{"gene":"NOXO1","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2003,"finding":"NOXO1 PX domain binds phosphatidylinositol lipids (PtdIns 3,5-P2, PtdIns 5-P, PtdIns 4-P) and mediates constitutive membrane co-localization with Nox1; a point mutation in the PX domain decreases lipid binding, causes cytosolic localization, and inhibits Nox1 activation. Co-transfection of Nox1, NOXO1, and NOXA1 reconstitutes ROS generation in HEK293 cells without agonist stimulation. NOXO1 lacks the autoinhibitory region and phosphorylation sites present in p47phox.","method":"Co-transfection/reconstitution in HEK293 cells, lipid-binding assays, site-directed mutagenesis of PX domain, co-localization imaging","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution in cells, lipid-binding assay, mutagenesis with functional readout, multiple orthogonal methods in single rigorous study","pmids":["14617635"],"is_preprint":false},{"year":2004,"finding":"Nox3 is strongly activated by NOXO1 alone (without NOXA1 or p67phox), unlike gp91phox or Nox1 which require both organizer and activator subunits. Nox3 regulation by p47phox (but not NOXO1) is PMA-dependent, consistent with the phosphorylation-regulated autoinhibitory region present in p47phox but absent in NOXO1. Deletion of the autoinhibitory region from p47phox renders it highly active without PMA but still requires an activator subunit.","method":"Cell-based reconstitution assay measuring superoxide production with various subunit combinations and mutants","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution assay with multiple subunit combinations and deletion mutants, clear mechanistic dissection","pmids":["15181005"],"is_preprint":false},{"year":2005,"finding":"NOXO1 has four splice forms (alpha, beta, gamma, delta) arising from alternative splicing of exon 3 in the PX domain region. NOXO1beta is the major form in colon and fetal liver; NOXO1gamma is the majority in testis. Both NOXO1beta and NOXO1gamma bind phosphoinositide lipids with the same specificity and affinity but differ functionally: NOXO1gamma shows poorer ability to activate Nox3 compared with NOXO1beta, while both activate Nox1 equally.","method":"RT-PCR/cloning of splice forms, purified PX domain lipid-binding assays, cell-based Nox1/Nox3 reconstitution assays","journal":"Gene","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro lipid-binding assay plus cell-based functional reconstitution, single lab with multiple orthogonal methods","pmids":["15949904"],"is_preprint":false},{"year":2006,"finding":"NOXO1 contains an intramolecular interaction between its bis-SH3 domain and its C-terminal proline-rich region (PRR); this interaction prevents the SH3 domains from binding p22phox and the PRR from associating with NOXA1. Arachidonic acid enhances SH3-mediated binding of NOXO1 to p22phox, and disruption of the intramolecular SH3-PRR interaction facilitates both p22phox binding and PRR-Noxa1 association required for Nox activation. NOXO1 also supports superoxide production in a cell-free system for gp91phox/Nox2 activation by arachidonic acid.","method":"Cell-free Nox2 reconstitution assay, pulldown/binding assays with domain mutants, arachidonic acid stimulation","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cell-free reconstitution plus domain-interaction pulldown with mechanistic mutagenesis, single lab multiple methods","pmids":["17126813"],"is_preprint":false},{"year":2008,"finding":"TNF-alpha induces NOXO1 transcription through an AP-1 binding site (−561 to −551 bp, agtAAGtcatg) in the NOXO1 promoter, via rapid activation of p38 MAPK and JNK1/2, followed by c-Jun/c-Fos phosphorylation and AP-1 complex formation. TNF-alpha-induced upregulation of superoxide production in T84 colon epithelial cells was abolished by NOXO1 siRNA knockdown.","method":"Promoter cloning and serial deletion/mutation analysis, AP-1 decoy oligonucleotides, siRNA knockdown, superoxide measurement","journal":"Free radical biology & medicine","confidence":"High","confidence_rationale":"Tier 2 / Moderate — promoter mutagenesis, AP-1 decoy, siRNA knockdown with functional ROS readout, multiple orthogonal methods","pmids":["18929641"],"is_preprint":false},{"year":2010,"finding":"The isolated tandem SH3 domains of NOXO1 bind to p22phox with high affinity, likely adopting a superSH3 domain conformation. The C-terminal tail of NOXO1 competes for SH3 binding to p22phox and thereby inhibits complex formation, suggesting autoinhibitory regulation of NOX1 activity. The molecular details of NOXO1-NOXA1 interaction differ significantly from those of the homologous p47phox-p67phox interaction.","method":"Isothermal titration calorimetry (ITC) measuring binding affinity between isolated domains and partner peptides/proteins","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Moderate — quantitative biophysical binding assay (ITC) with defined domain constructs, reveals autoinhibitory mechanism","pmids":["20454568"],"is_preprint":false},{"year":2013,"finding":"PMA stimulates NOXO1 phosphorylation at Ser-154 via protein kinase C in HEK293 and T84 colon epithelial cells. Phosphorylation at Ser-154 enhances NOXO1 binding to NOXA1 (+97%) and to p22phox C-terminal region (+384%), increases NOXO1 co-localization with p22phox, and is required for optimal ROS production by NOX1 (demonstrated by S154A and S154D mutants). Pulldown experiments show phospho-Ser154 first enhances NOXO1-NOXA1 binding, which then acts as a molecular switch enabling optimal NOXO1-p22phox interaction.","method":"Site-directed mutagenesis (S154A, S154D), pulldown assays, co-localization imaging, ROS measurement, endogenous phosphorylation confirmed in T84 cells","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis plus pulldown plus functional ROS assay plus endogenous validation, multiple orthogonal methods","pmids":["23322165"],"is_preprint":false},{"year":2013,"finding":"PKC directly phosphorylates NOXO1 at Thr341 in vitro; T341A substitution reduces PMA-induced NOXO1 phosphorylation and PMA-dependent Nox1 superoxide production. Phosphorylation of Thr341 promotes NOXO1 interaction with NOXA1, which is required for Nox1 activation. Additionally, Ser154 is phosphorylated in both resting and PMA-stimulated cells and may be the major PKA phosphorylation site in vitro, contributing to constitutive Nox1 activity.","method":"In vitro PKC kinase assay, alanine-substitution mutagenesis (T341A), pulldown assays for NOXO1-NOXA1 and NOXO1-p22phox interactions, superoxide measurement","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay plus mutagenesis plus interaction pulldowns plus functional superoxide readout","pmids":["23957209"],"is_preprint":false},{"year":2016,"finding":"Grb2 interacts with NOXO1 and recruits the Cbl E3 ubiquitin ligase, leading to proteasomal degradation of NOXO1 and reduced ROS production. EGF-mediated phosphorylation of NOXO1 causes its release from Grb2 and promotes its association with NOXA1 to stimulate ROS production. Grb2 overexpression decreases Nox1 activity; Grb2 knockdown increases Nox1 activity. CRISPR/Cas9-mediated NoxO1 knockout abrogates anchorage-independent growth and tumor formation in nude mice.","method":"Co-immunoprecipitation, CRISPR/Cas9 knockout, Grb2 overexpression/knockdown, soft agar colony assay, xenograft tumor assay, protein stability assay","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, CRISPR KO, gain/loss-of-function, multiple orthogonal methods, in vitro and in vivo validation","pmids":["26781991"],"is_preprint":false},{"year":2016,"finding":"At resting state, NOXO1 co-localizes with NOX1 (distinct