{"gene":"NCF2","run_date":"2026-04-29T11:37:56","timeline":{"discoveries":[{"year":1992,"finding":"p67phox (NCF2 gene product) is one of four components required to fully reconstitute NADPH oxidase activity in a cell-free system; no activity is observed in its absence, establishing it as an essential cytosolic activator of flavocytochrome b-245.","method":"Cell-free reconstitution with purified recombinant p67phox, p47phox, p21rac1, and cytochrome b-245","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with purified components, highly cited foundational study","pmids":["1512217"],"is_preprint":false},{"year":1994,"finding":"Rac1 (GTP-bound form) interacts directly with p67phox in a GTP-dependent manner; effector-site mutations in Rac that abolish NADPH oxidase activation also abolish binding to p67phox, identifying p67phox as the Rac effector in the NADPH oxidase complex.","method":"Co-immunoprecipitation, GTP-dependence assay, Rac effector-site mutagenesis, cell-free oxidase activity","journal":"Science","confidence":"High","confidence_rationale":"Tier 1–2 — direct binding with mutagenesis, replicated in multiple subsequent studies","pmids":["8036496"],"is_preprint":false},{"year":1994,"finding":"p67phox and p47phox translocate from cytosol to the plasma membrane/submembranous actin cytoskeleton upon neutrophil activation; translocation of p67phox and Rac2 to the cytoskeleton requires p47phox, but Rac2 translocation to the membrane can occur independently of p67phox.","method":"Subcellular fractionation of activated normal and CGD (p47phox-deficient) neutrophils, immunoblotting","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — loss-of-function in patient cells with defined phenotype, replicated across multiple labs","pmids":["8120032"],"is_preprint":false},{"year":1994,"finding":"Rac2 translocates to membranes independently of p47phox and p67phox but depends on flavocytochrome b558 for maximal membrane stability; Rac2 membrane binding is reduced ~75% in CGD neutrophils lacking gp91phox.","method":"Cell-free translocation assay with normal and CGD neutrophil membranes/cytosols; quantitative immunoblotting of Rac1 and Rac2 in intact stimulated CGD neutrophils","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple CGD genotypes tested, cell-free and intact-cell systems","pmids":["7982999"],"is_preprint":false},{"year":1994,"finding":"Deletion of either SH3 domain of p67phox dramatically reduces NADPH oxidase activity and membrane binding in intact B-cells; deletion of both SH3 domains completely abolishes membrane binding and oxidase activity, demonstrating that the SH3 domains are required for translocation and activity in whole cells but not in the cell-free system.","method":"Stable transfection of p67phox-deficient CGD B-cells with SH3 deletion mutants; whole-cell and cell-free oxidase assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — mutagenesis in reconstituted intact cells with multiple deletion variants","pmids":["8206939"],"is_preprint":false},{"year":1994,"finding":"p40phox forms a tight 1:1 complex with p67phox in neutrophil cytosol; p40phox is absent in CGD patients lacking p67phox, indicating that its stability depends on p67phox.","method":"Co-immunoprecipitation and column chromatography of neutrophil cytosol; gel analysis of CGD patient samples","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 2–3 — co-IP in native neutrophil cytosol, confirmed with patient cells, replicated by multiple groups","pmids":["8147882"],"is_preprint":false},{"year":1995,"finding":"p67phox (not p47phox) is required for electron flow from NADPH to the flavin (FAD) center of the oxidase; p47phox is additionally required for electron transfer beyond the flavin to the heme in cytochrome b-245 and thence to oxygen.","method":"Cell-free oxidase reconstitution with individually depleted cytosol fractions from CGD patients; spectrophotometric electron-flow assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro mechanistic dissection using defined components and patient-derived material","pmids":["7896790"],"is_preprint":false},{"year":1995,"finding":"p40phox interacts with p67phox via the C-terminal SH3 domain of p40phox binding to p47phox, and via the C-terminal portion of p40phox binding to the region between the two SH3 domains of p67phox; p40phox also binds p47phox but more weakly.","method":"Affinity-bead pulldown, surface plasmon resonance (Biacore) with truncation constructs","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal binding methods with domain mapping","pmids":["8760383"],"is_preprint":false},{"year":1995,"finding":"Two-hybrid and in vitro binding studies show p67phox interacts with p47phox via the C-terminal SH3 domain of p67phox binding to the polyproline motif of p47phox; p40phox interacts with p67phox via the region between p67phox's two SH3 domains, not via SH3-polyproline interactions.","method":"Yeast two-hybrid system; in vitro binding with domain constructs","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — two orthogonal methods, domain-level resolution","pmids":["7890694"],"is_preprint":false},{"year":1996,"finding":"p67phox interacts with Rac2 approximately 6-fold more strongly than with Rac1 in yeast two-hybrid assays; Rac effector-site mutants inactive in NADPH oxidase lose interaction with p67phox, and an activating Rac2 mutant shows increased affinity for p67phox.","method":"Yeast two-hybrid system; cell-free oxidase activity","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — two-hybrid with multiple mutants, consistent with biochemical data from other labs","pmids":["8550629"],"is_preprint":false},{"year":1996,"finding":"p67phox contains an NADPH-binding site (identified by NADPH dialdehyde crosslinking and competition with NADPH); this site participates in catalysis by the leukocyte NADPH oxidase.","method":"NADPH dialdehyde affinity labeling of cytosol and purified recombinant p67phox; cell-free oxidase reconstitution rescue assay","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1 — chemical crosslinking combined with reconstitution activity assay","pmids":["8770870"],"is_preprint":false},{"year":1996,"finding":"p67phox mediates translocation of p40phox and Rac1 from cytosol to membrane upon neutrophil stimulation; in p67phox-deficient CGD neutrophils, p40phox and Rac1 fail to translocate, whereas Rac2 and p47phox translocate normally.","method":"Subcellular fractionation and immunoblotting of stimulated normal and CGD (p67phox-deficient and p47phox-deficient) patient neutrophils","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 — loss-of-function patient cells with multiple component tracking","pmids":["8670049"],"is_preprint":false},{"year":1996,"finding":"A deletion of Lys58 in p67phox found in a CGD patient disrupts the interaction between p67phox and p21-Rac1, prevents translocation of p47phox and p67phox to the plasma membrane upon stimulation, and abolishes superoxide production — demonstrating that p67phox–Rac1 interaction is essential for oxidase complex assembly.","method":"CGD patient genetic analysis; co-immunoprecipitation of p67phox-Rac1; membrane translocation assay in patient neutrophils","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — naturally occurring disease mutation linked directly to disrupted protein–protein interaction and functional loss","pmids":["8879195"],"is_preprint":false},{"year":1997,"finding":"p67phox is phosphorylated on serine residues upon neutrophil activation by fMLP and PMA; PKC phosphorylates p67phox at the same tryptic peptide site in vitro and in intact cells via a PKC-dependent pathway; an additional PKC-independent pathway also phosphorylates p67phox.","method":"32P-labeling of intact neutrophils; phosphoamino acid analysis; 2D tryptic peptide mapping; in vitro kinase assay with PKC and GST-p67phox","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro kinase assay plus intact-cell phosphopeptide mapping with inhibitors","pmids":["9202043"],"is_preprint":false},{"year":1997,"finding":"Rac1 effector region (residues 26–45) mutations increase Kd for p67phox binding, showing this region mediates direct Rac1–p67phox interaction; insert region mutations (residues 124–135) elevate EC50 for oxidase activity without affecting p67phox binding, suggesting the insert region interacts with another component (possibly cytochrome b558).","method":"Fluorescence (MANT-GTP) direct binding assay; steady-state kinetic NADPH oxidase reconstitution; site-directed Rac1 mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — direct binding measurements plus kinetic reconstitution with multiple mutants","pmids":["9228059"],"is_preprint":false},{"year":1998,"finding":"An activation domain in p67phox (residues ~199–210, especially Val204) is essential for NADPH oxidase activity independently of p47phox or Rac binding; V204A mutation completely abolishes activity and acts as a dominant-negative by competing with wild-type p67phox for membrane translocation.","method":"C-terminal truncation series; single amino acid mutagenesis; cell-free oxidase reconstitution; dominant-negative competition assay; membrane translocation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — systematic mutagenesis with reconstitution and dominant-negative validation","pmids":["9642219"],"is_preprint":false},{"year":1998,"finding":"C-terminal truncations of both p47phox and p67phox relieve autoinhibition, allowing amphiphile-independent NADPH oxidase activation in vitro; the C-terminal regions of both proteins function as negative regulatory elements, and the N-terminal SH3 domain of p47phox is normally masked by intramolecular interaction with its own C-terminus.","method":"Cell-free oxidase activation with truncated recombinant proteins; p22phox binding assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with