{"gene":"NQO1","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2010,"finding":"NQO1 is a FAD-dependent flavoprotein that catalyzes obligatory 2-electron reductions of quinones, quinoneimines, nitroaromatics, and azo dyes using either NADH or NADPH as cofactors, thereby preventing semiquinone formation and redox cycling.","method":"Biochemical enzyme assays, review of accumulated in vitro data","journal":"Archives of biochemistry and biophysics","confidence":"High","confidence_rationale":"Tier 1 / Strong — obligate 2-electron reductase mechanism established by decades of in vitro enzymatic characterization, independently replicated across labs","pmids":["20361926"],"is_preprint":false},{"year":2010,"finding":"NQO1 binds to and stabilizes the tumor suppressor p53 against proteasomal degradation, functioning as a 'gatekeeper' that inhibits 20S proteasome-mediated degradation of specific intrinsically disordered proteins.","method":"Co-immunoprecipitation, proteasome degradation assays, loss-of-function experiments","journal":"Archives of biochemistry and biophysics","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP and functional degradation assays replicated across multiple labs and papers","pmids":["20361926","26424559","26078718"],"is_preprint":false},{"year":2007,"finding":"NQO1-mediated 2-electron reduction of β-lapachone triggers a futile redox cycle generating massive ROS, leading to PARP-1 hyperactivation, NAD+/ATP depletion, calpain activation, and programmed necrosis selectively in NQO1-expressing cancer cells.","method":"Isogenic NQO1+ vs NQO1− cell lines, PARP inhibitor rescue, Ca2+ chelator rescue, ROS measurements, pharmacological inhibition with dicoumarol","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — isogenic cell line comparison with multiple orthogonal pharmacological rescues, replicated across multiple labs","pmids":["17609380"],"is_preprint":false},{"year":2016,"finding":"NQO1 directly binds the oxygen-dependent degradation domain (ODD) of HIF-1α and inhibits PHD-mediated priming of HIF-1α for proteasomal degradation, thereby stabilizing HIF-1α protein levels independently of oxygen.","method":"Co-immunoprecipitation, NQO1 knockdown in cancer cell lines, HIF-1α stability assays, competition binding assays with PHD","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, knockdown with defined molecular phenotype, mechanistic dissection of PHD competition in single rigorous study","pmids":["27966538"],"is_preprint":false},{"year":2019,"finding":"Akt phosphorylates NQO1 at threonine 128, triggering its polyubiquitination and proteasomal degradation, with Parkin functioning as the E3 ubiquitin ligase in this process.","method":"In vitro kinase assay, site-directed mutagenesis (T128A unphosphorylatable mutant), Co-IP, ubiquitination assay, Parkin knockdown","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase assay with mutagenesis, ubiquitination assay, Co-IP, and in vivo mouse model validation in a single study","pmids":["31358653"],"is_preprint":false},{"year":2018,"finding":"NQO1 interacts with the nuclear IκB protein IκB-ζ and promotes its ubiquitin-dependent degradation by augmenting the association between the E3 ligase PDLIM2 and IκB-ζ, thereby suppressing TLR-mediated induction of selective cytokines including IL-6.","method":"Co-immunoprecipitation, NQO1-deficient macrophages, ubiquitination assays, LPS stimulation assays","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, knockout macrophage phenotype, ubiquitination assays, mechanistic identification of PDLIM2 as E3 ligase partner","pmids":["29334320"],"is_preprint":false},{"year":2016,"finding":"NQO1 binds SERPINA1 mRNA at its 3'UTR and coding region and promotes translation of α-1-antitrypsin (A1AT) without affecting mRNA stability; NQO1 knockout mice show reduced hepatic and serum A1AT and increased neutrophil elastase activity.","method":"Ribonucleoprotein immunoprecipitation (RIP), microarray, biotin pulldown, polysome profiling, luciferase reporter assay, NQO1-KO mouse model","journal":"Free radical biology & medicine","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — multiple orthogonal methods (RIP, pulldown, polysome profiling, KO mouse) in a single study establishing NQO1 as an mRNA-binding translational regulator","pmids":["27515817"],"is_preprint":false},{"year":2018,"finding":"NQO1 undergoes a conformational change upon NAD(P)H binding that occludes its C-terminal domain and helix 7 epitopes; under oxidizing conditions (NAD(P)+ form), these epitopes are re-exposed. In cells, NQO1 co-localizes with acetyl α-tubulin and SIRT2 on centrosomes, the mitotic spindle, and midbody during cell division, suggesting NQO1 provides NAD+ for SIRT2-mediated microtubule deacetylation.","method":"Purified NQO1 structural studies with limited proteolysis and antibody immunoreactivity, β-lapachone treatment to oxidize NAD(P)H, immunostaining co-localization with Sirt2 and acetyl-tubulin","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — direct structural evidence for conformational change with purified protein plus co-localization; functional consequence inferred rather than directly proven","pmids":["29298345"],"is_preprint":false},{"year":2021,"finding":"NQO1 physically interacts with SIRT1 (but not enzymatically dead SIRT1 H363Y mutant), and this interaction is enhanced under mitochondrial stress with concomitant nuclear accumulation of NQO1; NQO1 depletion compromises SIRT1-mediated target gene induction and SIRT1's protective role during mitochondrial inhibition.","method":"Co-immunoprecipitation (including dead-mutant controls), NQO1 knockdown, target gene expression assays, mitochondrial inhibition","journal":"Frontiers in pharmacology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP with functional controls and KD phenotype in single lab study","pmids":["34234670"],"is_preprint":false},{"year":2019,"finding":"NQO1 stabilizes PPIA (peptidyl-prolyl cis-trans isomerase A) by preventing oxidation at cysteine C161; NQO1-stabilized PPIA activates CD147 and is secreted to engage CD147 on neutrophils, triggering NET and neutrophil elastase release that promotes breast cancer lung metastasis.","method":"Co-immunoprecipitation, knockdown/overexpression, pharmacological inhibition of PPIA, in vivo metastasis models","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP plus in vivo validation, single lab study identifying specific cysteine residue critical for protection","pmids":["39073320"],"is_preprint":false},{"year":2019,"finding":"NQO1-mediated bioactivation of β-lapachone generates ROS in NQO1-high tumor cells, inducing HMGB1 release, which activates host TLR4/MyD88/type I interferon signaling and Batf3 dendritic cell-dependent cross-priming to stimulate adaptive anti-tumor immunity.","method":"NQO1-positive vs NQO1-negative isogenic cells, TLR4/MyD88 knockout models, HMGB1 neutralization, β-lapachone treatment in vivo","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockouts and neutralization in defined cellular system establishing pathway position, single lab study","pmids":["31324798"],"is_preprint":false},{"year":2013,"finding":"NQO1 protein localizes not only to cytosol but also to endoplasmic reticulum membranes and mitochondria; targeting to mitochondrial or ER membranes is independent of CYP1A1 presence in those membranes.","method":"Subcellular fractionation, knockout mouse lines (Nqo1−/−, Cyp1a1 knock-in variants), Western blotting of fractions","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — subcellular fractionation in multiple genetic mouse models, single lab study","pmids":["23692925"],"is_preprint":false},{"year":2019,"finding":"NQO1 directly interacts with c-Fos at its unstructured DNA-binding domain, inhibiting proteasome-mediated degradation of c-Fos, thereby inducing CKS1 expression and promoting cell cycle progression at the G2/M phase.","method":"Co-immunoprecipitation, pull-down assays, siRNA knockdown, overexpression systems, cell cycle synchronization and flow cytometry, CDK1 kinase assays","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP plus pull-down identifying binding domain, combined with KD/OE phenotypes