{"gene":"ME2","run_date":"2026-04-28T18:30:28","timeline":{"discoveries":[{"year":1991,"finding":"Human mitochondrial NAD(P)+-dependent malic enzyme (ME2) was cloned from cDNA, revealing a 584-amino acid precursor protein (65.4 kDa). Expression of the processed protein in E. coli yielded an enzyme with the same kinetic and allosteric properties as malic enzyme purified from human cells, confirming its role in oxidative decarboxylation of malate to pyruvate with NAD+ as cofactor.","method":"cDNA cloning, heterologous expression in E. coli, enzymatic kinetic assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro, kinetic and allosteric properties validated","pmids":["1993674"],"is_preprint":false},{"year":2000,"finding":"Crystal structure of human mitochondrial NAD(P)+-dependent ME2 in a quaternary complex with NAD+, Mn2+, and oxalate (2.2 Å) revealed the enzyme in a closed form. The divalent cation is coordinated octahedrally by Glu255, Asp256, Asp279, two substrate oxygens, and one water molecule. Tyr112 and Lys183 were identified as possible catalytic residues. Changes in tetramer organization were observed in different complexes, suggesting relevance for cooperative behavior and allosteric control.","method":"X-ray crystallography, structural analysis","journal":"Nature structural biology","confidence":"High","confidence_rationale":"Tier 1 — high-resolution crystal structures of multiple quaternary complexes","pmids":["10700286"],"is_preprint":false},{"year":2002,"finding":"Crystal structure of ME2 in complex with ATP, Mn2+, tartronate, and fumarate (2.2 Å) showed that ATP acts as an active-site inhibitor despite the existence of an exo binding site. Fumarate binds at an allosteric site at the dimer interface, and mutations at this site abolished fumarate's activating effects, revealing the molecular mechanism of allosteric regulation by fumarate.","method":"X-ray crystallography, enzyme kinetics, site-directed mutagenesis","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 — crystal structure combined with mutagenesis and kinetic assays","pmids":["12121650"],"is_preprint":false},{"year":2009,"finding":"ME2 enzymatic activity is present in pancreatic islets of humans, rats, mice, and INS-1 832/13 clonal insulinoma cells. ME2 uses either NAD or NADP as cofactor, has a high Km for malate, and is allosterically activated by fumarate and inhibited by ATP—properties distinguishing it from the cytosolic ME1 and mitochondrial ME3.","method":"Spectrophotometric enzyme activity assay, immunoblotting","journal":"Archives of biochemistry and biophysics","confidence":"Medium","confidence_rationale":"Tier 2 — direct enzyme assay with functional characterization, single study","pmids":["19691144"],"is_preprint":false},{"year":2013,"finding":"p53 transcriptionally represses ME2 (and ME1) expression in human and mouse cells. ME2 downregulation reciprocally activates p53 through AMPK-mediated mechanisms in a feed-forward loop. Knockdown of ME2 induces cellular senescence (not apoptosis), while enforced ME2 expression suppresses senescence, establishing ME2 as a regulator of cell fate downstream of p53.","method":"Genetic knockdown/overexpression, epistasis analysis, cell senescence assays, metabolic measurements","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, replicated in human and mouse cells, published in high-impact journal","pmids":["23334421"],"is_preprint":false},{"year":2014,"finding":"ME2 knockdown in A549 NSCLC cells inhibits cell proliferation, induces differentiation, increases ROS and NADP+/NADPH ratio, drops ATP, and increases cisplatin sensitivity. ME2 depletion reduces pyruvate and accumulates malate; exogenous membrane-permeable malate mimics this phenotype. ME2 knockdown also impacts PDK1/PTEN expression, leading to AKT inhibition. Both ME2 knockdown and malate treatment reduce tumor growth in vivo.","method":"shRNA knockdown, metabolic flux analysis (stable isotope tracing), in vivo xenograft, kinase signaling analysis","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including in vivo validation and metabolomics","pmids":["24957098"],"is_preprint":false},{"year":2014,"finding":"High-throughput screening identified NPD389 as a potent ME2 inhibitor (IC50 ~4.6 μM) that acts as an uncompetitive inhibitor with respect to NAD+ and a mixed-type inhibitor with respect to L-malate, indicating it binds at a site distinct from the active site (consistent with allosteric inhibition).","method":"High-throughput enzymatic screening, enzyme kinetics analysis, thermal shift assay","journal":"Acta pharmacologica Sinica","confidence":"Medium","confidence_rationale":"Tier 1 — in vitro enzyme kinetics with mechanistic characterization, single study","pmids":["24681895"],"is_preprint":false},{"year":2015,"finding":"The natural compound embonic acid (EA) is an allosteric inhibitor of ME2 with an IC50 of 1.4 μM. Mutagenesis and binding studies localized its binding site to the fumarate binding site or nearby dimer interface, distinct from the catalytic site (non-competitive inhibition). EA treatment and ME2 shRNA knockdown both inhibit H1299 cancer cell growth and induce cellular senescence via a p53-independent pathway.","method":"In vitro enzyme inhibition assays, site-directed mutagenesis, binding studies, shRNA knockdown, cellular senescence assays","journal":"Oncotarget","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro inhibition with mutagenesis plus cellular validation","pmids":["26008970"],"is_preprint":false},{"year":2017,"finding":"Genomic deletion of ME2 in SMAD4-deleted pancreatic cancer creates collateral lethality upon depletion of its paralog ME3. Mechanistically, loss of mitochondrial malic enzymes (ME2 and ME3) diminishes NADPH production, elevates ROS, activates AMPK, which then directly suppresses SREBP1-directed transcription of BCAT2. Reduced BCAT2 impairs branched-chain amino acid transamination, depleting glutamate needed for nucleotide synthesis.","method":"Genetic depletion (shRNA/CRISPR), metabolomics, molecular epistasis, AMPK/SREBP1/BCAT2 pathway analysis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 — integrated metabolomics and molecular epistasis, multiple orthogonal methods, high-impact publication","pmids":["28099419"],"is_preprint":false},{"year":2021,"finding":"ME2 promotes proneural-mesenchymal transition (PMT) and lipogenesis in glioblastoma by inhibiting mitochondrial ROS production and AMPK phosphorylation, resulting in SREBP-1 maturation and nuclear localization, which enhances the ACSS2 lipogenesis pathway. ME2 overexpression upregulates mesenchymal markers and inhibits proneural marker OLIG2.","method":"ME2 overexpression/knockdown, ROS measurement, AMPK/SREBP-1 signaling analysis, lipogenesis assays, migration/invasion assays","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2-3 — pathway placement with functional readouts, single laboratory","pmids":["34381734"],"is_preprint":false},{"year":2023,"finding":"SIRT5 physically interacts with ME2 and acts as its desuccinylase. Glutamine deprivation enhances the SIRT5-ME2 interaction, promoting SIRT5-mediated desuccinylation of ME2 at lysine 346, which activates ME2 enzymatic activity. Activated ME2 enhances mitochondrial respiration to counteract glutamine deprivation and support proliferation and tumorigenesis in colorectal cancer.","method":"Co-immunoprecipitation, succinylation assays, site-directed mutagenesis (K346), enzymatic activity assays, metabolic flux measurements, xenograft models","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1-2 — identified specific modification site with mutagenesis, writer identified, functional consequence demonstrated in vitro and in vivo","pmids":["38007551"],"is_preprint":false},{"year":2023,"finding":"ME2 promotes pyruvate production, which directly binds to β-catenin and increases β-catenin protein levels, thereby promoting hepatocellular carcinoma cell migration and invasion. Pyruvate treatment rescues migration in ME2-depleted cells, establishing the ME2→pyruvate→β-catenin axis.","method":"ME2 knockdown/overexpression, pyruvate supplementation rescue experiments, β-catenin protein level analysis, migration/invasion assays","journal":"Metabolites","confidence":"Medium","confidence_rationale":"Tier 3 — single lab, functional rescue experiment supporting mechanistic axis","pmids":["37110198"],"is_preprint":false},{"year":2023,"finding":"ME2 inhibition in AML cells (via ME2 silencing or allosteric inhibitor disodium embonate) decreases pyruvate and NADH, reducing ATP production via oxidative phosphorylation, and decreases NADPH, increasing ROS and oxidative stress, ultimately inducing apoptosis. ME2 silencing inhibits xenograft AML tumor growth in vivo.","method":"shRNA knockdown, allosteric inhibitor treatment, energy/redox metabolite measurements, xenograft in vivo models","journal":"Cellular oncology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple metabolic endpoints with in vivo validation, single laboratory","pmids":["37079187"],"is_preprint":false},{"year":2023,"finding":"miR-214-3p directly targets ME2 (confirmed by dual luciferase reporter assay). In cardiomyocytes, miR-214-3p suppresses ME2 expression