multiple truncation mutants","pmids":["9461621"],"is_preprint":false},{"year":1998,"finding":"The C-terminal SH3 domain of p67phox and the polyproline motif of p40phox constitute a PC motif-based interaction interface; mutagenesis of the PC motif in p40phox abolishes binding to p67phox.","method":"Yeast two-hybrid screening; in vitro binding with recombinant proteins; site-directed mutagenesis of PC motif","journal":"European journal of biochemistry","confidence":"High","confidence_rationale":"Tier 1–2 — two orthogonal methods with mutagenesis","pmids":["9490029"],"is_preprint":false},{"year":1998,"finding":"Rac1 binds to p67phox at residues 170–199 (N-terminal region); deletion of the C-terminal SH3 domain or polyproline motif of p67phox increases Rac1 binding ~8-fold, revealing a cryptic Rac-binding site; PAK kinase phosphorylates p67phox adjacent to the Rac-binding site, and this phosphorylation is enhanced by C-terminal SH3 or polyproline deletion.","method":"Pulldown binding assays with p67phox truncations/deletions; in vitro PAK kinase assay; NADPH oxidase inhibition assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2–3 — binding assays with multiple deletions, single lab","pmids":["9624165"],"is_preprint":false},{"year":1999,"finding":"The major phosphorylation site of p67phox is Thr233; mutagenesis of Thr233 to Ala abolishes in vitro phosphorylation; this site is phosphorylated by MAP kinase and cytosol in vitro and in stimulated neutrophils.","method":"Phosphopeptide mapping by cyanogen bromide digestion and HPLC-MS; Thr233Ala mutagenesis; in vitro kinase assays","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 — mass spectrometry site identification combined with mutagenesis confirmation","pmids":["9931304"],"is_preprint":false},{"year":2000,"finding":"Crystal structure of the N-terminal TPR domain of p67phox bound to Rac·GTP at 2.7 Å reveals a novel Rho effector interaction mode in which an insertion between TPR motifs mediates complex formation and GTPase specificity.","method":"X-ray crystallography of Rac·GTP–p67phox TPR domain complex","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 — crystal structure at atomic resolution","pmids":["11090627"],"is_preprint":false},{"year":2001,"finding":"Crystal structure of the active N-terminal fragment of p67phox (residues 1–213) at 1.8 Å resolution reveals four TPR motifs with the C-terminus folding back into a hydrophobic groove; an additional short C-terminal helix (residues 187–193) likely constitutes part of the activation domain; CGD mutations G78E and A128V map to this region, with A128V exhibiting temperature-sensitive activity.","method":"X-ray crystallography; temperature-shift cell-free oxidase reconstitution with CGD mutants","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — crystal structure combined with functional mutagenesis and disease variant characterization","pmids":["11262407"],"is_preprint":false},{"year":2002,"finding":"In the resting cytosolic complex, p67phox acts as the central bridging molecule that connects p40phox and p47phox; the complex contains one copy of each protein forming a 1:1:1 heterotrimer with an extended non-globular shape; isothermal titration calorimetry shows p67phox is the primary binding partner of p47phox in the resting state.","method":"Gel filtration; isothermal titration calorimetry (ITC); analytical ultracentrifugation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — multiple biophysical methods including ITC for thermodynamic characterization","pmids":["11796733"],"is_preprint":false},{"year":2002,"finding":"A prenylated chimera of p67phox(1–212) fused to full-length Rac1, when loaded with GTP, activates NADPH oxidase in the absence of p47phox and amphiphile by spontaneously associating with membranes, demonstrating that Rac serves as a membrane-targeting carrier for p67phox and that intrachimeric p67phox–Rac interaction induces a conformational change in the p67phox activation domain.","method":"Cell-free NADPH oxidase reconstitution with prenylated chimeric proteins; membrane association assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — reconstituted system with engineered chimeras plus prenylation, mechanistically dissects Rac function","pmids":["11896062"],"is_preprint":false},{"year":2003,"finding":"ERK2 phosphorylates p67phox primarily in its N-terminal fragment (residues 1–243), while p38MAPK phosphorylates sites in the C-terminal fragment (residues 244–526); a C-terminal phosphorylation site is cryptic in the intact protein due to an intramolecular interaction with the N-terminal TPR-rich region; both kinases phosphorylate p67phox in fMLP- and PMA-stimulated neutrophils.","method":"In vitro kinase assays with ERK2 and p38MAPK; phosphopeptide mapping; truncation constructs; MEK1/2 and p38MAPK inhibitors in intact neutrophils","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro kinase assays with domain mapping confirmed in intact cells with inhibitors","pmids":["12693948"],"is_preprint":false},{"year":2004,"finding":"Rac has a dual role in NADPH oxidase assembly: (1) tethering p67phox to the membrane via Rac's C-terminal basic residues, and (2) inducing an activating conformational change in p67phox (via intrachimeric p67phox–Rac interaction); inactive Rac moiety chimeras can be rescued by exogenous Rac1-GTP but not by exogenous p67phox, demonstrating the two functions are separable.","method":"Cell-free NADPH oxidase reconstitution with p67phox(1–212)–Rac1 chimeras bearing site-directed mutations in activation domain, Rac effector region, and C-terminal basic residues","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — systematic chimeric mutagenesis with reconstitution, complementation tests","pmids":["14761978"],"is_preprint":false},{"year":2004,"finding":"PKCdelta forms a complex with p67phox in activated monocytes and directly phosphorylates p67phox in vitro; rottlerin (PKCdelta inhibitor) and PKCdelta antisense both block p67phox phosphorylation and reduce superoxide production, identifying PKCdelta as a regulator of p67phox in human monocytes.","method":"Co-immunoprecipitation of PKCdelta–p67phox; in vitro kinase assay with recombinant PKCdelta; PKCdelta antisense knockdown; selective inhibitor studies; superoxide assay","journal":"Journal of leukocyte biology","confidence":"High","confidence_rationale":"Tier 1–2 — co-IP, in vitro kinase assay, KD with defined phenotype","pmids":["15591124"],"is_preprint":false},{"year":2005,"finding":"S100A8 (but not S100A9) interacts with p67phox and Rac in vitro; S100A8/A9 promotes NADPH oxidase activation by transferring arachidonic acid to the complex; S100A9 knockdown in NB4 cells and S100A9-/- mouse neutrophils show impaired oxidase activity, confirming functional relevance.","method":"Protein-protein interaction studies (pulldown); cell-free oxidase assay with S100A8/A9 mutant unable to bind arachidonic acid; S100A9 antibody neutralization; S100A9-/- mouse neutrophils; S100A9 siRNA in NB4 cells","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including KO mice and in vitro functional assays","pmids":["15642721"],"is_preprint":false},{"year":2006,"finding":"p67phox depends on p40phox (via PX domain-phosphatidylinositol 3-phosphate interaction) or p47phox (via PX domain-plasma membrane interaction) for membrane targeting; GFP-p67phox expressed alone does not translocate to membranes; mutations disrupting p67phox binding to p40phox or p47phox abolish p67phox translocation to their respective membrane compartments.","method":"Live-cell GFP imaging of translocation; mutations in PX and PB1 domains; co-expression in COS-7 cells","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — live imaging with mutagenesis in intact cells","pmids":["17122360"],"is_preprint":false},{"year":2008,"finding":"PKC phosphorylates gp91phox/NOX2 in stimulated neutrophils; this phosphorylation enhances diaphorase activity of the NOX2 flavoprotein domain and increases its binding to Rac2, p67phox, and p47phox, indicating a novel mechanism of NADPH oxidase regulation through phosphorylation-enhanced assembly.","method":"32P-labeling of intact neutrophils; in vitro PKC kinase assay on recombinant NOX2 domain; 2D tryptic peptide mapping; co-IP pulldown after phosphorylation; diaphorase activity assay","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro kinase assay with binding and activity readouts confirmed in intact cells","pmids":["19028840"],"is_preprint":false},{"year":2009,"finding":"SAXS analysis shows p67phox in solution is a very elongated multidomain protein with semi-flexible linkers, adopting a beads-on-a-string arrangement with evidence of intramolecular interactions that constrain the overall conformation; this architecture underlies its ability to interact simultaneously with all other NADPH oxidase components.","method":"Small-angle X-ray scattering (SAXS) of full-length p67phox","journal":"Journal of structural biology","confidence":"High","confidence_rationale":"Tier 1 — structural method with quantitative solution-state data","pmids":["19723583"],"is_preprint":false},{"year":2011,"finding":"A naturally occurring NCF2 missense mutation H389Q in the PB1 domain reduces binding efficiency to the Vav1 guanine nucleotide exchange factor and causes a ~2-fold decrease in ROS production stimulated via FcγR-elicited (Vav-dependent) pathway, linking this SLE-risk variant to a specific loss of Vav1-dependent oxidase activation.","method":"Computational modeling of NCF2–Vav1 interaction; site-directed mutagenesis at position 389; FcγR-stimulated ROS production assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — mutagenesis with functional ROS assay, supported by structural modeling","pmids":["22203994"],"is_preprint":false},{"year":2011,"finding":"A rare NCF2 missense variant found in