2 signaling in HCC, combined with the discovery that desloratadine inhibits NMT1 by binding Asn-246, provided the first clinically repurposable inhibitor–target pair and a defined NMT1→VILIP3→NFκB signaling axis.","evidence":"DARTS, SPR for target ID; metabolic labeling/MS for substrate ID; NMT1 KD/OE; CDX/PDO/PDX in vivo models","pmids":["36617552"],"confidence":"High","gaps":["Desloratadine selectivity for NMT1 vs. NMT2 not quantified","whether Asn-246 binding is allosteric or competitive unclear"]},{"year":2023,"claim":"NMT1-mediated myristoylation of ICAM-1 stabilizes the adhesion molecule by blocking FBXO4-mediated ubiquitination, linking NMT1 activity to cell adhesion and tumor cell migration suppression.","evidence":"NMT1 knockdown; co-IP; ubiquitination and half-life assays; wound-healing and Transwell migration","pmids":["37269961"],"confidence":"Medium","gaps":["ICAM-1 N-terminal myristoylation site not structurally confirmed","single-lab finding without independent replication"]},{"year":2024,"claim":"Sorbs2 was identified as a novel NMT1-binding partner whose association upon TNF-α stimulation paradoxically inhibits NMT1 catalytic activity despite increased NMT1 expression, revealing a post-translational regulatory mechanism for NMT1 in osteoblasts.","evidence":"IP/MS identification of Sorbs2; click-it myristoylation assay; RNAi in MC3T3-E1 osteoblastic cells","pmids":["38148048"],"confidence":"Medium","gaps":["Binding interface between Sorbs2 and NMT1 not mapped","functional consequence for osteoblast biology not fully defined","single lab, single cell type"]},{"year":2025,"claim":"NMT1 myristoylates both Gly2 and Lys3 of TMEM106B, directing it to lysosomes for degradation; non-myristoylated TMEM106B accumulates at the cell surface, establishing dual G- and K-myristoylation as a physiological trafficking determinant for a single substrate.","evidence":"Gly2/Lys3 mutagenesis; NMT1/2 overexpression and knockdown; live-cell imaging; lysosomal inhibitor rescue","pmids":["40451428"],"confidence":"High","gaps":["Relative contribution of NMT1 vs. NMT2 to TMEM106B modification not separated","structural basis for dual-site myristoylation unclear"]},{"year":2025,"claim":"HIF1α-driven NMT1 upregulation mediates CHP1 myristoylation, which facilitates PD-L1 membrane translocation and immune evasion in HNSCC, and pharmacological NMT1 inhibition by PCLX-001 enhances anti-PD-1 immunotherapy efficacy in vivo.","evidence":"HIF1α manipulation; click chemistry; co-IP of CHP1–PD-L1; membrane fractionation; xenograft mouse model with anti-PD-1 + PCLX-001","pmids":["40605065"],"confidence":"Medium","gaps":["CHP1–PD-L1 binding interface not structurally characterized","whether PCLX-001 effects are NMT1-specific or also involve NMT2 not distinguished"]},{"year":null,"claim":"Key unresolved questions include the full substrate repertoire that distinguishes NMT1 from NMT2, the structural basis for NMT1 substrate selectivity, the physiological scope of K-myristoylation, and whether NMT1-selective (vs. pan-NMT) inhibitors can be developed for therapeutic use.","evidence":"","pmids":[],"confidence":"Low","gaps":["NMT1 vs. NMT2 substrate discrimination rules not established","no high-resolution human NMT1 structure with endogenous substrates","in vivo essentiality of NMT1 in adult tissues not comprehensively characterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,2,4,9,15,16,17,21]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4,8]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,4,14,15,17,21]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[9]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,11,20]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,16,20]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[4,15]}],"complexes":[],"partners":["SORBS2","CHP1","VILIP3","ICAM-1","TMEM106B","BID"],"other_free_text":[]},"mechanistic_narrative":"NMT1 is a glycylpeptide N-tetradecanoyltransferase that catalyzes the cotranslational and posttranslational transfer of myristate from myristoyl-CoA to the N-terminal glycine α-amino group of substrate proteins, thereby governing their membrane association, subcellular trafficking, stability, and signaling competence. Its substrates span G-protein α-subunits, Src-family kinases, HIV Gag/Nef/matrix, BID, CHP1, VILIP3, ICAM-1, and TMEM106B; myristoylation of BID after caspase-8 cleavage redirects truncated BID to mitochondria to promote cytochrome c release and apoptosis [PMID:11099414], while myristoylation of CHP1 facilitates PD-L1 plasma-membrane translocation and immune evasion [PMID:40605065]. NMT1 additionally catalyzes a less efficient lysine ε-amino (K-myristoylation) reaction using a direct catalytic-base contact distinct from the water-mediated mechanism of canonical G-myristoylation [PMID:36181773]. Its enzymatic activity is negatively regulated by Sorbs2 binding upon TNF-α stimulation [PMID:38148048] and is pharmacologically targetable by small-molecule inhibitors including desloratadine (binding Asn-246) and PCLX-001 [PMID:36617552, PMID:40605065]."},"prefetch_data":{"uniprot":{"accession":"P30419","full_name":"Glycylpeptide N-tetradecanoyltransferase 1","aliases":["Myristoyl-CoA:protein N-myristoyltransferase 1","HsNMT1","NMT 1","Type I N-myristoyltransferase","Peptide N-myristoyltransferase 1","Protein-lysine myristoyltransferase NMT1"],"length_aa":496,"mass_kda":56.8,"function":"Adds a myristoyl group to the N-terminal glycine residue of certain cellular and viral proteins (PubMed:22865860, PubMed:25255805, PubMed:32686708, PubMed:34999170, PubMed:9353336, PubMed:9506952). Also able to mediate N-terminal lysine myristoylation of proteins: catalyzes myristoylation of ARF6 on both 'Gly-2' and 'Lys-3' (PubMed:32103017, PubMed:32111831). 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virology","url":"https://pubmed.ncbi.nlm.nih.gov/8139035","citation_count":484,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"11099414","id":"PMC_11099414","title":"Posttranslational N-myristoylation of BID as a molecular switch for targeting mitochondria and apoptosis.","date":"2000","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/11099414","citation_count":472,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15489334","id":"PMC_15489334","title":"The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).","date":"2004","source":"Genome research","url":"https://pubmed.ncbi.nlm.nih.gov/15489334","citation_count":438,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"26638075","id":"PMC_26638075","title":"A Dynamic Protein Interaction Landscape of the Human Centrosome-Cilium Interface.","date":"2015","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/26638075","citation_count":433,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"35271311","id":"PMC_35271311","title":"OpenCell: Endogenous tagging for the cartography of human cellular organization.","date":"2022","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/35271311","citation_count":432,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"26344197","id":"PMC_26344197","title":"Panorama of ancient metazoan macromolecular complexes.","date":"2015","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/26344197","citation_count":407,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"34079125","id":"PMC_34079125","title":"A proximity-dependent biotinylation map of a human cell.","date":"2021","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/34079125","citation_count":339,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"11463741","id":"PMC_11463741","title":"Structure--function relationships in HIV-1 Nef.","date":"2001","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/11463741","citation_count":317,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"2153964","id":"PMC_2153964","title":"G-protein alpha-subunit expression, myristoylation, and membrane association in COS cells.","date":"1990","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/2153964","citation_count":277,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"22863883","id":"PMC_22863883","title":"A high-throughput approach for measuring temporal changes in the interactome.","date":"2012","source":"Nature methods","url":"https://pubmed.ncbi.nlm.nih.gov/22863883","citation_count":273,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"14699046","id":"PMC_14699046","title":"Entropic switch regulates myristate exposure in the HIV-1 matrix protein.","date":"2003","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/14699046","citation_count":272,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"3917576","id":"PMC_3917576","title":"Amino terminal myristylation of the protein kinase p60src, a retroviral transforming protein.","date":"1985","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/3917576","citation_count":256,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"7527558","id":"PMC_7527558","title":"Amino-terminal basic residues of Src mediate membrane binding through electrostatic interaction with acidic phospholipids.","date":"1994","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/7527558","citation_count":223,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"10625670","id":"PMC_10625670","title":"Five members of a novel Ca(2+)-binding protein (CABP) subfamily with similarity to calmodulin.