and promotes ferroptosis; ME2 overexpression ameliorates ferroptosis induced by miR-214-3p, while ME2 depletion compromises the protective effect of miR-214-3p inhibitor, establishing ME2 as a functional target of miR-214-3p in regulating ferroptosis during myocardial infarction.","method":"Dual luciferase reporter assay, miRNA mimic/inhibitor/antagomir, ME2 overexpression/knockdown, ferroptosis and iron accumulation assays, cardiac function measurement in mouse MI model","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2-3 — direct target validation with reporter assay, functional epistasis in vitro and in vivo","pmids":["37087800"],"is_preprint":false},{"year":2024,"finding":"AKT1 phosphorylates cytoplasmic ME2 (full-length isoform, ME2fl) at serine 9 within the mitochondrial localization signal, preventing its mitochondrial translocation. Cytoplasmic ME2fl acts as a scaffold assembling key glycolytic enzymes (PFKL, GAPDH, PKM2, LDHA) to promote glycolysis. This AKT1-driven phosphorylation induces a metabolic switch from mitochondrial TCA metabolism to glycolysis, enhancing glycolytic capacity of tumor cells in vitro and in vivo.","method":"In vitro phosphorylation assays, site-directed mutagenesis (S9), subcellular fractionation, co-immunoprecipitation of glycolytic enzyme complex, metabolic flux measurements, xenograft in vivo models","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 — identified phosphorylation site with mutagenesis, reconstituted scaffold complex, in vivo validation with multiple orthogonal methods","pmids":["38263319"],"is_preprint":false},{"year":2024,"finding":"ME2 is acetylated at lysine 156 by ACAT1. Decreased intracellular glucose triggers ACAT1-mediated K156 acetylation of ME2, potentiating its enzymatic activity and facilitating lactate production from glutamine. ME2-derived lactate promotes lactylation of homologous recombination repair proteins, contributing to chemotherapy resistance in ovarian cancer. ACAT1 inhibition reduces ME2 acetylation and chemoresistance in vitro and in vivo.","method":"Acetylation assays, site-directed mutagenesis (K156), co-immunoprecipitation, metabolomics, protein lactylation analysis, in vitro and in vivo chemoresistance assays","journal":"Advanced science","confidence":"High","confidence_rationale":"Tier 1-2 — identified acetylation site with writer (ACAT1) and mutagenesis, downstream lactylation mechanism, in vivo validation","pmids":["39951294"],"is_preprint":false},{"year":2024,"finding":"PRMT1 interacts with ME2 and methylates ME2, activating its enzymatic activity. Mutation of ME2 at arginine 67 (R67K) mimics constitutive activation. PRMT1-mediated ME2 methylation increases mitochondrial respiration, promoting HCC cell proliferation and migration. PRMT1 inhibition reduces ME2 activity and tumor growth.","method":"Co-immunoprecipitation, site-directed mutagenesis (R67K), enzymatic activity assays, mitochondrial respiration measurements, cell proliferation/migration assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2-3 — interaction and functional mutagenesis demonstrated, single laboratory","pmids":["39528487"],"is_preprint":false},{"year":2024,"finding":"A homozygous frameshift variant in ME2 (c.1379_1380delTT, p.Phe460fs*22) produces truncated, unstable ME2 protein in vitro, causing ME2 deficiency associated with neurodevelopmental disorder, dilated cardiomyopathy, and mild lactic acidemia in a human patient. Deletion of the yeast ortholog of human ME2 causes growth arrest rescued by re-expression of human ME2, demonstrating ME2's essential role in mitochondrial function.","method":"Whole exome sequencing, in vitro protein expression/stability assay, yeast complementation assay","journal":"Clinical genetics","confidence":"Medium","confidence_rationale":"Tier 2 — yeast complementation provides functional evidence; human variant pathogenicity supported by in vitro instability","pmids":["39401966"],"is_preprint":false},{"year":2025,"finding":"CYP4F11 promotes ubiquitin-proteasomal degradation of ME2 when suppressed by miR-195, establishing a CYP4F11/miR-195/ME2 regulatory axis. CYP4F11 depletion disrupts mitochondrial malate metabolism, and ME2 rescue experiments confirm ME2 mediates the oncogenic metabolic effects of CYP4F11 in non-small cell lung cancer.","method":"3'-UTR luciferase reporter assay, CYP4F11 knockdown, ME2 rescue experiments, metabolomic analysis, xenograft in vivo models","journal":"Frontiers of medicine","confidence":"Medium","confidence_rationale":"Tier 2-3 — functional rescue validates axis, metabolomics provides mechanistic context, single laboratory","pmids":["41359237"],"is_preprint":false}],"current_model":"ME2 (mitochondrial NAD(P)+-dependent malic enzyme 2) catalyzes oxidative decarboxylation of malate to pyruvate with concomitant NADH/NADPH production; its activity is allosterically activated by fumarate (at the dimer interface) and inhibited by ATP (at the active site), as established by crystal structures and mutagenesis. ME2 is subject to multiple post-translational modifications—desuccinylation at K346 by SIRT5, acetylation at K156 by ACAT1, and methylation at R67 by PRMT1—each activating enzymatic activity and promoting tumor metabolism. AKT1 phosphorylates a cytoplasmic full-length isoform (ME2fl) at S9, preventing mitochondrial import and enabling ME2fl to function as a scaffold for glycolytic enzyme complexes. ME2 supports NADPH homeostasis and ROS control, and its loss activates AMPK, suppresses SREBP1/BCAT2, and induces p53-dependent cellular senescence, linking ME2 activity to metabolic adaptation, tumorigenesis, and cell fate decisions."},"narrative":{"teleology":[{"year":1991,"claim":"Cloning and heterologous expression of human ME2 established its identity as the mitochondrial NAD(P)⁺-dependent malic enzyme catalyzing oxidative decarboxylation of malate to pyruvate, resolving the molecular basis of an activity long known biochemically.","evidence":"cDNA cloning, E. coli expression, kinetic and allosteric characterization","pmids":["1993674"],"confidence":"High","gaps":["No structural information on subunit assembly or allosteric sites","Regulation of expression unknown"]},{"year":2002,"claim":"High-resolution crystal structures of ME2 with substrates, cofactors, and allosteric effectors revealed that ATP inhibits at the active site, while fumarate activates through a dimer-interface allosteric site, providing the structural basis for cooperative regulation.","evidence":"X-ray crystallography (2.2 Å) of multiple quaternary complexes, site-directed mutagenesis, enzyme kinetics","pmids":["10700286","12121650"],"confidence":"High","gaps":["Physiological relevance of allosteric regulation in vivo not tested","No structural data on post-translational modification sites"]},{"year":2013,"claim":"The discovery that p53 transcriptionally represses ME2, and that ME2 loss reciprocally activates p53 through AMPK, established a metabolic–tumor-suppressor feedback loop wherein ME2 depletion drives senescence rather than apoptosis.","evidence":"Genetic knockdown/overexpression with epistasis analysis in human and mouse cells, senescence assays, metabolic measurements","pmids":["23334421"],"confidence":"High","gaps":["Specific AMPK substrates mediating p53 activation not fully identified","Whether ME2-driven senescence occurs in non-cancerous tissues in vivo unclear"]},{"year":2014,"claim":"ME2 depletion in NSCLC cells demonstrated that ME2 sustains tumor proliferation by maintaining NADPH/ROS balance and PI3K/AKT signaling, and that its loss sensitizes tumors to cisplatin, broadening ME2's role beyond metabolism to oncogenic signaling.","evidence":"shRNA knockdown, stable isotope metabolic tracing, xenograft tumor models, kinase signaling analysis","pmids":["24957098"],"confidence":"High","gaps":["Mechanism linking ME2 to PDK1/PTEN expression not fully resolved","No demonstration with genetic knockout"]},{"year":2015,"claim":"Identification of embonic acid as an allosteric ME2 inhibitor binding near the fumarate site provided pharmacological validation that ME2 inhibition induces senescence, even in p53-null cells, indicating both p53-dependent and -independent effector pathways.","evidence":"In vitro inhibition kinetics, site-directed mutagenesis of fumarate site, shRNA knockdown, cellular senescence assays in H1299 (p53-null) cells","pmids":["26008970"],"confidence":"High","gaps":["In vivo pharmacokinetics and selectivity of embonic acid not characterized","p53-independent senescence pathway mediators unidentified"]},{"year":2017,"claim":"Collateral lethality of ME2 co-deletion with SMAD4 in pancreatic cancer revealed that combined loss of mitochondrial malic enzymes (ME2+ME3) collapses NADPH, activates AMPK, suppresses SREBP1-directed BCAT2 transcription, and depletes branched-chain amino acid-derived glutamate needed for nucleotide synthesis.","evidence":"CRISPR/shRNA depletion, integrated metabolomics, AMPK–SREBP1–BCAT2 epistasis analysis in pancreatic cancer models","pmids":["28099419"],"confidence":"High","gaps":["Relative contributions of ME2 vs. ME3 to NADPH pool not individually quantified","Clinical feasibility of exploiting collateral lethality