from p47phox co-localizing with NOX2) in endothelial cells. RNAi of NOXO1 reduces superoxide and increases NO in response to oscillatory shear stress (OSS) and corrects OSS-induced eNOS uncoupling, while having no effect under laminar shear stress (LSS). This identifies the NOX1-NOXO1 complex as the mediator of OSS-induced eNOS uncoupling.","method":"Immunocytochemical co-localization, RNAi knockdown, superoxide/NO measurements, eNOS uncoupling assay under defined flow conditions","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown with mechanistic readout, co-localization, single lab with multiple readouts","pmids":["26826128"],"is_preprint":false},{"year":2016,"finding":"NoxO1 deletion in lung endothelial cells attenuates ADAM17 (α-secretase) activity and Notch intracellular domain release, leading to reduced Notch signaling and a tip-cell phenotype with increased angiogenesis. NoxO1 overexpression promotes ADAM17 oxidation and maximal α-secretase activity, mechanistically linking NoxO1-derived ROS to ADAM17 activation and Notch-dependent stalk cell fate.","method":"NoxO1 knockout mouse, spheroid outgrowth assay, retinal angiogenesis imaging, femoral artery ligation model, ADAM17 activity assay, Notch signaling readouts in NoxO1-/- LECs","journal":"Arteriosclerosis, thrombosis, and vascular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with defined cellular phenotype plus enzyme activity assay, single lab","pmids":["27283741"],"is_preprint":false},{"year":2018,"finding":"NoxO1 knockout in mice reduces superoxide production in colon crypts without compensatory p47phox upregulation, and results in increased proliferative capacity and decreased apoptosis of colon epithelial cells, demonstrating a role for NoxO1-dependent ROS in limiting colon epithelial proliferation.","method":"NoxO1 knockout mouse, superoxide measurement in colon crypts, proliferation and apoptosis assays, DSS colitis and AOM/DSS colon cancer models","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with defined cellular and in vivo phenotypic readouts, single lab","pmids":["29867954"],"is_preprint":false},{"year":2021,"finding":"CYLD (cylindromatosis deubiquitinase) binds NoxO1, promotes its ubiquitination, and reduces NoxO1 protein half-life, thereby destabilizing NoxO1 and suppressing excessive ROS generation. CRISPR/Cas9-mediated CYLD knockout in PC-3 prostate cancer cells promotes proliferation, migration, colony formation, and invasion, and increases tumor growth in xenograft mice, consistent with CYLD acting as a NoxO1 destabilizer.","method":"Genome-wide CRISPR/Cas9 DUB-knockout library screen, Co-IP for CYLD-NoxO1 binding, ubiquitination assay, protein half-life measurement, CYLD KO xenograft tumor model","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR screen plus Co-IP plus ubiquitination assay plus in vivo xenograft, single lab","pmids":["34742871"],"is_preprint":false},{"year":2023,"finding":"A D-box mutation in NOXO1 (mut1) increases NoxO1 translocation from the membrane-soluble fraction to a cytoskeletal-insoluble fraction, causes a filamentous subcellular phenotype, and associates with intermediate filaments keratin 18 and vimentin; this redistribution correlates with increased Nox1-dependent NADPH oxidase activity and ROS production.","method":"Expression of wt and D-box mutant NOXO1 in cell lines, subcellular fractionation, co-immunoprecipitation with cytoskeletal proteins, ROS measurement","journal":"International journal of molecular sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single Co-IP/fractionation approach, no mutagenesis rescue or in vitro reconstitution","pmids":["36902094"],"is_preprint":false},{"year":2024,"finding":"NoxO1 interacts with Erbin (ErbB2 interacting protein) as identified by BioID proximity labeling; NoxO1 co-localizes with EGFR and Erbin, and EGF treatment disrupts NoxO1-EGFR co-localization. NoxO1 overexpression delays EGF-mediated kinase activation and inhibits EGF-induced wound closure, while NoxO1 KO has the opposite effect, indicating a Nox1-independent scaffolding role for NoxO1 in EGFR signaling.","method":"BioID proximity labeling, co-localization imaging, NoxO1 overexpression and CRISPR KO, EGF receptor phosphorylation kinetics, wound closure assay","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — BioID plus co-localization plus gain/loss-of-function with functional readout, single lab multiple methods","pmids":["39426288"],"is_preprint":false},{"year":2024,"finding":"The level of ROS production by the Nox1-centered NADPH oxidase complex is dose-dependently regulated by NoxO1 concentration: increasing NoxO1 above stoichiometric levels progressively controls complex activity, indicating that ROS output is regulated by the NoxO1:Nox1:NoxA1 ratio rather than a strict 1:1:1 stoichiometry.","method":"Stable HEK293 cell lines constitutively expressing Nox1 and NoxA1 transfected with increasing NoxO1 concentrations; ROS measurement","journal":"Antioxidants (Basel, Switzerland)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single functional assay, no biochemical interaction or structural validation","pmids":["39334772"],"is_preprint":false},{"year":2025,"finding":"NoxO1 overexpression expands early endosomes and lysosomes, increases EGFR internalization rates while reducing degradative sorting and lysosomal cargo processing. Mechanistically, NoxO1 activates transcription factor EB (TFEB), the master regulator of lysosomal biogenesis, in an Erbin-dependent but ROS-independent manner. Proximity ligation assays show spatial association of NoxO1, Erbin, EGFR, and TFEB as a multi-protein complex. Genetic ablation of Erbin abolishes NoxO1-induced increases in early endosome (EEA1) and lysosome (LAMP1) markers.","method":"NoxO1 overexpression and KO cell lines, mathematical ODE modeling of EGFR trafficking, fluorescent cargo (EGF, BSA) trafficking assays, proximity ligation assay, Erbin KO, EEA1/LAMP1 marker quantification, TFEB activity assay","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (PLA, KO rescue, trafficking assays, mathematical modeling), single lab","pmids":["41406574"],"is_preprint":false}],"current_model":"NOXO1 is a scaffold organizer protein that constitutively localizes to membranes via PX domain binding to phosphoinositide lipids, where it assembles the NOX1 (and NOX3) NADPH oxidase complex by bridging p22phox (through its tandem SH3 domains) and NOXA1 (through its proline-rich region); its activity is regulated by PKC-mediated phosphorylation at Ser154 and Thr341 (enhancing p22phox and NOXA1 interactions), by intramolecular SH3-PRR autoinhibition, by Grb2/Cbl-mediated proteasomal degradation and CYLD-mediated ubiquitination, and by TNF-alpha/AP-1-driven transcriptional induction; beyond ROS production, NOXO1 also acts as an adaptor protein that interacts with Erbin to modulate EGFR signaling, endosomal trafficking, and TFEB-dependent lysosomal biogenesis in a ROS-independent manner."