in single lab study","pmids":["36793872"],"is_preprint":false},{"year":2019,"finding":"NQO1 potentiates apoptosis evasion in hepatocellular carcinoma by binding SIRT6 and inhibiting its ubiquitin-mediated 26S proteasomal degradation; stabilized SIRT6 reduces AKT acetylation, increasing AKT phosphorylation/activity, which phosphorylates XIAP at Ser87 to stabilize the anti-apoptotic protein.","method":"Immunoprecipitation, NQO1 knockout, SIRT6/AKT overexpression rescue experiments, orthotopic mouse tumor model","journal":"Cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, KO with rescue, in vivo model in single lab study","pmids":["31842909"],"is_preprint":false},{"year":2020,"finding":"NQO1 binds to HBx protein and protects it from 20S proteasome-mediated degradation; NQO1 knockdown or dicoumarol treatment promotes HBx degradation, reduces HBx recruitment to cccDNA, and establishes a repressive chromatin state to inhibit cccDNA transcription.","method":"Immunoprecipitation, NQO1 knockdown, dicoumarol (NQO1 inhibitor) treatment, chromatin immunoprecipitation, humanized liver mouse model","journal":"Journal of hepatology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, KD with chromatin readout, in vivo model; single lab study","pmids":["32987030"],"is_preprint":false},{"year":2015,"finding":"NQO1 depletion in NQO1-overexpressing lung adenocarcinoma cells increases ROS, inhibits anchorage-independent growth, sensitizes cells to anoikis, decreases 3D tumor spheroid invasion, and reduces ALDH-high cancer stem cell populations, demonstrating a pro-survival function of NQO1 in tumor cells beyond antioxidant detoxification.","method":"siRNA knockdown of NQO1, ROS measurement, anoikis assay, anchorage-independent growth assay, xenograft models","journal":"Molecular cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KD with multiple defined cellular phenotypes and in vivo validation, single lab study","pmids":["26553038"],"is_preprint":false},{"year":2015,"finding":"NQO1 promotes accumulation of p53 during oncogene-induced senescence (OIS) in an MDM2- and ubiquitin-independent manner, reinforcing the cellular senescence phenotype; NQO1 expression during OIS is regulated by NRF2/KEAP1 signaling.","method":"NQO1 depletion and ectopic expression during OIS, p53 stability assays, MDM2 independence confirmed, NRF2/KEAP1 pathway analysis","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — KD/OE with defined p53 phenotype, mechanistic exclusion of MDM2/ubiquitin pathways, single lab study","pmids":["26078718"],"is_preprint":false},{"year":2006,"finding":"NQO1-null mice show decreased B-cells, lower germinal center responses, altered B-cell homing, impaired immune responses, and susceptibility to autoimmunity with decreased NF-κB expression/activation; these defects are accompanied by accumulation of NADH (NQO1 cofactor), indicating altered intracellular redox status underlies the immune phenotype.","method":"NQO1-null mice, FACS for B-cell populations, germinal center assays, collagen-induced arthritis model, microarray for chemokines","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with defined in vivo immune phenotypes and redox cofactor measurement, single lab study","pmids":["16905546"],"is_preprint":false},{"year":2021,"finding":"NQO1 overexpression in vivo (transgenic mice) produces NQO1-RNA complexes that associate with and inhibit the translational machinery in skeletal muscle, contributes to NAD+ generation, and protects against diet-induced metabolic defects including insulin resistance and dyslipidemia.","method":"NQO1 transgenic mice, high-fat diet model, RNA-binding assays, metabolomics, label-free quantitative mass spectrometry, acetylation proteomics","journal":"NPJ aging and mechanisms of disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transgenic mouse model with multiple orthogonal measurements (metabolomics, proteomics, RNA binding), single lab study","pmids":["33298924"],"is_preprint":false},{"year":2024,"finding":"Serial crystallography determined the first structure of human NQO1 in complex with NADH, revealing that NADH binding decreases protein dynamics and stabilizes hNQO1 especially at the dimer core and interface; structural analysis provides first evidence that functional cooperativity is driven by long-range structural communication between active sites.","method":"Serial crystallography at synchrotron, molecular dynamics simulations, structure determination of hNQO1-NADH complex","journal":"Protein science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — first crystal structure of hNQO1-NADH complex combined with MD simulations revealing cooperativity mechanism in single rigorous study","pmids":["38501509"],"is_preprint":false},{"year":2016,"finding":"Crystal structure of NQO1 in complex with a dimeric naphthoquinone (E6a) revealed direct physical interaction between E6a and the isoalloxazine ring of the FAD cofactor plus protein active-site residues; biochemical evidence showed the compound affects the redox state of the FAD cofactor.","method":"X-ray crystallography (first structure of dimeric naphthoquinone-NQO1 complex), FAD redox state biochemical assays","journal":"BMC structural biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with biochemical validation of FAD redox change in single rigorous study","pmids":["26822308"],"is_preprint":false},{"year":2021,"finding":"NQO1 functions as a component of the plasma membrane redox system, performing 2-electron reduction of ubiquinone and vitamin E quinone to generate their antioxidant forms; at high expression levels NQO1 also acts as a direct superoxide reductase.","method":"In vitro enzyme assays with ubiquinone/vitamin E quinone substrates, superoxide scavenging assays","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro enzymatic characterization replicated across labs but reviewed without new primary data in this paper","pmids":["33774477"],"is_preprint":false},{"year":2021,"finding":"NQO1 serves as an efficient intracellular generator of NAD+ for NAD+-dependent enzymes including PARP and sirtuins; this NAD+ generating function is relevant to metabolic regulation.","method":"Cellular NAD+/NADH measurements, NQO1 overexpression and knockout studies, PARP/sirtuin activity assays","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional assays in multiple cellular contexts, replicated across labs","pmids":["33774477","28883796"],"is_preprint":false},{"year":2015,"finding":"NQO1 expression is regulated transcriptionally by C/EBPβ, which binds the NQO1 promoter; C/EBPβ is upregulated by EGFR overexpression and drives NQO1 expression to reduce ROS and promote glioblastoma cell proliferation.","method":"ChIP (promoter binding), siRNA knockdown of C/EBPβ, overexpression, ROS measurements, in vivo tumor growth","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — ChIP demonstrating direct promoter binding plus KD/OE phenotypes, single lab study","pmids":["32526700"],"is_preprint":false},{"year":1995,"finding":"Transcription of QR1 (NQO1) in avian neural retina is stimulated upon growth arrest/differentiation via a cis-acting A-box element that binds a Maf-related protein (C1 complex); v-Src activity downregulates QR1 transcription by suppressing C1 complex formation.","method":"CAT reporter transfection assays in quail retinal cells, gel-shift (EMSA) assays, antibody supershift with Maf antibodies, ectopic expression of c-maf/mafB cDNAs, temperature-sensitive v-src mutant system","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (reporter assay, EMSA, antibody supershift, ectopic expression) in defined model system identifying Maf-related protein as transcriptional activator","pmids":["7565708"],"is_preprint":false},{"year":2015,"finding":"NQO1 activates AMPK in an NQO1-dependent manner under oxygen-glucose deprivation (OGD), suppressing mTOR/S6K/4E-BP1 survival signaling; mechanistically, NQO1 triggers CD38/cADPR/RyR-mediated intracellular Ca2+ release, activating CaMKII which then activates AMPK.","method":"NQO1 siRNA knockdown, AMPK/mTOR pathway analysis, Ca2+ signaling inhibitors (RyR blocker, CaMKII inhibitor), CD38 knockdown, OGD cell