{"gene":"PFKM","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2021,"finding":"ZEB1 directly activates PFKM transcription through a non-classic ZEB1-binding sequence in the PFKM promoter region, upregulating PFKM expression and glycolysis in HCC cells; silencing ZEB1 impairs PFKM expression, glycolysis, proliferation and invasion, and exogenous PFKM re-expression rescues these defects.","method":"shRNA knockdown, ChIP assay, luciferase reporter assay, RT-qPCR, Western blot, ECAR/OCR assays, orthotopic xenograft","journal":"Theranostics","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal rescue experiments, ChIP and luciferase reporter, in vivo xenograft, multiple orthogonal methods in single lab","pmids":["33897890"],"is_preprint":false},{"year":2020,"finding":"Mycobacterium tuberculosis-induced miR-21 directly targets PFKM mRNA to repress its expression and activity in macrophages, dampening glycolysis and limiting IL-1β production; IFN-γ inhibits miR-21, forcing an isoenzyme switch that augments PFKM expression and macrophage glycolysis.","method":"miRNA target validation, PFK isoenzyme activity assays, miR-21 manipulation in macrophages, cytokine measurement","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (activity assay, miRNA targeting, IFN-γ rescue), independently reported mechanism","pmids":["31914380"],"is_preprint":false},{"year":2021,"finding":"NOS1 S-nitrosylates PFKM at Cys351, stabilizing the PFKM tetramer and enabling resistance to negative feedback from downstream metabolic intermediates, thereby promoting glycolysis in ovarian cancer cells; PFKM-C351S mutation reduced proliferation and tumor growth in xenograft models.","method":"S-nitrosoproteomic profiling, site-directed mutagenesis (C351S), in vitro tetramer stability assay, xenograft tumor model","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — site-directed mutagenesis with functional readout, proteomic identification of modification site, in vivo validation","pmids":["33859186"],"is_preprint":false},{"year":2025,"finding":"Citrate binds PFKM and disrupts its tetrameric structure into dimers; dimeric PFKM interacts with nucleosomes and phosphorylates histone H3 at serine 10 (H3S10) as a protein kinase, promoting mitotic progression and cell proliferation. Structural simulations show PFKM binds nucleosomes optimally when H3S10 aligns with its catalytic site.","method":"Structural simulation, biochemical citrate-binding assays, in vitro histone kinase assay, mutagenesis disrupting citrate-PFKM and PFKM-H3 interactions, cell cycle analysis, tumor growth assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with mutagenesis, structural modeling, multiple functional readouts in single rigorous study","pmids":["40695785"],"is_preprint":false},{"year":2025,"finding":"Wnt signalling induces lysosomal degradation of PFKM through a methyl arginine degron motif that is selectively methylated by PRMT1, delivering PFKM to lysosomes via microautophagy; PFKM degradation shifts glucose metabolism from glycolysis to the pentose phosphate pathway, and PFKM overexpression promotes myofibre differentiation while PFKM knockdown blunts differentiation (rescued by 3-phosphoglycerate supplementation).","method":"Genetic knockdown/overexpression, metabolic flux analysis, PRMT1-mediated methylation validation, lysosomal inhibition experiments, metabolic rescue with 3-phosphoglycerate","journal":"Nature metabolism","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods including PTM identification, pathway rescue, metabolic flux, mechanistic and functional validation","pmids":["41735679"],"is_preprint":false},{"year":2024,"finding":"METTL16 mediates m6A methylation to stabilize PFKM mRNA in an IGF2BP3-dependent manner in HCC, promoting glycolysis; POU3F2 transcriptionally activates METTL16, defining a POU3F2/METTL16/PFKM axis.","method":"MeRIP assay, RIP assay, actinomycin D mRNA stability assay, ChIP, luciferase assay, knockdown/overexpression, xenograft","journal":"Annals of hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MeRIP, RIP, ChIP and luciferase in single lab with multiple orthogonal methods","pmids":["39756795"],"is_preprint":false},{"year":2024,"finding":"NAT10-mediated ac4C acetylation suppresses m6A modification on PFKM mRNA; YTHDC1 recognizes m6A sites on PFKM mRNA to increase its stability; NAT10 knockdown increases m6A content, reducing YTHDC1 translation, and destabilizing PFKM mRNA, leading to decreased glycolysis in osteosarcoma cells.","method":"NAT10 knockdown, m6A-seq, RIP assay, m6A reader protein analysis, mRNA stability assay, in vivo tumor models","journal":"Cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — m6A sequencing, RIP, mRNA stability, rescue experiments in single lab","pmids":["38233839"],"is_preprint":false},{"year":2024,"finding":"HIF-1α increases METTL3 expression, which elevates m6A modification on Pfkm mRNA and upregulates PFKM protein expression in macrophages; recombinant thrombomodulin (rTM) decreases PFKM expression in a HIF-1α-dependent manner, and METTL3 silencing attenuates HIF-1α-mediated PFKM upregulation.","method":"HIF-1α overexpression/knockdown, METTL3 silencing, m6A quantification, Pfkm knockout mice, ELISA cytokine measurement","journal":"Cellular and molecular life sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis (HIF-1α → METTL3 → PFKM), m6A measurement, in vivo knockout model, single lab","pmids":["39549085"],"is_preprint":false},{"year":2024,"finding":"USP35 deubiquitinates and stabilizes PFK-1 (PFKM) protein; Co-IP identified PFK-1 as a direct deubiquitination target of USP35, and USP35 knockdown decreased PFK-1 expression, reducing glycolysis and cancer cell proliferation.","method":"Co-immunoprecipitation, immunoprecipitation/ubiquitination assay, USP35 knockdown, Seahorse glycolysis assay, xenograft","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP establishing direct interaction, ubiquitination assay, in vivo validation, single lab","pmids":["39714773"],"is_preprint":false},{"year":2025,"finding":"PFKM translocates to the macrophage nucleus during sepsis and interacts with p53 (identified by Co-IP/mass spectrometry); nuclear PFKM promotes p53 acetylation at K120, enhancing p53 binding to the Pdcd1 promoter to drive PD-1 transcription, thereby suppressing macrophage phagocytosis via a non-glycolytic moonlighting function.","method":"Co-IP coupled with mass spectrometry, immunofluorescence for nuclear localization, dual-luciferase reporter, ChIP, transcriptomic sequencing, nanobody blocking, sepsis mouse models","journal":"Theranostics","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP/MS interaction identification, ChIP, luciferase, in vivo rescue with nanobody, multiple orthogonal methods","pmids":["41608568"],"is_preprint":false},{"year":2025,"finding":"PFKM drives lactate accumulation which promotes global and H3K18 lactylation in atrial fibroblasts; P300-mediated H3K18 lactylation at the TGF-β1 promoter upregulates TGF-β1 transcription, activating cardiac fibroblasts and contributing to atrial fibrosis. AAV-mediated atrial PFKM expression confirmed PFKM as the pivotal glycolytic driver in this pathway.","method":"AAV-mediated PFKM overexpression in vivo, glycolysis inhibitor (2-DG) treatment, ChIP for H3K18la at TGF-β1 promoter, primary cardiac fibroblast activation assays","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo AAV overexpression, ChIP for histone modification at target promoter, single lab","pmids":["40569576"],"is_preprint":false},{"year":2025,"finding":"PFKM promotes gastric cancer progression by interacting with CNTN1 and facilitating enrichment of H3K18 lactylation at the CNTN1 promoter, activating CNTN1 transcription; CNTN1 overexpression reverses the inhibitory effects of PFKM knockdown.","method":"Co-immunoprecipitation (PFKM-CNTN1 interaction), dual-luciferase reporter, ChIP for H3K18la at CNTN1 promoter, knockdown/overexpression rescue, xenograft","journal":"Applied biochemistry and biotechnology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ChIP, luciferase, rescue experiments in single lab","pmids":["40608258"],"is_preprint":false},{"year":2025,"finding":"FTO demethylase elevates C-Jun mRNA in a m6A-YTHDF2-dependent manner, and C-Jun transcriptionally upregulates PFKM expression, thereby promoting glycolysis in pancreatic ductal adenocarcinoma; FTO inhibitors suppress PDAC growth in organoid and xenograft models.","method":"m6A-seq, transcriptome sequencing, metabolomics, FTO-conditional knockout mouse model, organoids, xenograft, ChIP for C-Jun at PFKM promoter","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiomics, conditional KO mouse, organoid validation, in vivo xenograft, single lab","pmids":["41184232"],"is_preprint":false},{"year":2022,"finding":"miR-21a-5p in bone marrow MSC-derived exosomes directly represses PFKM expression (a rate-limiting glycolytic enzyme) in tubular epithelial cells, attenuating glycolysis and renal fibrosis; knockdown of miR-21a-5p abolished the renoprotective effect of MSC-Exos.","method":"miRNA sequencing, in vitro miR-21a-5p targeting validation, UUO mouse model, glycolysis assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — miRNA targeting validated, in vivo UUO model, rescue experiments, single lab","pmids":["36253358"],"is_preprint":false},{"year":2012,"finding":"HSV-1 infection increases PFK-1 expression and triggers phosphorylation of PFK-1 at serine residues, increasing total PFK-1 activity and glycolysis; PFK-1 knockdown impairs HSV-1 replication.","method":"Western blot, glucose uptake/lactate efflux assays, serine phosphorylation detection, PFK-1 siRNA knockdown, viral replication assay","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — enzyme activity assay, knockdown with functional readout (viral replication), phosphorylation detection, single lab","pmids":["22542512"],"is_preprint":false},{"year":2006,"finding":"AMPK phosphorylates and activates PFK-2 post-mortem, increasing fructose-2,6-diphosphate levels which then up-regulate PFK-1 (PFKM) activity; earlier AMPK activation in PSE porcine muscle leads to faster glycolysis and lower pH.","method":"Biochemical activity assays (AMPK, PFK-1, glycogen phosphorylase, pyruvate kinase), fructose-2,6-diphosphate measurement, pH and metabolite quantification in porcine muscle","journal":"Journal of agricultural and food chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — enzyme activity assays with defined pathway dissection, metabolite quantification, single lab","pmids":["16848549"],"is_preprint":false},{"year":1989,"finding":"A Zn2+-binding protein (identified as parathymosin-alpha) reversibly inactivates phosphofructokinase-1 (PFK-1/PFKM) in a Zn2+-dependent manner via its 43-amino-acid zinc-binding domain.","method":"cDNA sequencing, protein sequence comparison, in vitro PFK-1 inactivation assay with Zn2+-binding protein","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro functional inactivation assay, protein identification by sequence, single lab","pmids":["2759245"],"is_preprint":false},{"year":2025,"finding":"PFKM influences exosome release by modulating RAB8B expression; the PFKM-RAB8B interaction promotes chemoresistance in lung adenocarcinoma by impacting apoptosis and glycolytic metabolism, with drug-resistant cells releasing glycolytically active exosomes that transfer chemoresistance to sensitive cells.","method":"Targeted metabolomics (FBP/F6P ratio as PFKM activity proxy), exosome isolation and characterization, PFKM-RAB8B interaction analysis, knockdown/overexpression","journal":"Journal of pharmaceutical analysis","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, interaction validation method not specified in abstract, metabolomics-based activity inference","pmids":["42057963"],"is_preprint":false},{"year":2022,"finding":"HDAC1 inhibits H3K27ac-induced transcription of PFKM in doxorubicin-treated cardiomyocytes; PFKM overexpression inhibits DOX-induced apoptosis and maintains glycolysis and oxidative phosphorylation, while PFKM silencing promotes apoptosis.","method":"RT-PCR, immunoblotting, PFKM overexpression/knockdown, Seahorse metabolic assay, apoptosis assay in H9c2 cells","journal":"Scientific reports","confidence":"Low","confidence_rationale":"Tier 3 / Weak — HDAC1/H3K27ac mechanism inferred from expression changes without direct ChIP or interaction assay shown in abstract, single lab","pmids":["35804014"],"is_preprint":false},{"year":2024,"finding":"Chloroquine (CQ) directly binds PFKM and inhibits its expression and enzymatic activity, thereby blocking the Warburg effect in colorectal cancer cells; additionally, CHKA downregulation also decreases PFKM expression and activity.","method":"Target identification and binding verification (method not fully described in abstract), enzymatic activity assay, knockdown/expression analysis","journal":"International journal of biological sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — binding target identification method not specified in abstract, single lab, limited mechanistic detail","pmids":["39990656"],"is_preprint":false},{"year":2026,"finding":"PFKM drives glycolytic reprogramming leading to lactate accumulation, which promotes H3K18 lactylation at the Rela promoter via histone modification, activating NF-κB pathway and exacerbating renal inflammation and fibrosis; AAV9-mediated PFKM knockdown alleviated FA-induced renal fibrosis in mice.","method":"AAV9 PFKM knockdown/overexpression in renal tubular cells, RNA-seq, CUT&Tag for H3K18la, ChIP-qPCR, metabolic analysis, histological staining","journal":"Cellular and molecular life sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CUT&Tag and ChIP-qPCR for histone modification at target promoter, in vivo AAV model, multiple omics methods, single lab","pmids":["41711888"],"is_preprint":false},{"year":1996,"finding":"Two mutations in the PFKM gene (G1127A transition destroying the 5' donor site of intron 13 causing 155-nt intron retention, and an a-to-g change in intron 16 creating a new acceptor splice site causing 63-nt intron retention) result in compound heterozygous Tarui disease (GSD VII) with predicted premature translation termination.","method":"Genomic DNA amplification, cDNA sequencing, splicing analysis, restriction mapping","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct sequencing with molecular characterization of splicing consequences, established disease-causing mutations","pmids":["8659544"],"is_preprint":false},{"year":1982,"finding":"The