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10625670","citation_count":221,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":24878,"output_tokens":3847,"usd":0.06617},"stage2":{"model":"claude-opus-4-6","input_tokens":7319,"output_tokens":5653,"usd":0.26688},"total_usd":0.65795,"stage1_batch_id":"msgbatch_012ZiiCUBfB62NCTQd4Bk1Jo","stage2_batch_id":"msgbatch_01Xuqq9aUHoQouS7UVGZNStZ","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":35805,"output_tokens":7879,"usd":0.1128},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":9560,"output_tokens":3744,"usd":0.2121}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1992,\n      \"finding\": \"Saccharomyces cerevisiae Nmt1p is an essential, monomeric enzyme whose minimal catalytic domain lies between residues Ile59–Phe96 and Gly451–Leu455; the amino-terminal 59 residues serve a non-catalytic targeting role allowing the cytosolic enzyme to access myristoyl-CoA pools derived from exogenous fatty acid activation by acyl-CoA synthetase.\",\n      \"method\": \"Amino- and carboxyl-terminal deletion mutagenesis assessed by in vitro enzyme assay, E. coli co-expression with eukaryotic substrates (Gpa1p, rat Goα), and yeast complementation of nmt1 null lethal allele\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro assay plus mutagenesis plus genetic complementation in single rigorous study\",\n      \"pmids\": [\"1429724\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"Structural elements determining the difference in peptide substrate specificity between yeast Nmt1p and human NMT were mapped using human/yeast chimeras: recognition of human Gzα (a substrate only for human NMT) requires elements distributed across the amino-terminal half through the region defined by Leu352–Lys410 of the human enzyme, and a fully functional peptide-binding site requires contributions from both amino- and carboxyl-terminal halves of the enzyme.\",\n      \"method\": \"E. coli co-expression of chimeric NMT enzymes with substrate G-protein alpha subunits, in vitro myristoylation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with chimeric mutagenesis defining substrate specificity determinants\",\n      \"pmids\": [\"1429724\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Cryptococcus neoformans NMT is essential for viability; under-myristoylation of a cellular ADP-ribosylation factor (Arf) contributes to the temperature-sensitive lethal phenotype of the conditional nmt487D allele, and this phenotype can be rescued by forced expression of human NMT.\",\n      \"method\": \"Homologous recombination to introduce conditional lethal NMT allele, genetic rescue with human NMT, protein myristoylation analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with allele-specific rescue and substrate identification in vivo\",\n      \"pmids\": [\"9575206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"A fully depeptidized inhibitor of C. neoformans NMT kills the fungus through an NMT-dependent mechanism, with Ki(app) of 1.8 µM for fungal NMT and 9 µM for human NMT; strain-selective killing was confirmed using isogenic strains differing only in whether they relied on fungal or human NMT.\",\n      \"method\": \"Kinetic inhibition studies with purified fungal and human NMTs, isogenic strain pair with differential NMT dependence, protein synthesis inhibition assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — kinetic characterization of purified enzyme plus genetically controlled strain pair\",\n      \"pmids\": [\"9575206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NMTs catalyze both N-terminal glycine α-amino group myristoylation (G-myristoylation) and lysine ε-amino group myristoylation (K-myristoylation); G-myristoylation uses a water-mediated interaction with the NMT catalytic base while K-myristoylation uses a direct interaction, resulting in much lower efficiency for K-myristoylation. K-myristoylation is restricted to post-translational events, and human proteomes show evolutionary depletion of NMT-dependent K-myristoylation motifs.\",\n      \"method\": \"Mass spectrometry, kinetic studies, in silico analysis, and X-ray crystallography of NMT–substrate complexes\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus kinetic assays plus MS, multiple orthogonal methods in single study\",\n      \"pmids\": [\"36181773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NMT1 myristoylates VILIP3 at its N-terminus; desloratadine binds to Asn-246 of NMT1 and inhibits its enzymatic activity, disrupting NMT1-mediated myristoylation of VILIP3 and subsequent NFκB/Bcl-2 signaling in hepatocellular carcinoma cells.\",\n      \"method\": \"Drug affinity responsive target stability (DARTS) and surface plasmon resonance (SPR) to identify NMT1 as drug target; metabolic labeling and mass spectrometry to identify VILIP3 as NMT1 substrate; NMT1 knockdown and overexpression in vitro and in vivo (CDX, PDO, PDX models)\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — target engagement assay, substrate identification by MS, and in vivo validation with multiple orthogonal methods\",\n      \"pmids\": [\"36617552\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Genetic inhibition of NMT1 in breast cancer cells promotes oxidative stress and ER stress, which cross-talk with mitochondria to increase ROS production; both oxidative and ER stress activate JNK pathway leading to autophagy. ROS negatively regulates NMT1 expression, forming a feedback loop.\",\n      \"method\": \"siRNA knockdown of NMT1 in breast cancer cell lines, in vitro and in vivo tumor models, ROS measurement, ER stress markers, JNK pathway activation assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — KD with defined pathway readouts but pathway placement based primarily on pharmacological inhibitors\",\n      \"pmids\": [\"30446635\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NMT1-mediated N-myristoylation differentially regulates ubiquitination and protein stability of two classes of substrates in a POTEE-dependent manner: NMT1 N-myristoylates NDP proteins (LXN, RPL29, FAU) leading to their destabilization via HIST1H4H-mediated ubiquitination facilitated by RPL7A, while NMT1 N-myristoylates NUP proteins (AHSG, ALB, TF) whose stability is maintained as HBB prevents their ubiquitination by HIST1H4H.\",\n      \"method\": \"Proteomics (iTRAQ), click chemistry N-myristoylation assay, liver-conditional NMT1 knockout mouse model, immunoprecipitation, ubiquitination assays\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — click chemistry for myristoylation, IP for interactions, KO mouse, but ubiquitination mechanism partially inferred\",\n      \"pmids\": [\"34136404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NMT1 N-myristoylates ICAM-1 at its N-terminus and prevents its ubiquitination and proteasomal degradation by inhibiting F-box protein 4 (FBXO4, a Ub E3 ligase), thereby prolonging ICAM-1 half-life and sustaining cell adhesion while suppressing tumor cell migration.\",\n      \"method\": \"NMT1 knockdown/overexpression, co-immunoprecipitation, protein half-life chase experiments, proteasome inhibition, identification of FBXO4 as E3 ligase\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP and KD/OE with pathway placement, single lab, moderate mechanistic follow-up\",\n      \"pmids\": [\"37269961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TMEM106B is myristoylated by NMT1/2 at its glycine 2 α-amino group and lysine 3 ε-amino group; myristoylation promotes lysosomal degradation of TMEM106B and controls its trafficking—non-myristoylated TMEM106B accumulates on the cell surface instead of lysosomes.\",\n      \"method\": \"NMT1/2 knockdown and inhibition, site-directed mutagenesis of myristoylation sites, subcellular fractionation, live-cell imaging, pulse-chase degradation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis of substrate sites combined with localization and degradation assays using multiple orthogonal methods\",\n      \"pmids\": [\"40451428\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Hypoxia-inducible factor-1α (HIF1α) upregulates NMT1, which mediates myristoylation of calcineurin B homologous protein 1 (CHP1); myristoylated CHP1 binds PD-L1 and facilitates its rapid translocation to the cell membrane, enhancing PD-L1-mediated immune evasion. NMT1 inhibitor PCLX-001 blocks CHP1 myristoylation and disrupts PD-L1 membrane localization.\",\n      \"method\": \"NMT1 inhibition (PCLX-001), myristoylation assays, co-immunoprecipitation of CHP1 and PD-L1, subcellular fractionation, HNSCC xenograft mouse model with anti-PD-1 combination\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus inhibitor pharmacology plus in vivo model, but single lab\",\n      \"pmids\": [\"40605065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TNF-α stimulation in osteoblasts induces binding between NMT1 and Sorbs2, a novel NMT1-interacting protein identified by immunoprecipitation and mass spectrometry; this NMT1–Sorbs2 interaction inhibits overall protein myristoylation.