patients with very early onset IBD reduces binding of p67phox to RAC2, providing a direct mechanistic link between partial oxidase impairment and intestinal inflammation.","method":"Direct sequencing; functional binding assay of variant p67phox–RAC2 interaction","journal":"Gut","confidence":"Medium","confidence_rationale":"Tier 2–3 — binding assay for variant, single lab, limited mechanistic depth","pmids":["21900546"],"is_preprint":false},{"year":2012,"finding":"NCF2/p67phox is a direct transcriptional target of p53; p53 binds to the NCF2 promoter in vivo; NCF2 knockdown by siRNA reduces ROS production and stimulates cell death, indicating p53-driven NCF2 expression generates ROS that promote cell survival against apoptosis.","method":"Microarray identification; luciferase reporter assay; chromatin immunoprecipitation (ChIP); siRNA knockdown with ROS and apoptosis readouts","journal":"Cell cycle","confidence":"High","confidence_rationale":"Tier 2 — ChIP plus reporter assay plus siRNA KD with functional phenotype","pmids":["23187810"],"is_preprint":false},{"year":1999,"finding":"IFN-γ-induced transcription of NCF2 (p67phox) requires cooperative recruitment of PU.1, IRF1, and ICSBP to a specific NCF2 cis element; this transcription factor complex then recruits the coactivator CBP (CREB-binding protein) to activate NCF2 transcription.","method":"Promoter deletion and mutation analysis; gel shift assays; reporter gene assays; co-IP of transcription factor complex with CBP","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — promoter mapping with mutagenesis and co-IP of activating complex","pmids":["10570299"],"is_preprint":false},{"year":2015,"finding":"TLR4/TLR2 signaling activates an IRAK–ERK pathway that drives ERK–p67phox interaction, p67phox translocation to membrane, and p67phox–Nox2 interaction, resulting in ROS generation that mediates IL-1β transcription and processing; disruption at any node (IRAK, ERK, p67phox, or Nox2 siRNA) blocks IL-1β production.","method":"Co-IP of ERK–p67phox and p67phox–Nox2 complexes; siRNA knockdown of Nox2; pharmacological inhibitors; membrane translocation assay; IL-1β reporter and ELISA","journal":"Cellular & molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2–3 — multiple co-IPs and siRNA data but pathway assigned primarily in a cell line","pmids":["26320741"],"is_preprint":false},{"year":2019,"finding":"M. tuberculosis PPE2 protein directly interacts with p67phox via its Trp236 residue; this interaction prevents p67phox and p47phox translocation to the membrane and reduces NADPH oxidase activity; Trp236Ala PPE2 mutant loses both p67phox binding and inhibition of ROS production.","method":"Co-IP of PPE2–p67phox; site-directed mutagenesis (Trp236Ala); membrane translocation assay; ROS quantification in macrophages; bacterial survival assay","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — co-IP plus mutagenesis plus translocation and activity phenotype","pmids":["31375544"],"is_preprint":false},{"year":2003,"finding":"p67phox translocation to forming phagosomes is deficient in neutrophils lacking p47phox, demonstrating that p47phox is required for stable phagosomal recruitment of p67phox; in X-linked CGD neutrophils lacking flavocytochrome b558, both p47phox and p67phox accumulate transiently but are shed from nascent phagosomes once particles are internalized, showing flavocytochrome b558 is required for stable retention of p67phox at the phagosome.","method":"Synchronized phagocytosis assay with immunofluorescence microscopy; immunoblotting of phagosome fractions from CGD patient neutrophils","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — direct imaging with defined genetic loss-of-function cells","pmids":["14623873"],"is_preprint":false}],"current_model":"NCF2-encoded p67phox is an essential cytosolic activator of the phagocyte NADPH oxidase that serves as the central scaffold of the resting p40phox–p47phox–p67phox heterotrimer; upon cell stimulation it is phosphorylated on multiple serine/threonine sites by PKC, ERK2, p38MAPK, and PKCδ, then translocated to the plasma membrane/phagosome via p47phox (plasma membrane) or p40phox (early endosomes) as carrier proteins, where its N-terminal TPR domain binds GTP-loaded Rac2 (Kd ~60 nM) to induce a conformational change in its activation domain (Val204-containing region) that directly stimulates electron transfer from NADPH to the FAD center of flavocytochrome b558, while its two SH3 domains contribute to stable membrane association and its PB1 domain mediates interaction with Vav1."},"narrative":{"teleology":[{"year":1992,"claim":"Establishing that p67phox is an indispensable component for reconstituting NADPH oxidase activity resolved the question of whether all four cytosolic/membrane factors are individually required, positioning p67phox as a direct catalytic activator rather than an accessory factor.","evidence":"Cell-free reconstitution with purified recombinant p67phox, p47phox, Rac1, and cytochrome b-245","pmids":["1512217"],"confidence":"High","gaps":["Mechanism of p67phox activation of electron transfer unknown","Which domain(s) of p67phox are catalytically essential not yet defined"]},{"year":1994,"claim":"Identification of p67phox as the direct effector of Rac GTPase answered which oxidase subunit reads the Rac activation signal, establishing the Rac–p67phox axis as the trigger for oxidase assembly.","evidence":"Co-immunoprecipitation with GTP-dependence and Rac effector-site mutagenesis; two-hybrid confirmation with Rac2 preference","pmids":["8036496","8550629"],"confidence":"High","gaps":["Structural basis of Rac–p67phox recognition unknown","Whether Rac2 selectivity matters in vivo not tested"]},{"year":1994,"claim":"Demonstrating that p67phox translocation to membranes requires p47phox, while p67phox itself carries p40phox and Rac1 to the membrane, defined a hierarchical assembly pathway in which p67phox acts as the central scaffold bridging upstream carriers and downstream effectors.","evidence":"Subcellular fractionation of stimulated normal and CGD (p47phox-deficient and p67phox-deficient) neutrophils","pmids":["8120032","8670049"],"confidence":"High","gaps":["Whether p40phox provides an alternative membrane-targeting route not yet explored","Phagosomal versus plasma membrane targeting not distinguished"]},{"year":1994,"claim":"Mapping the domain architecture of the p67phox-centered trimer — SH3 domains required for membrane association and activity, C-terminal SH3 binding p47phox, inter-SH3 region binding p40phox — established p67phox as the central bridging molecule of the resting cytosolic complex.","evidence":"SH3 deletion mutagenesis in reconstituted CGD B-cells; two-hybrid and in vitro binding with domain constructs; co-IP of native p40phox–p67phox complex","pmids":["8206939","7890694","8147882"],"confidence":"High","gaps":["Stoichiometry and shape of native complex not determined","Whether SH3 domains contact membrane targets directly unknown"]},{"year":1995,"claim":"Showing that p67phox is specifically required for NADPH→FAD electron transfer while p47phox is additionally needed for FAD→heme transfer dissected the catalytic roles of individual cytosolic subunits for the first time.","evidence":"Cell-free oxidase reconstitution with CGD patient cytosol selectively depleted of each subunit; spectrophotometric electron-flow assays","pmids":["7896790"],"confidence":"High","gaps":["Whether p67phox directly contacts the FAD-binding site of gp91phox unknown","Structural basis for activation domain function not defined"]},{"year":1996,"claim":"Discovery of an NADPH-binding site on p67phox itself suggested a substrate-channeling or regulatory role beyond simple protein–protein scaffolding, adding catalytic complexity to the activation mechanism.","evidence":"NADPH dialdehyde affinity labeling of purified recombinant p67phox; competition with NADPH; cell-free rescue assay","pmids":["8770870"],"confidence":"High","gaps":["Physiological significance of p67phox NADPH binding versus gp91phox NADPH binding not resolved","No structural data for NADPH-binding site"]},{"year":1996,"claim":"A CGD patient mutation (ΔLys58) that disrupts p67phox–Rac1 interaction and abolishes superoxide production provided the first human genetic proof that the Rac–p67phox interaction is essential for oxidase function in vivo.","evidence":"Patient genetic analysis; co-IP of mutant p67phox with Rac1; membrane translocation and oxidase activity in patient neutrophils","pmids":["8879195"],"confidence":"High","gaps":["Full spectrum of CGD-causing NCF2 mutations not catalogued","Whether partial loss of Rac binding causes intermediate phenotypes unknown"]},{"year":1997,"claim":"Identification of PKC-dependent and PKC-independent phosphorylation of p67phox on serine residues upon neutrophil activation opened the question of how post-translational modifications regulate oxidase assembly dynamics.","evidence":"32P-labeling of intact neutrophils; 2D tryptic peptide mapping; in vitro PKC kinase assay","pmids":["9202043"],"confidence":"High","gaps":["Specific phosphorylation sites not yet mapped","Functional consequences of phosphorylation on oxidase activity not tested"]},{"year":1998,"claim":"Identification of an activation domain (residues ~199–210, Val204 critical) that is essential for catalysis independently of Rac or p47phox binding defined the minimal catalytic surface on p67phox and provided a dominant-negative tool.","evidence":"Systematic truncation and single amino acid mutagenesis; cell-free oxidase reconstitution; dominant-negative competition assay","pmids":["9642219"],"confidence":"High","gaps":["How Val204 region contacts flavocytochrome b558 structurally unknown","Whether activation domain undergoes conformational change upon Rac binding not shown"]},{"year":1999,"claim":"Mapping Thr233 as the