},"narrative":{"mechanistic_narrative":"NOXO1 is a scaffold organizer that assembles and regulates the NOX1 (and NOX3) NADPH oxidase complex to drive localized reactive oxygen species production [PMID:14617635, PMID:15181005]. It binds phosphoinositide lipids (PtdIns 3,5-P2, PtdIns 5-P, PtdIns 4-P) through its PX domain to constitutively localize at membranes alongside NOX1; PX domain mutation displaces NOXO1 to the cytosol and abolishes NOX1 activation [PMID:14617635]. NOXO1 bridges the membrane subunit p22phox through its tandem (superSH3) domains and the activator NOXA1 through its C-terminal proline-rich region, and unlike its homolog p47phox it lacks an autoinhibitory region and supports agonist-independent ROS generation; this distinction allows NOXO1 alone to activate NOX3 [PMID:14617635, PMID:15181005, PMID:20454568]. Its assembly is gated by an intramolecular SH3-PRR autoinhibitory interaction that, when disrupted (e.g. by arachidonic acid), licenses both p22phox binding and NOXA1 association [PMID:17126813, PMID:20454568]. PKC phosphorylates NOXO1 at Ser154 and Thr341, enhancing NOXA1 and p22phox binding as a molecular switch for optimal NOX1 ROS output [PMID:23322165, PMID:23957209]. NOXO1 abundance is controlled both transcriptionally—TNF-alpha induces it via a p38/JNK-AP-1 axis on the NOXO1 promoter [PMID:18929641]—and post-translationally, with Grb2/Cbl-directed proteasomal degradation and CYLD-mediated ubiquitination limiting NOXO1 levels and ROS [PMID:26781991, PMID:34742871]. Through NOX1-derived ROS, NOXO1 regulates colon epithelial proliferation [PMID:29867954], oscillatory-shear-stress-induced eNOS uncoupling [PMID:26826128], and ADAM17/Notch signaling during angiogenesis [PMID:27283741], and its loss abrogates anchorage-independent growth and tumor formation [PMID:26781991]. Beyond ROS, NOXO1 functions as a ROS-independent adaptor: it associates with Erbin and EGFR to modulate EGFR signaling and internalization, and activates TFEB to expand endosomal and lysosomal compartments in an Erbin-dependent manner [PMID:39426288, PMID:41406574].","teleology":[{"year":2003,"claim":"Established how NOXO1 is targeted to membranes and what its role is in NOX1 activation, addressing whether NOX1 needs a dedicated organizer like the phagocyte oxidase needs p47phox.","evidence":"Co-transfection/reconstitution in HEK293 cells with lipid-binding assays and PX-domain mutagenesis","pmids":["14617635"],"confidence":"High","gaps":["Did not define how p22phox and NOXA1 are physically bridged","Constitutive (agonist-independent) activity left open how the complex is normally controlled"]},{"year":2004,"claim":"Distinguished NOX3 regulation from NOX1/NOX2 by showing NOXO1 alone suffices to activate NOX3 and that NOXO1 lacks the phosphorylation-regulated autoinhibitory region of p47phox.","evidence":"Cell-based reconstitution with multiple subunit combinations and p47phox deletion mutants","pmids":["15181005"],"confidence":"High","gaps":["Did not explain why NOX3 uniquely bypasses an activator subunit","Physiological context of NOX3 regulation not addressed"]},{"year":2005,"claim":"Showed alternative splicing of the PX-region generates functionally distinct NOXO1 isoforms with tissue-specific expression, indicating regulation through isoform choice.","evidence":"RT-PCR/cloning, purified PX-domain lipid binding, and Nox1/Nox3 reconstitution assays","pmids":["15949904"],"confidence":"High","gaps":["Structural basis for isoform-specific NOX3 activation unresolved","In vivo relevance of isoform distribution not tested"]},{"year":2006,"claim":"Identified an intramolecular SH3-PRR autoinhibitory interaction relieved by arachidonic acid, providing a switch for assembly despite the absence of the p47phox autoinhibitory region.","evidence":"Cell-free NOX2 reconstitution and domain-mutant pulldown assays with arachidonic acid stimulation","pmids":["17126813"],"confidence":"High","gaps":["Physiological trigger of arachidonic-acid-mediated relief unclear","Did not quantify binding affinities of the autoinhibited state"]},{"year":2010,"claim":"Quantified the tandem-SH3 (superSH3) high-affinity binding to p22phox and confirmed C-terminal-tail competition as an autoinhibitory brake on NOX1 assembly.","evidence":"Isothermal titration calorimetry with defined domain constructs and partner peptides","pmids":["20454568"],"confidence":"High","gaps":["No full-length structure of the assembled complex","Did not link biophysics to cellular activation kinetics"]},{"year":2008,"claim":"Defined transcriptional control of NOXO1, showing TNF-alpha drives expression through a p38/JNK-AP-1 promoter axis to amplify epithelial superoxide.","evidence":"Promoter mutagenesis, AP-1 decoy oligonucleotides, and siRNA knockdown with superoxide readout in T84 cells","pmids":["18929641"],"confidence":"High","gaps":["Did not connect induced NOXO1 levels to a downstream physiological outcome","Other promoter elements not characterized"]},{"year":2013,"claim":"Resolved the activation switch by showing PKC phosphorylation at Ser154 and Thr341 enhances NOXA1 and p22phox binding to optimize NOX1 ROS output.","evidence":"Phospho-site mutagenesis (S154A/D, T341A), in vitro PKC kinase assay, pulldowns, co-localization, and ROS measurement with endogenous validation","pmids":["23322165","23957209"],"confidence":"High","gaps":["Upstream signals activating PKC toward NOXO1 in vivo not mapped","Relative contributions of PKC vs PKA to constitutive activity not fully separated"]},{"year":2016,"claim":"Identified post-translational destabilization of NOXO1 via Grb2/Cbl and linked NOXO1 to oncogenic growth, establishing it as a tumor-promoting node.","evidence":"Reciprocal Co-IP, CRISPR/Cas9 knockout, Grb2 gain/loss-of-function, soft-agar and xenograft assays","pmids":["26781991"],"confidence":"High","gaps":["Cbl ubiquitination sites on NOXO1 not mapped","Whether degradation is constitutive or signal-triggered in tumors unclear"]},{"year":2016,"claim":"Connected the NOX1-NOXO1 complex to vascular dysfunction and angiogenesis, showing it mediates oscillatory-shear-stress eNOS uncoupling and ROS-dependent ADAM17/Notch signaling.","evidence":"RNAi and NoxO1 knockout mouse models with co-localization, superoxide/NO measurement, eNOS uncoupling, retinal angiogenesis, and ADAM17 activity assays","pmids":["26826128","27283741"],"confidence":"Medium","gaps":["Direct ROS targets on eNOS and ADAM17 not molecularly defined","Single-lab models for each phenotype"]},{"year":2018,"claim":"Demonstrated in vivo that NoxO1-dependent ROS restrains colon epithelial proliferation and promotes apoptosis, defining a physiological homeostatic role.","evidence":"NoxO1 knockout mouse with crypt superoxide measurement, proliferation/apoptosis assays, and colitis/cancer models","pmids":["29867954"],"confidence":"Medium","gaps":["Downstream ROS-sensitive effectors controlling proliferation not identified","No compensatory p47phox response examined further"]},{"year":2021,"claim":"Identified CYLD as a deubiquitinase that paradoxically promotes NoxO1 ubiquitination and degradation, adding a second post-translational brake on ROS in cancer.","evidence":"Genome-wide DUB CRISPR screen, Co-IP, ubiquitination and half-life assays, and CYLD-KO xenograft model in PC-3 cells","pmids":["34742871"],"confidence":"Medium","gaps":["Mechanism by which a DUB promotes ubiquitination not resolved","Relationship to the Grb2/Cbl pathway unclear"]},{"year":2023,"claim":"Linked a D-box motif to NoxO1 subcellular partitioning, showing mutation redistributes it to a cytoskeletal fraction associated with keratin 18/vimentin and elevated NOX1 activity.","evidence":"Wild-type vs D-box mutant expression, subcellular fractionation, Co-IP with cytoskeletal proteins, ROS measurement","pmids":["36902094"],"confidence":"Low","gaps":["Single Co-IP/fractionation approach without rescue or reconstitution","Functional significance of cytoskeletal association not established","Whether the D-box mediates regulated degradation untested"]},{"year":2024,"claim":"Revealed a ROS-independent adaptor function, showing NoxO1 associates with Erbin and EGFR to modulate EGFR kinase activation and cell migration.","evidence":"BioID proximity labeling, co-localization, NoxO1 overexpression/CRISPR KO, EGFR phosphorylation