\",\n      \"method\": \"Click-it myristoylation assay, RNA interference, mass spectrometry, co-immunoprecipitation, immunocytochemistry, western blotting in murine osteoblastic cells (MC3T3-E1)\",\n      \"journal\": \"In vivo (Athens, Greece)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP/MS identification of binding partner, single lab, mechanism of inhibition not fully defined\",\n      \"pmids\": [\"38148048\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In rat cerebral ischemia, NMT1 is downregulated in the penumbra leading to accumulation of its substrate myristic acid (MA); elevated MA reduces expression of VPS15 (PI3K regulatory subunit 4) to exacerbate ischemia injury, placing NMT1 upstream of an NMT1/MA/VPS15 signaling pathway.\",\n      \"method\": \"NMT1 knockdown in rat ischemia-reperfusion model, intraperitoneal MA injection, transcranial direct current stimulation (BCtDCS) to modulate MA levels, western blotting for VPS15\",\n      \"journal\": \"Experimental neurology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — in vivo knockdown with pathway placement but mechanism connecting MA to VPS15 not biochemically reconstituted\",\n      \"pmids\": [\"40221008\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NMT1 (and NMT2) are required for N-terminal myristoylation of the mammarenavirus matrix Z protein and the stable signal peptide (SSP); the pan-NMT inhibitor DDD85464 reduces Z-mediated virus budding activity and GP2-mediated fusion activity and promotes proteasome-mediated degradation of the Z protein, demonstrating that host NMT1/NMT2 enzymatic activity is required for mammarenavirus multiplication.\",\n      \"method\": \"Pharmacological NMT inhibition with validated pan-NMT inhibitor DDD85464, Z protein budding assay, GP2-mediated fusion assay, proteasome inhibition rescue experiment, antiviral assays with LCMV, JUNV, and LASV\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — validated on-target inhibitor with multiple functional readouts, but preprint, single lab\",\n      \"pmids\": [\"bio_10.1101_2024.08.01.606235\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"Human NMT1 is a monomeric N-myristoyltransferase enzyme (EC 2.3.1.97) that co- and post-translationally transfers myristate from myristoyl-CoA to the α-amino group of N-terminal glycine residues (and less efficiently to lysine ε-amino groups via a mechanistically distinct direct interaction with the catalytic base) of diverse substrate proteins including ARF family GTPases, VILIP3, ICAM-1, CHP1, TMEM106B, and viral Z proteins/SSPs; myristoylation of these substrates controls their membrane targeting, protein stability (by modulating ubiquitination), subcellular trafficking, and downstream signaling cascades (NFκB/Bcl-2, JNK, PI3K/AKT, and immune checkpoint pathways), and NMT1 activity is itself regulated by HIF1α-driven transcription, ROS feedback, and protein–protein interactions such as binding to Sorbs2.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries only from KEEP papers.\n\n**Classification:**\n\n**KEEP (canonical human/mammalian NMT1 - N-myristoyltransferase 1):**\n- [5] PMID:36617552 - NMT1 enzymatic activity, VILIP3 substrate, HCC\n- [6] PMID:1429724 - Yeast Nmt1p catalytic domain, substrate specificity (ortholog)\n- [8] PMID:9575206 - C. neoformans NMT (ortholog, fungicidal)\n- [9] PMID:30446635 - NMT1 inhibition, breast cancer, JNK/ROS/ER stress\n- [17] PMID:34136404 - NMT1/POTEE/ubiquitination in liver cancer\n- [21] PMID:36181773 - NMT structure, K-myristoylation vs G-myristoylation mechanism\n- [22] PMID:10470373 - NMT subcellular localization, mammary cells\n- [24] PMID:37269961 - NMT1/ICAM-1, ubiquitination, adhesion/migration\n- [33] PMID:40605065 - NMT1/CHP1/PD-L1, hypoxia, HIF1α\n- [35] PMID:40451428 - TMEM106B myristoylation by NMT1/2, trafficking\n- [36] PMID:38148048 - NMT1/Sorbs2 binding, TNF-α inhibition of myristoylation\n- [41] PMID:40221008 - NMT1/MA/VPS15 pathway, cerebral ischemia\n- [44] bio_10.1101_2024.08.01.606235 - NMT1/2 myristoylate mammarenavirus Z and SSP\n- [45] bio_10.1101_2025.06.20.660686 - NMT inhibitors antiviral\n- [3] PMID:1594577 - S. cerevisiae NMT1 gene (ortholog context, PWP1)\n- Gene2pubmed curated:\n  - PMID:11099414 - Posttranslational N-myristoylation of BID by NMT\n  - PMID:2788277 - HIV Gag myristoylation\n  - PMID:2405382 - HIV-1 myristoylation/replication\n  - PMID:8139035 - HIV Gag membrane targeting domain\n  - PMID:14699046 - HIV-1 matrix myristyl switch\n  - PMID:3917576 - p60src myristylation\n  - PMID:2153964 - G-protein alpha subunit myristoylation\n  - PMID:7527558 - Src membrane binding\n  - PMID:10625670 - CaBP myristoylation\n  - PMID:11463741 - HIV Nef myristoylation\n\n**EXCLUDE (alias collision/off-target):**\n- [1] PMID:2358444 - S. pombe nmt1 = thiamine biosynthesis gene, NOT N-myristoyltransferase (SYMBOL COLLISION - different protein)\n- [2] PMID:8422997 - S. pombe nmt1 promoter/thiamine\n- [7] PMID:34200747 - NMT = noradrenergic-to-mesenchymal transition (alias collision)\n- [10] PMID:24248932 - Plant N-methyltransferase (Catharanthus roseus, different enzyme)\n- [11] PMID:9606189 - S. pombe nmt1/nmt2 thiamine genes\n- [12] PMID:8613682 - NMT-1 rat yolk sac tumor cell line (alias collision)\n- [13] PMID:7992507 - S. pombe nmt2 thiamine gene\n- [14] PMID:8163491 - S. pombe nmt1 promoter regulation\n- [15] PMID:29777000 - Arabidopsis NMT1/NMT3 phosphoethanolamine N-methyltransferases (plant, different enzyme)\n- [16] PMID:7507475 - Rat yolk sac tumor NMT-1 cell line\n- [18] PMID:21295003 - S. pombe nmt1 promoter tool\n- [19] PMID:33805223 - Hs-NMT QSAR (computational only)\n- [20] PMID:13680155 - Neurospora nmt-1 thiamine gene\n- [23] PMID:15644961 - Wheat NMT (plant NMT enzyme - but this IS N-myristoyltransferase from a plant... keep? The abstract says it's NMT EC 2.3.1.97 and validates activity in E.coli. However, it's a plant/monocot NMT - borderline but the function IS compatible with mammalian NMT1. I'll KEEP for the enzymatic activity validation.)\n- [25] PMID:25801050 - S. pombe nmt promoter tool\n- [26] PMID:34333213 - ANCR/miR-4731-5p/NMT1 axis - this does describe NMT1 but is mainly expression/lncRNA; no direct mechanistic experiment on NMT1 protein itself - EXCLUDE (no mechanistic finding about NMT1 protein)\n- [27] PMID:31664711 - Neurospora NMT1 riboswitch (different gene)\n- [28] PMID:8533474 - S. cerevisiae chromosome XIV / thiamine nmt1 homologue\n- [29] PMID:16874521 - S. pombe nmt1 promoter regulation\n- [30] PMID:36002266 - Plasmodium NMT (computational docking only)\n- [31] PMID:37486304 - NMT1 riboswitch in bacteria (different gene/RNA)\n- [32] PMID:35732157 - NMT1/PI3K/AKT in NSCLC - only Western blot/expression correlation, no direct mechanistic experiment on NMT1\n- [34] PMID:33937538 - NMT = nanoparticle mineral trioxide (alias collision)\n- [37] PMID:38975119 - NMT indicator = mercury sensor (alias collision)\n- [38] PMID:36542915 - Leishmania NMT computational\n- [39] PMID:40933604 - Leishmania NMT computational\n- [40] PMID:31161515 - NMT labeling method for bacteria (tool paper, no mechanistic finding about NMT1)\n- [42] PMID:41406521 - NMT = non-invasive micro-test technology (alias collision)\n- [43] PMID:24908050 - Bacterial NMT1/THI5 thiamine protein (different gene)\n- [46] bio_10.1101_2025.07.10.663842 - NMT = macaque brain MRI standard space (alias collision)\n- [47] bio_10.1101_2024.11.22.624841 - NMT = noradrenergic-mesenchymal transition (alias collision)\n- [48] bio_10.1101_2024.06.22.600193 - S. pombe nmt1 promoter tool\n- [49] bio_10.1101_2025.03.24.644888 - NMT = neural machine translation (alias collision)\n- [50] bio_10.1101_2025.01.07.631647 - NMT = non-invasive micro-test (alias collision)\n- [51] bio_10.1101_2024.07.28.605509 - Plant MIA N-methyltransferases (different enzyme)\n- Gene2pubmed: PMID:17081983, 22658674, 12477932, 16964243, 18187620, 15302935, 26186194, 28514442, 26496610, 32296183, 29507755, 21873635, 22190034, 33961781, 26638075, 35271311, 26344197, 34079125 - large-scale proteomics/interactome screens; NMT1 appears incidentally, no specific mechanistic findings about NMT1 itself. EXCLUDE.\n\nNow extracting discoveries from KEEP papers:\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1985,\n      \"finding\": \"The transforming protein p60src of Rous sarcoma virus is N-terminally myristoylated at its amino-terminal glycine residue, establishing that myristoylation of N-terminal glycine is a posttranslational modification catalyzed by NMT on signaling kinases and retroviral transforming proteins.\",\n      \"method\": \"Isolation and sequencing of tryptic peptide from p60src; metabolic labeling with myristic acid\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct biochemical identification of myristoylated peptide, foundational paper replicated broadly\",\n      \"pmids\": [\"3917576\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1989,\n      \"finding\": \"Myristoylation of the N-terminal glycine of HIV-1 Gag precursor Pr55gag, catalyzed by cellular NMT, is essential for virus particle assembly and budding; G2A mutation abolishing myristoylation prevents virus particle release.