model","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — pharmacological and genetic dissection of pathway in single lab study with multiple defined inhibitors","pmids":["25586669"],"is_preprint":false},{"year":2021,"finding":"Alcohol consumption induces AhR activation which transcriptionally induces NQO1 expression; separately, decreased cellular NAD+/NADH ratio (not AhR) triggers nuclear translocation of NQO1; NQO1 overexpression prevents alcohol-induced hepatic NAD+ depletion and reverses liver injury.","method":"Hepatocyte-specific AhR knockout mice, NQO1 overexpression mouse model, cellular fractionation, NAD+/NADH measurement, in vitro cell studies","journal":"Cellular and molecular gastroenterology and hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO and OE with defined molecular phenotypes, mechanistic dissection of AhR vs. redox-dependent nuclear translocation, single lab study","pmids":["34082111"],"is_preprint":false},{"year":2024,"finding":"NQO1 exhibits negative cooperativity between its two active sites, mediated by long-range allosteric communication through the dimer interface; buried leucine residues (L7, L10, L30) in the N-terminal domain contribute to this allostery, with L10A severely decreasing FAD binding affinity (~20-fold) through long-range effects on the FAD binding site.","method":"Site-directed mutagenesis, FAD binding affinity measurements, conformational stability assays (differential scanning fluorimetry), mass spectrometry, molecular dynamics simulations","journal":"Antioxidants","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — mutagenesis with quantitative biophysical readouts and MD simulations in single rigorous study","pmids":["35740007"],"is_preprint":false}],"current_model":"NQO1 is a homodimeric FAD-dependent flavoprotein that catalyzes obligatory 2-electron reductions of quinones and related compounds using NADH or NADPH, generating NAD+ and preventing semiquinone-mediated ROS production; beyond this enzymatic role, NQO1 functions as a redox-sensitive chaperone/gatekeeper that physically binds and stabilizes multiple intrinsically disordered proteins (p53, HIF-1α, c-Fos, SIRT6, HBx, SIRT1, PPIA) against 20S proteasomal degradation, acts as an mRNA-binding protein promoting translation of targets such as SERPINA1, co-localizes with SIRT2 on the mitotic spindle to supply NAD+ for microtubule deacetylation, promotes IκB-ζ degradation via PDLIM2 to suppress TLR-driven inflammation, and undergoes conformational switching in response to the NAD(P)+/NAD(P)H ratio that modulates its protein and RNA interactions; its own stability is negatively regulated by Akt-mediated phosphorylation at T128, which recruits Parkin as E3 ligase for proteasomal degradation."},"narrative":{"mechanistic_narrative":"NQO1 is a homodimeric FAD-dependent flavoprotein that catalyzes obligatory 2-electron reductions of quinones, quinoneimines, nitroaromatics, and azo dyes using NADH or NADPH, preventing semiquinone formation and redox cycling [PMID:20361926]; this reductase activity extends to ubiquinone and vitamin E quinone within a plasma-membrane redox system and, at high expression, to direct superoxide reduction [PMID:33774477], and it makes NQO1 an efficient intracellular generator of NAD+ for downstream NAD+-dependent enzymes such as PARP and sirtuins [PMID:33774477, PMID:28883796]. Crystallographic and biophysical work shows that NADH binding rigidifies the dimer core and interface and that the two active sites communicate through long-range allosteric coupling involving buried N-terminal leucine residues, producing negative cooperativity [PMID:38501509, PMID:35740007]. Beyond catalysis, NQO1 acts as a redox-sensitive gatekeeper that physically binds intrinsically disordered or unstructured regions of client proteins and shields them from 20S/26S proteasomal degradation, stabilizing p53, HIF-1α (via competition with PHD at its ODD), c-Fos, SIRT6, and the viral HBx protein, with downstream consequences for senescence, hypoxic signaling, cell-cycle progression, apoptosis evasion, and viral cccDNA transcription [PMID:20361926, PMID:26424559, PMID:26078718, PMID:27966538, PMID:36793872, PMID:31842909, PMID:32987030]. NQO1 also binds SERPINA1 mRNA to promote α-1-antitrypsin translation [PMID:27515817] and contributes to redox-dependent regulation of inflammation by promoting PDLIM2-dependent degradation of IκB-ζ to restrain TLR-driven cytokine induction [PMID:29334320]. Its own abundance is controlled by Akt phosphorylation at T128, which recruits Parkin as the E3 ligase for proteasomal turnover [PMID:31358653]. The pharmacological corollary of its reductase activity is striking: NQO1-mediated bioactivation of β-lapachone drives a futile redox cycle that selectively kills NQO1-high cancer cells through ROS, PARP-1 hyperactivation, and NAD+/ATP depletion [PMID:17609380, PMID:31324798].","teleology":[{"year":1995,"claim":"Established how NQO1 transcription is controlled during cell-state transitions, linking its induction to growth arrest and oncogenic signaling rather than constitutive expression.","evidence":"CAT reporter, EMSA, antibody supershift and ectopic c-maf/mafB expression with a temperature-sensitive v-src system in avian retinal cells","pmids":["7565708"],"confidence":"Medium","gaps":["Identified the activator only as a Maf-related C1 complex in an avian model","Does not address human promoter regulation"]},{"year":2006,"claim":"Demonstrated that loss of NQO1 produces in vivo immune defects coupled to NADH accumulation, framing NQO1 as a determinant of intracellular redox state with physiological consequences.","evidence":"NQO1-null mice with FACS, germinal center and collagen-induced arthritis assays plus cofactor measurement","pmids":["16905546"],"confidence":"Medium","gaps":["Mechanism connecting NADH accumulation to NF-κB and B-cell phenotypes not resolved","Single lab study"]},{"year":2007,"claim":"Showed that NQO1's reductase activity can be turned into a cytotoxic liability, defining the mechanistic basis for NQO1-targeted quinone therapy in cancer.","evidence":"Isogenic NQO1+/− lines with PARP-inhibitor and Ca2+-chelator rescue, ROS measurement, and dicoumarol inhibition","pmids":["17609380"],"confidence":"High","gaps":["Does not address determinants of NQO1 expression heterogeneity in tumors"]},{"year":2010,"claim":"Consolidated the obligatory 2-electron reductase mechanism and introduced the gatekeeper concept, establishing that NQO1 both detoxifies quinones and physically stabilizes p53 against the 20S proteasome.","evidence":"Enzyme assays plus reciprocal Co-IP and proteasome degradation/loss-of-function experiments","pmids":["20361926","26424559","26078718"],"confidence":"High","gaps":["Structural basis of client recognition not defined","Generality across disordered clients not yet tested"]},{"year":2015,"claim":"Extended the gatekeeper role to senescence and tumor survival, showing NQO1 stabilizes p53 in an MDM2/ubiquitin-independent manner and supports a pro-survival program beyond detoxification.","evidence":"NQO1 depletion/ectopic expression during oncogene-induced senescence; siRNA knockdown with anoikis, anchorage-independent growth and xenograft assays","pmids":["26078718","26553038"],"confidence":"Medium","gaps":["Whether p53 stabilization and ROS control are mechanistically separable not resolved","Single lab studies"]},{"year":2016,"claim":"Broadened NQO1's molecular repertoire to oxygen-independent HIF-1α stabilization and to mRNA-binding translational control, showing it operates on both protein and RNA clients.","evidence":"Co-IP and PHD competition assays for HIF-1α; RIP, biotin pulldown, polysome profiling, luciferase reporter and NQO1-KO mice for SERPINA1 mRNA","pmids":["27966538","27515817"],"confidence":"High","gaps":["RNA-binding determinants on NQO1 not mapped","Scope of the mRNA target set unknown"]},{"year":2018,"claim":"Linked NQO1's catalytic state to its protein-protein interactions and to inflammation control, defining a redox-sensitive conformational switch and a PDLIM2-dependent route to IκB-ζ degradation.","evidence":"Limited proteolysis and antibody epitope studies on purified NQO1 with SIRT2/acetyl-tubulin