{"gene":"G6PD","run_date":"2026-06-09T23:54:44","timeline":{"discoveries":[{"year":2014,"finding":"G6PD is negatively regulated by acetylation on lysine 403 (K403), an evolutionarily conserved residue. K403-acetylated G6PD is incapable of forming active dimers and displays complete loss of activity. SIRT2 deacetylates K403 to activate G6PD in response to oxidative stress. KAT9/ELP3 was identified as a potential acetyltransferase of G6PD.","method":"Acetylation-mimetic mutants, in vitro enzymatic activity assays, Co-IP, knockdown/rescue experiments in cells and mouse erythrocytes, SIRT2 depletion","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods including mutagenesis, in vitro activity assays, and cellular rescue; replicated across cell lines and erythrocytes","pmids":["24769394"],"is_preprint":false},{"year":2016,"finding":"SIRT5 deglutarylates G6PD, activating the enzyme and increasing NADPH production. Knockdown or knockout of SIRT5 leads to inhibition of G6PD activity, decreased NADPH, lowered GSH, and increased cellular susceptibility to oxidative stress.","method":"SIRT5 KO/knockdown, enzymatic activity assays, NADPH and GSH measurements, ROS quantification","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genetic and biochemical validation including KO and activity assays, replicated with multiple orthogonal methods","pmids":["27113762"],"is_preprint":false},{"year":2015,"finding":"G6PD is dynamically modified by O-linked β-N-acetylglucosamine (O-GlcNAcylation) in response to hypoxia. This glycosylation activates G6PD activity, increases glucose flux through the PPP, and promotes nucleotide/lipid biosynthesis and antioxidant defense. Blocking G6PD glycosylation reduces cancer cell proliferation in vitro and impairs tumor growth in vivo.","method":"O-GlcNAc modification assays, enzymatic activity assays, metabolic flux analysis, glycosylation site mutagenesis, in vivo xenograft models","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — direct biochemical identification of modification site, functional rescue by mutagenesis, in vitro and in vivo validation","pmids":["26399441"],"is_preprint":false},{"year":2013,"finding":"PTEN protein directly binds G6PD and prevents formation of the active G6PD dimer, thereby inhibiting PPP flux. Tcl1, acting via hnRNPK, promotes G6PD pre-mRNA splicing and protein expression. PTEN also forms a complex with hnRNPK to inhibit G6PD pre-mRNA splicing. PTEN inactivates Tcl1 via GSK3β-mediated phosphorylation.","method":"Co-immunoprecipitation, mass spectrometry, molecular biology assays, PPP flux measurements, epistasis analysis","journal":"Gut","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, mass spectrometry, multiple orthogonal functional assays in a single study","pmids":["24352616"],"is_preprint":false},{"year":2016,"finding":"SIRT2 promotes G6PD deacetylation at K403, activating G6PD to increase NADPH production and support leukaemia cell proliferation. Chemical inhibition of SIRT2 suppresses G6PD activity and reduces leukaemia cell but not normal hematopoietic cell proliferation.","method":"SIRT2 knockdown, K403 acetylation-mimetic mutants, enzymatic activity assays, colony formation assays, SIRT2 inhibitors, patient AML sample analysis","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — site-specific mutagenesis, enzymatic assays, pharmacological validation, corroborated by independent paper (PMID 24769394)","pmids":["27586085"],"is_preprint":false},{"year":2020,"finding":"Aldolase B (Aldob) directly binds G6PD and inhibits its enzymatic activity, suppressing PPP metabolism. Aldob potentiates p53-mediated inhibition of G6PD by forming an Aldob-G6PD-p53 complex. This scaffolding effect is independent of Aldob enzymatic activity.","method":"Direct binding assays, Co-IP, enzymatic activity assays, Aldob KO mouse model, Aldob/G6PD re-expression rescue experiments, pharmacological G6PD inhibition","journal":"Nature cancer","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct protein-protein interaction demonstrated, KO mouse model with rescue, multiple orthogonal methods","pmids":["35122041"],"is_preprint":false},{"year":2021,"finding":"c-Src tyrosine kinase directly interacts with and phosphorylates G6PD at Tyr112, enhancing catalytic activity by decreasing Km and increasing Kcat for glucose-6-phosphate substrate, thereby augmenting PPP flux for NADPH and ribose-5-phosphate production.","method":"Co-IP, in vitro kinase assay, Km/Kcat kinetic analysis, site-directed mutagenesis, metabolic flux assays, clinical CRC sample correlation","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with kinetic characterization, mutagenesis validation, single lab","pmids":["33686238"],"is_preprint":false},{"year":2019,"finding":"G6PD is ubiquitinated on K366 and K403 by the VHL E3 ubiquitin ligase, which directly binds G6PD, leading to G6PD proteasomal degradation under high glucose conditions and resulting in ROS accumulation and podocyte injury.","method":"Co-IP demonstrating VHL-G6PD interaction, site-directed mutagenesis of ubiquitination sites, Western blot, G6PD overexpression rescue, siRNA knockdown, G6PD-deficient mouse kidney analysis","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct Co-IP, site-specific mutagenesis of ubiquitination sites K366/K403, in vivo mouse validation, multiple orthogonal methods","pmids":["30785802"],"is_preprint":false},{"year":2004,"finding":"G6PD is indispensable for definitive erythropoiesis after the embryonic-adult hemoglobin switch. G6PD-null ES cells differentiate normally into primitive erythroid cells but definitive erythrocytes undergo apoptosis that is prevented only by restoration of G6PD activity.","method":"G6PD-null mouse ES cell differentiation (embryoid body system), apoptosis assays, reducing agents rescue, caspase inhibitor rescue, G6PD re-expression rescue","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic null model with specific rescue by G6PD re-expression, multiple rescue conditions tested","pmids":["15271799"],"is_preprint":false},{"year":2016,"finding":"HSPB1 (Hsp27) enhances the binding between G6PD and SIRT2, leading to deacetylation and activation of G6PD, thereby sustaining cellular NADPH and pentose production in response to oxidative stress or DNA damage.","method":"Co-IP, enzymatic activity assays, NADPH measurement, siRNA knockdown, HSPB1 overexpression","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — reciprocal Co-IP demonstrating HSPB1-G6PD-SIRT2 complex, enzymatic activity validation, single lab, single study","pmids":["27711253"],"is_preprint":false},{"year":2017,"finding":"PAK4 interacts with G6PD and increases G6PD activity via enhancing Mdm2-mediated p53 ubiquitination and degradation (as p53 suppresses G6PD), thereby promoting glucose intake, NADPH production, lipid biosynthesis and colon cancer cell proliferation.","method":"Co-IP, G6PD enzymatic activity assays, metabolic measurements, p53 ubiquitination assays, siRNA knockdown, clinical correlation","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, epistasis via p53 pathway, enzymatic activity assays, single lab with multiple orthogonal methods","pmids":["28542136"],"is_preprint":false},{"year":2021,"finding":"TSP50 binds G6PD in the cytoplasm and activates G6PD activity by inhibiting acetylation of G6PD at K171. TSP50 also promotes the binding of G6PD to SIRT2. K171 acetylation of G6PD is required for TSP50-induced cell proliferation and tumor formation.","method":"LC-MS/MS, Co-IP, GST pull-down, site-specific mutation of K171, enzymatic activity assays, cell proliferation and tumor formation assays","journal":"Cell proliferation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pull-down and Co-IP, site-specific mutagenesis, multiple methods in single lab","pmids":["33630390"],"is_preprint":false},{"year":2023,"finding":"Acetylation at K89 activates G6PD while acetylation at K403 inhibits G6PD. K403 acetylation-dependent inactivation is explained by structural distortion of the dimeric structure and active site. K403 acetylation also leads to K95/97 ubiquitylation and Y503 phosphorylation of G6PD, interaction with p53, and induction of early apoptotic events.","method":"Site-specifically acetylated G6PD via genetic code expansion, enzymatic activity assays, structural studies, mass spectrometry-based PTM analysis, p53 Co-IP, apoptosis assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — site-specific acetylation with genetic code expansion (reconstitution-level), structural validation, multiple PTM and signaling readouts in single rigorous study","pmids":["37798264"],"is_preprint":false},{"year":2019,"finding":"Small molecule AG1 