\",\n      \"method\": \"Site-directed mutagenesis of Gag glycine-2; metabolic labeling; virion morphogenesis assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis with functional virological readout, replicated across multiple HIV studies\",\n      \"pmids\": [\"2788277\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"Myristoylation of G-protein alpha subunits (Gi1, Gi2, Gi3, Go, Gt, Gz but not Gs) by cellular NMT is required for their stable membrane association; mutation of the N-terminal glycine to alanine in Gαo abolishes both myristoylation and membrane binding.\",\n      \"method\": \"Metabolic [3H]myristate labeling of COS cell transfectants; site-directed mutagenesis; subcellular fractionation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis plus subcellular fractionation in mammalian cells, multiple substrates tested\",\n      \"pmids\": [\"2153964\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"Myristoylation of HIV-1 Pr55gag by cellular NMT is required for stable membrane association and virus assembly; non-myristoylated Gag accumulates in cells but is not incorporated into virions, though it can still be cleaved by HIV protease in vitro, demonstrating myristate is not required for protease recognition.\",\n      \"method\": \"G-to-A mutagenesis of Gag; metabolic labeling; complementation in trans; in vitro protease cleavage assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis, in vitro assay, and complementation\",\n      \"pmids\": [\"2405382\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"The minimal catalytic domain of S. cerevisiae Nmt1p lies between residues Ile59–Phe96 (N-terminal boundary) and Gly451–Leu455 (C-terminal boundary); the N-terminal 59 residues play a non-catalytic targeting role enabling access to myristoyl-CoA pools from exogenous fatty acid. Peptide substrate specificity differences between yeast Nmt1p and human NMT involve elements distributed across both halves of the enzyme.\",\n      \"method\": \"Deletion mutagenesis; co-expression with substrates in E. coli; in vitro enzyme assay; yeast complementation of nmt1 null allele; human/yeast chimeric enzymes\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro assay plus mutagenesis and genetic rescue, ortholog of human NMT1\",\n      \"pmids\": [\"1429724\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"The membrane-targeting domain of HIV-1 Gag consists of a bipartite signal: the myristoylated N-terminal 14 amino acids (added by NMT) and an adjacent highly basic region that binds acidic phospholipids. Both myristate and the basic region are required for efficient membrane binding and virion formation.\",\n      \"method\": \"Gag-Src chimera constructs; in vitro membrane binding assay; mutagenesis of basic residues; virion formation assay in vivo\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — reconstituted membrane binding with chimeras and mutagenesis, in vivo confirmation\",\n      \"pmids\": [\"8139035\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Membrane binding of pp60c-src requires both NMT-mediated N-terminal myristoylation and six basic residues that interact electrostatically with acidic phospholipids; removal of either element drastically reduces membrane affinity (Kd shifts from ~0.6 µM to ~2 mM for neutral bilayers).\",\n      \"method\": \"In vitro lipid vesicle binding assay; mutagenesis of basic residues; metabolic labeling; transformation assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — quantitative in vitro reconstitution with mutagenesis and in vivo confirmation\",\n      \"pmids\": [\"7527558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Cryptococcus neoformans NMT is an essential enzyme whose inhibition causes under-myristoylation of ADP ribosylation factor (Arf) and cell death; this fungicidal effect of a depeptidized NMT inhibitor is NMT-dependent, as demonstrated using isogenic strains expressing either fungal or human NMT.\",\n      \"method\": \"Homologous recombination to introduce conditional lethal nmt allele; strain-specific inhibitor sensitivity assay; kinetic studies of purified fungal vs. human NMT; protein synthesis inhibition assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — genetic and biochemical evidence with isogenic strains and purified enzyme kinetics\",\n      \"pmids\": [\"9575206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"NMT is targeted to membranes in mammary epithelial cells via interaction with ribosomal proteins, and its subcellular localization is an important variable in the regulation of cellular proliferation; NMT expression and membrane/cytosol distribution are differentially regulated during normal mammary gland development and in breast cancer transformation.\",\n      \"method\": \"Subcellular fractionation; expression analysis during pregnancy/lactation and malignant transformation; comparison of normal vs. cancerous human breast tissue\",\n      \"journal\": \"Advances in enzyme regulation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — fractionation without functional manipulation; single lab, partial mechanistic follow-up\",\n      \"pmids\": [\"10470373\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"BID undergoes posttranslational (non-cotranslational) N-myristoylation by NMT after caspase-8 cleavage exposes a glycine residue; this myristoylation enables BID fragments to target mitochondria and enhances cytochrome c release and apoptosis, functioning as a molecular activating switch.\",\n      \"method\": \"In vitro myristoylation assay; liposome binding assay with defined lipid compositions; mitochondria targeting assay; cytochrome c release assay; mutagenesis of glycine acceptor\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution, mutagenesis, and functional apoptosis assay; highly cited\",\n      \"pmids\": [\"11099414\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"CaBP1 and CaBP2, novel Ca²⁺-binding proteins expressed in the retina, contain consensus sequences for N-terminal myristoylation and are fatty acid acylated in vitro by NMT; their subcellular localization patterns depend on whether they are myristoylated.\",\n      \"method\": \"In vitro myristoylation assay; heterologous expression with subcellular localization analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro NMT assay plus localization, single lab\",\n      \"pmids\": [\"10625670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"HIV-1 Nef is N-terminally myristoylated by cellular NMT; this myristoylation is required for membrane association of Nef and is critical for Nef-mediated CD4 downregulation and viral pathogenesis.\",\n      \"method\": \"Structure-function analysis; mutagenesis of N-terminal glycine; metabolic labeling; CD4 downregulation assays\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis with functional assays, extensively reviewed and replicated\",\n      \"pmids\": [\"11463741\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The HIV-1 matrix protein (myr-MA) adopts myristate-exposed and myristate-sequestered states that are in equilibrium; myristate exposure is coupled to trimerization (not mechanically induced conformational change), constituting an entropic myristyl switch. This switch regulates Gag targeting to the plasma membrane during assembly.\",\n      \"method\": \"NMR structural studies of myr-MA in both states; sedimentation equilibrium analytical ultracentrifugation; equilibrium constant measurements for monomer-trimer transition\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure plus biophysical quantification of equilibrium, mechanistically novel finding\",\n      \"pmids\": [\"14699046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Genetic inhibition of NMT1 in breast cancer cells promotes ROS accumulation and ER stress, which cross-talk to produce more ROS. Both oxidative stress and ER stress activate the JNK pathway, leading to autophagy that suppresses breast cancer progression. ROS negatively regulate NMT1 expression, forming a feedback loop.\",\n      \"method\": \"shRNA knockdown of NMT1; ROS measurement; ER stress markers; JNK pathway analysis; autophagy assays; in vitro and in vivo xenograft models\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KD with multiple pathway readouts, single lab\",\n      \"pmids\": [\"30446635\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NMT1-mediated N-myristoylation differentially regulates two protein classes in liver cancer: NDP proteins (LXN, RPL29, FAU) are destabilized by myristoylation via HIST1H4H-mediated ubiquitination facilitated by RPL7A, while NUP proteins (AHSG, ALB, TF) are stabilized by myristoylation because HBB prevents their ubiquitination by HIST1H4H. Both effects depend on NMT1 and POTEE.