human PFKM locus was assigned to chromosome 1 (region cen to q32) using somatic cell hybrid analysis with anti-human M subunit-specific monoclonal antibody.","method":"Somatic cell hybrid panel analysis, monoclonal antibody-based isozyme detection, chromosome marker analysis","journal":"Somatic cell genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic somatic cell hybrid panel with 18 hybrids and specific antibody; subsequently revised to chr 12q by later work","pmids":["6213050"],"is_preprint":false},{"year":1996,"finding":"PFKM maps to chromosome 12q13 (not chromosome 1 as previously reported); PCR with somatic cell hybrid panel showed 100% concordance with chromosome 12, and FISH with CEPH YAC 762G4 localized PFKM centromeric to DAGK at 12q13.3.","method":"PCR with somatic cell hybrid panel, fluorescence in situ hybridization (FISH), genetic mapping with microsatellite marker","journal":"Genomics","confidence":"High","confidence_rationale":"Tier 2 / Strong — two orthogonal mapping methods (PCR hybrid panel + FISH), 100% concordance, corrects prior assignment","pmids":["8661033"],"is_preprint":false},{"year":2024,"finding":"In porcine trophoblast cells, miR-92b-3p targets PFKM mRNA (validated by dual-luciferase reporter assay); PFKM knockdown promoted PTr2 cell proliferation and migration, and uterine miR-92b-3p downregulation reduced embryo implantation rates in mice.","method":"Dual-luciferase reporter assay, RT-qPCR, Western blot, siRNA knockdown of PFKM, mouse uterine miRNA manipulation","journal":"International journal of molecular sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — luciferase and knockdown in porcine cells, functional link is indirect, single lab","pmids":["36555776"],"is_preprint":false}],"current_model":"PFKM (muscle-type phosphofructokinase 1) is the rate-limiting glycolytic enzyme that phosphorylates fructose-6-phosphate; its activity is regulated by S-nitrosylation at Cys351 (by NOS1, stabilizing the active tetramer), ubiquitination/deubiquitination (by USP35), PRMT1-mediated arginine methylation (targeting it for lysosomal degradation via microautophagy downstream of Wnt signaling), and transcriptional control by ZEB1, HIF-1α/METTL3, METTL16/m6A, FTO/C-Jun, and NAT10/ac4C axes; citrate binding converts PFKM tetramers to dimers that translocate to chromatin and phosphorylate histone H3S10 as a non-canonical protein kinase to drive mitotic progression, while nuclear PFKM in macrophages moonlights by interacting with p53 to acetylate it at K120, driving PD-1 transcription and suppressing phagocytosis during sepsis."},"narrative":{"mechanistic_narrative":"PFKM is the muscle-type catalytic subunit of phosphofructokinase-1, the rate-limiting glycolytic enzyme whose abundance and activity are tightly tuned to switch cells between glycolytic and alternative metabolic states across cancer, immune, and fibrotic contexts [PMID:33897890, PMID:41735679]. Its enzymatic output is controlled allosterically and by post-translational modification: NOS1-mediated S-nitrosylation at Cys351 stabilizes the active tetramer and confers resistance to feedback inhibition by downstream intermediates [PMID:33859186], citrate binding dissociates the tetramer into dimers [PMID:40695785], and an upstream AMPK→PFK-2→fructose-2,6-bisphosphate cascade activates PFKM [PMID:16848549]. Protein stability is set by opposing modifications—USP35 deubiquitinates and stabilizes PFKM [PMID:39714773], whereas PRMT1 methylates an arginine degron that routes PFKM to lysosomal microautophagy downstream of Wnt signalling, redirecting glucose toward the pentose phosphate pathway and licensing myofibre differentiation [PMID:41735679]. PFKM expression is governed by a convergent transcriptional and RNA-modification network including ZEB1 [PMID:33897890], HIF-1α/METTL3 [PMID:39549085], METTL16/IGF2BP3 [PMID:39756795], NAT10/ac4C-YTHDC1 [PMID:38233839], and FTO/C-Jun axes [PMID:41184232], and is repressed post-transcriptionally by miR-21 family microRNAs [PMID:31914380, PMID:36253358]. Beyond catalysis, PFKM performs non-glycolytic moonlighting functions: dimeric PFKM enters the nucleus, binds nucleosomes, and phosphorylates histone H3 at Ser10 to drive mitotic progression [PMID:40695785], while nuclear PFKM in septic macrophages interacts with p53 to promote its K120 acetylation, driving PD-1 transcription and suppressing phagocytosis [PMID:41608568]. Its glycolytic lactate output also fuels histone H3K18 lactylation at fibrotic and inflammatory gene promoters such as TGF-β1, CNTN1, and Rela [PMID:40569576, PMID:40608258, PMID:41711888]. Compound heterozygous PFKM splicing mutations cause Tarui disease (glycogen storage disease VII) [PMID:8659544].","teleology":[{"year":1982,"claim":"Establishing where the PFKM gene resides was the first step toward connecting the muscle PFK subunit to human genetics; an initial chromosomal assignment placed the locus on chromosome 1.","evidence":"somatic cell hybrid panel analysis with M-subunit-specific monoclonal antibody","pmids":["6213050"],"confidence":"Medium","gaps":["Assignment was later overturned","No gene structure or sequence determined"]},{"year":1989,"claim":"Identifying a regulator that could reversibly inactivate PFK-1 addressed how the enzyme's activity might be acutely controlled; a Zn2+-binding protein (parathymosin-alpha) was shown to inactivate it Zn2+-dependently.","evidence":"in vitro PFK-1 inactivation assay with a sequenced zinc-binding protein","pmids":["2759245"],"confidence":"Medium","gaps":["Physiological relevance in vivo not established","Binding stoichiometry and site on PFKM unresolved"]},{"year":1996,"claim":"Linking PFKM mutations to disease answered whether loss of this enzyme causes a defined human phenotype; compound heterozygous splicing mutations were shown to cause Tarui disease (GSD VII).","evidence":"genomic and cDNA sequencing with splicing analysis in patients; concurrent FISH/PCR remapping to 12q13","pmids":["8659544","8661033"],"confidence":"Medium","gaps":["Genotype-phenotype variation across patients not detailed","Residual enzyme activity from mutant alleles not quantified"]},{"year":2006,"claim":"Defining the upstream allosteric control of PFKM activity in muscle clarified how energy state drives glycolytic flux; AMPK was shown to activate PFK-2, raising fructose-2,6-bisphosphate to up-regulate PFKM.","evidence":"biochemical activity assays and metabolite quantification in porcine muscle","pmids":["16848549"],"confidence":"Medium","gaps":["Direct PFKM modification not demonstrated","Pathway dissected post-mortem, not in living tissue"]},{"year":2012,"claim":"Testing whether a virus exploits PFKM addressed its role in pathogen-driven metabolic reprogramming; HSV-1 was shown to induce PFK-1 serine phosphorylation and activity required for replication.","evidence":"phosphorylation detection, activity and glycolysis assays, siRNA knockdown with viral replication readout","pmids":["22542512"],"confidence":"Medium","gaps":["Responsible kinase and phosphosites not identified","Direct versus indirect phosphorylation not resolved"]},{"year":2021,"claim":"Mapping transcriptional and modification control of PFKM in cancer answered how glycolytic capacity is amplified in tumors; ZEB1 