major phosphorylation site and identifying MAP kinase as the responsible kinase provided a specific molecular handle for studying phospho-regulation of p67phox.","evidence":"Phosphopeptide mapping by CNBr digestion/HPLC-MS; Thr233Ala mutagenesis; in vitro kinase assay","pmids":["9931304"],"confidence":"High","gaps":["Functional consequence of Thr233 phosphorylation on oxidase activation not determined","Additional phosphorylation sites not yet mapped"]},{"year":2000,"claim":"The crystal structure of the p67phox TPR domain–Rac·GTP complex at 2.7 Å revealed a novel Rho effector recognition mode mediated by an insertion between TPR motifs, explaining GTPase specificity and providing a structural basis for CGD mutations in this region.","evidence":"X-ray crystallography of Rac·GTP–p67phox(1–203) complex","pmids":["11090627"],"confidence":"High","gaps":["Full-length p67phox structure not available","How Rac binding transmits conformational change to the activation domain structurally unresolved"]},{"year":2001,"claim":"The 1.8 Å structure of the active N-terminal fragment revealed the four-TPR fold, mapped CGD mutations G78E and A128V to the hydrophobic core, and identified a C-terminal helix (187–193) as part of the activation domain, connecting structure to disease-causing loss of function.","evidence":"X-ray crystallography; temperature-shift cell-free oxidase assay with CGD mutants","pmids":["11262407"],"confidence":"High","gaps":["Structure of activation domain in contact with flavocytochrome b558 not determined","No structure of full-length p67phox"]},{"year":2002,"claim":"Biophysical characterization of the 1:1:1 p40phox–p67phox–p47phox heterotrimer with p67phox as the central bridge, combined with chimeric Rac–p67phox demonstrating Rac's dual role as membrane anchor and conformational activator, provided an integrated model of oxidase assembly.","evidence":"ITC, gel filtration, analytical ultracentrifugation for trimer; cell-free reconstitution with prenylated p67phox–Rac1 chimeras","pmids":["11796733","11896062"],"confidence":"High","gaps":["Structure of complete ternary cytosolic complex not solved","Kinetics of assembly in intact cells not measured"]},{"year":2003,"claim":"Demonstration that ERK2 phosphorylates the N-terminal region and p38MAPK the C-terminal region of p67phox, with a cryptic C-terminal site unmasked by domain rearrangement, revealed compartmentalized phospho-regulation linked to intramolecular conformational control.","evidence":"In vitro kinase assays with truncation constructs; MEK1/2 and p38MAPK inhibitors in intact neutrophils","pmids":["12693948"],"confidence":"High","gaps":["Identity of C-terminal phosphorylation sites not mapped","Whether phosphorylation at these sites regulates oxidase activity directly not tested"]},{"year":2003,"claim":"Showing that p47phox is required for stable phagosomal recruitment of p67phox and that flavocytochrome b558 is needed for retention of both subunits on nascent phagosomes extended the assembly hierarchy to the phagosome compartment.","evidence":"Synchronized phagocytosis with immunofluorescence in CGD patient neutrophils","pmids":["14623873"],"confidence":"High","gaps":["Role of p40phox in phagosomal targeting of p67phox not assessed here","Whether retention mechanism involves direct p67phox–cytochrome b558 interaction unknown"]},{"year":2004,"claim":"Identification of PKCδ as a direct kinase for p67phox in monocytes, with functional consequences for superoxide production, expanded the kinase network regulating p67phox beyond conventional PKC and MAPKs.","evidence":"Co-IP of PKCδ–p67phox; in vitro kinase assay; PKCδ antisense knockdown; rottlerin inhibitor; superoxide assay","pmids":["15591124"],"confidence":"High","gaps":["PKCδ phosphorylation sites on p67phox not identified","Whether PKCδ regulates p67phox translocation or catalytic activation not distinguished"]},{"year":2006,"claim":"Live-cell imaging established that p67phox has no intrinsic membrane-targeting capacity and reaches the plasma membrane via p47phox and endosomes via p40phox, resolving the question of how p67phox accesses distinct membrane compartments.","evidence":"GFP-p67phox live imaging with PX and PB1 domain mutations in COS-7 cells","pmids":["17122360"],"confidence":"High","gaps":["Whether p40phox-mediated endosomal route is essential for antimicrobial ROS not tested in phagocytes","Contribution of Rac-mediated membrane tethering in intact cells not assessed"]},{"year":2011,"claim":"Linking the NCF2 PB1-domain variant H389Q to reduced Vav1 binding and impaired FcγR-stimulated ROS production connected a lupus-risk allele to a specific defect in the Vav1-dependent arm of oxidase activation.","evidence":"Site-directed mutagenesis at position 389; computational modeling; FcγR-stimulated ROS assay","pmids":["22203994"],"confidence":"High","gaps":["In vivo relevance in patient cells not demonstrated","Whether other PB1-domain partners are affected not tested"]},{"year":2012,"claim":"Demonstrating that p53 directly activates NCF2 transcription revealed a link between tumor-suppressor signaling and oxidase-derived ROS, with NCF2 knockdown reducing ROS and enhancing apoptosis.","evidence":"ChIP of p53 on NCF2 promoter; luciferase reporter; siRNA knockdown with ROS and apoptosis readouts","pmids":["23187810"],"confidence":"High","gaps":["Whether p53-driven NCF2 expression is relevant in phagocytes or primarily in tumor cells not clarified","Downstream ROS targets mediating survival not identified"]},{"year":2019,"claim":"Identification of M. tuberculosis PPE2 as a direct p67phox-binding virulence factor that blocks oxidase assembly exemplified pathogen subversion of the Rac–p67phox activation axis.","evidence":"Co-IP of PPE2–p67phox; Trp236Ala mutagenesis abolishing binding; membrane translocation and ROS assays in macrophages","pmids":["31375544"],"confidence":"High","gaps":["Which domain of p67phox is targeted by PPE2 not mapped","In vivo significance during TB infection not established"]},{"year":null,"claim":"A full-length structure of p67phox in complex with flavocytochrome b558 is lacking, leaving the precise contact surface through which the activation domain stimulates FAD reduction unresolved; the functional consequences of individual phosphorylation events and the integrated regulatory logic of multi-site phosphorylation remain to be determined.","evidence":"","pmids":[],"confidence":"Low","gaps":["No full-length p67phox structure or p67phox–cytochrome b558 complex structure available","Functional roles of individual phosphorylation sites (beyond Thr233) on oxidase kinetics not characterized","How intramolecular conformational changes couple Rac binding to activation domain rearrangement structurally unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,6,22,23]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[6,15,25]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,5,22]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,28,37]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[28]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,6,12,27,36,37]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,25,31,35]}],"complexes":["NADPH oxidase (NOX2 complex)","p40phox–p67phox–p47phox cytosolic heterotrimer"],"partners":["NCF1","NCF4","RAC2","RAC1","CYBB","VAV1","PRKCQ","S100A8"],"other_free_text":[]},"mechanistic_narrative":"NCF2 encodes p67phox, an essential cytosolic activator of the phagocyte NADPH oxidase (NOX2) that functions as the central scaffold of the resting p40phox–p47phox–p67phox heterotrimer and as the direct catalytic activator of electron transfer from NADPH to the FAD center of flavocytochrome b558 [PMID:1512217, PMID:7896790]. Its N-terminal TPR domain binds GTP-loaded Rac2 (preferentially over Rac1), inducing a conformational change in the activation domain (centered on Val204) that stimulates oxidase catalysis, while its C-terminal SH3 domain anchors the trimer via interaction with p47phox and its PB1 domain mediates interaction with p40phox and the guanine nucleotide exchange factor Vav1 [PMID:11090627, PMID:9642219, PMID:7890694, PMID:22203994]. p67phox lacks intrinsic membrane-targeting capacity and depends on p47phox for plasma-membrane/phagosomal recruitment and on p40phox for endosomal recruitment; upon stimulation, it is phosphorylated by PKC, ERK2, p38 MAPK, and PKCδ on multiple serine/threonine residues including Thr233, modulating oxidase assembly and activity [PMID:17122360, PMID:9202043, PMID:12693948, PMID:15591124, PMID:9931304]. Loss-of-function mutations in NCF2 cause autosomal recessive chronic granulomatous disease (CGD), and coding variants that impair Rac2 or Vav1 binding are linked to very early onset inflammatory bowel disease and systemic lupus erythematosus susceptibility [PMID:8879195, PMID:21900546, PMID:22203994]."