\",\n      \"method\": \"Conditional liver-specific NMT1 knockout mice; click chemistry N-myristoylation assay; iTRAQ proteomics; co-immunoprecipitation; ubiquitination assays\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse model, proteomics, and mechanistic ubiquitination assays; single lab\",\n      \"pmids\": [\"34136404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NMT catalyzes both G-myristoylation (N-terminal glycine α-amino group, efficient) and K-myristoylation (lysine ε-amino group, poor efficiency); G-myristoylation uses a water-mediated interaction with the NMT catalytic base while K-myristoylation uses direct interaction. Evolutionary pressure has depleted K-myristoylation motifs in humans. K-myristoylation likely only results from post-translational events.\",\n      \"method\": \"Mass spectrometry; enzyme kinetics; in silico analysis; X-ray crystallography of NMT complexes\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus kinetics and MS, multiple orthogonal methods in single study\",\n      \"pmids\": [\"36181773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NMT1 myristoylates VILIP3 at its N-terminus; desloratadine binds Asn-246 in NMT1 and inhibits its enzymatic activity, blocking VILIP3 myristoylation and suppressing downstream NFκB/Bcl-2 signaling to reduce HCC tumor growth.\",\n      \"method\": \"DARTS and SPR assays to identify NMT1 as desloratadine target; metabolic labeling and mass spectrometry to identify VILIP3 as NMT1 substrate; NMT1 knockdown/overexpression; CDX/PDO/PDX models\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct target identification by SPR, substrate identification by metabolic labeling/MS, multiple in vivo models\",\n      \"pmids\": [\"36617552\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NMT1 N-myristoylates the N-terminus of ICAM-1 and prevents its ubiquitination by inhibiting the Ub E3 ligase FBXO4 (F-box protein 4), thereby stabilizing ICAM-1 and sustaining cell adhesion while suppressing tumor cell migration.\",\n      \"method\": \"NMT1 knockdown; co-immunoprecipitation; ubiquitination assay; protein half-life measurement; wound healing and Transwell migration assay\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — co-IP and ubiquitination assays with functional migration readout; single lab\",\n      \"pmids\": [\"37269961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TNF-α stimulation of osteoblasts increases NMT1 expression but paradoxically inhibits overall protein myristoylation; Sorbs2 was identified as a novel NMT1 binding protein by immunoprecipitation/mass spectrometry, and TNF-α-induced Sorbs2–NMT1 binding inhibits myristoylation activity.\",\n      \"method\": \"Click-it myristoylation assay; RNA interference; immunoprecipitation/mass spectrometry; immunocytochemistry; western blotting in MC3T3-E1 osteoblastic cells\",\n      \"journal\": \"In vivo\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — biochemical identification of Sorbs2 as NMT1 interactor with functional myristoylation readout; single lab\",\n      \"pmids\": [\"38148048\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NMT1 and NMT2 myristoylate the mammarenavirus matrix Z protein and stable signal peptide (SSP) at their N-terminal glycines; pharmacological NMT inhibition with DDD85464 reduces Z-mediated budding, impairs GP2-mediated fusion activity, and promotes proteasomal degradation of Z protein, exerting potent antiviral activity against LCMV, JUNV, and LASV.\",\n      \"method\": \"NMT inhibitor DDD85464 treatment; Z budding assay; GP2 fusion assay; proteasome inhibition rescue; antiviral titer assays for multiple arenaviruses\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — validated on-target NMT inhibitor with multiple functional assays; preprint, single lab\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Hypoxia-inducible factor HIF1α upregulates NMT1, which mediates myristoylation of calcineurin B homologous protein 1 (CHP1); myristoylated CHP1 binds PD-L1 and facilitates its rapid translocation to the cell membrane, enhancing immune evasion. NMT1 inhibitor PCLX-001 blocks CHP1 myristoylation and disrupts excessive PD-L1 membrane localization.\",\n      \"method\": \"HIF1α overexpression/knockdown; NMT1 knockdown; click chemistry myristoylation assay; co-immunoprecipitation of CHP1 and PD-L1; membrane fractionation; HNSCC xenograft mouse model with anti-PD-1 + PCLX-001 combination\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP, fractionation, functional assays and in vivo model; single lab\",\n      \"pmids\": [\"40605065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TMEM106B is myristoylated by NMT1/2 at both its Gly2 α-amino group (G-myristoylation) and Lys3 ε-amino group (K-myristoylation); myristoylation promotes lysosomal degradation of TMEM106B and regulates its trafficking, as non-myristoylated TMEM106B accumulates at the cell surface rather than lysosomes.\",\n      \"method\": \"Mutagenesis of Gly2 and Lys3; NMT1/2 overexpression and knockdown; subcellular fractionation; live-cell imaging; lysosomal inhibitor experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis, NMT1/2 manipulation, subcellular localization with functional consequence; multiple orthogonal methods\",\n      \"pmids\": [\"40451428\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NMT1-mediated myristoylation in the ischemic brain regulates VPS15 (PI3K regulatory subunit 4) expression: ischemia-induced downregulation of NMT1 increases myristic acid (MA) levels in the penumbra, and elevated MA reduces VPS15 expression to exacerbate ischemia injury. The NMT1/MA/VPS15 pathway operates in the ischemic cerebral cortex.\",\n      \"method\": \"NMT1 knockdown in rat cerebral ischemia-reperfusion model; intraperitoneal MA injection; BCtDCS intervention; VPS15 expression analysis; infarct volume measurement\",\n      \"journal\": \"Experimental neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo rat model with NMT1 KD and substrate manipulation; single lab, pathway placement\",\n      \"pmids\": [\"40221008\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NMT1 is a monomeric N-myristoyltransferase (EC 2.3.1.97) that cotranslationally and posttranslationally transfers myristate from myristoyl-CoA to N-terminal glycine residues (and, with lower efficiency, to lysine ε-amino groups via direct catalytic base contact) of substrate proteins including G-protein α-subunits, Src-family kinases, HIV Gag/Nef/matrix, BID, VILIP3, CHP1, ICAM-1, and TMEM106B, thereby regulating their membrane association, subcellular trafficking, stability (through modulation of ubiquitination), and downstream signaling (NFκB/Bcl-2, JNK, PI3K/VPS15); its activity is inhibited by Sorbs2 binding upon TNF-α stimulation and by small-molecule inhibitors (e.g., desloratadine binding Asn-246, PCLX-001), and its catalytic domain spans residues Ile59–Leu455 with the N-terminal 59 residues providing non-catalytic targeting to myristoyl-CoA pools.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"NMT1 is an essential, monomeric N-myristoyltransferase (EC 2.3.1.97) that co- and post-translationally transfers myristate from myristoyl-CoA to the α-amino group of N-terminal glycine residues of diverse substrate proteins, and less efficiently to lysine ε-amino groups via a mechanistically distinct direct interaction with the catalytic base [PMID:36181773, PMID:1429724]. By myristoylating substrates such as ARF GTPases, VILIP3, ICAM-1, CHP1, and TMEM106B, NMT1 controls their membrane targeting, subcellular trafficking, protein stability (through modulation of ubiquitin-dependent degradation), and downstream signaling including NFκB/Bcl-2, JNK/autophagy, and PD-L1-mediated immune evasion pathways [PMID:9575206, PMID:36617552, PMID:37269961, PMID:40451428, PMID:40605065]. NMT1 activity is itself regulated transcriptionally by HIF1α and post-translationally through a ROS-dependent negative feedback loop and protein–protein interactions [PMID:30446635, PMID:40605065]. Substrate specificity is determined by elements distributed across both halves of the enzyme, with the N-terminal ~59 residues serving a non-catalytic targeting function [PMID:1429724].\",\n  \"teleology\": [\n    {\n      \"year\": 1992,\n      \"claim\": \"Defining the minimal catalytic architecture and substrate-specificity determinants of NMT resolved how the enzyme recognizes diverse substrates and where catalytic versus targeting functions reside within its primary sequence.\",\n      \"evidence\": \"Deletion mutagenesis, human/yeast chimeras, in vitro myristoylation assays, and yeast complementation of nmt1-null lethal allele\",\n      \"pmids\": [\"1429724\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No crystal structure of human NMT1 at this stage\",\n        \"Substrate scope in human cells undefined\",\n        \"Regulation of NMT1 expression and activity unknown\"\n      ]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Establishing that NMT is essential for viability in a pathogenic fungus and that an ADP-ribosylation factor (Arf) is a critical in vivo substrate demonstrated the biological necessity of myristoylation for membrane trafficking and validated NMT as an antifungal drug target selectively inhibitable over human NMT.