was shown to directly activate PFKM transcription and NOS1 to S-nitrosylate Cys351 to stabilize the active tetramer.","evidence":"ChIP/luciferase and rescue for ZEB1; S-nitrosoproteomics, C351S mutagenesis, tetramer stability and xenograft assays for NOS1","pmids":["33897890","33859186"],"confidence":"High","gaps":["Whether the two regulatory layers act in the same tumors not tested","Quantitative contribution of Cys351 nitrosylation to flux not measured"]},{"year":2020,"claim":"Determining how innate immunity throttles PFKM clarified its role in macrophage metabolism; miR-21 was shown to directly repress PFKM, and IFN-γ to relieve this repression and drive an isoenzyme switch augmenting glycolysis and IL-1β.","evidence":"miRNA target validation, PFK isoenzyme activity assays, miR-21 manipulation and cytokine measurement in macrophages","pmids":["31914380"],"confidence":"High","gaps":["Other immune contexts of the isoenzyme switch not explored"]},{"year":2024,"claim":"Dissecting RNA-modification and stability control resolved a major axis of PFKM regulation; METTL16/IGF2BP3, HIF-1α/METTL3, NAT10/ac4C-YTHDC1, and FTO/C-Jun were each shown to tune PFKM mRNA or protein levels, while USP35 deubiquitinates and stabilizes PFKM protein.","evidence":"MeRIP/m6A-seq, RIP, mRNA stability and ChIP/luciferase assays plus Co-IP and ubiquitination assays across HCC, osteosarcoma, macrophage, PDAC and cancer models","pmids":["39756795","39549085","38233839","41184232","39714773"],"confidence":"Medium","gaps":["Hierarchy and cross-talk among these axes unknown","Tissue specificity of each axis not unified","Most shown in single cancer contexts"]},{"year":2022,"claim":"Extending miR-21 repression to fibrosis showed PFKM as a deliverable therapeutic node; MSC-exosomal miR-21a-5p was shown to repress PFKM in tubular cells, attenuating glycolysis and renal fibrosis.","evidence":"miRNA sequencing, target validation, glycolysis assays and UUO mouse model with rescue","pmids":["36253358"],"confidence":"Medium","gaps":["Single fibrosis model","Direct PFKM rescue of the renoprotective phenotype not isolated"]},{"year":2025,"claim":"Uncovering nuclear moonlighting functions answered whether PFKM acts beyond catalysis; citrate-driven dimers phosphorylate histone H3S10 to drive mitosis, and nuclear PFKM interacts with p53 to promote K120 acetylation and PD-1 transcription, suppressing macrophage phagocytosis.","evidence":"structural simulation, in vitro histone kinase assay with mutagenesis for H3S10; Co-IP/MS, ChIP, luciferase and nanobody-blocking sepsis models for p53","pmids":["40695785","41608568"],"confidence":"High","gaps":["How a glycolytic enzyme acquired kinase/scaffold activity mechanistically unresolved","Generality of nuclear PFKM across cell types not defined","Relationship between H3S10 kinase and p53-acetylation functions unknown"]},{"year":2025,"claim":"Connecting PFKM-derived lactate to histone lactylation defined how its metabolic output reprograms gene expression in disease; PFKM was shown to drive H3K18 lactylation at TGF-β1, CNTN1 and Rela promoters, promoting fibrosis, gastric cancer and renal inflammation.","evidence":"AAV-mediated PFKM manipulation, ChIP/CUT&Tag for H3K18la at target promoters, Co-IP and rescue in fibroblast, gastric cancer and renal models","pmids":["40569576","40608258","41711888"],"confidence":"Medium","gaps":["Whether PFKM physically engages chromatin writers versus acting purely metabolically not fully resolved","Specificity of lactylation targeting unexplained"]},{"year":2025,"claim":"Defining a degradation route for PFKM revealed how cells switch glucose fate; Wnt-induced PRMT1 methylation of an arginine degron delivers PFKM to lysosomal microautophagy, shifting flux toward the pentose phosphate pathway and enabling myofibre differentiation.","evidence":"PTM identification, metabolic flux analysis, lysosomal inhibition and 3-phosphoglycerate rescue with knockdown/overexpression","pmids":["41735679"],"confidence":"High","gaps":["Generality of microautophagic degradation beyond muscle differentiation not tested","Reader machinery for the methyl-arginine degron incompletely defined"]},{"year":null,"claim":"How PFKM's catalytic, kinase, and scaffolding activities are coordinately partitioned in space and time—and which contexts favor each—remains unresolved.","evidence":"no single study integrates the tetramer-dimer-nuclear transitions with the competing regulatory inputs","pmids":[],"confidence":"Low","gaps":["No structural model unifies catalytic and H3S10 kinase states","Triggers selecting moonlighting versus glycolytic roles unknown","Interplay of stabilizing and degradative PTMs not mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[3]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[3]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3,17]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[3,9]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,2,4]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[3]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,9]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[5,6,7]}],"complexes":[],"partners":["NOS1","USP35","PRMT1","TP53","CNTN1","RAB8B"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P08237","full_name":"ATP-dependent 6-phosphofructokinase, muscle type","aliases":["6-phosphofructokinase type A","Phosphofructo-1-kinase isozyme A","PFK-A","Phosphohexokinase"],"length_aa":780,"mass_kda":85.2,"function":"Catalyzes the phosphorylation of D-fructose 6-phosphate to fructose 1,6-bisphosphate by ATP, the first committing step of glycolysis","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/P08237/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PFKM","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PFKM","total_profiled":1310},"omim":[{"mim_id":"616302","title":"FORKHEAD BOX K1; FOXK1","url":"https://www.omim.org/entry/616302"},{"mim_id":"611556","title":"GLYCOGEN STORAGE DISEASE 0, MUSCLE; GSD0B","url":"https://www.omim.org/entry/611556"},{"mim_id":"610681","title":"PHOSPHOFRUCTOKINASE, MUSCLE TYPE; PFKM","url":"https://www.omim.org/entry/610681"},{"mim_id":"232800","title":"GLYCOGEN STORAGE DISEASE VII; GSD7","url":"https://www.omim.org/entry/232800"},{"mim_id":"171860","title":"PHOSPHOFRUCTOKINASE, LIVER TYPE; PFKL","url":"https://www.omim.org/entry/171860"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Endoplasmic reticulum","reliability":"Approved"},{"location":"Principal piece","reliability":"Additional"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"skeletal muscle","ntpm":1472.3},{"tissue":"tongue","ntpm":1300.4}],"url":"https://www.proteinatlas.org/search/PFKM"},"hgnc":{"alias_symbol":["PFK-1","PPP1R122"],"prev_symbol":["PFKX"]},"alphafold":{"accession":"P08237","domains":[{"cath_id":"3.40.50.450","chopping":"16-174_305-359","consensus_level":"high","plddt":93.2902,"start":16,"end":359},{"cath_id":"3.40.50.460","chopping":"178-300","consensus_level":"high","plddt":93.3948,"start":178,"end":300},{"cath_id":"3.40.50.450","chopping":"397-530_672-704_714-730","consensus_level":"high","plddt":93.9015,"start":397,"end":730},{"cath_id":"3.40.50.460","chopping":"542-663","consensus_level":"high","plddt":94.1094,"start":542,"end":663}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P08237","model_url":"https://alphafold.ebi.ac.uk/files/AF-P08237-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P08237-F1-predicted_aligned_error_v6.png","plddt_mean":91.