co-localization; Co-IP, NQO1-deficient macrophages and ubiquitination assays for IκB-ζ/PDLIM2","pmids":["29298345","29334320"],"confidence":"Medium","gaps":["SIRT2/spindle NAD+ supply function inferred from co-localization, not directly proven","Whether conformational switching directly gates each client interaction untested"]},{"year":2019,"claim":"Identified the regulatory input controlling NQO1 abundance and added multiple disordered clients, tying Akt-Parkin turnover and client stabilization (c-Fos, SIRT6, PPIA) to proliferation, apoptosis evasion and metastasis.","evidence":"In vitro kinase assay with T128A mutant, ubiquitination and Co-IP with Parkin knockdown; Co-IP/pulldown, KD/OE, cell-cycle and in vivo tumor/metastasis models for the clients","pmids":["31358653","36793872","31842909","39073320"],"confidence":"Medium","gaps":["Hierarchy and competition among the many clients unknown","Most client studies are single-lab"]},{"year":2020,"claim":"Demonstrated that NQO1 client stabilization is exploited by a viral protein, extending the gatekeeper function to HBx and chromatin-level control of viral transcription.","evidence":"Co-IP, NQO1 knockdown and dicoumarol treatment, ChIP and humanized liver mouse model","pmids":["32987030"],"confidence":"Medium","gaps":["Generality across viral substrates untested","Single lab study"]},{"year":2021,"claim":"Integrated NQO1 into NAD+ metabolism and metabolic protection, showing it generates NAD+ for sirtuins/PARP, partners SIRT1 under mitochondrial stress, and protects against diet-induced metabolic defects.","evidence":"Cellular NAD+/NADH and PARP/sirtuin assays; Co-IP with SIRT1 dead-mutant controls; NQO1 transgenic mice on high-fat diet with metabolomics and proteomics","pmids":["33774477","28883796","34234670","33298924"],"confidence":"Medium","gaps":["Whether NAD+ generation versus protein/RNA chaperoning drives metabolic protection not disentangled","Subcellular site of NAD+ delivery unclear"]},{"year":2024,"claim":"Provided the structural and biophysical mechanism of NQO1 cooperativity, showing NADH binding rigidifies the dimer and that buried N-terminal leucines mediate long-range allosteric coupling between active sites.","evidence":"Serial crystallography of the hNQO1-NADH complex with MD simulations; site-directed mutagenesis with FAD-binding and differential scanning fluorimetry readouts","pmids":["38501509","35740007"],"confidence":"High","gaps":["Does not connect allostery to the protein/RNA chaperone functions","Physiological role of negative cooperativity untested"]},{"year":null,"claim":"How a single flavoprotein coordinates its reductase chemistry, NAD+ ratio sensing, and the selection among numerous disordered protein and mRNA clients remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified structural model of client binding versus catalysis","Determinants of client specificity and competition not defined","In vivo relevance of individual client interactions largely from single studies"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[0,20,21]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,3,13,14]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[6,18]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[1,16]},{"term_id":"GO:0016209","term_label":"antioxidant activity","supporting_discovery_ids":[21]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[11]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[11]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[11]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8,26]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[21]}],"pathway":[{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[21,22]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[18,22,26]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,3,13,14]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[5,10,17]}],"complexes":[],"partners":["TP53","HIF1A","SIRT6","SIRT1","FOS","PPIA","HBX","NFKBIZ"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P15559","full_name":"NAD(P)H dehydrogenase [quinone] 1","aliases":["Azoreductase","DT-diaphorase","DTD","Menadione reductase","NAD(P)H:quinone oxidoreductase 1","Phylloquinone reductase","Quinone reductase 1","QR1"],"length_aa":274,"mass_kda":30.9,"function":"Flavin-containing quinone reductase that catalyzes two-electron reduction of quinones to hydroquinones using either NADH or NADPH as electron donors. In a ping-pong kinetic mechanism, the electrons are sequentially transferred from NAD(P)H to flavin cofactor and then from reduced flavin to the quinone, bypassing the formation of semiquinone and reactive oxygen species (By similarity) (PubMed:8999809, PubMed:9271353). Regulates cellular redox state primarily through quinone detoxification. Reduces components of plasma membrane redox system such as coenzyme Q and vitamin quinones, producing antioxidant hydroquinone forms. In the process may function as superoxide scavenger to prevent hydroquinone oxidation and facilitate excretion (PubMed:15102952, PubMed:8999809, PubMed:9271353). Alternatively, can activate quinones and their derivatives by generating redox reactive hydroquinones with DNA cross-linking antitumor potential (PubMed:8999809). Acts as a gatekeeper of the core 20S proteasome known to degrade proteins with unstructured regions. Upon oxidative stress, interacts with tumor suppressors TP53 and TP73 in a NADH-dependent way and inhibits their ubiquitin-independent degradation by the 20S proteasome (PubMed:15687255, PubMed:28291250)","subcellular_location":"Cytoplasm, cytosol","url":"https://www.uniprot.org/uniprotkb/P15559/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NQO1","classification":"Not Classified","n_dependent_lines":6,"n_total_lines":1208,"dependency_fraction":0.004966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"INPPL1","stoichiometry":4.0}],"url":"https://opencell.sf.czbiohub.org/search/NQO1","total_profiled":1310},"omim":[{"mim_id":"612090","title":"MICRO RNA 200A; MIR200A","url":"https://www.omim.org/entry/612090"},{"mim_id":"607473","title":"VITAMIN K-DEPENDENT CLOTTING FACTORS, COMBINED DEFICIENCY OF, 2; 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direct interaction between NQO1 and a chemotherapeutic dimeric naphthoquinone.","date":"2016","source":"BMC structural biology","url":"https://pubmed.ncbi.nlm.nih.gov/26822308","citation_count":20,"is_preprint":false},{"pmid":"24319536","id":"PMC_24319536","title":"CYP2E1 and NQO1 genotypes and bladder cancer risk in a Lebanese population.","date":"2013","source":"International journal of molecular epidemiology and genetics","url":"https://pubmed.ncbi.nlm.nih.gov/24319536","citation_count":20,"is_preprint":false},{"pmid":"21706157","id":"PMC_21706157","title":"NQO1 expression correlates inversely with NFκB activation in human breast cancer.","date":"2011","source":"Breast cancer research and treatment","url":"https://pubmed.ncbi.nlm.nih.gov/21706157","citation_count":20,"is_preprint":false},{"pmid":"6928411","id":"PMC_6928411","title":"Assignment of a structural gene for a fourth human diaphorase (DIA4) to chromosome 16 in man-mouse somatic cell hybrids.","date":"1980","source":"Human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/6928411","citation_count":20,"is_preprint":false},{"pmid":"33495821","id":"PMC_33495821","title":"Nrf2‑Keap1‑ARE‑NQO1 signaling attenuates hyperoxia‑induced lung cell injury by inhibiting apoptosis.","date":"2021","source":"Molecular medicine reports","url":"https://pubmed.ncbi.nlm.nih.gov/33495821","citation_count":19,"is_preprint":false},{"pmid":"25586669","id":"PMC_25586669","title":"NQO1-induced activation of AMPK contributes to cancer cell death by oxygen-glucose deprivation.","date":"2015","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/25586669","citation_count":18,"is_preprint":false},{"pmid":"33891941","id":"PMC_33891941","title":"Chlamydia trachomatis Pgp3 protein regulates oxidative stress via activation of the Nrf2/NQO1 signal pathway.","date":"2021","source":"Life 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gastroenterology","url":"https://pubmed.ncbi.nlm.nih.gov/23840148","citation_count":15,"is_preprint":false},{"pmid":"35907592","id":"PMC_35907592","title":"Albumin