\",\n      \"evidence\": \"Conditional lethal allele in C. neoformans rescued by human NMT; selective inhibitor with differential Ki for fungal vs. human enzyme tested in isogenic strains\",\n      \"pmids\": [\"9575206\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Full substrate repertoire of NMT1 in human cells uncharacterized\",\n        \"Structural basis of species-selective inhibition not resolved\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"NMT1 knockdown in cancer cells was shown to trigger oxidative stress and ER stress converging on JNK-mediated autophagy, with ROS feeding back to suppress NMT1 expression, revealing that NMT1 is not just a housekeeping enzyme but is embedded in stress-response circuitry.\",\n      \"evidence\": \"siRNA knockdown in breast cancer cells, ROS measurement, ER stress markers, JNK pathway inhibitors, xenograft models\",\n      \"pmids\": [\"30446635\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Pathway placement relies partly on pharmacological inhibitors rather than genetic epistasis\",\n        \"Identity of myristoylated substrates mediating the stress response not determined\",\n        \"ROS-NMT1 feedback mechanism at transcriptional/post-transcriptional level unresolved\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstration that NMT1-mediated myristoylation differentially channels substrate proteins toward ubiquitin-dependent stabilization or degradation established a general principle that myristoylation is a sorting signal for protein quality control.\",\n      \"evidence\": \"iTRAQ proteomics, click chemistry myristoylation assays, liver-conditional NMT1 knockout mouse, ubiquitination assays\",\n      \"pmids\": [\"34136404\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Proposed role of HIST1H4H as ubiquitin ligase adaptor is unconventional and not reconstituted in vitro\",\n        \"Generality of NDP/NUP substrate classification beyond liver unknown\",\n        \"Direct structural basis for myristoylation-dependent ubiquitination not defined\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolving that NMTs catalyze both canonical N-terminal glycine (G-) myristoylation and lysine ε-amino (K-) myristoylation through mechanistically distinct catalytic base interactions explained why K-myristoylation is rare and post-translational, and revealed evolutionary depletion of K-myristoylation motifs in the human proteome.\",\n      \"evidence\": \"X-ray crystallography of NMT–substrate complexes, kinetic assays, mass spectrometry, in silico proteome analysis\",\n      \"pmids\": [\"36181773\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Physiological substrates of K-myristoylation in vivo largely unidentified\",\n        \"Regulatory contexts favoring K- over G-myristoylation not characterized\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of VILIP3 and ICAM-1 as NMT1 substrates whose stability and signaling (NFκB/Bcl-2 for VILIP3; FBXO4-mediated ubiquitination for ICAM-1) depend on myristoylation expanded the functional repertoire of NMT1 from membrane targeting to direct regulation of protein turnover and cell adhesion.\",\n      \"evidence\": \"DARTS/SPR for NMT1 target engagement (VILIP3); metabolic labeling and MS; Co-IP identifying FBXO4 as E3 ligase for ICAM-1; KD/OE with half-life chase experiments\",\n      \"pmids\": [\"36617552\", \"37269961\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"ICAM-1 myristoylation site not structurally confirmed\",\n        \"FBXO4 mechanism inhibited by myristoylation not biochemically reconstituted\",\n        \"Whether VILIP3 myristoylation effect on NFκB is direct or indirect unclear\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identification of Sorbs2 as a TNFα-induced NMT1-binding protein that inhibits global myristoylation revealed a previously unknown protein-level regulatory mechanism for NMT1 activity.\",\n      \"evidence\": \"Co-immunoprecipitation and mass spectrometry in murine osteoblastic cells, click-it myristoylation assay after TNFα stimulation\",\n      \"pmids\": [\"38148048\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Single Co-IP/MS identification without reciprocal validation or domain mapping\",\n        \"Mechanism by which Sorbs2 binding inhibits NMT1 catalytic activity unknown\",\n        \"Relevance beyond osteoblasts not tested\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"NMT1-dependent dual myristoylation of TMEM106B at Gly2 and Lys3 was shown to be required for lysosomal trafficking and degradation of TMEM106B, directly connecting the G- and K-myristoylation activities discovered structurally in 2022 to a physiological trafficking decision.\",\n      \"evidence\": \"NMT1/2 knockdown and inhibition, site-directed mutagenesis of myristoylation sites, subcellular fractionation, live-cell imaging, pulse-chase degradation assays\",\n      \"pmids\": [\"40451428\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Relative contribution of NMT1 vs NMT2 to TMEM106B myristoylation not resolved\",\n        \"Whether dual myristoylation at adjacent residues is common among NMT substrates unknown\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"HIF1α-driven upregulation of NMT1 was shown to promote CHP1 myristoylation, enabling CHP1 to bind PD-L1 and shuttle it to the plasma membrane for immune evasion, establishing NMT1 as a druggable node in immune checkpoint regulation.\",\n      \"evidence\": \"PCLX-001 NMT1 inhibition, Co-IP of CHP1–PD-L1, subcellular fractionation, HNSCC xenograft with anti-PD-1 combination therapy\",\n      \"pmids\": [\"40605065\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-lab study; CHP1-PD-L1 interaction awaits independent confirmation\",\n        \"Whether NMT2 compensates in this pathway not addressed\",\n        \"Structural basis of myristoylated CHP1–PD-L1 interaction not determined\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The full scope of the NMT1-specific substrate repertoire in human tissues, the structural basis for NMT1 vs NMT2 substrate partitioning, and the in vivo consequence of NMT1-selective versus pan-NMT inhibition remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No systematic comparison of NMT1- vs NMT2-specific substrates across tissues\",\n        \"No NMT1-selective small-molecule inhibitor characterized\",\n        \"Phenotype of NMT1-specific knockout in adult mammals not fully described beyond liver\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 4, 5, 7, 8, 9, 10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"VILIP3\",\n      \"ICAM-1\",\n      \"CHP1\",\n      \"TMEM106B\",\n      \"Sorbs2\",\n      \"ARF\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\nWait, I made an error in the pathway field — I used a GO term instead of Reactome terms. Let me fix that.\n\n```json\n{\n  \"mechanistic_narrative\": \"NMT1 is an essential, monomeric N-myristoyltransferase (EC 2.3.1.97) that co- and post-translationally transfers myristate from myristoyl-CoA to the α-amino group of N-terminal glycine residues of diverse substrate proteins, and less efficiently to lysine ε-amino groups via a mechanistically distinct direct interaction with the catalytic base [PMID:36181773, PMID:1429724]. By myristoylating substrates such as ARF GTPases, VILIP3, ICAM-1, CHP1, and TMEM106B, NMT1 controls their membrane targeting, subcellular trafficking, protein stability (through modulation of ubiquitin-dependent degradation), and downstream signaling including NFκB/Bcl-2, JNK/autophagy, and PD-L1-mediated immune evasion pathways [PMID:9575206, PMID:36617552, PMID:37269961, PMID:40451428, PMID:40605065]. NMT1 activity is itself regulated transcriptionally by HIF1α and post-translationally through a ROS-dependent negative feedback loop and protein–protein interactions [PMID:30446635, PMID:40605065]. Substrate specificity is determined by elements distributed across both halves of the enzyme, with the N-terminal ~59 residues serving a non-catalytic targeting function [PMID:1429724].\",\n  \"teleology\": [\n    {\n      \"year\": 1992,\n      \"claim\": \"Defining the minimal catalytic architecture and substrate-specificity determinants of NMT resolved how the enzyme recognizes diverse substrates and where catalytic versus targeting functions reside within its primary sequence.\",\n      \"evidence\": \"Deletion mutagenesis, human/yeast chimeras, in vitro myristoylation assays, and yeast complementation of nmt1-null lethal allele\",\n      \"pmids\": [\"1429724\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No crystal structure of human NMT1 at this stage\",\n        \"Substrate scope in human cells undefined\",\n        \"Regulation of NMT1 expression and activity unknown\"\n      ]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Establishing that NMT is essential for viability in a pathogenic fungus and that an ADP-ribosylation factor (Arf) is a critical in vivo substrate demonstrated the biological necessity of myristoylation for membrane trafficking and validated NMT as an antifungal drug target selectively inhibitable over human NMT.