activates G6PD by promoting oligomerization (dimer formation) at the structural NADP+ binding sites bridging the dimer interface. The mechanism is noncovalent and the disulfide in AG1 is not required for activation.","method":"Biochemical activity assays, structure-activity relationship analysis, oligomerization assays, site mapping at dimer interface","journal":"ChemMedChem","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical reconstitution of activation mechanism, SAR analysis, single lab","pmids":["31183991"],"is_preprint":false},{"year":2023,"finding":"Quercetin directly binds G6PD and inhibits its enzymatic activity by competitively abrogating NADP+ binding in the catalytic domain, reducing intracellular NADPH and causing degradation of EGFRT790M.","method":"Direct binding assays, competitive inhibition kinetics, NADPH measurement, EGFRT790M degradation assays, cell-based studies","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding demonstrated, competitive inhibition mechanism characterized, single lab","pmids":["37950872"],"is_preprint":false},{"year":2021,"finding":"FDX1 interacts with G6PD and reduces its protein stability, decreasing G6PD activity and NADPH/GSH levels, thereby enhancing cuproptosis in endometriosis cells.","method":"Co-IP demonstrating FDX1-G6PD interaction, G6PD stability assays, NADPH/GSH measurements, cuproptosis assays, mouse model","journal":"Apoptosis","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP, functional activity assays, single lab, single study","pmids":["37119432"],"is_preprint":false},{"year":2016,"finding":"BAG3 directly interacts with G6PD and suppresses PPP flux and de novo DNA synthesis in hepatocellular carcinoma cells. The growth defect from BAG3 overexpression is rescued by enforced G6PD expression. BAG3 elevation did not cause reduction in cellular NADPH, indicating the inhibitory effect is specifically on nucleotide synthesis via PPP.","method":"Co-IP, G6PD rescue overexpression, PPP flux measurement, DNA synthesis assay, nucleoside supplementation rescue","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct Co-IP, rescue by G6PD overexpression, specific pathway dissection via nucleoside supplementation, single lab","pmids":["26621836"],"is_preprint":false},{"year":2017,"finding":"G6PD variant pathogenicity is largely determined by a trade-off between protein stability and catalytic activity. Structural mutations at the dimer interface or structural NADP+ binding site cause instability and severe clinical phenotypes, while mutations affecting catalytic residues reduce activity without destabilizing the enzyme.","method":"Bioinformatic structural analysis, biochemical characterization of G6PD variants (stability, activity assays), multidimensional analysis of clinical variant data","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — systematic biochemical characterization of multiple variants with structural context, single lab but multiple methods","pmids":["28297664"],"is_preprint":false},{"year":2014,"finding":"G6PD variants with different clinical phenotypes show differing protein stability. Structural rigidity underlies mutation effects on protein stability and folding. Class I (most severe) variants remain thermolabile even with increasing NADP+, whereas Class II and III variants become more thermostable with NADP+, indicating that Class I mutations affect the structural NADP+ binding region.","method":"Overexpression and purification of G6PD variants, kinetic constants (kcat), T50 thermal stability assay, protein yield analysis, NADP+ thermostabilization assays","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical characterization with multiple stability assays, single lab, multiple variants tested","pmids":["25407525"],"is_preprint":false},{"year":2008,"finding":"G6PD deficiency leads to loss of cellular control of protein glutathionylation. G6PD-deficient cells show increased protein glutathionylation and loss of Ku protein function upon oxidant treatment (HEDS), due to decreased NADPH, protein thiols, and GSH. Reintroduction of the G6PD gene restores normal phenotype.","method":"Comparison of G6PD-deficient (E89) vs wild-type (K1) cells, oxidant challenge, protein glutathionylation assay (ELISA), Ku protein-DNA binding assay, NADPH/GSH measurement, G6PD rescue by gene reintroduction","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic null vs wild-type with gene rescue, multiple functional readouts, single lab","pmids":["17516514"],"is_preprint":false},{"year":2021,"finding":"G6PD defect in brown adipocytes impairs thermogenic function through excessive cytosolic ROS accumulation, leading to ERK activation, which suppresses thermogenic gene expression. Antioxidant treatment or ERK inhibition restores thermogenic activity in G6PD-deficient mice.","method":"G6PD-deficient mutant mice, cold exposure, ROS measurement, thermogenic gene expression analysis, ERK inhibitor treatment, antioxidant administration","journal":"Diabetes","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic mouse model with pharmacological epistasis, multiple orthogonal interventions, single lab","pmids":["34521642"],"is_preprint":false},{"year":2023,"finding":"JAK2 phosphorylates G6PD at Y437 under IL-6 treatment, accentuating G6PD enzymatic activity by promoting G6PD binding with its substrate G6P, leading to increased PPP flux and nucleotide biosynthesis to support tumor cell proliferation.","method":"Co-IP, enzymatic activity assays, site-directed mutagenesis of Y437, BrdU proliferation assay, colony formation assay, xenograft model, patient sample correlation","journal":"Molecular metabolism","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, site-specific mutagenesis, enzymatic kinetics, in vivo xenograft validation, single lab","pmids":["37949355"],"is_preprint":false},{"year":2024,"finding":"CDK5 phosphorylates G6PD at Thr-91, facilitating assembly of inactive G6PD monomers into active dimers under oxidative stress in breast cancer cells. CDK5 inhibition abrogates G6PD phosphorylation and synergistically sensitizes breast cancer cells to PARP inhibitor Olaparib.","method":"Kinase assays, site-directed mutagenesis, dimerization assays, CDK5 inhibitors, xenograft models, patient tissue analysis","journal":"Acta pharmaceutica Sinica B","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinase assay with site-specific mutagenesis, dimerization mechanistic link, in vivo validation, single lab","pmids":["40370560"],"is_preprint":false},{"year":2024,"finding":"BHMT deficiency activates G6PD by decreasing arginine methylation of G6PD at arginine 246. BHMT directly regulates methylation of G6PD, and pharmacological inhibition of G6PD attenuates BHMT-deficiency-driven hepatocarcinogenesis.","method":"Co-IP, proteomics, metabolomics, site-directed mutagenesis of R246, specific antibodies against methylated G6PD, Bhmt KO mouse models, G6PD inhibitor rescue","journal":"Science China. Life sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, site-specific mutagenesis, specific modification antibodies, KO mouse model, single lab","pmids":["38679670"],"is_preprint":false},{"year":2014,"finding":"HDAC inhibitors selectively enhance G6PD transcription among all 16 glycolytic/PPP pathway genes through enhanced recruitment of transcription factor Sp1, commensurate recruitment of histone acetyltransferases and deacetylases, increased histone acetylation, and RNA polymerase II recruitment to the G6PD locus, restoring enzymatic activity in G6PD-deficient nucleated cells.","method":"HDACi treatment, ChIP assays for Sp1/histone acetylation/Pol II, transcriptional analysis, G6PD enzymatic activity in patient B cells and erythroid precursors","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-validated chromatin mechanism, functional enzymatic rescue in patient-derived cells, single lab","pmids":["24805191"],"is_preprint":false},{"year":2021,"finding":"G6PD inhibits ferroptosis in hepatocellular carcinoma through cytochrome P450 oxidoreductase (POR). Knockdown of G6PD upregulates POR, which mediates the suppression of HCC cell growth and ferroptosis sensitivity.","method":"siRNA knockdown of G6PD, ferroptosis assays, POR expression analysis, in vivo xenograft tumor growth","journal":"Cellular signalling","confidence":"Low","confidence_rationale":"Tier 3 / Weak — knockdown with functional phenotype but pathway mechanism between G6PD and POR not directly demonstrated biochemically, single lab, single method per claim","pmids":["34325001"],"is_preprint":false},{"year":2020,"finding":"NF-κB p65 and pSTAT3 synergistically drive G6PD overexpression in clear cell RCC. p65 directly binds the G6PD