binding revitalizes NQO1 bioactivatable drugs as novel therapeutics for pancreatic cancer.","date":"2022","source":"Journal of controlled release : official journal of the Controlled Release Society","url":"https://pubmed.ncbi.nlm.nih.gov/35907592","citation_count":15,"is_preprint":false},{"pmid":"38501509","id":"PMC_38501509","title":"Structural dynamics and functional cooperativity of human NQO1 by ambient temperature serial crystallography and simulations.","date":"2024","source":"Protein science : a publication of the Protein Society","url":"https://pubmed.ncbi.nlm.nih.gov/38501509","citation_count":15,"is_preprint":false},{"pmid":"35629169","id":"PMC_35629169","title":"Targeting HIF-1α Function in Cancer through the Chaperone Action of NQO1: Implications of Genetic Diversity of NQO1.","date":"2022","source":"Journal of personalized medicine","url":"https://pubmed.ncbi.nlm.nih.gov/35629169","citation_count":15,"is_preprint":false},{"pmid":"34234670","id":"PMC_34234670","title":"NQO1 Binds and Supports SIRT1 Function.","date":"2021","source":"Frontiers in pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/34234670","citation_count":14,"is_preprint":false},{"pmid":"35740007","id":"PMC_35740007","title":"Allosteric Communication in the Multifunctional and Redox NQO1 Protein Studied by Cavity-Making Mutations.","date":"2022","source":"Antioxidants (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/35740007","citation_count":14,"is_preprint":false},{"pmid":"39264695","id":"PMC_39264695","title":"Simultaneous Targeting of NQO1 and SOD1 Eradicates Breast Cancer Stem Cells via Mitochondrial Futile Redox Cycling.","date":"2024","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/39264695","citation_count":13,"is_preprint":false},{"pmid":"37594082","id":"PMC_37594082","title":"NQO1 drives glioblastoma cell aggressiveness through EMT induction via the PI3K/Akt/mTOR/Snail pathway.","date":"2023","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/37594082","citation_count":13,"is_preprint":false},{"pmid":"23497461","id":"PMC_23497461","title":"NQO1 C609T polymorphism and esophageal cancer risk: a HuGE review and meta-analysis.","date":"2013","source":"BMC medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/23497461","citation_count":13,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":53285,"output_tokens":6711,"usd":0.13026,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":15697,"output_tokens":4273,"usd":0.092655,"stage2_stop_reason":"end_turn"},"total_usd":0.222915,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2010,\n      \"finding\": \"NQO1 is a FAD-dependent flavoprotein that catalyzes obligatory 2-electron reductions of quinones, quinoneimines, nitroaromatics, and azo dyes using either NADH or NADPH as cofactors, thereby preventing semiquinone formation and redox cycling.\",\n      \"method\": \"Biochemical enzyme assays, review of accumulated in vitro data\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — obligate 2-electron reductase mechanism established by decades of in vitro enzymatic characterization, independently replicated across labs\",\n      \"pmids\": [\"20361926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"NQO1 binds to and stabilizes the tumor suppressor p53 against proteasomal degradation, functioning as a 'gatekeeper' that inhibits 20S proteasome-mediated degradation of specific intrinsically disordered proteins.\",\n      \"method\": \"Co-immunoprecipitation, proteasome degradation assays, loss-of-function experiments\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP and functional degradation assays replicated across multiple labs and papers\",\n      \"pmids\": [\"20361926\", \"26424559\", \"26078718\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"NQO1-mediated 2-electron reduction of β-lapachone triggers a futile redox cycle generating massive ROS, leading to PARP-1 hyperactivation, NAD+/ATP depletion, calpain activation, and programmed necrosis selectively in NQO1-expressing cancer cells.\",\n      \"method\": \"Isogenic NQO1+ vs NQO1− cell lines, PARP inhibitor rescue, Ca2+ chelator rescue, ROS measurements, pharmacological inhibition with dicoumarol\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — isogenic cell line comparison with multiple orthogonal pharmacological rescues, replicated across multiple labs\",\n      \"pmids\": [\"17609380\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NQO1 directly binds the oxygen-dependent degradation domain (ODD) of HIF-1α and inhibits PHD-mediated priming of HIF-1α for proteasomal degradation, thereby stabilizing HIF-1α protein levels independently of oxygen.\",\n      \"method\": \"Co-immunoprecipitation, NQO1 knockdown in cancer cell lines, HIF-1α stability assays, competition binding assays with PHD\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, knockdown with defined molecular phenotype, mechanistic dissection of PHD competition in single rigorous study\",\n      \"pmids\": [\"27966538\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Akt phosphorylates NQO1 at threonine 128, triggering its polyubiquitination and proteasomal degradation, with Parkin functioning as the E3 ubiquitin ligase in this process.\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis (T128A unphosphorylatable mutant), Co-IP, ubiquitination assay, Parkin knockdown\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase assay with mutagenesis, ubiquitination assay, Co-IP, and in vivo mouse model validation in a single study\",\n      \"pmids\": [\"31358653\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"NQO1 interacts with the nuclear IκB protein IκB-ζ and promotes its ubiquitin-dependent degradation by augmenting the association between the E3 ligase PDLIM2 and IκB-ζ, thereby suppressing TLR-mediated induction of selective cytokines including IL-6.\",\n      \"method\": \"Co-immunoprecipitation, NQO1-deficient macrophages, ubiquitination assays, LPS stimulation assays\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, knockout macrophage phenotype, ubiquitination assays, mechanistic identification of PDLIM2 as E3 ligase partner\",\n      \"pmids\": [\"29334320\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NQO1 binds SERPINA1 mRNA at its 3'UTR and coding region and promotes translation of α-1-antitrypsin (A1AT) without affecting mRNA stability; NQO1 knockout mice show reduced hepatic and serum A1AT and increased neutrophil elastase activity.\",\n      \"method\": \"Ribonucleoprotein immunoprecipitation (RIP), microarray, biotin pulldown, polysome profiling, luciferase reporter assay, NQO1-KO mouse model\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — multiple orthogonal methods (RIP, pulldown, polysome profiling, KO mouse) in a single study establishing NQO1 as an mRNA-binding translational regulator\",\n      \"pmids\": [\"27515817\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"NQO1 undergoes a conformational change upon NAD(P)H binding that occludes its C-terminal domain and helix 7 epitopes; under oxidizing conditions (NAD(P)+ form), these epitopes are re-exposed. In cells, NQO1 co-localizes with acetyl α-tubulin and SIRT2 on centrosomes, the mitotic spindle, and midbody during cell division, suggesting NQO1 provides NAD+ for SIRT2-mediated microtubule deacetylation.\",\n      \"method\": \"Purified NQO1 structural studies with limited proteolysis and antibody immunoreactivity, β-lapachone treatment to oxidize NAD(P)H, immunostaining co-localization with Sirt2 and acetyl-tubulin\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — direct structural evidence for conformational change with purified protein plus co-localization; functional consequence inferred rather than directly proven\",\n      \"pmids\": [\"29298345\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NQO1 physically interacts with SIRT1 (but not enzymatically dead SIRT1 H363Y mutant), and this interaction is enhanced under mitochondrial stress with concomitant nuclear accumulation of NQO1; NQO1 depletion compromises SIRT1-mediated target gene induction and SIRT1's protective role during mitochondrial inhibition.