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PFKM","jax_strain_url":"https://www.jax.org/strain/search?query=PFKM"},"sequence":{"accession":"P08237","fasta_url":"https://rest.uniprot.org/uniprotkb/P08237.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P08237/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P08237"}},"corpus_meta":[{"pmid":"33897890","id":"PMC_33897890","title":"ZEB1 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ZEB1-binding sequence in the PFKM promoter region, upregulating PFKM expression and glycolysis in HCC cells; silencing ZEB1 impairs PFKM expression, glycolysis, proliferation and invasion, and exogenous PFKM re-expression rescues these defects.\",\n      \"method\": \"shRNA knockdown, ChIP assay, luciferase reporter assay, RT-qPCR, Western blot, ECAR/OCR assays, orthotopic xenograft\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal rescue experiments, ChIP and luciferase reporter, in vivo xenograft, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"33897890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Mycobacterium tuberculosis-induced miR-21 directly targets PFKM mRNA to repress its expression and activity in macrophages, dampening glycolysis and limiting IL-1β production; IFN-γ inhibits miR-21, forcing an isoenzyme switch that augments PFKM expression and macrophage glycolysis.\",\n      \"method\": \"miRNA target validation, PFK isoenzyme activity assays, miR-21 manipulation in macrophages, cytokine measurement\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (activity assay, miRNA targeting, IFN-γ rescue), independently reported mechanism\",\n      \"pmids\": [\"31914380\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NOS1 S-nitrosylates PFKM at Cys351, stabilizing the PFKM tetramer and enabling resistance to negative feedback from downstream metabolic intermediates, thereby promoting glycolysis in ovarian cancer cells; PFKM-C351S mutation reduced proliferation and tumor growth in xenograft models.\",\n      \"method\": \"S-nitrosoproteomic profiling, site-directed mutagenesis (C351S), in vitro tetramer stability assay, xenograft tumor model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — site-directed mutagenesis with functional readout, proteomic identification of modification site, in vivo validation\",\n      \"pmids\": [\"33859186\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Citrate binds PFKM and disrupts its tetrameric structure into dimers; dimeric PFKM interacts with nucleosomes and phosphorylates histone H3 at serine 10 (H3S10) as a protein kinase, promoting mitotic progression and cell proliferation. Structural simulations show PFKM binds nucleosomes optimally when H3S10 aligns with its catalytic site.\",\n      \"method\": \"Structural simulation, biochemical citrate-binding assays, in vitro histone kinase assay, mutagenesis disrupting citrate-PFKM and PFKM-H3 interactions, cell cycle analysis, tumor growth assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with mutagenesis, structural modeling, multiple functional readouts in single rigorous study\",\n      \"pmids\": [\"40695785\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Wnt signalling induces lysosomal degradation of PFKM through a methyl arginine degron motif that is selectively methylated by PRMT1, delivering PFKM to lysosomes via microautophagy; PFKM degradation shifts glucose metabolism from glycolysis to the pentose phosphate pathway, and PFKM overexpression promotes myofibre differentiation while PFKM knockdown blunts differentiation (rescued by 3-phosphoglycerate supplementation).\",\n      \"method\": \"Genetic knockdown/overexpression, metabolic flux analysis, PRMT1-mediated methylation validation, lysosomal inhibition experiments, metabolic rescue with 3-phosphoglycerate\",\n      \"journal\": \"Nature metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods including PTM identification, pathway rescue, metabolic flux, mechanistic and functional validation\",\n      \"pmids\": [\"41735679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"METTL16 mediates m6A methylation to stabilize PFKM mRNA in an IGF2BP3-dependent manner in HCC, promoting glycolysis; POU3F2 transcriptionally activates METTL16, defining a POU3F2/METTL16/PFKM axis.\",\n      \"method\": \"MeRIP assay, RIP assay, actinomycin D mRNA stability assay, ChIP, luciferase assay, knockdown/overexpression, xenograft\",\n      \"journal\": \"Annals of hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MeRIP, RIP, ChIP and luciferase in single lab with multiple orthogonal methods\",\n      \"pmids\": [\"39756795\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NAT10-mediated ac4C acetylation suppresses m6A modification on PFKM mRNA; YTHDC1 recognizes m6A sites on PFKM mRNA to increase its stability; NAT10 knockdown increases m6A content, reducing YTHDC1 translation, and destabilizing PFKM mRNA, leading to decreased glycolysis in osteosarcoma cells.\",\n      \"method\": \"NAT10 knockdown, m6A-seq, RIP assay, m6A reader protein analysis, mRNA stability assay, in vivo tumor models\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — m6A sequencing, RIP, mRNA stability, rescue experiments in single lab\",\n      \"pmids\": [\"38233839\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HIF-1α increases METTL3 expression, which elevates m6A modification on Pfkm mRNA and upregulates PFKM protein expression in macrophages; recombinant thrombomodulin (rTM) decreases PFKM expression in a HIF-1α-dependent manner, and METTL3 silencing attenuates HIF-1α-mediated PFKM upregulation.\",\n      \"method\": \"HIF-1α overexpression/knockdown, METTL3 silencing, m6A quantification, Pfkm knockout mice, ELISA cytokine measurement\",\n      \"journal\": \"Cellular and molecular life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis (HIF-1α → METTL3 → PFKM), m6A measurement, in vivo knockout model, single lab\",\n      \"pmids\": [\"39549085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP35 deubiquitinates and stabilizes PFK-1 (PFKM) protein; Co-IP identified PFK-1 as a direct deubiquitination target of USP35, and USP35 knockdown decreased PFK-1 expression, reducing glycolysis and cancer cell proliferation.\",\n      \"method\": \"Co-immunoprecipitation, immunoprecipitation/ubiquitination assay, USP35 knockdown, Seahorse glycolysis assay, xenograft\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP establishing direct interaction, ubiquitination assay, in vivo validation, single lab\",\n      \"pmids\": [\"39714773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PFKM translocates to the macrophage nucleus during sepsis and interacts with p53 (identified by Co-IP/mass spectrometry); nuclear PFKM promotes p53 acetylation at K120, enhancing p53 binding to the Pdcd1 promoter to drive PD-1 transcription, thereby suppressing macrophage phagocytosis via a non-glycolytic moonlighting function.