kinetics, wound-closure assay","pmids":["39426288"],"confidence":"Medium","gaps":["Direct vs proximity-only nature of NoxO1-EGFR interaction not resolved","Structural basis of the Erbin interaction unknown"]},{"year":2024,"claim":"Indicated that ROS output is set by the NoxO1:Nox1:NoxA1 ratio rather than fixed stoichiometry, reframing NoxO1 abundance as a rheostat.","evidence":"Stable Nox1/NoxA1 HEK293 lines transfected with increasing NoxO1 and ROS measurement","pmids":["39334772"],"confidence":"Low","gaps":["Single functional assay without biochemical or structural validation","No measurement of complex stoichiometry directly"]},{"year":2025,"claim":"Extended the ROS-independent adaptor role to organelle biogenesis, showing NoxO1 activates TFEB via Erbin to expand endosomes/lysosomes and alter EGFR trafficking.","evidence":"Overexpression/KO lines, ODE trafficking models, cargo trafficking assays, proximity ligation assay, Erbin KO, EEA1/LAMP1 quantification, TFEB activity assay","pmids":["41406574"],"confidence":"Medium","gaps":["Mechanism by which NoxO1-Erbin activates TFEB not defined","Direct membership of NoxO1 in the trafficking complex inferred from PLA proximity"]},{"year":null,"claim":"How NOXO1's two arms—NADPH oxidase organizer and ROS-independent Erbin/EGFR/TFEB adaptor—are coordinated within a cell, and what structural state of full-length NOXO1 governs the switch between them, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No full-length structure of NOXO1 in either functional state","Whether oxidase scaffolding and EGFR/TFEB adaptor functions are mutually exclusive or simultaneous is unknown","Physiological signals selecting between the two roles undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,5,6]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[0,2]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[6,7,15]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[16]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,9]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[16]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[16]}],"pathway":[{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[0,4,11]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[10,14]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[16]}],"complexes":["NOX1 NADPH oxidase complex","NOX3 NADPH oxidase complex"],"partners":["P22PHOX","NOXA1","NOX1","GRB2","CBL","CYLD","ERBIN","EGFR"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8NFA2","full_name":"NADPH oxidase organizer 1","aliases":["NADPH oxidase regulatory protein","Nox organizer 1","Nox-organizing protein 1","SH3 and PX domain-containing protein 5"],"length_aa":376,"mass_kda":41.3,"function":"Constitutively potentiates the superoxide-generating activity of NOX1 and NOX3 and is required for the biogenesis of otoconia/otolith, which are crystalline structures of the inner ear involved in the perception of gravity. Isoform 3 is more potent than isoform 1 in activating NOX3. Together with NOXA1, may also substitute to NCF1/p47phox and NCF2/p67phox in supporting the phagocyte NOX2/gp91phox superoxide-generating activity","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q8NFA2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NOXO1","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/NOXO1","total_profiled":1310},"omim":[{"mim_id":"611256","title":"NADPH OXIDASE ORGANIZER 1; NOXO1","url":"https://www.omim.org/entry/611256"},{"mim_id":"611255","title":"NADPH OXIDASE ACTIVATOR 1; NOXA1","url":"https://www.omim.org/entry/611255"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"brain","ntpm":5.4},{"tissue":"intestine","ntpm":8.1},{"tissue":"lymphoid tissue","ntpm":4.4},{"tissue":"testis","ntpm":4.6}],"url":"https://www.proteinatlas.org/search/NOXO1"},"hgnc":{"alias_symbol":["P41NOXA","P41NOXB","P41NOXC","SH3PXD5","SNX28"],"prev_symbol":[]},"alphafold":{"accession":"Q8NFA2","domains":[{"cath_id":"3.30.1520.10","chopping":"8-77_90-143","consensus_level":"high","plddt":78.8306,"start":8,"end":143},{"cath_id":"2.30.30.40","chopping":"167-223","consensus_level":"high","plddt":67.2828,"start":167,"end":223},{"cath_id":"2.30.30.40","chopping":"242-301","consensus_level":"high","plddt":72.1967,"start":242,"end":301}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NFA2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NFA2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NFA2-F1-predicted_aligned_error_v6.png","plddt_mean":63.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NOXO1","jax_strain_url":"https://www.jax.org/strain/search?query=NOXO1"},"sequence":{"accession":"Q8NFA2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8NFA2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8NFA2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NFA2"}},"corpus_meta":[{"pmid":"14617635","id":"PMC_14617635","title":"NOXO1, regulation of lipid binding, localization, and activation of Nox1 by the Phox homology (PX) domain.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/14617635","citation_count":182,"is_preprint":false},{"pmid":"15181005","id":"PMC_15181005","title":"Nox3 regulation by NOXO1, p47phox, and p67phox.","date":"2004","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15181005","citation_count":129,"is_preprint":false},{"pmid":"15469954","id":"PMC_15469954","title":"Helicobacter pylori lipopolysaccharide activates Rac1 and transcription of NADPH oxidase Nox1 and its organizer NOXO1 in guinea pig gastric mucosal cells.","date":"2004","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/15469954","citation_count":117,"is_preprint":false},{"pmid":"23975421","id":"PMC_23975421","title":"TNF-α/TNFR1 signaling promotes gastric tumorigenesis through induction of Noxo1 and Gna14 in tumor cells.","date":"2013","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/23975421","citation_count":116,"is_preprint":false},{"pmid":"22549734","id":"PMC_22549734","title":"The p47phox- and NADPH oxidase organiser 1 (NOXO1)-dependent activation of NADPH oxidase 1 (NOX1) mediates endothelial nitric oxide synthase (eNOS) uncoupling and endothelial dysfunction in a streptozotocin-induced murine model of diabetes.","date":"2012","source":"Diabetologia","url":"https://pubmed.ncbi.nlm.nih.gov/22549734","citation_count":114,"is_preprint":false},{"pmid":"18929641","id":"PMC_18929641","title":"Tumor necrosis factor alpha activates transcription of the NADPH oxidase organizer 1 (NOXO1) gene and upregulates superoxide production in colon epithelial cells.","date":"2008","source":"Free radical biology & medicine","url":"https://pubmed.ncbi.nlm.nih.gov/18929641","citation_count":53,"is_preprint":false},{"pmid":"20454568","id":"PMC_20454568","title":"Regulation of NOXO1 activity through reversible interactions with p22 and NOXA1.","date":"2010","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/20454568","citation_count":51,"is_preprint":false},{"pmid":"15949904","id":"PMC_15949904","title":"Alternative mRNA splice forms of NOXO1: differential tissue expression and regulation of Nox1 and Nox3.","date":"2005","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/15949904","citation_count":46,"is_preprint":false},{"pmid":"26781991","id":"PMC_26781991","title":"NADPH Oxidase 1 Activity and ROS Generation Are Regulated by Grb2/Cbl-Mediated Proteasomal Degradation of NoxO1 in Colon Cancer Cells.","date":"2016","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/26781991","citation_count":45,"is_preprint":false},{"pmid":"29195137","id":"PMC_29195137","title":"The NADPH organizers NoxO1 and p47phox are both mediators of diabetes-induced vascular