\",\n      \"evidence\": \"Conditional lethal allele in C. neoformans rescued by human NMT; selective inhibitor with differential Ki for fungal vs. human enzyme tested in isogenic strains\",\n      \"pmids\": [\"9575206\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Full substrate repertoire of NMT1 in human cells uncharacterized\",\n        \"Structural basis of species-selective inhibition not resolved\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"NMT1 knockdown in cancer cells was shown to trigger oxidative stress and ER stress converging on JNK-mediated autophagy, with ROS feeding back to suppress NMT1 expression, revealing that NMT1 is embedded in stress-response circuitry beyond housekeeping lipidation.\",\n      \"evidence\": \"siRNA knockdown in breast cancer cells, ROS measurement, ER stress markers, JNK pathway inhibitors, xenograft models\",\n      \"pmids\": [\"30446635\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Pathway placement relies partly on pharmacological inhibitors rather than genetic epistasis\",\n        \"Identity of myristoylated substrates mediating the stress response not determined\",\n        \"ROS-NMT1 feedback mechanism at transcriptional/post-transcriptional level unresolved\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstration that NMT1-mediated myristoylation differentially channels substrate proteins toward ubiquitin-dependent stabilization or degradation established a general principle that myristoylation is a sorting signal for protein quality control.\",\n      \"evidence\": \"iTRAQ proteomics, click chemistry myristoylation assays, liver-conditional NMT1 knockout mouse, ubiquitination assays\",\n      \"pmids\": [\"34136404\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Proposed role of HIST1H4H as ubiquitin ligase adaptor is unconventional and not reconstituted in vitro\",\n        \"Generality of NDP/NUP substrate classification beyond liver unknown\",\n        \"Direct structural basis for myristoylation-dependent ubiquitination not defined\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolving that NMTs catalyze both canonical N-terminal glycine (G-) myristoylation and lysine ε-amino (K-) myristoylation through mechanistically distinct catalytic base interactions explained why K-myristoylation is rare and post-translational, and revealed evolutionary depletion of K-myristoylation motifs in the human proteome.\",\n      \"evidence\": \"X-ray crystallography of NMT–substrate complexes, kinetic assays, mass spectrometry, in silico proteome analysis\",\n      \"pmids\": [\"36181773\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Physiological substrates of K-myristoylation in vivo largely unidentified\",\n        \"Regulatory contexts favoring K- over G-myristoylation not characterized\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of VILIP3 and ICAM-1 as NMT1 substrates whose stability and signaling depend on myristoylation expanded the functional repertoire of NMT1 from membrane targeting to direct regulation of protein turnover and cell adhesion.\",\n      \"evidence\": \"DARTS/SPR for NMT1 target engagement (VILIP3); metabolic labeling and MS; Co-IP identifying FBXO4 as E3 ligase for ICAM-1; KD/OE with half-life chase experiments\",\n      \"pmids\": [\"36617552\", \"37269961\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"ICAM-1 myristoylation site not structurally confirmed\",\n        \"FBXO4 mechanism inhibited by myristoylation not biochemically reconstituted\",\n        \"Whether VILIP3 myristoylation effect on NFκB is direct or indirect unclear\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identification of Sorbs2 as a TNFα-induced NMT1-binding protein that inhibits global myristoylation revealed a previously unknown protein-level regulatory mechanism for NMT1 activity.\",\n      \"evidence\": \"Co-immunoprecipitation and mass spectrometry in murine osteoblastic cells, click-it myristoylation assay after TNFα stimulation\",\n      \"pmids\": [\"38148048\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Single Co-IP/MS identification without reciprocal validation or domain mapping\",\n        \"Mechanism by which Sorbs2 binding inhibits NMT1 catalytic activity unknown\",\n        \"Relevance beyond osteoblasts not tested\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"NMT1-dependent dual myristoylation of TMEM106B at Gly2 and Lys3 was shown to be required for lysosomal trafficking and degradation, directly connecting the G- and K-myristoylation activities discovered structurally in 2022 to a physiological trafficking decision.\",\n      \"evidence\": \"NMT1/2 knockdown and inhibition, site-directed mutagenesis of myristoylation sites, subcellular fractionation, live-cell imaging, pulse-chase degradation assays\",\n      \"pmids\": [\"40451428\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Relative contribution of NMT1 vs NMT2 to TMEM106B myristoylation not resolved\",\n        \"Whether dual myristoylation at adjacent residues is common among NMT substrates unknown\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"HIF1α-driven upregulation of NMT1 was shown to promote CHP1 myristoylation, enabling CHP1 to bind PD-L1 and shuttle it to the plasma membrane for immune evasion, establishing NMT1 as a druggable node in immune checkpoint regulation.\",\n      \"evidence\": \"PCLX-001 NMT1 inhibition, Co-IP of CHP1–PD-L1, subcellular fractionation, HNSCC xenograft with anti-PD-1 combination therapy\",\n      \"pmids\": [\"40605065\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-lab study; CHP1-PD-L1 interaction awaits independent confirmation\",\n        \"Whether NMT2 compensates in this pathway not addressed\",\n        \"Structural basis of myristoylated CHP1–PD-L1 interaction not determined\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The full scope of the NMT1-specific substrate repertoire in human tissues, the structural basis for NMT1 vs NMT2 substrate partitioning, and the in vivo consequence of NMT1-selective versus pan-NMT inhibition remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No systematic comparison of NMT1- vs NMT2-specific substrates across tissues\",\n        \"No NMT1-selective small-molecule inhibitor characterized\",\n        \"Phenotype of NMT1-specific knockout in adult mammals not fully described beyond liver\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 4, 5, 7, 8, 9, 10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [4, 5, 7, 8, 9, 10]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [9, 10]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"VILIP3\",\n      \"ICAM-1\",\n      \"CHP1\",\n      \"TMEM106B\",\n      \"Sorbs2\",\n      \"ARF\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"NMT1 is a glycylpeptide N-tetradecanoyltransferase that catalyzes the cotranslational and posttranslational transfer of myristate from myristoyl-CoA to the N-terminal glycine α-amino group of substrate proteins, thereby governing their membrane association, subcellular trafficking, stability, and signaling competence. Its substrates span G-protein α-subunits, Src-family kinases, HIV Gag/Nef/matrix, BID, CHP1, VILIP3, ICAM-1, and TMEM106B; myristoylation of BID after caspase-8 cleavage redirects truncated BID to mitochondria to promote cytochrome c release and apoptosis [PMID:11099414], while myristoylation of CHP1 facilitates PD-L1 plasma-membrane translocation and immune evasion [PMID:40605065]. NMT1 additionally catalyzes a less efficient lysine ε-amino (K-myristoylation) reaction using a direct catalytic-base contact distinct from the water-mediated mechanism of canonical G-myristoylation [PMID:36181773]. Its enzymatic activity is negatively regulated by Sorbs2 binding upon TNF-α stimulation [PMID:38148048] and is pharmacologically targetable by small-molecule inhibitors including desloratadine (binding Asn-246) and PCLX-001 [PMID:36617552, PMID:40605065].\",\n  \"teleology\": [\n    {\n      \"year\": 1985,\n      \"claim\": \"The identification of N-terminal myristate on p60src established that NMT-catalyzed myristoylation is a defined covalent modification of signaling kinases and retroviral transforming proteins, opening the field of protein myristoylation.\",\n      \"evidence\": \"Tryptic peptide sequencing and metabolic [3H]myristate labeling of p60src from Rous sarcoma virus\",\n      \"pmids\": [\"3917576\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"NMT enzyme not yet purified or cloned\", \"substrate scope unknown\"]\n    },\n    {\n      \"year\": 1990,\n      \"claim\": \"Mutagenesis of the N-terminal glycine of HIV-1 Gag and Gα subunits demonstrated that NMT-mediated myristoylation is required for stable membrane association, viral assembly, and G-protein signaling, establishing myristoylation as a functional membrane-targeting signal rather than an inert modification.\",\n      \"evidence\": \"G2A mutagenesis of HIV-1 Gag and Gαo; metabolic labeling; subcellular fractionation and virion assembly assays in mammalian cells\",\n      \"pmids\": [\"2788277\", \"2153964\", \"2405382\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of myristate vs. adjacent basic residues not yet dissected\", \"no crystal structure of NMT–substrate complex\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Deletion and chimera mapping of yeast Nmt1p defined the minimal catalytic domain (Ile59–Leu455) and revealed that the N-terminal 59 residues play a non-catalytic role in accessing myristoyl-CoA pools, establishing the domain architecture conserved in human NMT1.