promoter, and p65/pSTAT3 form a complex that occupies the pSTAT3-binding site on the G6PD promoter to facilitate transcription.","method":"ChIP assay, Co-IP of p65/pSTAT3 complex, luciferase reporter assay, NF-κB activator/inhibitor treatment, xenograft model","journal":"Cancer cell international","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP, Co-IP, and reporter assays in single study, single lab","pmids":["33041664"],"is_preprint":false},{"year":2015,"finding":"HBV X protein (HBx) stimulates G6PD expression via Nrf2 activation. HBx associates with UBA and PB1 domains of adaptor protein p62, augmenting interaction between p62 and Nrf2 repressor Keap1 to form an HBx-p62-Keap1 complex that sequesters Keap1 from Nrf2, leading to Nrf2 activation and consequent G6PD transcription.","method":"Co-IP, domain mapping, G6PD promoter analysis, Nrf2 pathway analysis, G6PD expression and activity assays in HBV-infected cells","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain mapping, functional G6PD expression readout, single lab","pmids":["26583321"],"is_preprint":false},{"year":2021,"finding":"ALKBH5 acts as an m6A eraser that demethylates the G6PD transcript, enhancing G6PD mRNA stability and promoting G6PD translation, thereby activating the pentose phosphate pathway and supporting glioma cell proliferation.","method":"m6A-qRT-PCR, ALKBH5 gain/loss-of-function, G6PD mRNA stability assays, G6PD expression and activity analysis","journal":"Neurochemical research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — m6A-specific assays, gain/loss-of-function, mRNA stability measurement, single lab","pmids":["34297301"],"is_preprint":false},{"year":2024,"finding":"METTL14-mediated m6A modification of G6PD mRNA is recognized by IGF2BP2, which enhances G6PD mRNA stability, thereby upregulating G6PD expression post-transcriptionally and promoting lung adenocarcinoma tumor growth and metastasis.","method":"RNA sequencing, MeRIP-sequencing, METTL14 knockdown/overexpression, IGF2BP2 interaction with G6PD mRNA, G6PD expression and stability assays, in vivo xenograft model","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MeRIP-seq, RIP for IGF2BP2-G6PD mRNA interaction, mRNA stability assay, in vivo validation, single lab","pmids":["39138186"],"is_preprint":false},{"year":1996,"finding":"A 20-kb human G6PD construct containing only 2.5 kb upstream and 2.0 kb downstream flanking sequence is sufficient to drive high-level, constitutive, tissue-appropriate G6PD expression in transgenic mice, with steady-state mRNA levels accounting for tissue-to-tissue variation in enzyme activity.","method":"Transgenic mouse generation, enzyme activity assays across tissues, mRNA Northern blot","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transgenic mouse model with multiple tissues, mRNA quantitation, single lab","pmids":["8964507"],"is_preprint":false}],"current_model":"G6PD is the rate-limiting enzyme of the pentose phosphate pathway that produces NADPH by oxidizing glucose-6-phosphate; its activity is tightly regulated by multiple post-translational modifications—including inhibitory acetylation (K403, removed by SIRT2; K171) and activating phosphorylation (Y112 by c-Src, Y437 by JAK2, T91 by CDK5), inhibitory glutarylation (removed by SIRT5), activating O-GlcNAcylation, inhibitory arginine methylation (R246, by BHMT), and inhibitory ubiquitination (K366/K403 by VHL E3 ligase)—as well as by direct protein–protein interactions with PTEN, Aldob-p53 complexes, BAG3, FDX1, HSPB1, and PAK4/p53/Mdm2, all of which modulate its capacity to form catalytically active dimers and thereby control NADPH-dependent redox homeostasis, nucleotide biosynthesis, and cell survival."},"narrative":{"mechanistic_narrative":"G6PD is the rate-limiting enzyme of the pentose phosphate pathway, oxidizing glucose-6-phosphate to generate NADPH and ribose-5-phosphate that drive redox homeostasis, nucleotide and lipid biosynthesis, and cell survival [PMID:26399441, PMID:15271799]. Catalysis requires assembly of inactive monomers into active dimers, and a recurring theme across the literature is that this dimerization step is the regulatory fulcrum: stability and activity trade off through mutations at the dimer interface and structural NADP+ site, where the most severe clinical variants destabilize the enzyme while catalytic-residue mutations reduce activity without destabilization [PMID:28297664, PMID:25407525]. A dense network of post-translational modifications tunes dimer formation in opposite directions. Inhibitory inputs include acetylation at K403, which structurally distorts the dimer interface and active site to abolish activity (reversed by the deacetylase SIRT2) [PMID:24769394, PMID:27586085, PMID:37798264], glutarylation (reversed by SIRT5) [PMID:27113762], arginine methylation at R246 controlled by BHMT [PMID:38679670], and VHL-mediated ubiquitination at K366/K403 that targets G6PD for proteasomal degradation [PMID:30785802]. Activating inputs include O-GlcNAcylation under hypoxia [PMID:26399441], tyrosine phosphorylation at Y112 by c-Src and Y437 by JAK2 that improve substrate binding and kinetics [PMID:33686238, PMID:37949355], and CDK5 phosphorylation at T91 that promotes dimer assembly under oxidative stress [PMID:40370560]. G6PD activity is further set by direct protein partners that govern the dimer: PTEN, Aldolase B (acting as a scaffold with p53), BAG3, and FDX1 each bind G6PD and suppress its activity or stability, whereas HSPB1 and TSP50 promote its SIRT2-dependent activation [PMID:24352616, PMID:35122041, PMID:27711253, PMID:33630390, PMID:37119432, PMID:26621836]. Expression is independently set at the transcriptional and post-transcriptional level by Sp1/chromatin acetylation, NF-κB p65/pSTAT3, Nrf2 (engaged by HBV HBx), and m6A regulators ALKBH5, METTL14, and IGF2BP2 [PMID:24805191, PMID:33041664, PMID:26583321, PMID:34297301, PMID:39138186]. Functionally, G6PD-derived NADPH is indispensable for definitive erythropoiesis, preventing apoptosis of definitive erythrocytes [PMID:15271799], and for limiting protein glutathionylation and ROS-driven damage [PMID:17516514, PMID:34521642]. Across cancers these regulatory inputs converge to elevate PPP flux supporting proliferation [PMID:26399441, PMID:33686238, PMID:37949355].","teleology":[{"year":2004,"claim":"Establishing that G6PD-derived NADPH is not merely housekeeping but specifically required for a defined developmental program addressed why the enzyme is physiologically essential.","evidence":"G6PD-null ES cell differentiation in embryoid bodies with apoptosis assays and rescue by reducing agents, caspase inhibitors, and G6PD re-expression","pmids":["15271799"],"confidence":"High","gaps":["Does not define which downstream NADPH-dependent process protects definitive erythrocytes","Primitive erythropoiesis tolerance mechanism not explained"]},{"year":2008,"claim":"Linking G6PD deficiency to loss of redox buffering connected enzyme activity to a concrete protein-protective output.","evidence":"G6PD-deficient vs wild-type cells under oxidant challenge with glutathionylation, Ku DNA-binding, and NADPH/GSH readouts plus gene rescue","pmids":["17516514"],"confidence":"Medium","gaps":["Generality beyond Ku not established","Single cell-line pair"]},{"year":2013,"claim":"Identifying PTEN as a direct binder that blocks active-dimer formation revealed that protein-protein interactions, not just substrate supply, gate PPP flux.","evidence":"Co-IP, mass spectrometry, PPP flux measurements, and epistasis with the Tcl1/hnRNPK splicing axis","pmids":["24352616"],"confidence":"High","gaps":["Structural basis of dimer blockade not resolved","Quantitative contribution versus PTEN's phosphatase functions unclear"]},{"year":2014,"claim":"Pinpointing K403 acetylation as a dimer-disrupting inhibitory mark reversed by SIRT2 defined the first PTM switch controlling G6PD activity.","evidence":"Acetylation-mimetic mutants, in vitro activity assays, Co-IP, and rescue in cells and mouse erythrocytes with SIRT2 depletion","pmids":["24769394"],"confidence":"High","gaps":["Physiological acetyltransferase only proposed (KAT9/ELP3)","Stoichiometry of K403 acetylation in vivo unknown"]},{"year":2014,"claim":"Demonstrating selective transcriptional upregulation of G6PD by HDAC inhibitors and tissue-appropriate constitutive expression from a compact locus established how G6PD levels are set, separate from activity regulation.","evidence":"HDACi with ChIP for Sp1/histone acetylation/Pol II and enzyme rescue in patient cells; transgenic mouse with a 20-kb G6PD construct and tissue mRNA/activity quantitation","pmids":["24805191","8964507"],"confidence":"Medium","gaps":["Specific