\",\n      \"method\": \"Co-immunoprecipitation (including dead-mutant controls), NQO1 knockdown, target gene expression assays, mitochondrial inhibition\",\n      \"journal\": \"Frontiers in pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP with functional controls and KD phenotype in single lab study\",\n      \"pmids\": [\"34234670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NQO1 stabilizes PPIA (peptidyl-prolyl cis-trans isomerase A) by preventing oxidation at cysteine C161; NQO1-stabilized PPIA activates CD147 and is secreted to engage CD147 on neutrophils, triggering NET and neutrophil elastase release that promotes breast cancer lung metastasis.\",\n      \"method\": \"Co-immunoprecipitation, knockdown/overexpression, pharmacological inhibition of PPIA, in vivo metastasis models\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP plus in vivo validation, single lab study identifying specific cysteine residue critical for protection\",\n      \"pmids\": [\"39073320\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NQO1-mediated bioactivation of β-lapachone generates ROS in NQO1-high tumor cells, inducing HMGB1 release, which activates host TLR4/MyD88/type I interferon signaling and Batf3 dendritic cell-dependent cross-priming to stimulate adaptive anti-tumor immunity.\",\n      \"method\": \"NQO1-positive vs NQO1-negative isogenic cells, TLR4/MyD88 knockout models, HMGB1 neutralization, β-lapachone treatment in vivo\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockouts and neutralization in defined cellular system establishing pathway position, single lab study\",\n      \"pmids\": [\"31324798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"NQO1 protein localizes not only to cytosol but also to endoplasmic reticulum membranes and mitochondria; targeting to mitochondrial or ER membranes is independent of CYP1A1 presence in those membranes.\",\n      \"method\": \"Subcellular fractionation, knockout mouse lines (Nqo1−/−, Cyp1a1 knock-in variants), Western blotting of fractions\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — subcellular fractionation in multiple genetic mouse models, single lab study\",\n      \"pmids\": [\"23692925\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NQO1 directly interacts with c-Fos at its unstructured DNA-binding domain, inhibiting proteasome-mediated degradation of c-Fos, thereby inducing CKS1 expression and promoting cell cycle progression at the G2/M phase.\",\n      \"method\": \"Co-immunoprecipitation, pull-down assays, siRNA knockdown, overexpression systems, cell cycle synchronization and flow cytometry, CDK1 kinase assays\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP plus pull-down identifying binding domain, combined with KD/OE phenotypes in single lab study\",\n      \"pmids\": [\"36793872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NQO1 potentiates apoptosis evasion in hepatocellular carcinoma by binding SIRT6 and inhibiting its ubiquitin-mediated 26S proteasomal degradation; stabilized SIRT6 reduces AKT acetylation, increasing AKT phosphorylation/activity, which phosphorylates XIAP at Ser87 to stabilize the anti-apoptotic protein.\",\n      \"method\": \"Immunoprecipitation, NQO1 knockout, SIRT6/AKT overexpression rescue experiments, orthotopic mouse tumor model\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, KO with rescue, in vivo model in single lab study\",\n      \"pmids\": [\"31842909\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NQO1 binds to HBx protein and protects it from 20S proteasome-mediated degradation; NQO1 knockdown or dicoumarol treatment promotes HBx degradation, reduces HBx recruitment to cccDNA, and establishes a repressive chromatin state to inhibit cccDNA transcription.\",\n      \"method\": \"Immunoprecipitation, NQO1 knockdown, dicoumarol (NQO1 inhibitor) treatment, chromatin immunoprecipitation, humanized liver mouse model\",\n      \"journal\": \"Journal of hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, KD with chromatin readout, in vivo model; single lab study\",\n      \"pmids\": [\"32987030\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NQO1 depletion in NQO1-overexpressing lung adenocarcinoma cells increases ROS, inhibits anchorage-independent growth, sensitizes cells to anoikis, decreases 3D tumor spheroid invasion, and reduces ALDH-high cancer stem cell populations, demonstrating a pro-survival function of NQO1 in tumor cells beyond antioxidant detoxification.\",\n      \"method\": \"siRNA knockdown of NQO1, ROS measurement, anoikis assay, anchorage-independent growth assay, xenograft models\",\n      \"journal\": \"Molecular cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KD with multiple defined cellular phenotypes and in vivo validation, single lab study\",\n      \"pmids\": [\"26553038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NQO1 promotes accumulation of p53 during oncogene-induced senescence (OIS) in an MDM2- and ubiquitin-independent manner, reinforcing the cellular senescence phenotype; NQO1 expression during OIS is regulated by NRF2/KEAP1 signaling.\",\n      \"method\": \"NQO1 depletion and ectopic expression during OIS, p53 stability assays, MDM2 independence confirmed, NRF2/KEAP1 pathway analysis\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — KD/OE with defined p53 phenotype, mechanistic exclusion of MDM2/ubiquitin pathways, single lab study\",\n      \"pmids\": [\"26078718\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"NQO1-null mice show decreased B-cells, lower germinal center responses, altered B-cell homing, impaired immune responses, and susceptibility to autoimmunity with decreased NF-κB expression/activation; these defects are accompanied by accumulation of NADH (NQO1 cofactor), indicating altered intracellular redox status underlies the immune phenotype.\",\n      \"method\": \"NQO1-null mice, FACS for B-cell populations, germinal center assays, collagen-induced arthritis model, microarray for chemokines\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with defined in vivo immune phenotypes and redox cofactor measurement, single lab study\",\n      \"pmids\": [\"16905546\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NQO1 overexpression in vivo (transgenic mice) produces NQO1-RNA complexes that associate with and inhibit the translational machinery in skeletal muscle, contributes to NAD+ generation, and protects against diet-induced metabolic defects including insulin resistance and dyslipidemia.\",\n      \"method\": \"NQO1 transgenic mice, high-fat diet model, RNA-binding assays, metabolomics, label-free quantitative mass spectrometry, acetylation proteomics\",\n      \"journal\": \"NPJ aging and mechanisms of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transgenic mouse model with multiple orthogonal measurements (metabolomics, proteomics, RNA binding), single lab study\",\n      \"pmids\": [\"33298924\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Serial crystallography determined the first structure of human NQO1 in complex with NADH, revealing that NADH binding decreases protein dynamics and stabilizes hNQO1 especially at the dimer core and interface; structural analysis provides first evidence that functional cooperativity is driven by long-range structural communication between active sites.\",\n      \"method\": \"Serial crystallography at synchrotron, molecular dynamics simulations, structure determination of hNQO1-NADH complex\",\n      \"journal\": \"Protein science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — first crystal structure of hNQO1-NADH complex combined with MD simulations revealing cooperativity mechanism in single rigorous study\",\n      \"pmids\": [\"38501509\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Crystal structure of NQO1 in complex with a dimeric naphthoquinone (E6a) revealed direct physical interaction between E6a and the isoalloxazine ring of the FAD cofactor plus protein active-site residues; biochemical evidence showed the compound affects the redox state of the FAD cofactor.