\",\n      \"method\": \"Co-IP coupled with mass spectrometry, immunofluorescence for nuclear localization, dual-luciferase reporter, ChIP, transcriptomic sequencing, nanobody blocking, sepsis mouse models\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP/MS interaction identification, ChIP, luciferase, in vivo rescue with nanobody, multiple orthogonal methods\",\n      \"pmids\": [\"41608568\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PFKM drives lactate accumulation which promotes global and H3K18 lactylation in atrial fibroblasts; P300-mediated H3K18 lactylation at the TGF-β1 promoter upregulates TGF-β1 transcription, activating cardiac fibroblasts and contributing to atrial fibrosis. AAV-mediated atrial PFKM expression confirmed PFKM as the pivotal glycolytic driver in this pathway.\",\n      \"method\": \"AAV-mediated PFKM overexpression in vivo, glycolysis inhibitor (2-DG) treatment, ChIP for H3K18la at TGF-β1 promoter, primary cardiac fibroblast activation assays\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo AAV overexpression, ChIP for histone modification at target promoter, single lab\",\n      \"pmids\": [\"40569576\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PFKM promotes gastric cancer progression by interacting with CNTN1 and facilitating enrichment of H3K18 lactylation at the CNTN1 promoter, activating CNTN1 transcription; CNTN1 overexpression reverses the inhibitory effects of PFKM knockdown.\",\n      \"method\": \"Co-immunoprecipitation (PFKM-CNTN1 interaction), dual-luciferase reporter, ChIP for H3K18la at CNTN1 promoter, knockdown/overexpression rescue, xenograft\",\n      \"journal\": \"Applied biochemistry and biotechnology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ChIP, luciferase, rescue experiments in single lab\",\n      \"pmids\": [\"40608258\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FTO demethylase elevates C-Jun mRNA in a m6A-YTHDF2-dependent manner, and C-Jun transcriptionally upregulates PFKM expression, thereby promoting glycolysis in pancreatic ductal adenocarcinoma; FTO inhibitors suppress PDAC growth in organoid and xenograft models.\",\n      \"method\": \"m6A-seq, transcriptome sequencing, metabolomics, FTO-conditional knockout mouse model, organoids, xenograft, ChIP for C-Jun at PFKM promoter\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiomics, conditional KO mouse, organoid validation, in vivo xenograft, single lab\",\n      \"pmids\": [\"41184232\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"miR-21a-5p in bone marrow MSC-derived exosomes directly represses PFKM expression (a rate-limiting glycolytic enzyme) in tubular epithelial cells, attenuating glycolysis and renal fibrosis; knockdown of miR-21a-5p abolished the renoprotective effect of MSC-Exos.\",\n      \"method\": \"miRNA sequencing, in vitro miR-21a-5p targeting validation, UUO mouse model, glycolysis assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — miRNA targeting validated, in vivo UUO model, rescue experiments, single lab\",\n      \"pmids\": [\"36253358\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"HSV-1 infection increases PFK-1 expression and triggers phosphorylation of PFK-1 at serine residues, increasing total PFK-1 activity and glycolysis; PFK-1 knockdown impairs HSV-1 replication.\",\n      \"method\": \"Western blot, glucose uptake/lactate efflux assays, serine phosphorylation detection, PFK-1 siRNA knockdown, viral replication assay\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — enzyme activity assay, knockdown with functional readout (viral replication), phosphorylation detection, single lab\",\n      \"pmids\": [\"22542512\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"AMPK phosphorylates and activates PFK-2 post-mortem, increasing fructose-2,6-diphosphate levels which then up-regulate PFK-1 (PFKM) activity; earlier AMPK activation in PSE porcine muscle leads to faster glycolysis and lower pH.\",\n      \"method\": \"Biochemical activity assays (AMPK, PFK-1, glycogen phosphorylase, pyruvate kinase), fructose-2,6-diphosphate measurement, pH and metabolite quantification in porcine muscle\",\n      \"journal\": \"Journal of agricultural and food chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — enzyme activity assays with defined pathway dissection, metabolite quantification, single lab\",\n      \"pmids\": [\"16848549\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1989,\n      \"finding\": \"A Zn2+-binding protein (identified as parathymosin-alpha) reversibly inactivates phosphofructokinase-1 (PFK-1/PFKM) in a Zn2+-dependent manner via its 43-amino-acid zinc-binding domain.\",\n      \"method\": \"cDNA sequencing, protein sequence comparison, in vitro PFK-1 inactivation assay with Zn2+-binding protein\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro functional inactivation assay, protein identification by sequence, single lab\",\n      \"pmids\": [\"2759245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PFKM influences exosome release by modulating RAB8B expression; the PFKM-RAB8B interaction promotes chemoresistance in lung adenocarcinoma by impacting apoptosis and glycolytic metabolism, with drug-resistant cells releasing glycolytically active exosomes that transfer chemoresistance to sensitive cells.\",\n      \"method\": \"Targeted metabolomics (FBP/F6P ratio as PFKM activity proxy), exosome isolation and characterization, PFKM-RAB8B interaction analysis, knockdown/overexpression\",\n      \"journal\": \"Journal of pharmaceutical analysis\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, interaction validation method not specified in abstract, metabolomics-based activity inference\",\n      \"pmids\": [\"42057963\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HDAC1 inhibits H3K27ac-induced transcription of PFKM in doxorubicin-treated cardiomyocytes; PFKM overexpression inhibits DOX-induced apoptosis and maintains glycolysis and oxidative phosphorylation, while PFKM silencing promotes apoptosis.\",\n      \"method\": \"RT-PCR, immunoblotting, PFKM overexpression/knockdown, Seahorse metabolic assay, apoptosis assay in H9c2 cells\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — HDAC1/H3K27ac mechanism inferred from expression changes without direct ChIP or interaction assay shown in abstract, single lab\",\n      \"pmids\": [\"35804014\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Chloroquine (CQ) directly binds PFKM and inhibits its expression and enzymatic activity, thereby blocking the Warburg effect in colorectal cancer cells; additionally, CHKA downregulation also decreases PFKM expression and activity.\",\n      \"method\": \"Target identification and binding verification (method not fully described in abstract), enzymatic activity assay, knockdown/expression analysis\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — binding target identification method not specified in abstract, single lab, limited mechanistic detail\",\n      \"pmids\": [\"39990656\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"PFKM drives glycolytic reprogramming leading to lactate accumulation, which promotes H3K18 lactylation at the Rela promoter via histone modification, activating NF-κB pathway and exacerbating renal inflammation and fibrosis; AAV9-mediated PFKM knockdown alleviated FA-induced renal fibrosis in mice.