},"prefetch_data":{"uniprot":{"accession":"P19878","full_name":"Neutrophil cytosol factor 2","aliases":["67 kDa neutrophil oxidase factor","NADPH oxidase activator 2","Neutrophil NADPH oxidase factor 2","p67-phox"],"length_aa":526,"mass_kda":59.8,"function":"Subunit of the phagocyte NADPH oxidase complex that mediates the transfer of electrons from cytosolic NADPH to O2 to produce the superoxide anion (O2(-)) (PubMed:12207919, PubMed:38355798). In the activated complex, electrons are first transferred from NADPH to flavin adenine dinucleotide (FAD) and subsequently transferred via two heme molecules to molecular oxygen, producing superoxide through an outer-sphere reaction (PubMed:38355798). Activation of the NADPH oxidase complex is initiated by the assembly of cytosolic subunits of the NADPH oxidase complex with the core NADPH oxidase complex to form a complex at the plasma membrane or phagosomal membrane (PubMed:38355798). This activation process is initiated by phosphorylation dependent binding of the cytosolic NCF1/p47-phox subunit to the C-terminus of CYBA/p22-phox (By similarity)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/P19878/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NCF2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/NCF2","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"},{"mim_id":"608701","title":"NICOTINAMIDE NUCLEOTIDE ADENYLYLTRANSFERASE 2; NMNAT2","url":"https://www.omim.org/entry/608701"},{"mim_id":"608515","title":"NEUTROPHIL CYTOSOLIC FACTOR 2; NCF2","url":"https://www.omim.org/entry/608515"},{"mim_id":"608512","title":"NEUTROPHIL CYTOSOLIC FACTOR 1; NCF1","url":"https://www.omim.org/entry/608512"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"bone marrow","ntpm":119.1},{"tissue":"lung","ntpm":61.2},{"tissue":"lymphoid tissue","ntpm":73.9}],"url":"https://www.proteinatlas.org/search/NCF2"},"hgnc":{"alias_symbol":["p67phox","NOXA2"],"prev_symbol":[]},"alphafold":{"accession":"P19878","domains":[{"cath_id":"1.25.40.10","chopping":"2-167","consensus_level":"medium","plddt":96.2499,"start":2,"end":167},{"cath_id":"2.30.30.40","chopping":"242-295","consensus_level":"high","plddt":85.9719,"start":242,"end":295},{"cath_id":"3.10.20.90","chopping":"352-427","consensus_level":"high","plddt":85.6316,"start":352,"end":427},{"cath_id":"2.30.30.40","chopping":"459-512","consensus_level":"high","plddt":89.5426,"start":459,"end":512}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P19878","model_url":"https://alphafold.ebi.ac.uk/files/AF-P19878-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P19878-F1-predicted_aligned_error_v6.png","plddt_mean":78.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NCF2","jax_strain_url":"https://www.jax.org/strain/search?query=NCF2"},"sequence":{"accession":"P19878","fasta_url":"https://rest.uniprot.org/uniprotkb/P19878.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P19878/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P19878"}},"corpus_meta":[{"pmid":"8036496","id":"PMC_8036496","title":"Interaction of Rac with p67phox and regulation of phagocytic NADPH oxidase activity.","date":"1994","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/8036496","citation_count":340,"is_preprint":false},{"pmid":"12716910","id":"PMC_12716910","title":"Novel human homologues of p47phox and p67phox participate in activation of superoxide-producing NADPH oxidases.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12716910","citation_count":315,"is_preprint":false},{"pmid":"11090627","id":"PMC_11090627","title":"Structure of the TPR domain of p67phox in complex with Rac.GTP.","date":"2000","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/11090627","citation_count":260,"is_preprint":false},{"pmid":"1512217","id":"PMC_1512217","title":"Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components: p67-phox, p47-phox, p21rac1, and cytochrome b-245.","date":"1992","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/1512217","citation_count":256,"is_preprint":false},{"pmid":"12657628","id":"PMC_12657628","title":"Proteins homologous to p47phox and p67phox support superoxide production by NAD(P)H oxidase 1 in colon epithelial cells.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12657628","citation_count":241,"is_preprint":false},{"pmid":"19723583","id":"PMC_19723583","title":"NADPH oxidase activator p67(phox) behaves in solution as a multidomain protein with semi-flexible linkers.","date":"2009","source":"Journal of structural biology","url":"https://pubmed.ncbi.nlm.nih.gov/19723583","citation_count":239,"is_preprint":false},{"pmid":"7982999","id":"PMC_7982999","title":"Rac translocates independently of the neutrophil NADPH oxidase components p47phox and p67phox. Evidence for its interaction with flavocytochrome b558.","date":"1994","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7982999","citation_count":209,"is_preprint":false},{"pmid":"9719063","id":"PMC_9719063","title":"Angiotensin II induces p67phox mRNA expression and NADPH oxidase superoxide generation in rabbit aortic adventitial fibroblasts.","date":"1998","source":"Hypertension (Dallas, Tex. : 1979)","url":"https://pubmed.ncbi.nlm.nih.gov/9719063","citation_count":192,"is_preprint":false},{"pmid":"9642219","id":"PMC_9642219","title":"Regulation of the neutrophil respiratory burst oxidase. Identification of an activation domain in p67(phox).","date":"1998","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9642219","citation_count":188,"is_preprint":false},{"pmid":"14514646","id":"PMC_14514646","title":"Translocation of glomerular p47phox and p67phox by protein kinase C-beta activation is required for oxidative stress in diabetic nephropathy.","date":"2003","source":"Diabetes","url":"https://pubmed.ncbi.nlm.nih.gov/14514646","citation_count":182,"is_preprint":false},{"pmid":"8120032","id":"PMC_8120032","title":"Cytosolic guanine nucleotide-binding protein Rac2 operates in vivo as a component of the neutrophil respiratory burst oxidase. Transfer of Rac2 and the cytosolic oxidase components p47phox and p67phox to the submembranous actin cytoskeleton during oxidase activation.","date":"1994","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8120032","citation_count":167,"is_preprint":false},{"pmid":"11045958","id":"PMC_11045958","title":"Upregulation of p67(phox) and gp91(phox) in aortas from angiotensin II-infused mice.","date":"2000","source":"American journal of physiology. Heart and circulatory physiology","url":"https://pubmed.ncbi.nlm.nih.gov/11045958","citation_count":151,"is_preprint":false},{"pmid":"22203994","id":"PMC_22203994","title":"Lupus-associated causal mutation in neutrophil cytosolic factor 2 (NCF2) brings unique insights to the structure and function of NADPH oxidase.","date":"2011","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/22203994","citation_count":149,"is_preprint":false},{"pmid":"21900546","id":"PMC_21900546","title":"NADPH oxidase complex and IBD candidate gene studies: identification of a rare variant in NCF2 that results in reduced binding to RAC2.","date":"2011","source":"Gut","url":"https://pubmed.ncbi.nlm.nih.gov/21900546","citation_count":147,"is_preprint":false},{"pmid":"15642721","id":"PMC_15642721","title":"The arachidonic acid-binding protein S100A8/A9 promotes NADPH oxidase activation by interaction with p67phox and Rac-2.","date":"2005","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/15642721","citation_count":145,"is_preprint":false},{"pmid":"17041146","id":"PMC_17041146","title":"A p67Phox-like regulator is recruited to control hyphal branching in a fungal-grass mutualistic symbiosis.","date":"2006","source":"The Plant cell","url":"https://pubmed.ncbi.nlm.nih.gov/17041146","citation_count":140,"is_preprint":false},{"pmid":"19028840","id":"PMC_19028840","title":"Regulation of the phagocyte NADPH oxidase activity: phosphorylation of gp91phox/NOX2 by protein kinase C enhances its diaphorase activity and binding to Rac2, p67phox, and p47phox.","date":"2008","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/19028840","citation_count":137,"is_preprint":false},{"pmid":"7703387","id":"PMC_7703387","title":"The expression of NADPH oxidase components in human glomerular mesangial cells: detection of protein and mRNA for p47phox, p67phox, and p22phox.","date":"1995","source":"Journal of the American Society of Nephrology : JASN","url":"https://pubmed.ncbi.nlm.nih.gov/7703387","citation_count":133,"is_preprint":false},{"pmid":"11796733","id":"PMC_11796733","title":"Architecture of the p40-p47-p67phox complex in the resting state of the NADPH oxidase. A central role for p67phox.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11796733","citation_count":131,"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":"22326221","id":"PMC_22326221","title":"Increased expression of NAD(P)H oxidase subunit p67(phox) in the renal medulla contributes to excess oxidative stress and salt-sensitive hypertension.","date":"2012","source":"Cell metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/22326221","citation_count":123,"is_preprint":false},{"pmid":"9202043","id":"PMC_9202043","title":"Phosphorylation of the respiratory burst oxidase subunit p67(phox) during human neutrophil activation. Regulation by protein kinase C-dependent and independent pathways.","date":"1997","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9202043","citation_count":122,"is_preprint":false},{"pmid":"29483646","id":"PMC_29483646","title":"LINC01410-miR-532-NCF2-NF-kB feedback loop promotes gastric cancer angiogenesis and metastasis.","date":"2018","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/29483646","citation_count":121,"is_preprint":false},{"pmid":"1918085","id":"PMC_1918085","title":"Characterization of neutrophil NADPH oxidase factors p47-phox and p67-phox from recombinant baculoviruses.","date":"1991","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/1918085","citation_count":119,"is_preprint":false},{"pmid":"14761978","id":"PMC_14761978","title":"Dual role of Rac in the assembly of NADPH oxidase, tethering to the membrane and activation of p67phox: a study based on mutagenesis of p67phox-Rac1 chimeras.","date":"2004","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/14761978","citation_count":112,"is_preprint":false},{"pmid":"9228059","id":"PMC_9228059","title":"Rac binding to p67(phox). Structural basis for interactions of the Rac1 effector region and insert region with components of the respiratory burst oxidase.","date":"1997","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9228059","citation_count":111,"is_preprint":false},{"pmid":"8439286","id":"PMC_8439286","title":"Relationship between phosphorylation and translocation to the plasma membrane of p47phox and p67phox and activation of the NADPH oxidase in normal and Ca(2+)-depleted human