regulatory elements driving tissue variation not mapped","Whether HDACi rescue translates clinically untested in these studies"]},{"year":2015,"claim":"Showing O-GlcNAcylation activates G6PD under hypoxia connected nutrient/stress sensing to PPP-driven biosynthesis and tumor growth.","evidence":"O-GlcNAc assays, activity assays, metabolic flux, site mutagenesis, and xenograft models","pmids":["26399441"],"confidence":"High","gaps":["Enzymes adding/removing the modification on G6PD not identified","Interplay with other activating PTMs unresolved"]},{"year":2016,"claim":"Identifying glutarylation reversed by SIRT5 and the HSPB1-enhanced SIRT2 axis expanded the deacylation control of G6PD and showed PTM activation is scaffolded.","evidence":"SIRT5 KO/knockdown with activity, NADPH, GSH, ROS readouts; reciprocal Co-IP of HSPB1-G6PD-SIRT2 with activity assays","pmids":["27113762","27711253"],"confidence":"Medium","gaps":["Glutarylation site(s) on G6PD not pinpointed","HSPB1 axis from a single study"]},{"year":2016,"claim":"Confirming SIRT2-mediated K403 deacetylation sustains leukemia proliferation with a tumor-selective vulnerability advanced G6PD regulation toward therapeutic relevance.","evidence":"SIRT2 knockdown, K403 mimetic mutants, activity and colony assays, SIRT2 inhibitors, and AML patient samples","pmids":["27586085"],"confidence":"High","gaps":["Basis of selectivity for leukemic over normal cells incomplete"]},{"year":2016,"claim":"Finding BAG3 suppresses PPP-dependent DNA synthesis without lowering NADPH separated G6PD's biosynthetic from redox outputs.","evidence":"Co-IP, G6PD rescue overexpression, PPP flux and DNA synthesis assays, nucleoside supplementation rescue","pmids":["26621836"],"confidence":"Medium","gaps":["Mechanism by which BAG3 differentially channels output unclear","Single lab"]},{"year":2017,"claim":"Defining a stability-versus-activity trade-off across clinical variants explained the structural logic of G6PD deficiency phenotypes.","evidence":"Bioinformatic structural analysis with biochemical characterization of variant stability and activity and clinical correlation","pmids":["28297664"],"confidence":"Medium","gaps":["No direct timeline link to specific Mendelian disease via causative-mutation rescue","Predictive accuracy for novel variants not validated"]},{"year":2019,"claim":"Mapping VHL-mediated ubiquitination at K366/K403 established degradation as a control point coupling glucose load to G6PD abundance and tissue injury.","evidence":"Co-IP, ubiquitination-site mutagenesis, knockdown/overexpression rescue, and G6PD-deficient mouse kidney analysis","pmids":["30785802"],"confidence":"High","gaps":["Crosstalk between K403 acetylation and K403 ubiquitination not resolved here","Signal triggering VHL recruitment to G6PD unclear"]},{"year":2019,"claim":"Showing a small molecule activates G6PD by bridging the structural NADP+ sites to drive dimerization validated the dimer interface as a druggable activation switch.","evidence":"Biochemical activity and oligomerization assays with SAR and dimer-interface site mapping (AG1)","pmids":["31183991"],"confidence":"Medium","gaps":["Cellular and in vivo efficacy not established in this study","Selectivity over variant enzymes untested"]},{"year":2020,"claim":"Establishing Aldolase B as an enzymatically independent scaffold that potentiates p53-mediated G6PD inhibition added a tumor-suppressive complex to the regulatory network.","evidence":"Direct binding and Co-IP, activity assays, Aldob KO mouse with re-expression rescue, and pharmacological G6PD inhibition","pmids":["35122041"],"confidence":"High","gaps":["How the Aldob-G6PD-p53 complex mechanically blocks dimers unresolved"]},{"year":2020,"claim":"Demonstrating p65/pSTAT3 cooperative transcriptional activation showed inflammatory signaling directly elevates G6PD in renal cancer.","evidence":"ChIP, Co-IP of the p65/pSTAT3 complex, luciferase reporter, and xenografts","pmids":["33041664"],"confidence":"Medium","gaps":["Generality across tumor types not tested","Single lab"]},{"year":2021,"claim":"Identifying c-Src Y112 phosphorylation that lowers Km and raises Kcat provided a kinetic mechanism for oncogenic G6PD activation.","evidence":"Co-IP, in vitro kinase assay with Km/Kcat analysis, site mutagenesis, flux assays, and CRC sample correlation","pmids":["33686238"],"confidence":"High","gaps":["In vivo contribution relative to other activating PTMs unclear"]},{"year":2021,"claim":"Convergent findings positioned G6PD-derived NADPH as a determinant of cell-death modalities (ferroptosis, cuproptosis) and brown-fat thermogenesis, broadening its physiological reach.","evidence":"G6PD knockdown with POR and ferroptosis readouts; FDX1 Co-IP with stability/NADPH/GSH and cuproptosis assays; G6PD-deficient mice with cold exposure, ROS, ERK inhibition and antioxidant rescue","pmids":["34325001","37119432","34521642"],"confidence":"Medium","gaps":["G6PD-POR link not biochemically demonstrated (Low confidence)","Direct molecular coupling of NADPH levels to each death pathway incomplete"]},{"year":2021,"claim":"Showing TSP50 inhibits K171 acetylation and promotes SIRT2 binding identified an additional acetylation site and a tumor-promoting activator.","evidence":"LC-MS/MS, Co-IP, GST pull-down, K171 mutagenesis, and proliferation/tumor formation assays","pmids":["33630390"],"confidence":"Medium","gaps":["K171 acetyltransferase not defined","Relationship to K403/K89 acetylation unresolved"]},{"year":2021,"claim":"Identifying ALKBH5-mediated m6A demethylation as a stabilizer of G6PD mRNA established post-transcriptional control of PPP capacity.","evidence":"m6A-qRT-PCR, ALKBH5 gain/loss-of-function, mRNA stability and activity assays","pmids":["34297301"],"confidence":"Medium","gaps":["m6A sites on G6PD transcript not mapped","Single tumor context"]},{"year":2023,"claim":"Site-specific reconstitution distinguishing activating K89 from inhibitory K403 acetylation, with structural and downstream signaling consequences, resolved how a single PTM type produces opposite effects and links G6PD to apoptosis.","evidence":"Genetic code expansion for site-specific acetylation, structural studies, MS-based PTM analysis, p53 Co-IP, and apoptosis assays","pmids":["37798264"],"confidence":"High","gaps":["Endogenous enzymes setting K89 versus K403 acetylation not identified","Physiological balance of opposing marks in tissues unknown"]},{"year":2023,"claim":"Defining JAK2 Y437 phosphorylation downstream of IL-6 and quercetin as a competitive NADP+-site inhibitor extended both physiological activation and pharmacological inhibition mechanisms.","evidence":"Co-IP, mutagenesis of Y437, kinetics, proliferation and xenograft assays; 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K403-acetylated G6PD is incapable of forming active dimers and displays complete loss of activity. SIRT2 deacetylates K403 to activate G6PD in response to oxidative stress. KAT9/ELP3 was identified as a potential acetyltransferase of G6PD.\",\n      \"method\": \"Acetylation-mimetic mutants, in vitro enzymatic activity assays, Co-IP, knockdown/rescue experiments in cells and mouse erythrocytes, SIRT2 depletion\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods including mutagenesis, in vitro activity assays, and cellular rescue; replicated across cell lines and erythrocytes\",\n      \"pmids\": [\"24769394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SIRT5 deglutarylates G6PD, activating the enzyme and increasing NADPH production. Knockdown or knockout of SIRT5 leads to inhibition of G6PD activity, decreased NADPH, lowered GSH, and increased cellular susceptibility to oxidative stress.\",\n      \"method\": \"SIRT5 KO/knockdown, enzymatic activity assays, NADPH and GSH measurements, ROS quantification\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal genetic and biochemical validation including KO and activity assays, replicated with multiple orthogonal methods\",\n      \"pmids\": [\"27113762\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"G6PD is dynamically modified by O-linked β-N-acetylglucosamine (O-GlcNAcylation) in response to hypoxia. This glycosylation activates G6PD activity, increases glucose flux through the PPP, and promotes nucleotide/lipid biosynthesis and antioxidant defense. Blocking G6PD glycosylation reduces cancer cell proliferation in vitro and impairs tumor growth in vivo.