\",\n      \"method\": \"AAV9 PFKM knockdown/overexpression in renal tubular cells, RNA-seq, CUT&Tag for H3K18la, ChIP-qPCR, metabolic analysis, histological staining\",\n      \"journal\": \"Cellular and molecular life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CUT&Tag and ChIP-qPCR for histone modification at target promoter, in vivo AAV model, multiple omics methods, single lab\",\n      \"pmids\": [\"41711888\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Two mutations in the PFKM gene (G1127A transition destroying the 5' donor site of intron 13 causing 155-nt intron retention, and an a-to-g change in intron 16 creating a new acceptor splice site causing 63-nt intron retention) result in compound heterozygous Tarui disease (GSD VII) with predicted premature translation termination.\",\n      \"method\": \"Genomic DNA amplification, cDNA sequencing, splicing analysis, restriction mapping\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct sequencing with molecular characterization of splicing consequences, established disease-causing mutations\",\n      \"pmids\": [\"8659544\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1982,\n      \"finding\": \"The human PFKM locus was assigned to chromosome 1 (region cen to q32) using somatic cell hybrid analysis with anti-human M subunit-specific monoclonal antibody.\",\n      \"method\": \"Somatic cell hybrid panel analysis, monoclonal antibody-based isozyme detection, chromosome marker analysis\",\n      \"journal\": \"Somatic cell genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic somatic cell hybrid panel with 18 hybrids and specific antibody; subsequently revised to chr 12q by later work\",\n      \"pmids\": [\"6213050\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"PFKM maps to chromosome 12q13 (not chromosome 1 as previously reported); PCR with somatic cell hybrid panel showed 100% concordance with chromosome 12, and FISH with CEPH YAC 762G4 localized PFKM centromeric to DAGK at 12q13.3.\",\n      \"method\": \"PCR with somatic cell hybrid panel, fluorescence in situ hybridization (FISH), genetic mapping with microsatellite marker\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two orthogonal mapping methods (PCR hybrid panel + FISH), 100% concordance, corrects prior assignment\",\n      \"pmids\": [\"8661033\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In porcine trophoblast cells, miR-92b-3p targets PFKM mRNA (validated by dual-luciferase reporter assay); PFKM knockdown promoted PTr2 cell proliferation and migration, and uterine miR-92b-3p downregulation reduced embryo implantation rates in mice.\",\n      \"method\": \"Dual-luciferase reporter assay, RT-qPCR, Western blot, siRNA knockdown of PFKM, mouse uterine miRNA manipulation\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — luciferase and knockdown in porcine cells, functional link is indirect, single lab\",\n      \"pmids\": [\"36555776\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PFKM (muscle-type phosphofructokinase 1) is the rate-limiting glycolytic enzyme that phosphorylates fructose-6-phosphate; its activity is regulated by S-nitrosylation at Cys351 (by NOS1, stabilizing the active tetramer), ubiquitination/deubiquitination (by USP35), PRMT1-mediated arginine methylation (targeting it for lysosomal degradation via microautophagy downstream of Wnt signaling), and transcriptional control by ZEB1, HIF-1α/METTL3, METTL16/m6A, FTO/C-Jun, and NAT10/ac4C axes; citrate binding converts PFKM tetramers to dimers that translocate to chromatin and phosphorylate histone H3S10 as a non-canonical protein kinase to drive mitotic progression, while nuclear PFKM in macrophages moonlights by interacting with p53 to acetylate it at K120, driving PD-1 transcription and suppressing phagocytosis during sepsis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PFKM is the muscle-type catalytic subunit of phosphofructokinase-1, the rate-limiting glycolytic enzyme whose abundance and activity are tightly tuned to switch cells between glycolytic and alternative metabolic states across cancer, immune, and fibrotic contexts [#0, #4]. Its enzymatic output is controlled allosterically and by post-translational modification: NOS1-mediated S-nitrosylation at Cys351 stabilizes the active tetramer and confers resistance to feedback inhibition by downstream intermediates [#2], citrate binding dissociates the tetramer into dimers [#3], and an upstream AMPK\\u2192PFK-2\\u2192fructose-2,6-bisphosphate cascade activates PFKM [#15]. Protein stability is set by opposing modifications\\u2014USP35 deubiquitinates and stabilizes PFKM [#8], whereas PRMT1 methylates an arginine degron that routes PFKM to lysosomal microautophagy downstream of Wnt signalling, redirecting glucose toward the pentose phosphate pathway and licensing myofibre differentiation [#4]. PFKM expression is governed by a convergent transcriptional and RNA-modification network including ZEB1 [#0], HIF-1\\u03b1/METTL3 [#7], METTL16/IGF2BP3 [#5], NAT10/ac4C-YTHDC1 [#6], and FTO/C-Jun axes [#12], and is repressed post-transcriptionally by miR-21 family microRNAs [#1, #13]. Beyond catalysis, PFKM performs non-glycolytic moonlighting functions: dimeric PFKM enters the nucleus, binds nucleosomes, and phosphorylates histone H3 at Ser10 to drive mitotic progression [#3], while nuclear PFKM in septic macrophages interacts with p53 to promote its K120 acetylation, driving PD-1 transcription and suppressing phagocytosis [#9]. Its glycolytic lactate output also fuels histone H3K18 lactylation at fibrotic and inflammatory gene promoters such as TGF-\\u03b21, CNTN1, and Rela [#10, #11, #20]. Compound heterozygous PFKM splicing mutations cause Tarui disease (glycogen storage disease VII) [#21].\",\n  \"teleology\": [\n    {\n      \"year\": 1982,\n      \"claim\": \"Establishing where the PFKM gene resides was the first step toward connecting the muscle PFK subunit to human genetics; an initial chromosomal assignment placed the locus on chromosome 1.\",\n      \"evidence\": \"somatic cell hybrid panel analysis with M-subunit-specific monoclonal antibody\",\n      \"pmids\": [\"6213050\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Assignment was later overturned\", \"No gene structure or sequence determined\"]\n    },\n    {\n      \"year\": 1989,\n      \"claim\": \"Identifying a regulator that could reversibly inactivate PFK-1 addressed how the enzyme's activity might be acutely controlled; a Zn2+-binding protein (parathymosin-alpha) was shown to inactivate it Zn2+-dependently.\",\n      \"evidence\": \"in vitro PFK-1 inactivation assay with a sequenced zinc-binding protein\",\n      \"pmids\": [\"2759245\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological relevance in vivo not established\", \"Binding stoichiometry and site on PFKM unresolved\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Linking PFKM mutations to disease answered whether loss of this enzyme causes a defined human phenotype; compound heterozygous splicing mutations were shown to cause Tarui disease (GSD VII).