dysfunction in mice.","date":"2017","source":"Redox biology","url":"https://pubmed.ncbi.nlm.nih.gov/29195137","citation_count":35,"is_preprint":false},{"pmid":"23322165","id":"PMC_23322165","title":"NOXO1 phosphorylation on serine 154 is critical for optimal NADPH oxidase 1 assembly and activation.","date":"2013","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/23322165","citation_count":35,"is_preprint":false},{"pmid":"26826128","id":"PMC_26826128","title":"Differential Roles of Protein Complexes NOX1-NOXO1 and NOX2-p47phox in Mediating Endothelial Redox Responses to Oscillatory and Unidirectional Laminar Shear Stress.","date":"2016","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/26826128","citation_count":35,"is_preprint":false},{"pmid":"29867954","id":"PMC_29867954","title":"NoxO1 Controls Proliferation of Colon Epithelial Cells.","date":"2018","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/29867954","citation_count":32,"is_preprint":false},{"pmid":"23957209","id":"PMC_23957209","title":"Phosphorylation of Noxo1 at threonine 341 regulates its interaction with Noxa1 and the superoxide-producing activity of Nox1.","date":"2013","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/23957209","citation_count":28,"is_preprint":false},{"pmid":"34742871","id":"PMC_34742871","title":"CYLD destabilizes NoxO1 protein by promoting ubiquitination and regulates prostate cancer progression.","date":"2021","source":"Cancer 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communications","url":"https://pubmed.ncbi.nlm.nih.gov/17126813","citation_count":21,"is_preprint":false},{"pmid":"32164269","id":"PMC_32164269","title":"NoxO1 Knockout Promotes Longevity in Mice.","date":"2020","source":"Antioxidants (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/32164269","citation_count":8,"is_preprint":false},{"pmid":"39334772","id":"PMC_39334772","title":"NoxO1 Determines the Level of ROS Formation by the Nox1-Centered NADPH Oxidase.","date":"2024","source":"Antioxidants (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/39334772","citation_count":6,"is_preprint":false},{"pmid":"28944893","id":"PMC_28944893","title":"Pigment epithelium‑derived factor protects human glomerular mesangial cells from diabetes via NOXO1‑iNOS suppression.","date":"2017","source":"Molecular medicine reports","url":"https://pubmed.ncbi.nlm.nih.gov/28944893","citation_count":6,"is_preprint":false},{"pmid":"39426288","id":"PMC_39426288","title":"NoxO1 regulates EGFR signaling by its interaction with Erbin.","date":"2024","source":"Redox biology","url":"https://pubmed.ncbi.nlm.nih.gov/39426288","citation_count":3,"is_preprint":false},{"pmid":"36902094","id":"PMC_36902094","title":"Overexpression of a Novel Noxo1 Mutant Increases Ros Production and Noxo1 Relocalisation.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/36902094","citation_count":3,"is_preprint":false},{"pmid":"37031566","id":"PMC_37031566","title":"Identification of a Noxo1 inhibitor by addition of a polyethylene glycol chain.","date":"2023","source":"Bioorganic & medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/37031566","citation_count":2,"is_preprint":false},{"pmid":"38015525","id":"PMC_38015525","title":"DNA methylation of ICAM4 and NOXO1 participate in the formation of uterine fibroids via regulating immune cell infiltration.","date":"2023","source":"Cellular and molecular biology (Noisy-le-Grand, France)","url":"https://pubmed.ncbi.nlm.nih.gov/38015525","citation_count":0,"is_preprint":false},{"pmid":"41406574","id":"PMC_41406574","title":"NoxO1 promotes endosome formation and reduces intracellular vesicle processing.","date":"2025","source":"Redox biology","url":"https://pubmed.ncbi.nlm.nih.gov/41406574","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16677,"output_tokens":5178,"usd":0.063851,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":13244,"output_tokens":5060,"usd":0.09636,"stage2_stop_reason":"end_turn"},"total_usd":0.160211,"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\": 2003,\n      \"finding\": \"NOXO1 PX domain binds phosphatidylinositol lipids (PtdIns 3,5-P2, PtdIns 5-P, PtdIns 4-P) and mediates constitutive membrane co-localization with Nox1; a point mutation in the PX domain decreases lipid binding, causes cytosolic localization, and inhibits Nox1 activation. Co-transfection of Nox1, NOXO1, and NOXA1 reconstitutes ROS generation in HEK293 cells without agonist stimulation. NOXO1 lacks the autoinhibitory region and phosphorylation sites present in p47phox.\",\n      \"method\": \"Co-transfection/reconstitution in HEK293 cells, lipid-binding assays, site-directed mutagenesis of PX domain, co-localization imaging\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution in cells, lipid-binding assay, mutagenesis with functional readout, multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"14617635\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Nox3 is strongly activated by NOXO1 alone (without NOXA1 or p67phox), unlike gp91phox or Nox1 which require both organizer and activator subunits. Nox3 regulation by p47phox (but not NOXO1) is PMA-dependent, consistent with the phosphorylation-regulated autoinhibitory region present in p47phox but absent in NOXO1. Deletion of the autoinhibitory region from p47phox renders it highly active without PMA but still requires an activator subunit.\",\n      \"method\": \"Cell-based reconstitution assay measuring superoxide production with various subunit combinations and mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution assay with multiple subunit combinations and deletion mutants, clear mechanistic dissection\",\n      \"pmids\": [\"15181005\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"NOXO1 has four splice forms (alpha, beta, gamma, delta) arising from alternative splicing of exon 3 in the PX domain region. NOXO1beta is the major form in colon and fetal liver; NOXO1gamma is the majority in testis. Both NOXO1beta and NOXO1gamma bind phosphoinositide lipids with the same specificity and affinity but differ functionally: NOXO1gamma shows poorer ability to activate Nox3 compared with NOXO1beta, while both activate Nox1 equally.\",\n      \"method\": \"RT-PCR/cloning of splice forms, purified PX domain lipid-binding assays, cell-based Nox1/Nox3 reconstitution assays\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro lipid-binding assay plus cell-based functional reconstitution, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"15949904\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"NOXO1 contains an intramolecular interaction between its bis-SH3 domain and its C-terminal proline-rich region (PRR); this interaction prevents the SH3 domains from binding p22phox and the PRR from associating with NOXA1. Arachidonic acid enhances SH3-mediated binding of NOXO1 to p22phox, and disruption of the intramolecular SH3-PRR interaction facilitates both p22phox binding and PRR-Noxa1 association required for Nox activation. NOXO1 also supports superoxide production in a cell-free system for gp91phox/Nox2 activation by arachidonic acid.\",\n      \"method\": \"Cell-free Nox2 reconstitution assay, pulldown/binding assays with domain mutants, arachidonic acid stimulation\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cell-free reconstitution plus domain-interaction pulldown with mechanistic mutagenesis, single lab multiple methods\",\n      \"pmids\": [\"17126813\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TNF-alpha induces NOXO1 transcription through an AP-1 binding site (−561 to −551 bp, agtAAGtcatg) in the NOXO1 promoter, via rapid activation of p38 MAPK and JNK1/2, followed by c-Jun/c-Fos phosphorylation and AP-1 complex formation. TNF-alpha-induced upregulation of superoxide production in T84 colon epithelial cells was abolished by NOXO1 siRNA knockdown.