neutrophils.","date":"1993","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/8439286","citation_count":111,"is_preprint":false},{"pmid":"8670049","id":"PMC_8670049","title":"Mechanisms of NADPH oxidase activation: translocation of p40phox, Rac1 and Rac2 from the cytosol to the membranes in human neutrophils lacking p47phox or p67phox.","date":"1996","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/8670049","citation_count":107,"is_preprint":false},{"pmid":"7890694","id":"PMC_7890694","title":"Mapping the domains of interaction of p40phox with both p47phox and p67phox of the neutrophil oxidase complex using the two-hybrid system.","date":"1995","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7890694","citation_count":101,"is_preprint":false},{"pmid":"10570299","id":"PMC_10570299","title":"Recruitment of CREB-binding protein by PU.1, IFN-regulatory factor-1, and the IFN consensus sequence-binding protein is necessary for IFN-gamma-induced p67phox and gp91phox expression.","date":"1999","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/10570299","citation_count":101,"is_preprint":false},{"pmid":"8147882","id":"PMC_8147882","title":"A novel cytosolic component, p40phox, of respiratory burst oxidase associates with p67phox and is absent in patients with chronic granulomatous disease who lack p67phox.","date":"1994","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/8147882","citation_count":95,"is_preprint":false},{"pmid":"12693948","id":"PMC_12693948","title":"Phosphorylation of the NADPH oxidase component p67(PHOX) by ERK2 and P38MAPK: selectivity of phosphorylated sites and existence of an intramolecular regulatory domain in the tetratricopeptide-rich region.","date":"2003","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12693948","citation_count":95,"is_preprint":false},{"pmid":"8550629","id":"PMC_8550629","title":"The Rac target NADPH oxidase p67phox interacts preferentially with Rac2 rather than Rac1.","date":"1996","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8550629","citation_count":92,"is_preprint":false},{"pmid":"7964505","id":"PMC_7964505","title":"156Pro-->Gln substitution in the light chain of cytochrome b558 of the human NADPH oxidase (p22-phox) leads to defective translocation of the cytosolic proteins p47-phox and p67-phox.","date":"1994","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/7964505","citation_count":91,"is_preprint":false},{"pmid":"8206939","id":"PMC_8206939","title":"Role of p67-phox SH3 domains in assembly of the NADPH oxidase system.","date":"1994","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8206939","citation_count":91,"is_preprint":false},{"pmid":"7896790","id":"PMC_7896790","title":"The cytosolic activating factors p47phox and p67phox have distinct roles in the regulation of electron flow in NADPH oxidase.","date":"1995","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7896790","citation_count":90,"is_preprint":false},{"pmid":"8257426","id":"PMC_8257426","title":"Activation of NADPH oxidase of human neutrophils involves the phosphorylation and the translocation of cytosolic p67phox.","date":"1993","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/8257426","citation_count":89,"is_preprint":false},{"pmid":"8182143","id":"PMC_8182143","title":"A point mutation in gp91-phox of cytochrome b558 of the human NADPH oxidase leading to defective translocation of the cytosolic proteins p47-phox and p67-phox.","date":"1994","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/8182143","citation_count":88,"is_preprint":false},{"pmid":"12101222","id":"PMC_12101222","title":"Cytosolic phospholipase A2 (cPLA2) regulation of human monocyte NADPH oxidase activity. cPLA2 affects translocation but not phosphorylation of p67(phox) and p47(phox).","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12101222","citation_count":86,"is_preprint":false},{"pmid":"10233905","id":"PMC_10233905","title":"Transient association of the nicotinamide adenine dinucleotide phosphate oxidase subunits p47phox and p67phox with phagosomes in neutrophils from patients with X-linked chronic granulomatous disease.","date":"1999","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/10233905","citation_count":79,"is_preprint":false},{"pmid":"16626305","id":"PMC_16626305","title":"Homocysteine stimulates phosphorylation of NADPH oxidase p47phox and p67phox subunits in monocytes via protein kinase Cbeta activation.","date":"2006","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/16626305","citation_count":79,"is_preprint":false},{"pmid":"7616102","id":"PMC_7616102","title":"Dissociation of Rac translocation from p47phox/p67phox movements in human neutrophils by tyrosine kinase inhibitors.","date":"1995","source":"Journal of leukocyte biology","url":"https://pubmed.ncbi.nlm.nih.gov/7616102","citation_count":77,"is_preprint":false},{"pmid":"7738010","id":"PMC_7738010","title":"A 68-kDa kinase and NADPH oxidase component p67phox are targets for Cdc42Hs and Rac1 in neutrophils.","date":"1995","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7738010","citation_count":75,"is_preprint":false},{"pmid":"17122360","id":"PMC_17122360","title":"A regulated adaptor function of p40phox: distinct p67phox membrane targeting by p40phox and by p47phox.","date":"2006","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/17122360","citation_count":74,"is_preprint":false},{"pmid":"8760383","id":"PMC_8760383","title":"Interactions between cytosolic components of the NADPH oxidase: p40phox interacts with both p67phox and p47phox.","date":"1996","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/8760383","citation_count":73,"is_preprint":false},{"pmid":"9064349","id":"PMC_9064349","title":"Involvement of p40phox in activation of phagocyte NADPH oxidase through association of its carboxyl-terminal, but not its amino-terminal, with p67phox.","date":"1996","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/9064349","citation_count":71,"is_preprint":false},{"pmid":"9461621","id":"PMC_9461621","title":"Anionic amphiphile-independent activation of the phagocyte NADPH oxidase in a cell-free system by p47phox and p67phox, both in C terminally truncated forms. Implication for regulatory Src homology 3 domain-mediated interactions.","date":"1998","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9461621","citation_count":71,"is_preprint":false},{"pmid":"9490029","id":"PMC_9490029","title":"The PC motif: a novel and evolutionarily conserved sequence involved in interaction between p40phox and p67phox, SH3 domain-containing cytosolic factors of the phagocyte NADPH oxidase.","date":"1998","source":"European journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9490029","citation_count":70,"is_preprint":false},{"pmid":"7744754","id":"PMC_7744754","title":"Interactions between the cytosolic components p47phox and p67phox of the human neutrophil NADPH oxidase that are not required for activation in the cell-free system.","date":"1995","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7744754","citation_count":67,"is_preprint":false},{"pmid":"9490028","id":"PMC_9490028","title":"Functional modules and expression of mouse p40(phox) and p67(phox), SH3-domain-containing proteins involved in the phagocyte NADPH oxidase complex.","date":"1998","source":"European journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9490028","citation_count":67,"is_preprint":false},{"pmid":"2393022","id":"PMC_2393022","title":"Genes for two autosomal recessive forms of chronic granulomatous disease assigned to 1q25 (NCF2) and 7q11.23 (NCF1).","date":"1990","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/2393022","citation_count":67,"is_preprint":false},{"pmid":"8063777","id":"PMC_8063777","title":"The superoxide-generating system of human neutrophils possesses a novel diaphorase activity. Evidence for distinct regulation of electron flow within NADPH oxidase by p67-phox and p47-phox.","date":"1994","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8063777","citation_count":67,"is_preprint":false},{"pmid":"9624165","id":"PMC_9624165","title":"Cryptic Rac-binding and p21(Cdc42Hs/Rac)-activated kinase phosphorylation sites of NADPH oxidase component p67(phox).","date":"1998","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9624165","citation_count":66,"is_preprint":false},{"pmid":"7982496","id":"PMC_7982496","title":"The functional expression of p47-phox and p67-phox may contribute to the generation of superoxide by an NADPH oxidase-like system in human fibroblasts.","date":"1994","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/7982496","citation_count":65,"is_preprint":false},{"pmid":"24163247","id":"PMC_24163247","title":"Allelic heterogeneity in NCF2 associated with systemic lupus erythematosus (SLE) susceptibility across four ethnic populations.","date":"2013","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/24163247","citation_count":64,"is_preprint":false},{"pmid":"15657040","id":"PMC_15657040","title":"Effects of p47phox C terminus phosphorylations on binding interactions with p40phox and p67phox. Structural and functional comparison of p40phox and p67phox SH3 domains.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15657040","citation_count":64,"is_preprint":false},{"pmid":"8879195","id":"PMC_8879195","title":"Disturbed interaction of p21-rac with mutated p67-phox causes chronic granulomatous disease.","date":"1996","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/8879195","citation_count":64,"is_preprint":false},{"pmid":"11262407","id":"PMC_11262407","title":"The active N-terminal region of p67phox. Structure at 1.8 A resolution and biochemical characterizations of the A128V mutant implicated in chronic granulomatous disease.