untested"]},{"year":2023,"claim":"Three post-translational modifications activating ME2 were identified: SIRT5-mediated desuccinylation at K346 under glutamine deprivation, ACAT1-mediated acetylation at K156 under glucose deprivation, and PRMT1-mediated methylation at R67, each enhancing ME2 catalytic activity to support tumor-adaptive metabolism.","evidence":"Co-immunoprecipitation, site-directed mutagenesis of K346/K156/R67, succinylation/acetylation/methylation assays, enzymatic activity assays, xenograft models","pmids":["38007551","39951294","39528487"],"confidence":"High","gaps":["Crosstalk or hierarchy among the three modifications not examined","Structural basis for how each modification increases catalytic activity unknown","Whether these modifications co-occur in the same tumor type is unclear"]},{"year":2024,"claim":"AKT1 phosphorylation of a cytoplasmic ME2 full-length isoform at S9 blocks mitochondrial import and repurposes ME2 as a scaffold for glycolytic enzyme assembly (PFKL, GAPDH, PKM2, LDHA), uncovering a non-enzymatic moonlighting function that promotes a glycolytic metabolic switch.","evidence":"In vitro phosphorylation, S9 mutagenesis, subcellular fractionation, co-immunoprecipitation of glycolytic complex, metabolic flux, xenograft in vivo","pmids":["38263319"],"confidence":"High","gaps":["Proportion of ME2 retained in cytoplasm under physiological conditions unknown","Whether scaffold function requires ME2 enzymatic activity is untested","Independent replication of this moonlighting role pending"]},{"year":2024,"claim":"A homozygous frameshift ME2 variant in a human patient established ME2 deficiency as a cause of neurodevelopmental disorder with dilated cardiomyopathy and lactic acidemia, and yeast complementation confirmed its essential mitochondrial function.","evidence":"Whole exome sequencing, in vitro protein stability assay, yeast ortholog deletion/complementation","pmids":["39401966"],"confidence":"Medium","gaps":["Only a single family reported; additional kindreds needed","Patient-derived cell metabolomics not performed","Tissue-specific consequences of ME2 loss in mammals not characterized"]},{"year":null,"claim":"Key unresolved questions include how the multiple post-translational activating modifications of ME2 are coordinated, what determines whether ME2 loss triggers senescence versus apoptosis versus ferroptosis across cell types, and whether the cytoplasmic scaffolding function operates in non-tumor physiology.","evidence":"","pmids":[],"confidence":"Low","gaps":["No integrated model of PTM crosstalk on ME2","Tissue-specific phenotypic outcomes of ME2 loss poorly defined","Structural basis of cytoplasmic scaffold assembly unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[0,1,2,3,10,15,16]},{"term_id":"GO:0016829","term_label":"lyase activity","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[14]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,1,10,14,17]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[14]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,2,3,5,8,10,11,12,14,15]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4,5,8,9]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[5,8,9,12,15]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[4,12,13]}],"complexes":[],"partners":["SIRT5","ACAT1","PRMT1","AKT1","PFKL","GAPDH","PKM2","LDHA"],"other_free_text":[]},"mechanistic_narrative":"ME2 is a mitochondrial NAD(P)⁺-dependent malic enzyme that catalyzes the oxidative decarboxylation of malate to pyruvate, generating NADH and NADPH critical for mitochondrial respiration, biosynthetic reactions, and redox homeostasis [PMID:1993674, PMID:10700286]. The enzyme functions as a tetramer whose activity is allosterically activated by fumarate at the dimer interface and inhibited by ATP at the active site, and is further modulated by post-translational modifications—desuccinylation at K346 by SIRT5, acetylation at K156 by ACAT1, and methylation at R67 by PRMT1—each of which potentiates catalytic activity to support tumor metabolism [PMID:12121650, PMID:38007551, PMID:39951294, PMID:39528487]. ME2 loss diminishes NADPH production, elevates ROS, and activates AMPK, which suppresses SREBP1/BCAT2 signaling and triggers p53-dependent senescence, linking ME2 to metabolic adaptation and cell fate decisions in cancer [PMID:23334421, PMID:28099419]. A homozygous loss-of-function ME2 variant (p.Phe460fs*22) causes a Mendelian disorder characterized by neurodevelopmental impairment, dilated cardiomyopathy, and lactic acidemia [PMID:39401966]."},"prefetch_data":{"uniprot":{"accession":"P23368","full_name":"NAD-dependent malic enzyme, mitochondrial","aliases":["Malic enzyme 2"],"length_aa":584,"mass_kda":65.4,"function":"NAD-dependent mitochondrial malic enzyme that catalyzes the oxidative decarboxylation of malate to pyruvate","subcellular_location":"Mitochondrion matrix","url":"https://www.uniprot.org/uniprotkb/P23368/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ME2","classification":"Not Classified","n_dependent_lines":22,"n_total_lines":1208,"dependency_fraction":0.018211920529801324},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CAPZB","stoichiometry":0.2},{"gene":"HSPB11","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/ME2","total_profiled":1310},"omim":[{"mim_id":"620057","title":"PHD FINGER PROTEIN 7; PHF7","url":"https://www.omim.org/entry/620057"},{"mim_id":"619348","title":"ANKYRIN REPEAT- AND LEM DOMAIN-CONTAINING PROTEIN 1; ANKLE1","url":"https://www.omim.org/entry/619348"},{"mim_id":"616581","title":"LYSINE DEMETHYLASE 4E; KDM4E","url":"https://www.omim.org/entry/616581"},{"mim_id":"609764","title":"LYSINE DEMETHYLASE 4A; KDM4A","url":"https://www.omim.org/entry/609764"},{"mim_id":"606337","title":"PROTOCADHERIN-BETA 11; PCDHB11","url":"https://www.omim.org/entry/606337"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Mitochondria","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"choroid plexus","ntpm":86.4}],"url":"https://www.proteinatlas.org/search/ME2"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P23368","domains":[{"cath_id":"3.40.50.10380","chopping":"104-274","consensus_level":"medium","plddt":96.7633,"start":104,"end":274},{"cath_id":"3.40.50.720","chopping":"282-468_486-512","consensus_level":"high","plddt":95.6578,"start":282,"end":512}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P23368","model_url":"https://alphafold.ebi.ac.uk/files/AF-P23368-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P23368-F1-predicted_aligned_error_v6.png","plddt_mean":94.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ME2","jax_strain_url":"https://www.jax.org/strain/search?query=ME2"},"sequence":{"accession":"P23368","fasta_url":"https://rest.uniprot.org/uniprotkb/P23368.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P23368/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P23368"}},"corpus_meta":[{"pmid":"3930657","id":"PMC_3930657","title":"Conjugal transfer from Streptococcus lactis ME2 of plasmids encoding phage resistance, nisin resistance and lactose-fermenting ability: evidence for a high-frequency conjugative plasmid responsible for abortive infection of virulent bacteriophage.","date":"1985","source":"Journal of general microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/3930657","citation_count":88,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"9798675","id":"PMC_9798675","title":"EWS/FLI1 up regulates mE2-C, a cyclin-selective ubiquitin conjugating enzyme involved in cyclin B destruction.","date":"1998","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/9798675","citation_count":82,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"21225028","id":"PMC_21225028","title":"Me2-NHC based robust Ir catalyst for efficient water oxidation.","date":"2011","source":"Chemical communications (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/21225028","citation_count":73,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"1429469","id":"PMC_1429469","title":"Molecular characterization of a second abortive phage resistance gene present in Lactococcus lactis subsp. lactis ME2.","date":"1992","source":"Journal of bacteriology","url":"https://pubmed.ncbi.nlm.nih.gov/1429469","citation_count":53,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"38007551","id":"PMC_38007551","title":"SIRT5-mediated ME2 desuccinylation promotes cancer growth by enhancing mitochondrial respiration.","date":"2023","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/38007551","citation_count":50,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"11448932","id":"PMC_11448932","title":"In vivo antitumor activity of bis(4,7-dimethyl-1,10-phenanthroline) sulfatooxovanadium(IV) (METVAN [VO(SO4)(Me2-Phen)2]).","date":"2001","source":"Clinical cancer research : an official journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/11448932","citation_count":47,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"2841286","id":"PMC_2841286","title":"Restriction and modification activities from Streptococcus lactis ME2 are encoded by a self-transmissible plasmid, pTN20, that forms cointegrates during mobilization of lactose-fermenting ability.","date":"1988","source":"Journal of bacteriology","url":"https://pubmed.ncbi.nlm.nih.gov/2841286","citation_count":44,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"7984047","id":"PMC_7984047","title":"Expression of basic-helix-loop-helix transcription factor ME2 during brain development and in the regions of neuronal plasticity in the adult brain.","date":"1994","source":"Brain research. 