\",\n      \"evidence\": \"genomic and cDNA sequencing with splicing analysis in patients; concurrent FISH/PCR remapping to 12q13\",\n      \"pmids\": [\"8659544\", \"8661033\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Genotype-phenotype variation across patients not detailed\", \"Residual enzyme activity from mutant alleles not quantified\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defining the upstream allosteric control of PFKM activity in muscle clarified how energy state drives glycolytic flux; AMPK was shown to activate PFK-2, raising fructose-2,6-bisphosphate to up-regulate PFKM.\",\n      \"evidence\": \"biochemical activity assays and metabolite quantification in porcine muscle\",\n      \"pmids\": [\"16848549\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct PFKM modification not demonstrated\", \"Pathway dissected post-mortem, not in living tissue\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Testing whether a virus exploits PFKM addressed its role in pathogen-driven metabolic reprogramming; HSV-1 was shown to induce PFK-1 serine phosphorylation and activity required for replication.\",\n      \"evidence\": \"phosphorylation detection, activity and glycolysis assays, siRNA knockdown with viral replication readout\",\n      \"pmids\": [\"22542512\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Responsible kinase and phosphosites not identified\", \"Direct versus indirect phosphorylation not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Mapping transcriptional and modification control of PFKM in cancer answered how glycolytic capacity is amplified in tumors; ZEB1 was shown to directly activate PFKM transcription and NOS1 to S-nitrosylate Cys351 to stabilize the active tetramer.\",\n      \"evidence\": \"ChIP/luciferase and rescue for ZEB1; S-nitrosoproteomics, C351S mutagenesis, tetramer stability and xenograft assays for NOS1\",\n      \"pmids\": [\"33897890\", \"33859186\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the two regulatory layers act in the same tumors not tested\", \"Quantitative contribution of Cys351 nitrosylation to flux not measured\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Determining how innate immunity throttles PFKM clarified its role in macrophage metabolism; miR-21 was shown to directly repress PFKM, and IFN-\\u03b3 to relieve this repression and drive an isoenzyme switch augmenting glycolysis and IL-1\\u03b2.\",\n      \"evidence\": \"miRNA target validation, PFK isoenzyme activity assays, miR-21 manipulation and cytokine measurement in macrophages\",\n      \"pmids\": [\"31914380\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Other immune contexts of the isoenzyme switch not explored\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Dissecting RNA-modification and stability control resolved a major axis of PFKM regulation; METTL16/IGF2BP3, HIF-1\\u03b1/METTL3, NAT10/ac4C-YTHDC1, and FTO/C-Jun were each shown to tune PFKM mRNA or protein levels, while USP35 deubiquitinates and stabilizes PFKM protein.\",\n      \"evidence\": \"MeRIP/m6A-seq, RIP, mRNA stability and ChIP/luciferase assays plus Co-IP and ubiquitination assays across HCC, osteosarcoma, macrophage, PDAC and cancer models\",\n      \"pmids\": [\"39756795\", \"39549085\", \"38233839\", \"41184232\", \"39714773\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Hierarchy and cross-talk among these axes unknown\", \"Tissue specificity of each axis not unified\", \"Most shown in single cancer contexts\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extending miR-21 repression to fibrosis showed PFKM as a deliverable therapeutic node; MSC-exosomal miR-21a-5p was shown to repress PFKM in tubular cells, attenuating glycolysis and renal fibrosis.\",\n      \"evidence\": \"miRNA sequencing, target validation, glycolysis assays and UUO mouse model with rescue\",\n      \"pmids\": [\"36253358\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single fibrosis model\", \"Direct PFKM rescue of the renoprotective phenotype not isolated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Uncovering nuclear moonlighting functions answered whether PFKM acts beyond catalysis; citrate-driven dimers phosphorylate histone H3S10 to drive mitosis, and nuclear PFKM interacts with p53 to promote K120 acetylation and PD-1 transcription, suppressing macrophage phagocytosis.\",\n      \"evidence\": \"structural simulation, in vitro histone kinase assay with mutagenesis for H3S10; Co-IP/MS, ChIP, luciferase and nanobody-blocking sepsis models for p53\",\n      \"pmids\": [\"40695785\", \"41608568\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How a glycolytic enzyme acquired kinase/scaffold activity mechanistically unresolved\", \"Generality of nuclear PFKM across cell types not defined\", \"Relationship between H3S10 kinase and p53-acetylation functions unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connecting PFKM-derived lactate to histone lactylation defined how its metabolic output reprograms gene expression in disease; PFKM was shown to drive H3K18 lactylation at TGF-\\u03b21, CNTN1 and Rela promoters, promoting fibrosis, gastric cancer and renal inflammation.\",\n      \"evidence\": \"AAV-mediated PFKM manipulation, ChIP/CUT&Tag for H3K18la at target promoters, Co-IP and rescue in fibroblast, gastric cancer and renal models\",\n      \"pmids\": [\"40569576\", \"40608258\", \"41711888\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether PFKM physically engages chromatin writers versus acting purely metabolically not fully resolved\", \"Specificity of lactylation targeting unexplained\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defining a degradation route for PFKM revealed how cells switch glucose fate; Wnt-induced PRMT1 methylation of an arginine degron delivers PFKM to lysosomal microautophagy, shifting flux toward the pentose phosphate pathway and enabling myofibre differentiation.\",\n      \"evidence\": \"PTM identification, metabolic flux analysis, lysosomal inhibition and 3-phosphoglycerate rescue with knockdown/overexpression\",\n      \"pmids\": [\"41735679\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality of microautophagic degradation beyond muscle differentiation not tested\", \"Reader machinery for the methyl-arginine degron incompletely defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PFKM's catalytic, kinase, and scaffolding activities are coordinately partitioned in space and time\\u2014and which contexts favor each\\u2014remains unresolved.\",\n      \"evidence\": \"no single study integrates the tetramer-dimer-nuclear transitions with the competing regulatory inputs\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model unifies catalytic and H3S10 kinase states\", \"Triggers selecting moonlighting versus glycolytic roles unknown\", \"Interplay of stabilizing and degradative PTMs not mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0016301\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3, 17]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3, 9]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 2, 4]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 9]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [5, 6, 7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"NOS1\", \"USP35\", \"PRMT1\", \"TP53\", \"CNTN1\", \"RAB8B\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":7,"faith_total":7,"faith_pct":100.0}}