\",\n      \"method\": \"O-GlcNAc modification assays, enzymatic activity assays, metabolic flux analysis, glycosylation site mutagenesis, in vivo xenograft models\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — direct biochemical identification of modification site, functional rescue by mutagenesis, in vitro and in vivo validation\",\n      \"pmids\": [\"26399441\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PTEN protein directly binds G6PD and prevents formation of the active G6PD dimer, thereby inhibiting PPP flux. Tcl1, acting via hnRNPK, promotes G6PD pre-mRNA splicing and protein expression. PTEN also forms a complex with hnRNPK to inhibit G6PD pre-mRNA splicing. PTEN inactivates Tcl1 via GSK3β-mediated phosphorylation.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, molecular biology assays, PPP flux measurements, epistasis analysis\",\n      \"journal\": \"Gut\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, mass spectrometry, multiple orthogonal functional assays in a single study\",\n      \"pmids\": [\"24352616\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SIRT2 promotes G6PD deacetylation at K403, activating G6PD to increase NADPH production and support leukaemia cell proliferation. Chemical inhibition of SIRT2 suppresses G6PD activity and reduces leukaemia cell but not normal hematopoietic cell proliferation.\",\n      \"method\": \"SIRT2 knockdown, K403 acetylation-mimetic mutants, enzymatic activity assays, colony formation assays, SIRT2 inhibitors, patient AML sample analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — site-specific mutagenesis, enzymatic assays, pharmacological validation, corroborated by independent paper (PMID 24769394)\",\n      \"pmids\": [\"27586085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Aldolase B (Aldob) directly binds G6PD and inhibits its enzymatic activity, suppressing PPP metabolism. Aldob potentiates p53-mediated inhibition of G6PD by forming an Aldob-G6PD-p53 complex. This scaffolding effect is independent of Aldob enzymatic activity.\",\n      \"method\": \"Direct binding assays, Co-IP, enzymatic activity assays, Aldob KO mouse model, Aldob/G6PD re-expression rescue experiments, pharmacological G6PD inhibition\",\n      \"journal\": \"Nature cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct protein-protein interaction demonstrated, KO mouse model with rescue, multiple orthogonal methods\",\n      \"pmids\": [\"35122041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"c-Src tyrosine kinase directly interacts with and phosphorylates G6PD at Tyr112, enhancing catalytic activity by decreasing Km and increasing Kcat for glucose-6-phosphate substrate, thereby augmenting PPP flux for NADPH and ribose-5-phosphate production.\",\n      \"method\": \"Co-IP, in vitro kinase assay, Km/Kcat kinetic analysis, site-directed mutagenesis, metabolic flux assays, clinical CRC sample correlation\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with kinetic characterization, mutagenesis validation, single lab\",\n      \"pmids\": [\"33686238\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"G6PD is ubiquitinated on K366 and K403 by the VHL E3 ubiquitin ligase, which directly binds G6PD, leading to G6PD proteasomal degradation under high glucose conditions and resulting in ROS accumulation and podocyte injury.\",\n      \"method\": \"Co-IP demonstrating VHL-G6PD interaction, site-directed mutagenesis of ubiquitination sites, Western blot, G6PD overexpression rescue, siRNA knockdown, G6PD-deficient mouse kidney analysis\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct Co-IP, site-specific mutagenesis of ubiquitination sites K366/K403, in vivo mouse validation, multiple orthogonal methods\",\n      \"pmids\": [\"30785802\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"G6PD is indispensable for definitive erythropoiesis after the embryonic-adult hemoglobin switch. G6PD-null ES cells differentiate normally into primitive erythroid cells but definitive erythrocytes undergo apoptosis that is prevented only by restoration of G6PD activity.\",\n      \"method\": \"G6PD-null mouse ES cell differentiation (embryoid body system), apoptosis assays, reducing agents rescue, caspase inhibitor rescue, G6PD re-expression rescue\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic null model with specific rescue by G6PD re-expression, multiple rescue conditions tested\",\n      \"pmids\": [\"15271799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HSPB1 (Hsp27) enhances the binding between G6PD and SIRT2, leading to deacetylation and activation of G6PD, thereby sustaining cellular NADPH and pentose production in response to oxidative stress or DNA damage.\",\n      \"method\": \"Co-IP, enzymatic activity assays, NADPH measurement, siRNA knockdown, HSPB1 overexpression\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — reciprocal Co-IP demonstrating HSPB1-G6PD-SIRT2 complex, enzymatic activity validation, single lab, single study\",\n      \"pmids\": [\"27711253\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PAK4 interacts with G6PD and increases G6PD activity via enhancing Mdm2-mediated p53 ubiquitination and degradation (as p53 suppresses G6PD), thereby promoting glucose intake, NADPH production, lipid biosynthesis and colon cancer cell proliferation.\",\n      \"method\": \"Co-IP, G6PD enzymatic activity assays, metabolic measurements, p53 ubiquitination assays, siRNA knockdown, clinical correlation\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, epistasis via p53 pathway, enzymatic activity assays, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"28542136\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TSP50 binds G6PD in the cytoplasm and activates G6PD activity by inhibiting acetylation of G6PD at K171. TSP50 also promotes the binding of G6PD to SIRT2. K171 acetylation of G6PD is required for TSP50-induced cell proliferation and tumor formation.\",\n      \"method\": \"LC-MS/MS, Co-IP, GST pull-down, site-specific mutation of K171, enzymatic activity assays, cell proliferation and tumor formation assays\",\n      \"journal\": \"Cell proliferation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pull-down and Co-IP, site-specific mutagenesis, multiple methods in single lab\",\n      \"pmids\": [\"33630390\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Acetylation at K89 activates G6PD while acetylation at K403 inhibits G6PD. K403 acetylation-dependent inactivation is explained by structural distortion of the dimeric structure and active site. K403 acetylation also leads to K95/97 ubiquitylation and Y503 phosphorylation of G6PD, interaction with p53, and induction of early apoptotic events.\",\n      \"method\": \"Site-specifically acetylated G6PD via genetic code expansion, enzymatic activity assays, structural studies, mass spectrometry-based PTM analysis, p53 Co-IP, apoptosis assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — site-specific acetylation with genetic code expansion (reconstitution-level), structural validation, multiple PTM and signaling readouts in single rigorous study\",\n      \"pmids\": [\"37798264\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Small molecule AG1 activates G6PD by promoting oligomerization (dimer formation) at the structural NADP+ binding sites bridging the dimer interface. The mechanism is noncovalent and the disulfide in AG1 is not required for activation.\",\n      \"method\": \"Biochemical activity assays, structure-activity relationship analysis, oligomerization assays, site mapping at dimer interface\",\n      \"journal\": \"ChemMedChem\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical reconstitution of activation mechanism, SAR analysis, single lab\",\n      \"pmids\": [\"31183991\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Quercetin directly binds G6PD and inhibits its enzymatic activity by competitively abrogating NADP+ binding in the catalytic domain, reducing intracellular NADPH and causing degradation of EGFRT790M.\",\n      \"method\": \"Direct binding assays, competitive inhibition kinetics, NADPH measurement, EGFRT790M degradation assays, cell-based studies\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding demonstrated, competitive inhibition mechanism characterized, single lab\",\n      \"pmids\": [\"37950872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"FDX1 interacts with G6PD and reduces its protein stability, decreasing G6PD activity and NADPH/GSH levels, thereby enhancing cuproptosis in endometriosis cells.\",\n      \"method\": \"Co-IP demonstrating FDX1-G6PD interaction, G6PD stability assays, NADPH/GSH measurements, cuproptosis assays, mouse model\",\n      \"journal\": \"Apoptosis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP, functional activity assays, single lab, single study\",\n      \"pmids\": [\"37119432\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"BAG3 directly interacts with G6PD and suppresses PPP flux and de novo DNA synthesis in hepatocellular carcinoma cells. The growth defect from BAG3 overexpression is rescued by enforced G6PD expression. BAG3 elevation did not cause reduction in cellular NADPH, indicating the inhibitory effect is specifically on nucleotide synthesis via PPP.