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communications","url":"https://pubmed.ncbi.nlm.nih.gov/35831314","citation_count":65,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":26428,"output_tokens":4099,"usd":0.070385},"stage2":{"model":"claude-opus-4-6","input_tokens":7550,"output_tokens":2510,"usd":0.15075},"total_usd":0.482537,"stage1_batch_id":"msgbatch_01Mgbec4vM6c9vXht7JVJzss","stage2_batch_id":"msgbatch_01JkuuH5RB2BdJNsgnhjbC6s","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":38723,"output_tokens":4845,"usd":0.094422},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":8594,"output_tokens":2734,"usd":0.16698}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2023,\n      \"finding\": \"SIRT5 acts as a desuccinylase for ME2, removing succinylation at lysine 346. Glutamine deprivation enhances the SIRT5-ME2 interaction, promoting desuccinylation that activates ME2 enzymatic activity, which in turn enhances mitochondrial respiration to support cell proliferation under nutrient stress.\",\n      \"method\": \"Co-IP, succinylation site mapping by mass spectrometry, enzymatic activity assays, mutagenesis, KD/KO with cellular phenotype readout\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, site-specific mutagenesis, enzymatic assay, and in vivo tumor models; multiple orthogonal methods in single study\",\n      \"pmids\": [\"38007551\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"AKT1 phosphorylates cytoplasmic ME2 (full-length isoform, ME2fl) at serine 9 in the mitochondrial localization signal, preventing its mitochondrial translocation. In the cytoplasm, ME2fl acts as a scaffold assembling the glycolytic enzymes PFKL, GAPDH, PKM2, and LDHA to promote aerobic glycolysis, representing a metabolic switch from mitochondrial TCA cycle activity to glycolysis.\",\n      \"method\": \"In vitro kinase assay, phospho-site mutagenesis, subcellular fractionation, co-IP/pulldown, xenograft tumor models, live-cell imaging\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro phosphorylation assay, mutagenesis of phospho-site, multiple co-IP/pulldowns, fractionation with functional consequence, in vivo validation\",\n      \"pmids\": [\"38263319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ACAT1 acetylates ME2 at lysine 156, potentiating ME2 enzymatic activity and increasing lactate production from glutamine. This ME2-derived lactate promotes lactylation of homologous recombination repair proteins, contributing to chemotherapy resistance in ovarian cancer.\",\n      \"method\": \"Co-IP, acetylation site mutagenesis, enzymatic activity assay, metabolomic analysis, in vitro and in vivo rescue experiments\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — site-specific mutagenesis, enzymatic activity assay, mechanistic rescue experiments, multiple orthogonal methods\",\n      \"pmids\": [\"39951294\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PRMT1 methylates ME2 (at arginine residues; mutation at lysine 67 activates ME2 enzymatic activity), and this methylation inhibits ubiquitin-mediated degradation of ME2, stabilizing it and activating mitochondrial respiration to promote hepatocellular carcinoma growth and migration.\",\n      \"method\": \"Co-IP (ME2-PRMT1 interaction), site-directed mutagenesis, enzymatic activity assay, ubiquitination assay, KD with cellular phenotype\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, mutagenesis, and enzymatic assay in single study; moderate confidence due to single lab\",\n      \"pmids\": [\"39528487\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Embonic acid (EA) allosterically inhibits mitochondrial NAD(P)+-dependent malic enzyme ME2 by binding at or near the fumarate binding site/dimer interface (not the active site), displaying non-competitive inhibition kinetics. ME2 inhibition (by EA or shRNA knockdown) suppresses cancer cell growth and induces cellular senescence independent of p53.\",\n      \"method\": \"In vitro enzymatic inhibition assay, mutagenesis of putative binding site, thermal shift assay, shRNA knockdown with cell growth phenotype\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic assay with mutagenesis, kinetic analysis, thermal shift binding confirmation, and cellular KD phenotype\",\n      \"pmids\": [\"26008970\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NPD389, identified by high-throughput screening, inhibits ME2 as an uncompetitive inhibitor with respect to NAD+ and a mixed-type inhibitor with respect to L-malate, binding in a fast-binding mode distinct from the substrate binding site.\",\n      \"method\": \"High-throughput enzymatic screening, enzyme kinetics analysis, thermal shift assay\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic assay with kinetics; single lab, no mutagenesis of binding site\",\n      \"pmids\": [\"24681895\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Mitochondrial ME2 uses either NAD or NADP as cofactor, has a high Km for malate, and is allosterically activated by fumarate and inhibited by ATP — kinetic properties distinct from ME1 (cytosolic) and ME3 (mitochondrial). Substantial ME2 enzymatic activity was demonstrated for the first time in human, rat, and mouse pancreatic islets and INS-1 cells.\",\n      \"method\": \"Spectrophotometric enzymatic assay using defined kinetic properties, immunoblotting for protein confirmation\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — direct enzymatic assay with kinetic characterization; single lab, biochemical characterization\",\n      \"pmids\": [\"19691144\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ME2 promotes liver cancer cell migration and invasion by producing pyruvate, which directly binds to β-catenin and increases β-catenin protein levels. Pyruvate treatment rescues migration in ME2-depleted cells, establishing pyruvate as the downstream effector.\",\n      \"method\": \"ME2 KD/OE with migration/invasion assay, pyruvate rescue experiment, β-catenin binding/protein level assays\",\n      \"journal\": \"Metabolites\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KD/OE with defined phenotype and metabolite rescue; single lab, moderate orthogonal evidence\",\n      \"pmids\": [\"37110198\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"miR-214-3p directly targets ME2 (validated by dual luciferase reporter assay), suppressing its expression. ME2 depletion enhances ferroptosis in cardiomyocytes under hypoxia; ME2 overexpression rescues ferroptosis induced by miR-214-3p mimics, placing ME2 downstream of miR-214-3p in a ferroptosis regulatory pathway in cardiomyocytes.\",\n      \"method\": \"Dual luciferase reporter assay, antagomir injection in MI mouse model, ME2 KD/OE with ferroptosis phenotype readout\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct luciferase validation of miR-target interaction, in vivo and in vitro functional rescue; single lab\",\n      \"pmids\": [\"37087800\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ME2 inhibition (by silencing or allosteric inhibitor disodium embonate) decreases pyruvate and NADH, reducing ATP production via oxidative phosphorylation, and decreases NADPH, increasing reactive oxygen species and oxidative stress leading to apoptosis in AML cells. ME2 silencing also inhibits xenotransplanted AML cell growth in vivo.\",\n      \"method\": \"shRNA silencing, allosteric inhibitor treatment, metabolite measurement, ROS assay, xenograft model\",\n      \"journal\": \"Cellular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KD with defined metabolic and cellular phenotypes, in vivo validation; single lab\",\n      \"pmids\": [\"37079187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ME2 inhibits mitochondrial reactive oxygen species production and AMPK phosphorylation, leading to SREBP-1 maturation and nuclear localization, thereby enhancing the ACSS2 lipogenesis pathway in glioblastoma cells.\",\n      \"method\": \"ME2 KD/OE with ROS measurement, AMPK phosphorylation assay, SREBP-1 localization, lipogenesis pathway analysis\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, single-method per pathway step, limited mechanistic follow-up\",\n      \"pmids\": [\"34381734\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"A dominant-negative form of murine E2-C (mE2-C, the cyclin-selective ubiquitin conjugase), created by mutating the catalytic cysteine to serine, inhibits in vitro ubiquitination and degradation of cyclin B in HeLa cell extracts, establishing mE2-C as required for cyclin B destruction.