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11262407","citation_count":62,"is_preprint":false},{"pmid":"11896062","id":"PMC_11896062","title":"A prenylated p67phox-Rac1 chimera elicits NADPH-dependent superoxide production by phagocyte membranes in the absence of an activator and of p47phox: conversion of a pagan NADPH oxidase to monotheism.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11896062","citation_count":60,"is_preprint":false},{"pmid":"8071369","id":"PMC_8071369","title":"Evidence for a readily dissociable complex of p47phox and p67phox in cytosol of unstimulated human neutrophils.","date":"1994","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8071369","citation_count":56,"is_preprint":false},{"pmid":"14623873","id":"PMC_14623873","title":"Continuous translocation of Rac2 and the NADPH oxidase component p67(phox) during phagocytosis.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/14623873","citation_count":56,"is_preprint":false},{"pmid":"18453614","id":"PMC_18453614","title":"Heme oxygenase-1 protects against neutrophil-mediated intestinal damage by down-regulation of neutrophil p47phox and p67phox activity and O2- production in a two-hit model of alcohol intoxication and burn injury.","date":"2008","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/18453614","citation_count":56,"is_preprint":false},{"pmid":"26320741","id":"PMC_26320741","title":"The IRAK-ERK-p67phox-Nox-2 axis mediates TLR4, 2-induced ROS production for IL-1β transcription and processing in monocytes.","date":"2015","source":"Cellular & molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/26320741","citation_count":55,"is_preprint":false},{"pmid":"11724569","id":"PMC_11724569","title":"Activation of the superoxide-generating NADPH oxidase by chimeric proteins consisting of segments of the cytosolic component p67(phox) and the small GTPase Rac1.","date":"2001","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11724569","citation_count":55,"is_preprint":false},{"pmid":"23187810","id":"PMC_23187810","title":"Identification of NCF2/p67phox as a novel p53 target gene.","date":"2012","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/23187810","citation_count":54,"is_preprint":false},{"pmid":"15591124","id":"PMC_15591124","title":"Protein kinase Cdelta regulates p67phox phosphorylation in human monocytes.","date":"2004","source":"Journal of leukocyte biology","url":"https://pubmed.ncbi.nlm.nih.gov/15591124","citation_count":54,"is_preprint":false},{"pmid":"11483597","id":"PMC_11483597","title":"SHP1 protein-tyrosine phosphatase inhibits gp91PHOX and p67PHOX expression by inhibiting interaction of PU.1, IRF1, interferon consensus sequence-binding protein, and CREB-binding protein with homologous Cis elements in the CYBB and NCF2 genes.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11483597","citation_count":53,"is_preprint":false},{"pmid":"8679714","id":"PMC_8679714","title":"Topological organization of the cytosolic activating complex of the superoxide-generating NADPH-oxidase. Pinpointing the sites of interaction between p47phoz, p67phox and p40phox using the two-hybrid system.","date":"1996","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/8679714","citation_count":47,"is_preprint":false},{"pmid":"10614785","id":"PMC_10614785","title":"P40phox associates with the neutrophil Triton X-100-insoluble cytoskeletal fraction and PMA-activated membrane skeleton: a comparative study with P67phox and P47phox.","date":"1999","source":"Journal of leukocyte biology","url":"https://pubmed.ncbi.nlm.nih.gov/10614785","citation_count":46,"is_preprint":false},{"pmid":"31375544","id":"PMC_31375544","title":"Mycobacterium tuberculosis PPE2 Protein Interacts with p67phox and Inhibits Reactive Oxygen Species Production.","date":"2019","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/31375544","citation_count":43,"is_preprint":false},{"pmid":"8948460","id":"PMC_8948460","title":"Mechanisms of NADPH oxidase activation in human neutrophils: p67phox is required for the translocation of rac 1 but not of rac 2 from cytosol to the membranes.","date":"1995","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/8948460","citation_count":43,"is_preprint":false},{"pmid":"16920193","id":"PMC_16920193","title":"Translocation of proteins homologous to human neutrophil p47phox and p67phox to the cell membrane in activated hemocytes of Galleria mellonella.","date":"2006","source":"Developmental and comparative immunology","url":"https://pubmed.ncbi.nlm.nih.gov/16920193","citation_count":41,"is_preprint":false},{"pmid":"16275890","id":"PMC_16275890","title":"Impaired NADPH oxidase activity in Rac2-deficient murine neutrophils does not result from defective translocation of p47phox and p67phox and can be rescued by exogenous arachidonic acid.","date":"2005","source":"Journal of leukocyte biology","url":"https://pubmed.ncbi.nlm.nih.gov/16275890","citation_count":40,"is_preprint":false},{"pmid":"8770870","id":"PMC_8770870","title":"The cytosolic subunit p67phox contains an NADPH-binding site that participates in catalysis by the leukocyte NADPH oxidase.","date":"1996","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/8770870","citation_count":40,"is_preprint":false},{"pmid":"8943869","id":"PMC_8943869","title":"Neutrophils from patients after burn injury express a deficiency of the oxidase components p47-phox and p67-phox.","date":"1996","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/8943869","citation_count":40,"is_preprint":false},{"pmid":"11333262","id":"PMC_11333262","title":"Fused p47phox and p67phox truncations efficiently reconstitute NADPH oxidase with higher activity and stability than the individual components.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11333262","citation_count":39,"is_preprint":false},{"pmid":"1321612","id":"PMC_1321612","title":"Reconstitution and characterization of the human neutrophil respiratory burst oxidase using recombinant p47-phox, p67-phox and plasma membrane.","date":"1992","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/1321612","citation_count":38,"is_preprint":false},{"pmid":"12121978","id":"PMC_12121978","title":"A 29-kDa protein associated with p67phox expresses both peroxiredoxin and phospholipase A2 activity and enhances superoxide anion production by a cell-free system of NADPH oxidase activity.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12121978","citation_count":38,"is_preprint":false},{"pmid":"37612524","id":"PMC_37612524","title":"M6A-mediated-upregulation of lncRNA BLACAT3 promotes bladder cancer angiogenesis and hematogenous metastasis through YBX3 nuclear shuttling and enhancing NCF2 transcription.","date":"2023","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/37612524","citation_count":37,"is_preprint":false},{"pmid":"8675631","id":"PMC_8675631","title":"Inhibitory effect of porcine surfactant on the respiratory burst oxidase in human neutrophils. Attenuation of p47phox and p67phox membrane translocation as the mechanism.","date":"1995","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/8675631","citation_count":37,"is_preprint":false},{"pmid":"10486263","id":"PMC_10486263","title":"Rac1 disrupts p67phox/p40phox binding: a novel role for Rac in NADPH oxidase activation.","date":"1999","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/10486263","citation_count":36,"is_preprint":false},{"pmid":"17138561","id":"PMC_17138561","title":"Activation of SHP2 protein-tyrosine phosphatase increases HoxA10-induced repression of the genes encoding gp91(PHOX) and p67(PHOX).","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17138561","citation_count":36,"is_preprint":false},{"pmid":"10598813","id":"PMC_10598813","title":"Autosomal recessive chronic granulomatous disease caused by novel mutations in NCF-2, the gene encoding the p67-phox component of phagocyte NADPH oxidase.","date":"1999","source":"Human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/10598813","citation_count":36,"is_preprint":false},{"pmid":"9425254","id":"PMC_9425254","title":"Roles for proline-rich regions of p47phox and p67phox in the phagocyte NADPH oxidase activation in vitro.","date":"1997","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/9425254","citation_count":35,"is_preprint":false},{"pmid":"11705402","id":"PMC_11705402","title":"A fusion protein between rac and p67phox (1-210) reconstitutes NADPH oxidase with higher activity and stability than the individual components.","date":"2001","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11705402","citation_count":35,"is_preprint":false},{"pmid":"9057660","id":"PMC_9057660","title":"Genetic correction of p67phox deficient chronic granulomatous disease using peripheral blood progenitor cells as a target for retrovirus mediated gene transfer.","date":"1997","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/9057660","citation_count":34,"is_preprint":false},{"pmid":"7903171","id":"PMC_7903171","title":"Characterization of the p67phox gene: genomic organization and restriction fragment length polymorphism analysis for prenatal diagnosis in chronic granulomatous disease.","date":"1993","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/7903171","citation_count":33,"is_preprint":false},{"pmid":"20529851","id":"PMC_20529851","title":"A prenylated p47phox-p67phox-Rac1 chimera is a Quintessential NADPH oxidase activator: membrane association and functional capacity.