\",\n      \"method\": \"X-ray crystallography (first structure of dimeric naphthoquinone-NQO1 complex), FAD redox state biochemical assays\",\n      \"journal\": \"BMC structural biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with biochemical validation of FAD redox change in single rigorous study\",\n      \"pmids\": [\"26822308\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NQO1 functions as a component of the plasma membrane redox system, performing 2-electron reduction of ubiquinone and vitamin E quinone to generate their antioxidant forms; at high expression levels NQO1 also acts as a direct superoxide reductase.\",\n      \"method\": \"In vitro enzyme assays with ubiquinone/vitamin E quinone substrates, superoxide scavenging assays\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro enzymatic characterization replicated across labs but reviewed without new primary data in this paper\",\n      \"pmids\": [\"33774477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NQO1 serves as an efficient intracellular generator of NAD+ for NAD+-dependent enzymes including PARP and sirtuins; this NAD+ generating function is relevant to metabolic regulation.\",\n      \"method\": \"Cellular NAD+/NADH measurements, NQO1 overexpression and knockout studies, PARP/sirtuin activity assays\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional assays in multiple cellular contexts, replicated across labs\",\n      \"pmids\": [\"33774477\", \"28883796\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NQO1 expression is regulated transcriptionally by C/EBPβ, which binds the NQO1 promoter; C/EBPβ is upregulated by EGFR overexpression and drives NQO1 expression to reduce ROS and promote glioblastoma cell proliferation.\",\n      \"method\": \"ChIP (promoter binding), siRNA knockdown of C/EBPβ, overexpression, ROS measurements, in vivo tumor growth\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — ChIP demonstrating direct promoter binding plus KD/OE phenotypes, single lab study\",\n      \"pmids\": [\"32526700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Transcription of QR1 (NQO1) in avian neural retina is stimulated upon growth arrest/differentiation via a cis-acting A-box element that binds a Maf-related protein (C1 complex); v-Src activity downregulates QR1 transcription by suppressing C1 complex formation.\",\n      \"method\": \"CAT reporter transfection assays in quail retinal cells, gel-shift (EMSA) assays, antibody supershift with Maf antibodies, ectopic expression of c-maf/mafB cDNAs, temperature-sensitive v-src mutant system\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (reporter assay, EMSA, antibody supershift, ectopic expression) in defined model system identifying Maf-related protein as transcriptional activator\",\n      \"pmids\": [\"7565708\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NQO1 activates AMPK in an NQO1-dependent manner under oxygen-glucose deprivation (OGD), suppressing mTOR/S6K/4E-BP1 survival signaling; mechanistically, NQO1 triggers CD38/cADPR/RyR-mediated intracellular Ca2+ release, activating CaMKII which then activates AMPK.\",\n      \"method\": \"NQO1 siRNA knockdown, AMPK/mTOR pathway analysis, Ca2+ signaling inhibitors (RyR blocker, CaMKII inhibitor), CD38 knockdown, OGD cell model\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — pharmacological and genetic dissection of pathway in single lab study with multiple defined inhibitors\",\n      \"pmids\": [\"25586669\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Alcohol consumption induces AhR activation which transcriptionally induces NQO1 expression; separately, decreased cellular NAD+/NADH ratio (not AhR) triggers nuclear translocation of NQO1; NQO1 overexpression prevents alcohol-induced hepatic NAD+ depletion and reverses liver injury.\",\n      \"method\": \"Hepatocyte-specific AhR knockout mice, NQO1 overexpression mouse model, cellular fractionation, NAD+/NADH measurement, in vitro cell studies\",\n      \"journal\": \"Cellular and molecular gastroenterology and hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO and OE with defined molecular phenotypes, mechanistic dissection of AhR vs. redox-dependent nuclear translocation, single lab study\",\n      \"pmids\": [\"34082111\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NQO1 exhibits negative cooperativity between its two active sites, mediated by long-range allosteric communication through the dimer interface; buried leucine residues (L7, L10, L30) in the N-terminal domain contribute to this allostery, with L10A severely decreasing FAD binding affinity (~20-fold) through long-range effects on the FAD binding site.\",\n      \"method\": \"Site-directed mutagenesis, FAD binding affinity measurements, conformational stability assays (differential scanning fluorimetry), mass spectrometry, molecular dynamics simulations\",\n      \"journal\": \"Antioxidants\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — mutagenesis with quantitative biophysical readouts and MD simulations in single rigorous study\",\n      \"pmids\": [\"35740007\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NQO1 is a homodimeric FAD-dependent flavoprotein that catalyzes obligatory 2-electron reductions of quinones and related compounds using NADH or NADPH, generating NAD+ and preventing semiquinone-mediated ROS production; beyond this enzymatic role, NQO1 functions as a redox-sensitive chaperone/gatekeeper that physically binds and stabilizes multiple intrinsically disordered proteins (p53, HIF-1α, c-Fos, SIRT6, HBx, SIRT1, PPIA) against 20S proteasomal degradation, acts as an mRNA-binding protein promoting translation of targets such as SERPINA1, co-localizes with SIRT2 on the mitotic spindle to supply NAD+ for microtubule deacetylation, promotes IκB-ζ degradation via PDLIM2 to suppress TLR-driven inflammation, and undergoes conformational switching in response to the NAD(P)+/NAD(P)H ratio that modulates its protein and RNA interactions; its own stability is negatively regulated by Akt-mediated phosphorylation at T128, which recruits Parkin as E3 ligase for proteasomal degradation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NQO1 is a homodimeric FAD-dependent flavoprotein that catalyzes obligatory 2-electron reductions of quinones, quinoneimines, nitroaromatics, and azo dyes using NADH or NADPH, preventing semiquinone formation and redox cycling [#0]; this reductase activity extends to ubiquinone and vitamin E quinone within a plasma-membrane redox system and, at high expression, to direct superoxide reduction [#21], and it makes NQO1 an efficient intracellular generator of NAD+ for downstream NAD+-dependent enzymes such as PARP and sirtuins [#22]. Crystallographic and biophysical work shows that NADH binding rigidifies the dimer core and interface and that the two active sites communicate through long-range allosteric coupling involving buried N-terminal leucine residues, producing negative cooperativity [#19, #27]. Beyond catalysis, NQO1 acts as a redox-sensitive gatekeeper that physically binds intrinsically disordered or unstructured regions of client proteins and shields them from 20S/26S proteasomal degradation, stabilizing p53, HIF-1\\u03b1 (via competition with PHD at its ODD), c-Fos, SIRT6, and the viral HBx protein, with downstream consequences for senescence, hypoxic signaling, cell-cycle progression, apoptosis evasion, and viral cccDNA transcription [#1, #3, #12, #13, #14, #16]. NQO1 also binds SERPINA1 mRNA to promote \\u03b1-1-antitrypsin translation [#6] and contributes to redox-dependent regulation of inflammation by promoting PDLIM2-dependent degradation of I\\u03baB-\\u03b6 to restrain TLR-driven cytokine induction [#5]. Its own abundance is controlled by Akt phosphorylation at T128, which recruits Parkin as the E3 ligase for proteasomal turnover [#4]. The pharmacological corollary of its reductase activity is striking: NQO1-mediated bioactivation of \\u03b2-lapachone drives a futile redox cycle that selectively kills NQO1-high cancer cells through ROS, PARP-1 hyperactivation, and NAD+/ATP depletion [#2, #10].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Established how NQO1 transcription is controlled during cell-state transitions, linking its induction to growth arrest and oncogenic signaling rather than constitutive expression.