\",\n      \"method\": \"Catalytic cysteine-to-serine mutagenesis, in vitro ubiquitination assay in HeLa extracts, cell cycle expression analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — active-site mutagenesis with in vitro ubiquitination assay; note this paper concerns mE2-C (UBE2C), not the malic enzyme ME2\",\n      \"pmids\": [\"9798675\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"ME2 (class A bHLH transcription factor) is expressed in specific brain regions during development and in regions of neuronal plasticity in adults (cerebral cortex, cerebellum, olfactory neuroepithelium, hippocampus), suggesting a regulatory role in neuronal developmental processes and plasticity.\",\n      \"method\": \"cDNA cloning, in situ hybridization for regional expression mapping\",\n      \"journal\": \"Brain research. Molecular brain research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — expression/localization characterization without direct functional mechanistic experiment; note this is the bHLH ME2, not malic enzyme ME2\",\n      \"pmids\": [\"7984047\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"ME2 (bHLH transcription factor) binds E-boxes as homodimers and heterodimers with distinct DNA-binding specificity compared to ME1a. Id2 forms heterodimers with ME2, abolishing its DNA-binding activity. Overexpression of Id2 in neuronal cells suppresses ME2 transcriptional activity.\",\n      \"method\": \"In vitro DNA-binding assays (EMSA), heterodimerization pulldown, in situ hybridization, Id2 overexpression with transcriptional activity readout\",\n      \"journal\": \"Brain research. Molecular brain research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct DNA-binding assay, heterodimerization demonstrated, functional suppression by Id2; note this is the bHLH ME2 transcription factor\",\n      \"pmids\": [\"7769987\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ME2 deficiency (homozygous frameshift variant causing truncated, unstable protein) is associated with neurodevelopmental disorder. Deletion of the yeast ortholog of human ME2 caused growth arrest rescued by overexpression of human ME2, establishing an essential role of ME2 in mitochondrial function.\",\n      \"method\": \"Whole exome sequencing, in vitro protein stability assay, yeast ortholog deletion with growth phenotype, human ME2 complementation rescue\",\n      \"journal\": \"Clinical genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — yeast ortholog deletion with human gene rescue establishes mitochondrial function; single study with genetic and functional evidence\",\n      \"pmids\": [\"39401966\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ME2 deletion in pancreatic cancer cells (which harbor SMAD4 homozygous deletion co-occurring with ME2 deletion) leads to upregulation of ME3 to compensate for ROS clearance. Co-delivery of siME3 and doxorubicin to ME2-deficient cells induces ROS accumulation and apoptosis, establishing ME2 as a component of tumor ROS defense through a collateral lethality mechanism.\",\n      \"method\": \"siRNA knockdown of ME3, ROS measurement, apoptosis assay in ME2-deficient cell lines, in vitro/in vivo nanostructure delivery\",\n      \"journal\": \"Nano letters\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — functional consequence of ME2 loss established indirectly through ME3 compensation; single lab, limited direct ME2 mechanistic data\",\n      \"pmids\": [\"39602246\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CYP4F11 prevents ubiquitin-proteasomal degradation of ME2; CYP4F11 knockdown leads to ME2 degradation, disrupting mitochondrial malate metabolism. miR-195 targets CYP4F11 to suppress this pathway, placing ME2 downstream of miR-195/CYP4F11 in NSCLC progression.\",\n      \"method\": \"CYP4F11 KD with ME2 protein level measurement, rescue experiments, metabolomic analysis, 3'-UTR luciferase assay for miR-195/CYP4F11\",\n      \"journal\": \"Frontiers of medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — indirect regulatory link to ME2 protein stability; mechanistic details of ubiquitination not fully characterized\",\n      \"pmids\": [\"41359237\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Mitochondrial malic enzyme 2 (ME2) catalyzes the oxidative decarboxylation of malate to pyruvate using NAD(P)+ as cofactor, is allosterically activated by fumarate and inhibited by ATP, and its enzymatic activity is regulated by multiple post-translational modifications: SIRT5-mediated desuccinylation at K346 activates ME2 to enhance mitochondrial respiration, ACAT1-mediated acetylation at K156 activates ME2 to support lactate production from glutamine for chemoresistance, and PRMT1-mediated methylation stabilizes ME2 by inhibiting ubiquitination; additionally, AKT1-mediated phosphorylation at S9 of the cytoplasmic full-length ME2 isoform prevents its mitochondrial import, redirecting it to function as a scaffold for glycolytic enzymes to promote aerobic glycolysis in tumor cells.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1991,\n      \"finding\": \"Human mitochondrial NAD(P)+-dependent malic enzyme (ME2) was cloned from cDNA, revealing a 584-amino acid precursor protein (65.4 kDa). Expression of the processed protein in E. coli yielded an enzyme with the same kinetic and allosteric properties as malic enzyme purified from human cells, confirming its role in oxidative decarboxylation of malate to pyruvate with NAD+ as cofactor.\",\n      \"method\": \"cDNA cloning, heterologous expression in E. coli, enzymatic kinetic assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro, kinetic and allosteric properties validated\",\n      \"pmids\": [\"1993674\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Crystal structure of human mitochondrial NAD(P)+-dependent ME2 in a quaternary complex with NAD+, Mn2+, and oxalate (2.2 Å) revealed the enzyme in a closed form. The divalent cation is coordinated octahedrally by Glu255, Asp256, Asp279, two substrate oxygens, and one water molecule. Tyr112 and Lys183 were identified as possible catalytic residues. Changes in tetramer organization were observed in different complexes, suggesting relevance for cooperative behavior and allosteric control.\",\n      \"method\": \"X-ray crystallography, structural analysis\",\n      \"journal\": \"Nature structural biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution crystal structures of multiple quaternary complexes\",\n      \"pmids\": [\"10700286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Crystal structure of ME2 in complex with ATP, Mn2+, tartronate, and fumarate (2.2 Å) showed that ATP acts as an active-site inhibitor despite the existence of an exo binding site. Fumarate binds at an allosteric site at the dimer interface, and mutations at this site abolished fumarate's activating effects, revealing the molecular mechanism of allosteric regulation by fumarate.\",\n      \"method\": \"X-ray crystallography, enzyme kinetics, site-directed mutagenesis\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure combined with mutagenesis and kinetic assays\",\n      \"pmids\": [\"12121650\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ME2 enzymatic activity is present in pancreatic islets of humans, rats, mice, and INS-1 832/13 clonal insulinoma cells. ME2 uses either NAD or NADP as cofactor, has a high Km for malate, and is allosterically activated by fumarate and inhibited by ATP—properties distinguishing it from the cytosolic ME1 and mitochondrial ME3.\",\n      \"method\": \"Spectrophotometric enzyme activity assay, immunoblotting\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct enzyme assay with functional characterization, single study\",\n      \"pmids\": [\"19691144\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"p53 transcriptionally represses ME2 (and ME1) expression in human and mouse cells. ME2 downregulation reciprocally activates p53 through AMPK-mediated mechanisms in a feed-forward loop. Knockdown of ME2 induces cellular senescence (not apoptosis), while enforced ME2 expression suppresses senescence, establishing ME2 as a regulator of cell fate downstream of p53.\",\n      \"method\": \"Genetic knockdown/overexpression, epistasis analysis, cell senescence assays, metabolic measurements\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, replicated in human and mouse cells, published in high-impact journal\",\n      \"pmids\": [\"23334421\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ME2 knockdown in A549 NSCLC cells inhibits cell proliferation, induces differentiation, increases ROS and NADP+/NADPH ratio, drops ATP, and increases cisplatin sensitivity. ME2 depletion reduces pyruvate and accumulates malate; exogenous membrane-permeable malate mimics this phenotype. ME2 knockdown also impacts PDK1/PTEN expression, leading to AKT inhibition. Both ME2 knockdown and malate treatment reduce tumor growth in vivo.\",\n      \"method\": \"shRNA knockdown, metabolic flux analysis (stable isotope tracing), in vivo xenograft, kinase signaling analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including in vivo validation and metabolomics\",\n      \"pmids\": [\"24957098\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"High-throughput screening identified NPD389 as a potent ME2 inhibitor (IC50 ~4.6 μM) that acts as an uncompetitive inhibitor with respect to NAD+ and a mixed-type inhibitor with respect to L-malate, indicating it binds at a site distinct from the active site (consistent with allosteric inhibition).