\",\n      \"method\": \"Co-IP, G6PD rescue overexpression, PPP flux measurement, DNA synthesis assay, nucleoside supplementation rescue\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct Co-IP, rescue by G6PD overexpression, specific pathway dissection via nucleoside supplementation, single lab\",\n      \"pmids\": [\"26621836\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"G6PD variant pathogenicity is largely determined by a trade-off between protein stability and catalytic activity. Structural mutations at the dimer interface or structural NADP+ binding site cause instability and severe clinical phenotypes, while mutations affecting catalytic residues reduce activity without destabilizing the enzyme.\",\n      \"method\": \"Bioinformatic structural analysis, biochemical characterization of G6PD variants (stability, activity assays), multidimensional analysis of clinical variant data\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — systematic biochemical characterization of multiple variants with structural context, single lab but multiple methods\",\n      \"pmids\": [\"28297664\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"G6PD variants with different clinical phenotypes show differing protein stability. Structural rigidity underlies mutation effects on protein stability and folding. Class I (most severe) variants remain thermolabile even with increasing NADP+, whereas Class II and III variants become more thermostable with NADP+, indicating that Class I mutations affect the structural NADP+ binding region.\",\n      \"method\": \"Overexpression and purification of G6PD variants, kinetic constants (kcat), T50 thermal stability assay, protein yield analysis, NADP+ thermostabilization assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical characterization with multiple stability assays, single lab, multiple variants tested\",\n      \"pmids\": [\"25407525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"G6PD deficiency leads to loss of cellular control of protein glutathionylation. G6PD-deficient cells show increased protein glutathionylation and loss of Ku protein function upon oxidant treatment (HEDS), due to decreased NADPH, protein thiols, and GSH. Reintroduction of the G6PD gene restores normal phenotype.\",\n      \"method\": \"Comparison of G6PD-deficient (E89) vs wild-type (K1) cells, oxidant challenge, protein glutathionylation assay (ELISA), Ku protein-DNA binding assay, NADPH/GSH measurement, G6PD rescue by gene reintroduction\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic null vs wild-type with gene rescue, multiple functional readouts, single lab\",\n      \"pmids\": [\"17516514\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"G6PD defect in brown adipocytes impairs thermogenic function through excessive cytosolic ROS accumulation, leading to ERK activation, which suppresses thermogenic gene expression. Antioxidant treatment or ERK inhibition restores thermogenic activity in G6PD-deficient mice.\",\n      \"method\": \"G6PD-deficient mutant mice, cold exposure, ROS measurement, thermogenic gene expression analysis, ERK inhibitor treatment, antioxidant administration\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic mouse model with pharmacological epistasis, multiple orthogonal interventions, single lab\",\n      \"pmids\": [\"34521642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"JAK2 phosphorylates G6PD at Y437 under IL-6 treatment, accentuating G6PD enzymatic activity by promoting G6PD binding with its substrate G6P, leading to increased PPP flux and nucleotide biosynthesis to support tumor cell proliferation.\",\n      \"method\": \"Co-IP, enzymatic activity assays, site-directed mutagenesis of Y437, BrdU proliferation assay, colony formation assay, xenograft model, patient sample correlation\",\n      \"journal\": \"Molecular metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, site-specific mutagenesis, enzymatic kinetics, in vivo xenograft validation, single lab\",\n      \"pmids\": [\"37949355\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CDK5 phosphorylates G6PD at Thr-91, facilitating assembly of inactive G6PD monomers into active dimers under oxidative stress in breast cancer cells. CDK5 inhibition abrogates G6PD phosphorylation and synergistically sensitizes breast cancer cells to PARP inhibitor Olaparib.\",\n      \"method\": \"Kinase assays, site-directed mutagenesis, dimerization assays, CDK5 inhibitors, xenograft models, patient tissue analysis\",\n      \"journal\": \"Acta pharmaceutica Sinica B\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinase assay with site-specific mutagenesis, dimerization mechanistic link, in vivo validation, single lab\",\n      \"pmids\": [\"40370560\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"BHMT deficiency activates G6PD by decreasing arginine methylation of G6PD at arginine 246. BHMT directly regulates methylation of G6PD, and pharmacological inhibition of G6PD attenuates BHMT-deficiency-driven hepatocarcinogenesis.\",\n      \"method\": \"Co-IP, proteomics, metabolomics, site-directed mutagenesis of R246, specific antibodies against methylated G6PD, Bhmt KO mouse models, G6PD inhibitor rescue\",\n      \"journal\": \"Science China. Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, site-specific mutagenesis, specific modification antibodies, KO mouse model, single lab\",\n      \"pmids\": [\"38679670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HDAC inhibitors selectively enhance G6PD transcription among all 16 glycolytic/PPP pathway genes through enhanced recruitment of transcription factor Sp1, commensurate recruitment of histone acetyltransferases and deacetylases, increased histone acetylation, and RNA polymerase II recruitment to the G6PD locus, restoring enzymatic activity in G6PD-deficient nucleated cells.\",\n      \"method\": \"HDACi treatment, ChIP assays for Sp1/histone acetylation/Pol II, transcriptional analysis, G6PD enzymatic activity in patient B cells and erythroid precursors\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-validated chromatin mechanism, functional enzymatic rescue in patient-derived cells, single lab\",\n      \"pmids\": [\"24805191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"G6PD inhibits ferroptosis in hepatocellular carcinoma through cytochrome P450 oxidoreductase (POR). Knockdown of G6PD upregulates POR, which mediates the suppression of HCC cell growth and ferroptosis sensitivity.\",\n      \"method\": \"siRNA knockdown of G6PD, ferroptosis assays, POR expression analysis, in vivo xenograft tumor growth\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — knockdown with functional phenotype but pathway mechanism between G6PD and POR not directly demonstrated biochemically, single lab, single method per claim\",\n      \"pmids\": [\"34325001\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NF-κB p65 and pSTAT3 synergistically drive G6PD overexpression in clear cell RCC. p65 directly binds the G6PD promoter, and p65/pSTAT3 form a complex that occupies the pSTAT3-binding site on the G6PD promoter to facilitate transcription.\",\n      \"method\": \"ChIP assay, Co-IP of p65/pSTAT3 complex, luciferase reporter assay, NF-κB activator/inhibitor treatment, xenograft model\",\n      \"journal\": \"Cancer cell international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP, Co-IP, and reporter assays in single study, single lab\",\n      \"pmids\": [\"33041664\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"HBV X protein (HBx) stimulates G6PD expression via Nrf2 activation. HBx associates with UBA and PB1 domains of adaptor protein p62, augmenting interaction between p62 and Nrf2 repressor Keap1 to form an HBx-p62-Keap1 complex that sequesters Keap1 from Nrf2, leading to Nrf2 activation and consequent G6PD transcription.\",\n      \"method\": \"Co-IP, domain mapping, G6PD promoter analysis, Nrf2 pathway analysis, G6PD expression and activity assays in HBV-infected cells\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain mapping, functional G6PD expression readout, single lab\",\n      \"pmids\": [\"26583321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ALKBH5 acts as an m6A eraser that demethylates the G6PD transcript, enhancing G6PD mRNA stability and promoting G6PD translation, thereby activating the pentose phosphate pathway and supporting glioma cell proliferation.