\",\n      \"method\": \"Promoter cloning and serial deletion/mutation analysis, AP-1 decoy oligonucleotides, siRNA knockdown, superoxide measurement\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter mutagenesis, AP-1 decoy, siRNA knockdown with functional ROS readout, multiple orthogonal methods\",\n      \"pmids\": [\"18929641\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The isolated tandem SH3 domains of NOXO1 bind to p22phox with high affinity, likely adopting a superSH3 domain conformation. The C-terminal tail of NOXO1 competes for SH3 binding to p22phox and thereby inhibits complex formation, suggesting autoinhibitory regulation of NOX1 activity. The molecular details of NOXO1-NOXA1 interaction differ significantly from those of the homologous p47phox-p67phox interaction.\",\n      \"method\": \"Isothermal titration calorimetry (ITC) measuring binding affinity between isolated domains and partner peptides/proteins\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — quantitative biophysical binding assay (ITC) with defined domain constructs, reveals autoinhibitory mechanism\",\n      \"pmids\": [\"20454568\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PMA stimulates NOXO1 phosphorylation at Ser-154 via protein kinase C in HEK293 and T84 colon epithelial cells. Phosphorylation at Ser-154 enhances NOXO1 binding to NOXA1 (+97%) and to p22phox C-terminal region (+384%), increases NOXO1 co-localization with p22phox, and is required for optimal ROS production by NOX1 (demonstrated by S154A and S154D mutants). Pulldown experiments show phospho-Ser154 first enhances NOXO1-NOXA1 binding, which then acts as a molecular switch enabling optimal NOXO1-p22phox interaction.\",\n      \"method\": \"Site-directed mutagenesis (S154A, S154D), pulldown assays, co-localization imaging, ROS measurement, endogenous phosphorylation confirmed in T84 cells\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis plus pulldown plus functional ROS assay plus endogenous validation, multiple orthogonal methods\",\n      \"pmids\": [\"23322165\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PKC directly phosphorylates NOXO1 at Thr341 in vitro; T341A substitution reduces PMA-induced NOXO1 phosphorylation and PMA-dependent Nox1 superoxide production. Phosphorylation of Thr341 promotes NOXO1 interaction with NOXA1, which is required for Nox1 activation. Additionally, Ser154 is phosphorylated in both resting and PMA-stimulated cells and may be the major PKA phosphorylation site in vitro, contributing to constitutive Nox1 activity.\",\n      \"method\": \"In vitro PKC kinase assay, alanine-substitution mutagenesis (T341A), pulldown assays for NOXO1-NOXA1 and NOXO1-p22phox interactions, superoxide measurement\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay plus mutagenesis plus interaction pulldowns plus functional superoxide readout\",\n      \"pmids\": [\"23957209\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Grb2 interacts with NOXO1 and recruits the Cbl E3 ubiquitin ligase, leading to proteasomal degradation of NOXO1 and reduced ROS production. EGF-mediated phosphorylation of NOXO1 causes its release from Grb2 and promotes its association with NOXA1 to stimulate ROS production. Grb2 overexpression decreases Nox1 activity; Grb2 knockdown increases Nox1 activity. CRISPR/Cas9-mediated NoxO1 knockout abrogates anchorage-independent growth and tumor formation in nude mice.\",\n      \"method\": \"Co-immunoprecipitation, CRISPR/Cas9 knockout, Grb2 overexpression/knockdown, soft agar colony assay, xenograft tumor assay, protein stability assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, CRISPR KO, gain/loss-of-function, multiple orthogonal methods, in vitro and in vivo validation\",\n      \"pmids\": [\"26781991\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"At resting state, NOXO1 co-localizes with NOX1 (distinct from p47phox co-localizing with NOX2) in endothelial cells. RNAi of NOXO1 reduces superoxide and increases NO in response to oscillatory shear stress (OSS) and corrects OSS-induced eNOS uncoupling, while having no effect under laminar shear stress (LSS). This identifies the NOX1-NOXO1 complex as the mediator of OSS-induced eNOS uncoupling.\",\n      \"method\": \"Immunocytochemical co-localization, RNAi knockdown, superoxide/NO measurements, eNOS uncoupling assay under defined flow conditions\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown with mechanistic readout, co-localization, single lab with multiple readouts\",\n      \"pmids\": [\"26826128\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NoxO1 deletion in lung endothelial cells attenuates ADAM17 (α-secretase) activity and Notch intracellular domain release, leading to reduced Notch signaling and a tip-cell phenotype with increased angiogenesis. NoxO1 overexpression promotes ADAM17 oxidation and maximal α-secretase activity, mechanistically linking NoxO1-derived ROS to ADAM17 activation and Notch-dependent stalk cell fate.\",\n      \"method\": \"NoxO1 knockout mouse, spheroid outgrowth assay, retinal angiogenesis imaging, femoral artery ligation model, ADAM17 activity assay, Notch signaling readouts in NoxO1-/- LECs\",\n      \"journal\": \"Arteriosclerosis, thrombosis, and vascular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with defined cellular phenotype plus enzyme activity assay, single lab\",\n      \"pmids\": [\"27283741\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"NoxO1 knockout in mice reduces superoxide production in colon crypts without compensatory p47phox upregulation, and results in increased proliferative capacity and decreased apoptosis of colon epithelial cells, demonstrating a role for NoxO1-dependent ROS in limiting colon epithelial proliferation.\",\n      \"method\": \"NoxO1 knockout mouse, superoxide measurement in colon crypts, proliferation and apoptosis assays, DSS colitis and AOM/DSS colon cancer models\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with defined cellular and in vivo phenotypic readouts, single lab\",\n      \"pmids\": [\"29867954\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CYLD (cylindromatosis deubiquitinase) binds NoxO1, promotes its ubiquitination, and reduces NoxO1 protein half-life, thereby destabilizing NoxO1 and suppressing excessive ROS generation. CRISPR/Cas9-mediated CYLD knockout in PC-3 prostate cancer cells promotes proliferation, migration, colony formation, and invasion, and increases tumor growth in xenograft mice, consistent with CYLD acting as a NoxO1 destabilizer.\",\n      \"method\": \"Genome-wide CRISPR/Cas9 DUB-knockout library screen, Co-IP for CYLD-NoxO1 binding, ubiquitination assay, protein half-life measurement, CYLD KO xenograft tumor model\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR screen plus Co-IP plus ubiquitination assay plus in vivo xenograft, single lab\",\n      \"pmids\": [\"34742871\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"A D-box mutation in NOXO1 (mut1) increases NoxO1 translocation from the membrane-soluble fraction to a cytoskeletal-insoluble fraction, causes a filamentous subcellular phenotype, and associates with intermediate filaments keratin 18 and vimentin; this redistribution correlates with increased Nox1-dependent NADPH oxidase activity and ROS production.