\",\n      \"method\": \"High-throughput enzymatic screening, enzyme kinetics analysis, thermal shift assay\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzyme kinetics with mechanistic characterization, single study\",\n      \"pmids\": [\"24681895\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The natural compound embonic acid (EA) is an allosteric inhibitor of ME2 with an IC50 of 1.4 μM. Mutagenesis and binding studies localized its binding site to the fumarate binding site or nearby dimer interface, distinct from the catalytic site (non-competitive inhibition). EA treatment and ME2 shRNA knockdown both inhibit H1299 cancer cell growth and induce cellular senescence via a p53-independent pathway.\",\n      \"method\": \"In vitro enzyme inhibition assays, site-directed mutagenesis, binding studies, shRNA knockdown, cellular senescence assays\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro inhibition with mutagenesis plus cellular validation\",\n      \"pmids\": [\"26008970\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Genomic deletion of ME2 in SMAD4-deleted pancreatic cancer creates collateral lethality upon depletion of its paralog ME3. Mechanistically, loss of mitochondrial malic enzymes (ME2 and ME3) diminishes NADPH production, elevates ROS, activates AMPK, which then directly suppresses SREBP1-directed transcription of BCAT2. Reduced BCAT2 impairs branched-chain amino acid transamination, depleting glutamate needed for nucleotide synthesis.\",\n      \"method\": \"Genetic depletion (shRNA/CRISPR), metabolomics, molecular epistasis, AMPK/SREBP1/BCAT2 pathway analysis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — integrated metabolomics and molecular epistasis, multiple orthogonal methods, high-impact publication\",\n      \"pmids\": [\"28099419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ME2 promotes proneural-mesenchymal transition (PMT) and lipogenesis in glioblastoma by inhibiting mitochondrial ROS production and AMPK phosphorylation, resulting in SREBP-1 maturation and nuclear localization, which enhances the ACSS2 lipogenesis pathway. ME2 overexpression upregulates mesenchymal markers and inhibits proneural marker OLIG2.\",\n      \"method\": \"ME2 overexpression/knockdown, ROS measurement, AMPK/SREBP-1 signaling analysis, lipogenesis assays, migration/invasion assays\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — pathway placement with functional readouts, single laboratory\",\n      \"pmids\": [\"34381734\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SIRT5 physically interacts with ME2 and acts as its desuccinylase. Glutamine deprivation enhances the SIRT5-ME2 interaction, promoting SIRT5-mediated desuccinylation of ME2 at lysine 346, which activates ME2 enzymatic activity. Activated ME2 enhances mitochondrial respiration to counteract glutamine deprivation and support proliferation and tumorigenesis in colorectal cancer.\",\n      \"method\": \"Co-immunoprecipitation, succinylation assays, site-directed mutagenesis (K346), enzymatic activity assays, metabolic flux measurements, xenograft models\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — identified specific modification site with mutagenesis, writer identified, functional consequence demonstrated in vitro and in vivo\",\n      \"pmids\": [\"38007551\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ME2 promotes pyruvate production, which directly binds to β-catenin and increases β-catenin protein levels, thereby promoting hepatocellular carcinoma cell migration and invasion. Pyruvate treatment rescues migration in ME2-depleted cells, establishing the ME2→pyruvate→β-catenin axis.\",\n      \"method\": \"ME2 knockdown/overexpression, pyruvate supplementation rescue experiments, β-catenin protein level analysis, migration/invasion assays\",\n      \"journal\": \"Metabolites\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, functional rescue experiment supporting mechanistic axis\",\n      \"pmids\": [\"37110198\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ME2 inhibition in AML cells (via ME2 silencing or allosteric inhibitor disodium embonate) decreases pyruvate and NADH, reducing ATP production via oxidative phosphorylation, and decreases NADPH, increasing ROS and oxidative stress, ultimately inducing apoptosis. ME2 silencing inhibits xenograft AML tumor growth in vivo.\",\n      \"method\": \"shRNA knockdown, allosteric inhibitor treatment, energy/redox metabolite measurements, xenograft in vivo models\",\n      \"journal\": \"Cellular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple metabolic endpoints with in vivo validation, single laboratory\",\n      \"pmids\": [\"37079187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"miR-214-3p directly targets ME2 (confirmed by dual luciferase reporter assay). In cardiomyocytes, miR-214-3p suppresses ME2 expression and promotes ferroptosis; ME2 overexpression ameliorates ferroptosis induced by miR-214-3p, while ME2 depletion compromises the protective effect of miR-214-3p inhibitor, establishing ME2 as a functional target of miR-214-3p in regulating ferroptosis during myocardial infarction.\",\n      \"method\": \"Dual luciferase reporter assay, miRNA mimic/inhibitor/antagomir, ME2 overexpression/knockdown, ferroptosis and iron accumulation assays, cardiac function measurement in mouse MI model\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — direct target validation with reporter assay, functional epistasis in vitro and in vivo\",\n      \"pmids\": [\"37087800\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"AKT1 phosphorylates cytoplasmic ME2 (full-length isoform, ME2fl) at serine 9 within the mitochondrial localization signal, preventing its mitochondrial translocation. Cytoplasmic ME2fl acts as a scaffold assembling key glycolytic enzymes (PFKL, GAPDH, PKM2, LDHA) to promote glycolysis. This AKT1-driven phosphorylation induces a metabolic switch from mitochondrial TCA metabolism to glycolysis, enhancing glycolytic capacity of tumor cells in vitro and in vivo.\",\n      \"method\": \"In vitro phosphorylation assays, site-directed mutagenesis (S9), subcellular fractionation, co-immunoprecipitation of glycolytic enzyme complex, metabolic flux measurements, xenograft in vivo models\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — identified phosphorylation site with mutagenesis, reconstituted scaffold complex, in vivo validation with multiple orthogonal methods\",\n      \"pmids\": [\"38263319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ME2 is acetylated at lysine 156 by ACAT1. Decreased intracellular glucose triggers ACAT1-mediated K156 acetylation of ME2, potentiating its enzymatic activity and facilitating lactate production from glutamine. ME2-derived lactate promotes lactylation of homologous recombination repair proteins, contributing to chemotherapy resistance in ovarian cancer. ACAT1 inhibition reduces ME2 acetylation and chemoresistance in vitro and in vivo.\",\n      \"method\": \"Acetylation assays, site-directed mutagenesis (K156), co-immunoprecipitation, metabolomics, protein lactylation analysis, in vitro and in vivo chemoresistance assays\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — identified acetylation site with writer (ACAT1) and mutagenesis, downstream lactylation mechanism, in vivo validation\",\n      \"pmids\": [\"39951294\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PRMT1 interacts with ME2 and methylates ME2, activating its enzymatic activity. Mutation of ME2 at arginine 67 (R67K) mimics constitutive activation. PRMT1-mediated ME2 methylation increases mitochondrial respiration, promoting HCC cell proliferation and migration. PRMT1 inhibition reduces ME2 activity and tumor growth.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis (R67K), enzymatic activity assays, mitochondrial respiration measurements, cell proliferation/migration assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — interaction and functional mutagenesis demonstrated, single laboratory\",\n      \"pmids\": [\"39528487\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"A homozygous frameshift variant in ME2 (c.1379_1380delTT, p.Phe460fs*22) produces truncated, unstable ME2 protein in vitro, causing ME2 deficiency associated with neurodevelopmental disorder, dilated cardiomyopathy, and mild lactic acidemia in a human patient. Deletion of the yeast ortholog of human ME2 causes growth arrest rescued by re-expression of human ME2, demonstrating ME2's essential role in mitochondrial function.\",\n      \"method\": \"Whole exome sequencing, in vitro protein expression/stability assay, yeast complementation assay\",\n      \"journal\": \"Clinical genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — yeast complementation provides functional evidence; human variant pathogenicity supported by in vitro instability\",\n      \"pmids\": [\"39401966\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CYP4F11 promotes ubiquitin-proteasomal degradation of ME2 when suppressed by miR-195, establishing a CYP4F11/miR-195/ME2 regulatory axis. CYP4F11 depletion disrupts mitochondrial malate metabolism, and ME2 rescue experiments confirm ME2 mediates the oncogenic metabolic effects of CYP4F11 in non-small cell lung cancer.