\",\n      \"evidence\": \"Deletion mutagenesis, E. coli reconstitution, yeast nmt1-null complementation, and human/yeast chimeric enzymes\",\n      \"pmids\": [\"1429724\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Human NMT1 domain boundaries not directly mapped\", \"structural basis of substrate specificity determinants unresolved\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Quantitative in vitro membrane-binding studies showed that NMT-added myristate cooperates with adjacent polybasic sequences to form a bipartite membrane-targeting signal on Src and HIV Gag, explaining how a single 14-carbon acyl chain achieves high-affinity membrane anchoring.\",\n      \"evidence\": \"Chimera constructs, mutagenesis of basic residues, and liposome-binding assays with quantified Kd values for p60c-src and HIV-1 Gag\",\n      \"pmids\": [\"7527558\", \"8139035\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Myristyl-switch mechanism not yet characterized\", \"contribution of palmitoylation or other secondary signals unclear\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"The discovery that caspase-8-cleaved BID undergoes posttranslational myristoylation by NMT, which redirects tBID to mitochondria and promotes cytochrome c release, demonstrated that NMT activity is not limited to cotranslational modification and directly controls apoptotic signaling.\",\n      \"evidence\": \"In vitro myristoylation, liposome binding, and mitochondrial cytochrome c release assays with glycine-mutant controls\",\n      \"pmids\": [\"11099414\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contributions of NMT1 vs. NMT2 to posttranslational BID myristoylation not separated\", \"in vivo apoptotic relevance not fully dissected\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"NMR studies of HIV-1 myristoylated matrix protein revealed an entropic myristyl switch coupled to protein trimerization, showing how NMT-installed myristate is regulated downstream of the transferase reaction to control membrane targeting in a conformationally gated manner.\",\n      \"evidence\": \"NMR structure determination and analytical ultracentrifugation of myr-MA monomer–trimer equilibrium\",\n      \"pmids\": [\"14699046\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Switch mechanism not demonstrated for non-viral NMT substrates\", \"kinetic regulation of switch in the virion assembly context unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"NMT1 knockdown in breast cancer cells established a feedback loop where reduced myristoylation triggers ROS accumulation and ER stress, activating JNK-mediated autophagy that suppresses tumor growth, placing NMT1 as a regulatory node integrating lipid modification with stress-response signaling.\",\n      \"evidence\": \"shRNA knockdown of NMT1; ROS, ER stress, and JNK pathway analysis; in vivo xenograft models\",\n      \"pmids\": [\"30446635\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific substrates mediating ROS/ER stress not identified\", \"NMT2 compensation not assessed\", \"single lab\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Liver-specific NMT1 knockout mice revealed that myristoylation differentially controls protein stability: NDP-class substrates are destabilized via HIST1H4H-mediated ubiquitination, while NUP-class substrates are stabilized because myristoylation prevents their ubiquitination, establishing that NMT1 modulates the ubiquitin–proteasome system.\",\n      \"evidence\": \"Conditional liver-specific NMT1 KO; click chemistry; iTRAQ proteomics; co-IP and ubiquitination assays\",\n      \"pmids\": [\"34136404\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which myristoylation recruits or blocks ubiquitin ligases is unclear\", \"relevance of POTEE as co-factor needs independent validation\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Structural and kinetic analysis demonstrated that NMT catalyzes both efficient G-myristoylation (water-mediated catalytic base) and inefficient K-myristoylation (direct catalytic base contact), revealing a dual-mechanism catalytic architecture and explaining evolutionary depletion of K-myristoylation motifs in humans.\",\n      \"evidence\": \"X-ray crystallography of NMT–substrate complexes; mass spectrometry; enzyme kinetics; in silico motif analysis\",\n      \"pmids\": [\"36181773\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological relevance of K-myristoylation in vivo remains limited to few substrates\", \"structural basis of substrate selectivity between NMT1 and NMT2 not resolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of VILIP3 as an NMT1 substrate whose myristoylation drives NFκB/Bcl-2 signaling in HCC, combined with the discovery that desloratadine inhibits NMT1 by binding Asn-246, provided the first clinically repurposable inhibitor–target pair and a defined NMT1→VILIP3→NFκB signaling axis.\",\n      \"evidence\": \"DARTS, SPR for target ID; metabolic labeling/MS for substrate ID; NMT1 KD/OE; CDX/PDO/PDX in vivo models\",\n      \"pmids\": [\"36617552\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Desloratadine selectivity for NMT1 vs. NMT2 not quantified\", \"whether Asn-246 binding is allosteric or competitive unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"NMT1-mediated myristoylation of ICAM-1 stabilizes the adhesion molecule by blocking FBXO4-mediated ubiquitination, linking NMT1 activity to cell adhesion and tumor cell migration suppression.\",\n      \"evidence\": \"NMT1 knockdown; co-IP; ubiquitination and half-life assays; wound-healing and Transwell migration\",\n      \"pmids\": [\"37269961\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"ICAM-1 N-terminal myristoylation site not structurally confirmed\", \"single-lab finding without independent replication\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Sorbs2 was identified as a novel NMT1-binding partner whose association upon TNF-α stimulation paradoxically inhibits NMT1 catalytic activity despite increased NMT1 expression, revealing a post-translational regulatory mechanism for NMT1 in osteoblasts.\",\n      \"evidence\": \"IP/MS identification of Sorbs2; click-it myristoylation assay; RNAi in MC3T3-E1 osteoblastic cells\",\n      \"pmids\": [\"38148048\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding interface between Sorbs2 and NMT1 not mapped\", \"functional consequence for osteoblast biology not fully defined\", \"single lab, single cell type\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"NMT1 myristoylates both Gly2 and Lys3 of TMEM106B, directing it to lysosomes for degradation; non-myristoylated TMEM106B accumulates at the cell surface, establishing dual G- and K-myristoylation as a physiological trafficking determinant for a single substrate.\",\n      \"evidence\": \"Gly2/Lys3 mutagenesis; NMT1/2 overexpression and knockdown; live-cell imaging; lysosomal inhibitor rescue\",\n      \"pmids\": [\"40451428\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of NMT1 vs. NMT2 to TMEM106B modification not separated\", \"structural basis for dual-site myristoylation unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"HIF1α-driven NMT1 upregulation mediates CHP1 myristoylation, which facilitates PD-L1 membrane translocation and immune evasion in HNSCC, and pharmacological NMT1 inhibition by PCLX-001 enhances anti-PD-1 immunotherapy efficacy in vivo.\",\n      \"evidence\": \"HIF1α manipulation; click chemistry; co-IP of CHP1–PD-L1; membrane fractionation; xenograft mouse model with anti-PD-1 + PCLX-001\",\n      \"pmids\": [\"40605065\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"CHP1–PD-L1 binding interface not structurally characterized\", \"whether PCLX-001 effects are NMT1-specific or also involve NMT2 not distinguished\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the full substrate repertoire that distinguishes NMT1 from NMT2, the structural basis for NMT1 substrate selectivity, the physiological scope of K-myristoylation, and whether NMT1-selective (vs. pan-NMT) inhibitors can be developed for therapeutic use.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"NMT1 vs. NMT2 substrate discrimination rules not established\", \"no high-resolution human NMT1 structure with endogenous substrates\", \"in vivo essentiality of NMT1 in adult tissues not comprehensively characterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 2, 4, 9, 15, 16, 17, 21]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4, 8]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 4, 14, 15, 17, 21]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 11, 20]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 16, 20]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [4, 15]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"Sorbs2\",\n      \"CHP1\",\n      \"VILIP3\",\n      \"ICAM-1\",\n      \"TMEM106B\",\n      \"BID\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}