\",\n      \"method\": \"Expression of wt and D-box mutant NOXO1 in cell lines, subcellular fractionation, co-immunoprecipitation with cytoskeletal proteins, ROS measurement\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single Co-IP/fractionation approach, no mutagenesis rescue or in vitro reconstitution\",\n      \"pmids\": [\"36902094\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NoxO1 interacts with Erbin (ErbB2 interacting protein) as identified by BioID proximity labeling; NoxO1 co-localizes with EGFR and Erbin, and EGF treatment disrupts NoxO1-EGFR co-localization. NoxO1 overexpression delays EGF-mediated kinase activation and inhibits EGF-induced wound closure, while NoxO1 KO has the opposite effect, indicating a Nox1-independent scaffolding role for NoxO1 in EGFR signaling.\",\n      \"method\": \"BioID proximity labeling, co-localization imaging, NoxO1 overexpression and CRISPR KO, EGF receptor phosphorylation kinetics, wound closure assay\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — BioID plus co-localization plus gain/loss-of-function with functional readout, single lab multiple methods\",\n      \"pmids\": [\"39426288\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The level of ROS production by the Nox1-centered NADPH oxidase complex is dose-dependently regulated by NoxO1 concentration: increasing NoxO1 above stoichiometric levels progressively controls complex activity, indicating that ROS output is regulated by the NoxO1:Nox1:NoxA1 ratio rather than a strict 1:1:1 stoichiometry.\",\n      \"method\": \"Stable HEK293 cell lines constitutively expressing Nox1 and NoxA1 transfected with increasing NoxO1 concentrations; ROS measurement\",\n      \"journal\": \"Antioxidants (Basel, Switzerland)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single functional assay, no biochemical interaction or structural validation\",\n      \"pmids\": [\"39334772\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NoxO1 overexpression expands early endosomes and lysosomes, increases EGFR internalization rates while reducing degradative sorting and lysosomal cargo processing. Mechanistically, NoxO1 activates transcription factor EB (TFEB), the master regulator of lysosomal biogenesis, in an Erbin-dependent but ROS-independent manner. Proximity ligation assays show spatial association of NoxO1, Erbin, EGFR, and TFEB as a multi-protein complex. Genetic ablation of Erbin abolishes NoxO1-induced increases in early endosome (EEA1) and lysosome (LAMP1) markers.\",\n      \"method\": \"NoxO1 overexpression and KO cell lines, mathematical ODE modeling of EGFR trafficking, fluorescent cargo (EGF, BSA) trafficking assays, proximity ligation assay, Erbin KO, EEA1/LAMP1 marker quantification, TFEB activity assay\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (PLA, KO rescue, trafficking assays, mathematical modeling), single lab\",\n      \"pmids\": [\"41406574\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NOXO1 is a scaffold organizer protein that constitutively localizes to membranes via PX domain binding to phosphoinositide lipids, where it assembles the NOX1 (and NOX3) NADPH oxidase complex by bridging p22phox (through its tandem SH3 domains) and NOXA1 (through its proline-rich region); its activity is regulated by PKC-mediated phosphorylation at Ser154 and Thr341 (enhancing p22phox and NOXA1 interactions), by intramolecular SH3-PRR autoinhibition, by Grb2/Cbl-mediated proteasomal degradation and CYLD-mediated ubiquitination, and by TNF-alpha/AP-1-driven transcriptional induction; beyond ROS production, NOXO1 also acts as an adaptor protein that interacts with Erbin to modulate EGFR signaling, endosomal trafficking, and TFEB-dependent lysosomal biogenesis in a ROS-independent manner.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NOXO1 is a scaffold organizer that assembles and regulates the NOX1 (and NOX3) NADPH oxidase complex to drive localized reactive oxygen species production [#0, #1]. It binds phosphoinositide lipids (PtdIns 3,5-P2, PtdIns 5-P, PtdIns 4-P) through its PX domain to constitutively localize at membranes alongside NOX1; PX domain mutation displaces NOXO1 to the cytosol and abolishes NOX1 activation [#0]. NOXO1 bridges the membrane subunit p22phox through its tandem (superSH3) domains and the activator NOXA1 through its C-terminal proline-rich region, and unlike its homolog p47phox it lacks an autoinhibitory region and supports agonist-independent ROS generation; this distinction allows NOXO1 alone to activate NOX3 [#0, #1, #5]. Its assembly is gated by an intramolecular SH3-PRR autoinhibitory interaction that, when disrupted (e.g. by arachidonic acid), licenses both p22phox binding and NOXA1 association [#3, #5]. PKC phosphorylates NOXO1 at Ser154 and Thr341, enhancing NOXA1 and p22phox binding as a molecular switch for optimal NOX1 ROS output [#6, #7]. NOXO1 abundance is controlled both transcriptionally—TNF-alpha induces it via a p38/JNK-AP-1 axis on the NOXO1 promoter [#4]—and post-translationally, with Grb2/Cbl-directed proteasomal degradation and CYLD-mediated ubiquitination limiting NOXO1 levels and ROS [#8, #12]. Through NOX1-derived ROS, NOXO1 regulates colon epithelial proliferation [#11], oscillatory-shear-stress-induced eNOS uncoupling [#9], and ADAM17/Notch signaling during angiogenesis [#10], and its loss abrogates anchorage-independent growth and tumor formation [#8]. Beyond ROS, NOXO1 functions as a ROS-independent adaptor: it associates with Erbin and EGFR to modulate EGFR signaling and internalization, and activates TFEB to expand endosomal and lysosomal compartments in an Erbin-dependent manner [#14, #16].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established how NOXO1 is targeted to membranes and what its role is in NOX1 activation, addressing whether NOX1 needs a dedicated organizer like the phagocyte oxidase needs p47phox.\",\n      \"evidence\": \"Co-transfection/reconstitution in HEK293 cells with lipid-binding assays and PX-domain mutagenesis\",\n      \"pmids\": [\"14617635\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define how p22phox and NOXA1 are physically bridged\", \"Constitutive (agonist-independent) activity left open how the complex is normally controlled\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Distinguished NOX3 regulation from NOX1/NOX2 by showing NOXO1 alone suffices to activate NOX3 and that NOXO1 lacks the phosphorylation-regulated autoinhibitory region of p47phox.\",\n      \"evidence\": \"Cell-based reconstitution with multiple subunit combinations and p47phox deletion mutants\",\n      \"pmids\": [\"15181005\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not explain why NOX3 uniquely bypasses an activator subunit\", \"Physiological context of NOX3 regulation not addressed\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showed alternative splicing of the PX-region generates functionally distinct NOXO1 isoforms with tissue-specific expression, indicating regulation through isoform choice.\",\n      \"evidence\": \"RT-PCR/cloning, purified PX-domain lipid binding, and Nox1/Nox3 reconstitution assays\",\n      \"pmids\": [\"15949904\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for isoform-specific NOX3 activation unresolved\", \"In vivo relevance of isoform distribution not tested\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identified an intramolecular SH3-PRR autoinhibitory interaction relieved by arachidonic acid, providing a switch for assembly despite the absence of the p47phox autoinhibitory region.