\",\n      \"method\": \"m6A-qRT-PCR, ALKBH5 gain/loss-of-function, G6PD mRNA stability assays, G6PD expression and activity analysis\",\n      \"journal\": \"Neurochemical research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — m6A-specific assays, gain/loss-of-function, mRNA stability measurement, single lab\",\n      \"pmids\": [\"34297301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"METTL14-mediated m6A modification of G6PD mRNA is recognized by IGF2BP2, which enhances G6PD mRNA stability, thereby upregulating G6PD expression post-transcriptionally and promoting lung adenocarcinoma tumor growth and metastasis.\",\n      \"method\": \"RNA sequencing, MeRIP-sequencing, METTL14 knockdown/overexpression, IGF2BP2 interaction with G6PD mRNA, G6PD expression and stability assays, in vivo xenograft model\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MeRIP-seq, RIP for IGF2BP2-G6PD mRNA interaction, mRNA stability assay, in vivo validation, single lab\",\n      \"pmids\": [\"39138186\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"A 20-kb human G6PD construct containing only 2.5 kb upstream and 2.0 kb downstream flanking sequence is sufficient to drive high-level, constitutive, tissue-appropriate G6PD expression in transgenic mice, with steady-state mRNA levels accounting for tissue-to-tissue variation in enzyme activity.\",\n      \"method\": \"Transgenic mouse generation, enzyme activity assays across tissues, mRNA Northern blot\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transgenic mouse model with multiple tissues, mRNA quantitation, single lab\",\n      \"pmids\": [\"8964507\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"G6PD is the rate-limiting enzyme of the pentose phosphate pathway that produces NADPH by oxidizing glucose-6-phosphate; its activity is tightly regulated by multiple post-translational modifications—including inhibitory acetylation (K403, removed by SIRT2; K171) and activating phosphorylation (Y112 by c-Src, Y437 by JAK2, T91 by CDK5), inhibitory glutarylation (removed by SIRT5), activating O-GlcNAcylation, inhibitory arginine methylation (R246, by BHMT), and inhibitory ubiquitination (K366/K403 by VHL E3 ligase)—as well as by direct protein–protein interactions with PTEN, Aldob-p53 complexes, BAG3, FDX1, HSPB1, and PAK4/p53/Mdm2, all of which modulate its capacity to form catalytically active dimers and thereby control NADPH-dependent redox homeostasis, nucleotide biosynthesis, and cell survival.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"G6PD is the rate-limiting enzyme of the pentose phosphate pathway, oxidizing glucose-6-phosphate to generate NADPH and ribose-5-phosphate that drive redox homeostasis, nucleotide and lipid biosynthesis, and cell survival [#2, #8]. Catalysis requires assembly of inactive monomers into active dimers, and a recurring theme across the literature is that this dimerization step is the regulatory fulcrum: stability and activity trade off through mutations at the dimer interface and structural NADP+ site, where the most severe clinical variants destabilize the enzyme while catalytic-residue mutations reduce activity without destabilization [#17, #18]. A dense network of post-translational modifications tunes dimer formation in opposite directions. Inhibitory inputs include acetylation at K403, which structurally distorts the dimer interface and active site to abolish activity (reversed by the deacetylase SIRT2) [#0, #4, #12], glutarylation (reversed by SIRT5) [#1], arginine methylation at R246 controlled by BHMT [#23], and VHL-mediated ubiquitination at K366/K403 that targets G6PD for proteasomal degradation [#7]. Activating inputs include O-GlcNAcylation under hypoxia [#2], tyrosine phosphorylation at Y112 by c-Src and Y437 by JAK2 that improve substrate binding and kinetics [#6, #21], and CDK5 phosphorylation at T91 that promotes dimer assembly under oxidative stress [#22]. G6PD activity is further set by direct protein partners that govern the dimer: PTEN, Aldolase B (acting as a scaffold with p53), BAG3, and FDX1 each bind G6PD and suppress its activity or stability, whereas HSPB1 and TSP50 promote its SIRT2-dependent activation [#3, #5, #9, #11, #15, #16]. Expression is independently set at the transcriptional and post-transcriptional level by Sp1/chromatin acetylation, NF-\\u03baB p65/pSTAT3, Nrf2 (engaged by HBV HBx), and m6A regulators ALKBH5, METTL14, and IGF2BP2 [#24, #26, #27, #28, #29]. Functionally, G6PD-derived NADPH is indispensable for definitive erythropoiesis, preventing apoptosis of definitive erythrocytes [#8], and for limiting protein glutathionylation and ROS-driven damage [#19, #20]. Across cancers these regulatory inputs converge to elevate PPP flux supporting proliferation [#2, #6, #21].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Establishing that G6PD-derived NADPH is not merely housekeeping but specifically required for a defined developmental program addressed why the enzyme is physiologically essential.\",\n      \"evidence\": \"G6PD-null ES cell differentiation in embryoid bodies with apoptosis assays and rescue by reducing agents, caspase inhibitors, and G6PD re-expression\",\n      \"pmids\": [\"15271799\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not define which downstream NADPH-dependent process protects definitive erythrocytes\", \"Primitive erythropoiesis tolerance mechanism not explained\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Linking G6PD deficiency to loss of redox buffering connected enzyme activity to a concrete protein-protective output.\",\n      \"evidence\": \"G6PD-deficient vs wild-type cells under oxidant challenge with glutathionylation, Ku DNA-binding, and NADPH/GSH readouts plus gene rescue\",\n      \"pmids\": [\"17516514\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Generality beyond Ku not established\", \"Single cell-line pair\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identifying PTEN as a direct binder that blocks active-dimer formation revealed that protein-protein interactions, not just substrate supply, gate PPP flux.\",\n      \"evidence\": \"Co-IP, mass spectrometry, PPP flux measurements, and epistasis with the Tcl1/hnRNPK splicing axis\",\n      \"pmids\": [\"24352616\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of dimer blockade not resolved\", \"Quantitative contribution versus PTEN's phosphatase functions unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Pinpointing K403 acetylation as a dimer-disrupting inhibitory mark reversed by SIRT2 defined the first PTM switch controlling G6PD activity.\",\n      \"evidence\": \"Acetylation-mimetic mutants, in vitro activity assays, Co-IP, and rescue in cells and mouse erythrocytes with SIRT2 depletion\",\n      \"pmids\": [\"24769394\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological acetyltransferase only proposed (KAT9/ELP3)\", \"Stoichiometry of K403 acetylation in vivo unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrating selective transcriptional upregulation of G6PD by HDAC inhibitors and tissue-appropriate constitutive expression from a compact locus established how G6PD levels are set, separate from activity regulation.\",\n      \"evidence\": \"HDACi with ChIP for Sp1/histone acetylation/Pol II and enzyme rescue in patient cells; transgenic mouse with a 20-kb G6PD construct and tissue mRNA/activity quantitation\",\n      \"pmids\": [\"24805191\", \"8964507\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific regulatory elements driving tissue variation not mapped\", \"Whether HDACi rescue translates clinically untested in these studies\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showing O-GlcNAcylation activates G6PD under hypoxia connected nutrient/stress sensing to PPP-driven biosynthesis and tumor growth.\",\n      \"evidence\": \"O-GlcNAc assays, activity assays, metabolic flux, site mutagenesis, and xenograft models\",\n      \"pmids\": [\"26399441\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Enzymes adding/removing the modification on G6PD not identified\", \"Interplay with other activating PTMs unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identifying glutarylation reversed by SIRT5 and the HSPB1-enhanced SIRT2 axis expanded the deacylation control of G6PD and showed PTM activation is scaffolded.