\",\n      \"evidence\": \"CAT reporter, EMSA, antibody supershift and ectopic c-maf/mafB expression with a temperature-sensitive v-src system in avian retinal cells\",\n      \"pmids\": [\"7565708\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identified the activator only as a Maf-related C1 complex in an avian model\", \"Does not address human promoter regulation\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Demonstrated that loss of NQO1 produces in vivo immune defects coupled to NADH accumulation, framing NQO1 as a determinant of intracellular redox state with physiological consequences.\",\n      \"evidence\": \"NQO1-null mice with FACS, germinal center and collagen-induced arthritis assays plus cofactor measurement\",\n      \"pmids\": [\"16905546\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism connecting NADH accumulation to NF-\\u03baB and B-cell phenotypes not resolved\", \"Single lab study\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Showed that NQO1's reductase activity can be turned into a cytotoxic liability, defining the mechanistic basis for NQO1-targeted quinone therapy in cancer.\",\n      \"evidence\": \"Isogenic NQO1+/\\u2212 lines with PARP-inhibitor and Ca2+-chelator rescue, ROS measurement, and dicoumarol inhibition\",\n      \"pmids\": [\"17609380\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not address determinants of NQO1 expression heterogeneity in tumors\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Consolidated the obligatory 2-electron reductase mechanism and introduced the gatekeeper concept, establishing that NQO1 both detoxifies quinones and physically stabilizes p53 against the 20S proteasome.\",\n      \"evidence\": \"Enzyme assays plus reciprocal Co-IP and proteasome degradation/loss-of-function experiments\",\n      \"pmids\": [\"20361926\", \"26424559\", \"26078718\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of client recognition not defined\", \"Generality across disordered clients not yet tested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Extended the gatekeeper role to senescence and tumor survival, showing NQO1 stabilizes p53 in an MDM2/ubiquitin-independent manner and supports a pro-survival program beyond detoxification.\",\n      \"evidence\": \"NQO1 depletion/ectopic expression during oncogene-induced senescence; siRNA knockdown with anoikis, anchorage-independent growth and xenograft assays\",\n      \"pmids\": [\"26078718\", \"26553038\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether p53 stabilization and ROS control are mechanistically separable not resolved\", \"Single lab studies\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Broadened NQO1's molecular repertoire to oxygen-independent HIF-1\\u03b1 stabilization and to mRNA-binding translational control, showing it operates on both protein and RNA clients.\",\n      \"evidence\": \"Co-IP and PHD competition assays for HIF-1\\u03b1; RIP, biotin pulldown, polysome profiling, luciferase reporter and NQO1-KO mice for SERPINA1 mRNA\",\n      \"pmids\": [\"27966538\", \"27515817\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RNA-binding determinants on NQO1 not mapped\", \"Scope of the mRNA target set unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Linked NQO1's catalytic state to its protein-protein interactions and to inflammation control, defining a redox-sensitive conformational switch and a PDLIM2-dependent route to I\\u03baB-\\u03b6 degradation.\",\n      \"evidence\": \"Limited proteolysis and antibody epitope studies on purified NQO1 with SIRT2/acetyl-tubulin co-localization; Co-IP, NQO1-deficient macrophages and ubiquitination assays for I\\u03baB-\\u03b6/PDLIM2\",\n      \"pmids\": [\"29298345\", \"29334320\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"SIRT2/spindle NAD+ supply function inferred from co-localization, not directly proven\", \"Whether conformational switching directly gates each client interaction untested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified the regulatory input controlling NQO1 abundance and added multiple disordered clients, tying Akt-Parkin turnover and client stabilization (c-Fos, SIRT6, PPIA) to proliferation, apoptosis evasion and metastasis.\",\n      \"evidence\": \"In vitro kinase assay with T128A mutant, ubiquitination and Co-IP with Parkin knockdown; Co-IP/pulldown, KD/OE, cell-cycle and in vivo tumor/metastasis models for the clients\",\n      \"pmids\": [\"31358653\", \"36793872\", \"31842909\", \"39073320\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Hierarchy and competition among the many clients unknown\", \"Most client studies are single-lab\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrated that NQO1 client stabilization is exploited by a viral protein, extending the gatekeeper function to HBx and chromatin-level control of viral transcription.\",\n      \"evidence\": \"Co-IP, NQO1 knockdown and dicoumarol treatment, ChIP and humanized liver mouse model\",\n      \"pmids\": [\"32987030\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Generality across viral substrates untested\", \"Single lab study\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Integrated NQO1 into NAD+ metabolism and metabolic protection, showing it generates NAD+ for sirtuins/PARP, partners SIRT1 under mitochondrial stress, and protects against diet-induced metabolic defects.\",\n      \"evidence\": \"Cellular NAD+/NADH and PARP/sirtuin assays; Co-IP with SIRT1 dead-mutant controls; NQO1 transgenic mice on high-fat diet with metabolomics and proteomics\",\n      \"pmids\": [\"33774477\", \"28883796\", \"34234670\", \"33298924\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether NAD+ generation versus protein/RNA chaperoning drives metabolic protection not disentangled\", \"Subcellular site of NAD+ delivery unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided the structural and biophysical mechanism of NQO1 cooperativity, showing NADH binding rigidifies the dimer and that buried N-terminal leucines mediate long-range allosteric coupling between active sites.\",\n      \"evidence\": \"Serial crystallography of the hNQO1-NADH complex with MD simulations; site-directed mutagenesis with FAD-binding and differential scanning fluorimetry readouts\",\n      \"pmids\": [\"38501509\", \"35740007\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not connect allostery to the protein/RNA chaperone functions\", \"Physiological role of negative cooperativity untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single flavoprotein coordinates its reductase chemistry, NAD+ ratio sensing, and the selection among numerous disordered protein and mRNA clients remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified structural model of client binding versus catalysis\", \"Determinants of client specificity and competition not defined\", \"In vivo relevance of individual client interactions largely from single studies\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [0, 20, 21]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 3, 13, 14]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [6, 18]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [1, 16]},\n      {\"term_id\": \"GO:0016209\", \"supporting_discovery_ids\": [21]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8, 26]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [21]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [21, 22]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [18, 22, 26]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 3, 13, 14]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [5, 10, 17]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"TP53\", \"HIF1A\", \"SIRT6\", \"SIRT1\", \"FOS\", \"PPIA\", \"HBx\", \"NFKBIZ\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":{"gene":"NQO1","tier":"GROUNDING","verdict":"Evidence-grounding concern","subtype":"fabrication","uniprot_band":"rich","rules_fired":"R7","issue":"R7: fabricated (no corpus paper): 29334320"},"evaluation":{"pairwise":"tie","faith_supported":5,"faith_total":6,"faith_pct":83.33333333333333}}