\",\n      \"evidence\": \"Cell-free NOX2 reconstitution and domain-mutant pulldown assays with arachidonic acid stimulation\",\n      \"pmids\": [\"17126813\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological trigger of arachidonic-acid-mediated relief unclear\", \"Did not quantify binding affinities of the autoinhibited state\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Quantified the tandem-SH3 (superSH3) high-affinity binding to p22phox and confirmed C-terminal-tail competition as an autoinhibitory brake on NOX1 assembly.\",\n      \"evidence\": \"Isothermal titration calorimetry with defined domain constructs and partner peptides\",\n      \"pmids\": [\"20454568\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No full-length structure of the assembled complex\", \"Did not link biophysics to cellular activation kinetics\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined transcriptional control of NOXO1, showing TNF-alpha drives expression through a p38/JNK-AP-1 promoter axis to amplify epithelial superoxide.\",\n      \"evidence\": \"Promoter mutagenesis, AP-1 decoy oligonucleotides, and siRNA knockdown with superoxide readout in T84 cells\",\n      \"pmids\": [\"18929641\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not connect induced NOXO1 levels to a downstream physiological outcome\", \"Other promoter elements not characterized\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Resolved the activation switch by showing PKC phosphorylation at Ser154 and Thr341 enhances NOXA1 and p22phox binding to optimize NOX1 ROS output.\",\n      \"evidence\": \"Phospho-site mutagenesis (S154A/D, T341A), in vitro PKC kinase assay, pulldowns, co-localization, and ROS measurement with endogenous validation\",\n      \"pmids\": [\"23322165\", \"23957209\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signals activating PKC toward NOXO1 in vivo not mapped\", \"Relative contributions of PKC vs PKA to constitutive activity not fully separated\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified post-translational destabilization of NOXO1 via Grb2/Cbl and linked NOXO1 to oncogenic growth, establishing it as a tumor-promoting node.\",\n      \"evidence\": \"Reciprocal Co-IP, CRISPR/Cas9 knockout, Grb2 gain/loss-of-function, soft-agar and xenograft assays\",\n      \"pmids\": [\"26781991\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cbl ubiquitination sites on NOXO1 not mapped\", \"Whether degradation is constitutive or signal-triggered in tumors unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Connected the NOX1-NOXO1 complex to vascular dysfunction and angiogenesis, showing it mediates oscillatory-shear-stress eNOS uncoupling and ROS-dependent ADAM17/Notch signaling.\",\n      \"evidence\": \"RNAi and NoxO1 knockout mouse models with co-localization, superoxide/NO measurement, eNOS uncoupling, retinal angiogenesis, and ADAM17 activity assays\",\n      \"pmids\": [\"26826128\", \"27283741\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ROS targets on eNOS and ADAM17 not molecularly defined\", \"Single-lab models for each phenotype\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrated in vivo that NoxO1-dependent ROS restrains colon epithelial proliferation and promotes apoptosis, defining a physiological homeostatic role.\",\n      \"evidence\": \"NoxO1 knockout mouse with crypt superoxide measurement, proliferation/apoptosis assays, and colitis/cancer models\",\n      \"pmids\": [\"29867954\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Downstream ROS-sensitive effectors controlling proliferation not identified\", \"No compensatory p47phox response examined further\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified CYLD as a deubiquitinase that paradoxically promotes NoxO1 ubiquitination and degradation, adding a second post-translational brake on ROS in cancer.\",\n      \"evidence\": \"Genome-wide DUB CRISPR screen, Co-IP, ubiquitination and half-life assays, and CYLD-KO xenograft model in PC-3 cells\",\n      \"pmids\": [\"34742871\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which a DUB promotes ubiquitination not resolved\", \"Relationship to the Grb2/Cbl pathway unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Linked a D-box motif to NoxO1 subcellular partitioning, showing mutation redistributes it to a cytoskeletal fraction associated with keratin 18/vimentin and elevated NOX1 activity.\",\n      \"evidence\": \"Wild-type vs D-box mutant expression, subcellular fractionation, Co-IP with cytoskeletal proteins, ROS measurement\",\n      \"pmids\": [\"36902094\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP/fractionation approach without rescue or reconstitution\", \"Functional significance of cytoskeletal association not established\", \"Whether the D-box mediates regulated degradation untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed a ROS-independent adaptor function, showing NoxO1 associates with Erbin and EGFR to modulate EGFR kinase activation and cell migration.\",\n      \"evidence\": \"BioID proximity labeling, co-localization, NoxO1 overexpression/CRISPR KO, EGFR phosphorylation kinetics, wound-closure assay\",\n      \"pmids\": [\"39426288\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs proximity-only nature of NoxO1-EGFR interaction not resolved\", \"Structural basis of the Erbin interaction unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Indicated that ROS output is set by the NoxO1:Nox1:NoxA1 ratio rather than fixed stoichiometry, reframing NoxO1 abundance as a rheostat.\",\n      \"evidence\": \"Stable Nox1/NoxA1 HEK293 lines transfected with increasing NoxO1 and ROS measurement\",\n      \"pmids\": [\"39334772\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single functional assay without biochemical or structural validation\", \"No measurement of complex stoichiometry directly\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended the ROS-independent adaptor role to organelle biogenesis, showing NoxO1 activates TFEB via Erbin to expand endosomes/lysosomes and alter EGFR trafficking.\",\n      \"evidence\": \"Overexpression/KO lines, ODE trafficking models, cargo trafficking assays, proximity ligation assay, Erbin KO, EEA1/LAMP1 quantification, TFEB activity assay\",\n      \"pmids\": [\"41406574\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which NoxO1-Erbin activates TFEB not defined\", \"Direct membership of NoxO1 in the trafficking complex inferred from PLA proximity\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How NOXO1's two arms—NADPH oxidase organizer and ROS-independent Erbin/EGFR/TFEB adaptor—are coordinated within a cell, and what structural state of full-length NOXO1 governs the switch between them, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No full-length structure of NOXO1 in either functional state\", \"Whether oxidase scaffolding and EGFR/TFEB adaptor functions are mutually exclusive or simultaneous is unknown\", \"Physiological signals selecting between the two roles undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 5, 6]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [6, 7, 15]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 9]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [16]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [16]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [0, 4, 11]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [10, 14]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [16]}\n    ],\n    \"complexes\": [\"NOX1 NADPH oxidase complex\", \"NOX3 NADPH oxidase complex\"],\n    \"partners\": [\"p22phox\", \"NOXA1\", \"NOX1\", \"Grb2\", \"Cbl\", \"CYLD\", \"Erbin\", \"EGFR\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}