\",\n      \"evidence\": \"SIRT5 KO/knockdown with activity, NADPH, GSH, ROS readouts; reciprocal Co-IP of HSPB1-G6PD-SIRT2 with activity assays\",\n      \"pmids\": [\"27113762\", \"27711253\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Glutarylation site(s) on G6PD not pinpointed\", \"HSPB1 axis from a single study\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Confirming SIRT2-mediated K403 deacetylation sustains leukemia proliferation with a tumor-selective vulnerability advanced G6PD regulation toward therapeutic relevance.\",\n      \"evidence\": \"SIRT2 knockdown, K403 mimetic mutants, activity and colony assays, SIRT2 inhibitors, and AML patient samples\",\n      \"pmids\": [\"27586085\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Basis of selectivity for leukemic over normal cells incomplete\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Finding BAG3 suppresses PPP-dependent DNA synthesis without lowering NADPH separated G6PD's biosynthetic from redox outputs.\",\n      \"evidence\": \"Co-IP, G6PD rescue overexpression, PPP flux and DNA synthesis assays, nucleoside supplementation rescue\",\n      \"pmids\": [\"26621836\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which BAG3 differentially channels output unclear\", \"Single lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defining a stability-versus-activity trade-off across clinical variants explained the structural logic of G6PD deficiency phenotypes.\",\n      \"evidence\": \"Bioinformatic structural analysis with biochemical characterization of variant stability and activity and clinical correlation\",\n      \"pmids\": [\"28297664\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct timeline link to specific Mendelian disease via causative-mutation rescue\", \"Predictive accuracy for novel variants not validated\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Mapping VHL-mediated ubiquitination at K366/K403 established degradation as a control point coupling glucose load to G6PD abundance and tissue injury.\",\n      \"evidence\": \"Co-IP, ubiquitination-site mutagenesis, knockdown/overexpression rescue, and G6PD-deficient mouse kidney analysis\",\n      \"pmids\": [\"30785802\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crosstalk between K403 acetylation and K403 ubiquitination not resolved here\", \"Signal triggering VHL recruitment to G6PD unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showing a small molecule activates G6PD by bridging the structural NADP+ sites to drive dimerization validated the dimer interface as a druggable activation switch.\",\n      \"evidence\": \"Biochemical activity and oligomerization assays with SAR and dimer-interface site mapping (AG1)\",\n      \"pmids\": [\"31183991\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cellular and in vivo efficacy not established in this study\", \"Selectivity over variant enzymes untested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Establishing Aldolase B as an enzymatically independent scaffold that potentiates p53-mediated G6PD inhibition added a tumor-suppressive complex to the regulatory network.\",\n      \"evidence\": \"Direct binding and Co-IP, activity assays, Aldob KO mouse with re-expression rescue, and pharmacological G6PD inhibition\",\n      \"pmids\": [\"35122041\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the Aldob-G6PD-p53 complex mechanically blocks dimers unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrating p65/pSTAT3 cooperative transcriptional activation showed inflammatory signaling directly elevates G6PD in renal cancer.\",\n      \"evidence\": \"ChIP, Co-IP of the p65/pSTAT3 complex, luciferase reporter, and xenografts\",\n      \"pmids\": [\"33041664\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Generality across tumor types not tested\", \"Single lab\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identifying c-Src Y112 phosphorylation that lowers Km and raises Kcat provided a kinetic mechanism for oncogenic G6PD activation.\",\n      \"evidence\": \"Co-IP, in vitro kinase assay with Km/Kcat analysis, site mutagenesis, flux assays, and CRC sample correlation\",\n      \"pmids\": [\"33686238\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo contribution relative to other activating PTMs unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Convergent findings positioned G6PD-derived NADPH as a determinant of cell-death modalities (ferroptosis, cuproptosis) and brown-fat thermogenesis, broadening its physiological reach.\",\n      \"evidence\": \"G6PD knockdown with POR and ferroptosis readouts; FDX1 Co-IP with stability/NADPH/GSH and cuproptosis assays; G6PD-deficient mice with cold exposure, ROS, ERK inhibition and antioxidant rescue\",\n      \"pmids\": [\"34325001\", \"37119432\", \"34521642\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"G6PD-POR link not biochemically demonstrated (Low confidence)\", \"Direct molecular coupling of NADPH levels to each death pathway incomplete\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showing TSP50 inhibits K171 acetylation and promotes SIRT2 binding identified an additional acetylation site and a tumor-promoting activator.\",\n      \"evidence\": \"LC-MS/MS, Co-IP, GST pull-down, K171 mutagenesis, and proliferation/tumor formation assays\",\n      \"pmids\": [\"33630390\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"K171 acetyltransferase not defined\", \"Relationship to K403/K89 acetylation unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identifying ALKBH5-mediated m6A demethylation as a stabilizer of G6PD mRNA established post-transcriptional control of PPP capacity.\",\n      \"evidence\": \"m6A-qRT-PCR, ALKBH5 gain/loss-of-function, mRNA stability and activity assays\",\n      \"pmids\": [\"34297301\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"m6A sites on G6PD transcript not mapped\", \"Single tumor context\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Site-specific reconstitution distinguishing activating K89 from inhibitory K403 acetylation, with structural and downstream signaling consequences, resolved how a single PTM type produces opposite effects and links G6PD to apoptosis.\",\n      \"evidence\": \"Genetic code expansion for site-specific acetylation, structural studies, MS-based PTM analysis, p53 Co-IP, and apoptosis assays\",\n      \"pmids\": [\"37798264\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous enzymes setting K89 versus K403 acetylation not identified\", \"Physiological balance of opposing marks in tissues unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defining JAK2 Y437 phosphorylation downstream of IL-6 and quercetin as a competitive NADP+-site inhibitor extended both physiological activation and pharmacological inhibition mechanisms.\",\n      \"evidence\": \"Co-IP, mutagenesis of Y437, kinetics, proliferation and xenograft assays; direct binding and competitive-inhibition kinetics for quercetin with EGFRT790M degradation\",\n      \"pmids\": [\"37949355\", \"37950872\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo selectivity of quercetin for G6PD uncertain\", \"Single lab per finding\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identifying CDK5 T91 phosphorylation as a dimer-promoting activator, BHMT-controlled R246 methylation as an inhibitor, and METTL14/IGF2BP2 m6A stabilization completed multiple converging regulatory axes feeding tumor metabolism.\",\n      \"evidence\": \"Kinase/dimerization assays and Olaparib synergy (CDK5); Co-IP, R246 mutagenesis, methyl-specific antibodies and Bhmt KO mice (BHMT); MeRIP-seq, IGF2BP2 RIP, and stability/xenograft assays (METTL14)\",\n      \"pmids\": [\"40370560\", \"38679670\", \"39138186\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Hierarchy and crosstalk among the many PTM and RNA-level inputs unresolved\", \"The R246 methyltransferase acting via BHMT not directly defined\", \"Each axis from a single study\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the dozen-plus competing PTMs, protein partners, transcriptional and m6A inputs are integrated in a given cell to set G6PD dimer state remains the central open question.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model of combinatorial PTM crosstalk on the dimer interface\", \"Endogenous writers for several modifications (K89, K171, R246, glutarylation, O-GlcNAc) unidentified\", \"Relative quantitative contribution of each regulator in normal physiology versus cancer unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [2, 6, 17]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [2, 6, 8]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [0, 1, 19]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [8, 12]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"PTEN\", \"SIRT2\", \"ALDOB\", \"BAG3\", \"FDX1\", \"HSPB1\", \"VHL\", \"PAK4\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}