\",\n      \"method\": \"3'-UTR luciferase reporter assay, CYP4F11 knockdown, ME2 rescue experiments, metabolomic analysis, xenograft in vivo models\",\n      \"journal\": \"Frontiers of medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — functional rescue validates axis, metabolomics provides mechanistic context, single laboratory\",\n      \"pmids\": [\"41359237\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ME2 (mitochondrial NAD(P)+-dependent malic enzyme 2) catalyzes oxidative decarboxylation of malate to pyruvate with concomitant NADH/NADPH production; its activity is allosterically activated by fumarate (at the dimer interface) and inhibited by ATP (at the active site), as established by crystal structures and mutagenesis. ME2 is subject to multiple post-translational modifications—desuccinylation at K346 by SIRT5, acetylation at K156 by ACAT1, and methylation at R67 by PRMT1—each activating enzymatic activity and promoting tumor metabolism. AKT1 phosphorylates a cytoplasmic full-length isoform (ME2fl) at S9, preventing mitochondrial import and enabling ME2fl to function as a scaffold for glycolytic enzyme complexes. ME2 supports NADPH homeostasis and ROS control, and its loss activates AMPK, suppresses SREBP1/BCAT2, and induces p53-dependent cellular senescence, linking ME2 activity to metabolic adaptation, tumorigenesis, and cell fate decisions.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"ME2 (mitochondrial malic enzyme 2) is a mitochondrial NAD(P)+-dependent oxidative decarboxylase that converts malate to pyruvate, generating NADH and NADPH to fuel oxidative phosphorylation and buffer reactive oxygen species, thereby occupying a central node in anaplerotic and redox metabolism [PMID:19691144, PMID:37079187]. ME2 is allosterically activated by fumarate and inhibited by ATP, and its activity is positively regulated by SIRT5-mediated desuccinylation at K346 and ACAT1-mediated acetylation at K156, while PRMT1-mediated methylation stabilizes ME2 protein by blocking ubiquitin-dependent degradation [PMID:38007551, PMID:39951294, PMID:39528487]. AKT1-mediated phosphorylation at S9 of a cytoplasmic full-length ME2 isoform blocks mitochondrial import and repurposes ME2 as a scaffold that assembles glycolytic enzymes PFKL, GAPDH, PKM2, and LDHA to promote aerobic glycolysis in tumor cells [PMID:38263319]. Homozygous loss-of-function variants in ME2 cause a neurodevelopmental disorder, and deletion of its yeast ortholog produces growth arrest rescued by human ME2 complementation, establishing an essential role in mitochondrial function [PMID:39401966].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Establishing ME2's enzymatic identity — demonstrating that ME2 uses both NAD+ and NADP+ with a high Km for malate and is allosterically activated by fumarate and inhibited by ATP — distinguished it kinetically from cytosolic ME1 and mitochondrial ME3.\",\n      \"evidence\": \"Spectrophotometric enzymatic assays with kinetic characterization in human, rat, and mouse pancreatic islets and INS-1 cells\",\n      \"pmids\": [\"19691144\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Crystal structure of human ME2 with fumarate/ATP bound not reported in this study\", \"Relative contribution of NAD vs NADP flux in vivo unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Discovery that ME2 can be inhibited by small molecules (NPD389, embonic acid) binding at sites distinct from the substrate pocket demonstrated the feasibility of allosteric pharmacological targeting and linked ME2 inhibition to cancer cell growth suppression and senescence.\",\n      \"evidence\": \"High-throughput enzymatic screening, enzyme kinetics, thermal shift assay, and shRNA knockdown with cellular phenotype in cancer lines\",\n      \"pmids\": [\"24681895\", \"26008970\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No co-crystal structure defining inhibitor binding pose\", \"Whether senescence induction requires p53 was excluded, but the downstream effector pathway remains undefined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Multiple studies converged to show that ME2's catalytic products — pyruvate, NADH, and NADPH — serve as critical downstream effectors: pyruvate sustains oxidative phosphorylation and stabilizes β-catenin to drive migration, while NADPH buffers ROS to prevent ferroptosis and apoptosis.\",\n      \"evidence\": \"ME2 KD/OE with metabolite rescue in liver cancer cells, ferroptosis measurement in cardiomyocytes, ROS/ATP quantification in AML cells, xenograft models\",\n      \"pmids\": [\"37110198\", \"37087800\", \"37079187\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct binding of pyruvate to β-catenin requires structural confirmation\", \"Relative importance of NADH vs NADPH generation by ME2 in different cell types not resolved\", \"Ferroptosis link established in single cardiomyocyte study\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of SIRT5-mediated desuccinylation at K346 as a nutrient-responsive activating modification revealed that ME2 activity is tuned by post-translational modification in response to glutamine deprivation to maintain mitochondrial respiration.\",\n      \"evidence\": \"Reciprocal Co-IP, mass spectrometry succinylation mapping, K346 mutagenesis, enzymatic activity assays, in vivo tumor models\",\n      \"pmids\": [\"38007551\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other sirtuins also desuccinylate ME2 untested\", \"Stoichiometry of succinylation in normal vs tumor tissue unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"AKT1-mediated phosphorylation at S9 of a full-length cytoplasmic ME2 isoform blocks mitochondrial import, uncovering a non-enzymatic scaffold function whereby cytoplasmic ME2 assembles glycolytic enzymes (PFKL, GAPDH, PKM2, LDHA) to promote aerobic glycolysis — a fundamentally new moonlighting role.\",\n      \"evidence\": \"In vitro kinase assay, phospho-site mutagenesis, subcellular fractionation, Co-IP/pulldown, xenograft tumor models\",\n      \"pmids\": [\"38263319\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the scaffold function requires ME2 catalytic activity or is purely structural is not resolved\", \"Regulation of the ME2 full-length isoform expression vs the canonical mitochondrial isoform not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Human genetic evidence linked homozygous ME2 loss-of-function to neurodevelopmental disorder, and yeast ortholog deletion rescued by human ME2 established its essential mitochondrial role across evolution.\",\n      \"evidence\": \"Whole exome sequencing, in vitro protein stability assay, yeast ortholog deletion with human complementation rescue\",\n      \"pmids\": [\"39401966\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single family reported; additional kindreds needed\", \"Neuronal-specific metabolic consequences of ME2 loss not characterized\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"PRMT1-mediated arginine methylation stabilizes ME2 by blocking ubiquitin-dependent degradation, adding protein stability regulation as another layer of ME2 control in hepatocellular carcinoma.\",\n      \"evidence\": \"Co-IP of ME2-PRMT1, site-directed mutagenesis, ubiquitination assay, enzymatic activity assay in HCC cells\",\n      \"pmids\": [\"39528487\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific arginine residue(s) methylated not conclusively mapped\", \"E3 ubiquitin ligase responsible for ME2 degradation not identified\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"ACAT1-mediated acetylation at K156 activates ME2 to increase glutamine-derived lactate, which drives lactylation of DNA repair proteins to confer chemoresistance — connecting ME2 post-translational regulation to epigenetic-like modifications and therapy response.\",\n      \"evidence\": \"Co-IP, K156 mutagenesis, enzymatic activity assay, metabolomics, in vitro/in vivo rescue in ovarian cancer models\",\n      \"pmids\": [\"39951294\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Deacetylase opposing ACAT1 at K156 not identified\", \"Whether K156 acetylation and K346 succinylation co-occur or are mutually exclusive unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The integration of multiple post-translational modifications (succinylation, acetylation, methylation, phosphorylation) on ME2 and how they combinatorially regulate its enzymatic activity, protein stability, and subcellular localization remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural basis for how modifications alter allosteric regulation\", \"E3 ubiquitin ligase(s) targeting ME2 not identified\", \"Crosstalk among PTMs (succinylation, acetylation, methylation, phosphorylation) not systematically tested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [0, 2, 4, 5, 6, 9]},\n      {\"term_id\": \"GO:0016829\", \"supporting_discovery_ids\": [6, 4]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 3, 6, 9, 14]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 2, 6, 7, 9]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [9, 8]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [8, 9]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [2, 7, 15]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"SIRT5\",\n      \"AKT1\",\n      \"ACAT1\",\n      \"PRMT1\",\n      \"PFKL\",\n      \"PKM2\",\n      \"LDHA\",\n      \"GAPDH\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"ME2 is a mitochondrial NAD(P)⁺-dependent malic enzyme that catalyzes the oxidative decarboxylation of malate to pyruvate, generating NADH and NADPH critical for mitochondrial respiration, biosynthetic reactions, and redox homeostasis [PMID:1993674, PMID:10700286]. The enzyme functions as a tetramer whose activity is allosterically activated by fumarate at the dimer interface and inhibited by ATP at the active site, and is further modulated by post-translational modifications—desuccinylation at K346 by SIRT5, acetylation at K156 by ACAT1, and methylation at R67 by PRMT1—each of which potentiates catalytic activity to support tumor metabolism [PMID:12121650, PMID:38007551, PMID:39951294, PMID:39528487]. ME2 loss diminishes NADPH production, elevates ROS, and activates AMPK, which suppresses SREBP1/BCAT2 signaling and triggers p53-dependent senescence, linking ME2 to metabolic adaptation and cell fate decisions in cancer [PMID:23334421, PMID:28099419]. A homozygous loss-of-function ME2 variant (p.Phe460fs*22) causes a Mendelian disorder characterized by neurodevelopmental impairment, dilated cardiomyopathy, and lactic acidemia [PMID:39401966].\",\n  \"teleology\": [\n    {\n      \"year\": 1991,\n      \"claim\": \"Cloning and heterologous expression of human ME2 established its identity as the mitochondrial NAD(P)⁺-dependent malic enzyme catalyzing oxidative decarboxylation of malate to pyruvate, resolving the molecular basis of an activity long known biochemically.\",\n      \"evidence\": \"cDNA cloning, E. coli expression, kinetic and allosteric characterization\",\n      \"pmids\": [\"1993674\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural information on subunit assembly or allosteric sites\", \"Regulation of expression unknown\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"High-resolution crystal structures of ME2 with substrates, cofactors, and allosteric effectors revealed that ATP inhibits at the active site, while fumarate activates through a dimer-interface allosteric site, providing the structural basis for cooperative regulation.\",\n      \"evidence\": \"X-ray crystallography (2.2 Å) of multiple quaternary complexes, site-directed mutagenesis, enzyme kinetics\",\n      \"pmids\": [\"10700286\", \"12121650\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological relevance of allosteric regulation in vivo not tested\", \"No structural data on post-translational modification sites\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"The discovery that p53 transcriptionally represses ME2, and that ME2 loss reciprocally activates p53 through AMPK, established a metabolic–tumor-suppressor feedback loop wherein ME2 depletion drives senescence rather than apoptosis.\",\n      \"evidence\": \"Genetic knockdown/overexpression with epistasis analysis in human and mouse cells, senescence assays, metabolic measurements\",\n      \"pmids\": [\"23334421\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific AMPK substrates mediating p53 activation not fully identified\", \"Whether ME2-driven senescence occurs in non-cancerous tissues in vivo unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"ME2 depletion in NSCLC cells demonstrated that ME2 sustains tumor proliferation by maintaining NADPH/ROS balance and PI3K/AKT signaling, and that its loss sensitizes tumors to cisplatin, broadening ME2's role beyond metabolism to oncogenic signaling.\",\n      \"evidence\": \"shRNA knockdown, stable isotope metabolic tracing, xenograft tumor models, kinase signaling analysis\",\n      \"pmids\": [\"24957098\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking ME2 to PDK1/PTEN expression not fully resolved\", \"No demonstration with genetic knockout\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identification of embonic acid as an allosteric ME2 inhibitor binding near the fumarate site provided pharmacological validation that ME2 inhibition induces senescence, even in p53-null cells, indicating both p53-dependent and -independent effector pathways.\",\n      \"evidence\": \"In vitro inhibition kinetics, site-directed mutagenesis of fumarate site, shRNA knockdown, cellular senescence assays in H1299 (p53-null) cells\",\n      \"pmids\": [\"26008970\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo pharmacokinetics and selectivity of embonic acid not characterized\", \"p53-independent senescence pathway mediators unidentified\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Collateral lethality of ME2 co-deletion with SMAD4 in pancreatic cancer revealed that combined loss of mitochondrial malic enzymes (ME2+ME3) collapses NADPH, activates AMPK, suppresses SREBP1-directed BCAT2 transcription, and depletes branched-chain amino acid-derived glutamate needed for nucleotide synthesis.\",\n      \"evidence\": \"CRISPR/shRNA depletion, integrated metabolomics, AMPK–SREBP1–BCAT2 epistasis analysis in pancreatic cancer models\",\n      \"pmids\": [\"28099419\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contributions of ME2 vs. ME3 to NADPH pool not individually quantified\", \"Clinical feasibility of exploiting collateral lethality untested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Three post-translational modifications activating ME2 were identified: SIRT5-mediated desuccinylation at K346 under glutamine deprivation, ACAT1-mediated acetylation at K156 under glucose deprivation, and PRMT1-mediated methylation at R67, each enhancing ME2 catalytic activity to support tumor-adaptive metabolism.\",\n      \"evidence\": \"Co-immunoprecipitation, site-directed mutagenesis of K346/K156/R67, succinylation/acetylation/methylation assays, enzymatic activity assays, xenograft models\",\n      \"pmids\": [\"38007551\", \"39951294\", \"39528487\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crosstalk or hierarchy among the three modifications not examined\", \"Structural basis for how each modification increases catalytic activity unknown\", \"Whether these modifications co-occur in the same tumor type is unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"AKT1 phosphorylation of a cytoplasmic ME2 full-length isoform at S9 blocks mitochondrial import and repurposes ME2 as a scaffold for glycolytic enzyme assembly (PFKL, GAPDH, PKM2, LDHA), uncovering a non-enzymatic moonlighting function that promotes a glycolytic metabolic switch.\",\n      \"evidence\": \"In vitro phosphorylation, S9 mutagenesis, subcellular fractionation, co-immunoprecipitation of glycolytic complex, metabolic flux, xenograft in vivo\",\n      \"pmids\": [\"38263319\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Proportion of ME2 retained in cytoplasm under physiological conditions unknown\", \"Whether scaffold function requires ME2 enzymatic activity is untested\", \"Independent replication of this moonlighting role pending\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"A homozygous frameshift ME2 variant in a human patient established ME2 deficiency as a cause of neurodevelopmental disorder with dilated cardiomyopathy and lactic acidemia, and yeast complementation confirmed its essential mitochondrial function.\",\n      \"evidence\": \"Whole exome sequencing, in vitro protein stability assay, yeast ortholog deletion/complementation\",\n      \"pmids\": [\"39401966\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Only a single family reported; additional kindreds needed\", \"Patient-derived cell metabolomics not performed\", \"Tissue-specific consequences of ME2 loss in mammals not characterized\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include how the multiple post-translational activating modifications of ME2 are coordinated, what determines whether ME2 loss triggers senescence versus apoptosis versus ferroptosis across cell types, and whether the cytoplasmic scaffolding function operates in non-tumor physiology.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No integrated model of PTM crosstalk on ME2\", \"Tissue-specific phenotypic outcomes of ME2 loss poorly defined\", \"Structural basis of cytoplasmic scaffold assembly unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [0, 1, 2, 3, 10, 15, 16]},\n      {\"term_id\": \"GO:0016829\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 1, 10, 14, 17]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [14]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 2, 3, 5, 8, 10, 11, 12, 14, 15]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4, 5, 8, 9]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [5, 8, 9, 12, 15]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [4, 12, 13]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"SIRT5\",\n      \"ACAT1\",\n      \"PRMT1\",\n      \"AKT1\",\n      \"PFKL\",\n      \"GAPDH\",\n      \"PKM2\",\n      \"LDHA\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}