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20529851","citation_count":33,"is_preprint":false},{"pmid":"11752058","id":"PMC_11752058","title":"Modulation of microglial superoxide production by alpha-tocopherol in vitro: attenuation of p67(phox) translocation by a protein phosphatase-dependent pathway.","date":"2001","source":"Journal of neurochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11752058","citation_count":33,"is_preprint":false},{"pmid":"9931304","id":"PMC_9931304","title":"The major phosphorylation site of the NADPH oxidase component p67phox is Thr233.","date":"1999","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/9931304","citation_count":32,"is_preprint":false},{"pmid":"18523285","id":"PMC_18523285","title":"Platelet-activating factor-mediated endosome formation causes membrane translocation of p67phox and p40phox that requires recruitment and activation of p38 MAPK, Rab5a, and phosphatidylinositol 3-kinase in human neutrophils.","date":"2008","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/18523285","citation_count":32,"is_preprint":false},{"pmid":"27279484","id":"PMC_27279484","title":"Role of Nox4 and p67phox subunit of Nox2 in ROS production in response to increased tubular flow in the mTAL of Dahl salt-sensitive rats.","date":"2016","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/27279484","citation_count":30,"is_preprint":false},{"pmid":"25489057","id":"PMC_25489057","title":"Null mutation of the nicotinamide adenine dinucleotide phosphate-oxidase subunit p67phox protects the Dahl-S rat from salt-induced reductions in medullary blood flow and glomerular filtration rate.","date":"2014","source":"Hypertension (Dallas, Tex. : 1979)","url":"https://pubmed.ncbi.nlm.nih.gov/25489057","citation_count":29,"is_preprint":false},{"pmid":"16210632","id":"PMC_16210632","title":"HoxA10 represses transcription of the gene encoding p67phox in phagocytic cells.","date":"2005","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/16210632","citation_count":29,"is_preprint":false},{"pmid":"17548354","id":"PMC_17548354","title":"Tripartite chimeras comprising functional domains derived from the cytosolic NADPH oxidase components p47phox, p67phox, and Rac1 elicit activator-independent superoxide production by phagocyte membranes: an essential role for anionic membrane phospholipids.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17548354","citation_count":29,"is_preprint":false},{"pmid":"21784060","id":"PMC_21784060","title":"The NADPH oxidase cytosolic component p67phox is constitutively phosphorylated in human neutrophils: Regulation by a protein tyrosine kinase, MEK1/2 and phosphatases 1/2A.","date":"2011","source":"Biochemical pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/21784060","citation_count":29,"is_preprint":false},{"pmid":"15814300","id":"PMC_15814300","title":"Expression of NOX-I, gp91phox, p47phox and P67phox in the aorta segments above and below coarctation.","date":"2005","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/15814300","citation_count":28,"is_preprint":false},{"pmid":"7803798","id":"PMC_7803798","title":"A mutation located at the 5' splice junction sequence of intron 3 in the p67phox gene causes the lack of p67phox mRNA in a patient with chronic granulomatous disease.","date":"1995","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/7803798","citation_count":27,"is_preprint":false},{"pmid":"11483614","id":"PMC_11483614","title":"Transcriptional regulation of the p67phox gene: role of AP-1 in concert with myeloid-specific transcription factors.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11483614","citation_count":26,"is_preprint":false},{"pmid":"23821607","id":"PMC_23821607","title":"Evolutionary dynamics of the human NADPH oxidase genes CYBB, CYBA, NCF2, and NCF4: functional implications.","date":"2013","source":"Molecular biology and evolution","url":"https://pubmed.ncbi.nlm.nih.gov/23821607","citation_count":26,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":53800,"output_tokens":8533,"usd":0.144698},"stage2":{"model":"claude-opus-4-6","input_tokens":12498,"output_tokens":4662,"usd":0.26856},"total_usd":0.413258,"stage1_batch_id":"msgbatch_012ZiiCUBfB62NCTQd4Bk1Jo","stage2_batch_id":"msgbatch_01Xuqq9aUHoQouS7UVGZNStZ","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1992,\n      \"finding\": \"p67phox (NCF2 gene product) is one of four components required to fully reconstitute NADPH oxidase activity in a cell-free system; no activity is observed in its absence, establishing it as an essential cytosolic activator of flavocytochrome b-245.\",\n      \"method\": \"Cell-free reconstitution with purified recombinant p67phox, p47phox, p21rac1, and cytochrome b-245\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with purified components, highly cited foundational study\",\n      \"pmids\": [\"1512217\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Rac1 (GTP-bound form) interacts directly with p67phox in a GTP-dependent manner; effector-site mutations in Rac that abolish NADPH oxidase activation also abolish binding to p67phox, identifying p67phox as the Rac effector in the NADPH oxidase complex.\",\n      \"method\": \"Co-immunoprecipitation, GTP-dependence assay, Rac effector-site mutagenesis, cell-free oxidase activity\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct binding with mutagenesis, replicated in multiple subsequent studies\",\n      \"pmids\": [\"8036496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"p67phox and p47phox translocate from cytosol to the plasma membrane/submembranous actin cytoskeleton upon neutrophil activation; translocation of p67phox and Rac2 to the cytoskeleton requires p47phox, but Rac2 translocation to the membrane can occur independently of p67phox.\",\n      \"method\": \"Subcellular fractionation of activated normal and CGD (p47phox-deficient) neutrophils, immunoblotting\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function in patient cells with defined phenotype, replicated across multiple labs\",\n      \"pmids\": [\"8120032\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Rac2 translocates to membranes independently of p47phox and p67phox but depends on flavocytochrome b558 for maximal membrane stability; Rac2 membrane binding is reduced ~75% in CGD neutrophils lacking gp91phox.\",\n      \"method\": \"Cell-free translocation assay with normal and CGD neutrophil membranes/cytosols; quantitative immunoblotting of Rac1 and Rac2 in intact stimulated CGD neutrophils\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple CGD genotypes tested, cell-free and intact-cell systems\",\n      \"pmids\": [\"7982999\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Deletion of either SH3 domain of p67phox dramatically reduces NADPH oxidase activity and membrane binding in intact B-cells; deletion of both SH3 domains completely abolishes membrane binding and oxidase activity, demonstrating that the SH3 domains are required for translocation and activity in whole cells but not in the cell-free system.\",\n      \"method\": \"Stable transfection of p67phox-deficient CGD B-cells with SH3 deletion mutants; whole-cell and cell-free oxidase assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis in reconstituted intact cells with multiple deletion variants\",\n      \"pmids\": [\"8206939\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"p40phox forms a tight 1:1 complex with p67phox in neutrophil cytosol; p40phox is absent in CGD patients lacking p67phox, indicating that its stability depends on p67phox.\",\n      \"method\": \"Co-immunoprecipitation and column chromatography of neutrophil cytosol; gel analysis of CGD patient samples\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2–3 — co-IP in native neutrophil cytosol, confirmed with patient cells, replicated by multiple groups\",\n      \"pmids\": [\"8147882\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"p67phox (not p47phox) is required for electron flow from NADPH to the flavin (FAD) center of the oxidase; p47phox is additionally required for electron transfer beyond the flavin to the heme in cytochrome b-245 and thence to oxygen.\",\n      \"method\": \"Cell-free oxidase reconstitution with individually depleted cytosol fractions from CGD patients; spectrophotometric electron-flow assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro mechanistic dissection using defined components and patient-derived material\",\n      \"pmids\": [\"7896790\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"p40phox interacts with p67phox via the C-terminal SH3 domain of p40phox binding to p47phox, and via the C-terminal portion of p40phox binding to the region between the two SH3 domains of p67phox; p40phox also binds p47phox but more weakly.\",\n      \"method\": \"Affinity-bead pulldown, surface plasmon resonance (Biacore) with truncation constructs\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal binding methods with domain mapping\",\n      \"pmids\": [\"8760383\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Two-hybrid and in vitro binding studies show p67phox interacts with p47phox via the C-terminal SH3 domain of p67phox binding to the polyproline motif of p47phox; p40phox interacts with p67phox via the region between p67phox's two SH3 domains, not via SH3-polyproline interactions.\",\n      \"method\": \"Yeast two-hybrid system; in vitro binding with domain constructs\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — two orthogonal methods, domain-level resolution\",\n      \"pmids\": [\"7890694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"p67phox interacts with Rac2 approximately 6-fold more strongly than with Rac1 in yeast two-hybrid assays; Rac effector-site mutants inactive in NADPH oxidase lose interaction with p67phox, and an activating Rac2 mutant shows increased affinity for p67phox.\",\n      \"method\": \"Yeast two-hybrid system; cell-free oxidase activity\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — two-hybrid with multiple mutants, consistent with biochemical data from other labs\",\n      \"pmids\": [\"8550629\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"p67phox contains an NADPH-binding site (identified by NADPH dialdehyde crosslinking and competition with NADPH); this site participates in catalysis by the leukocyte NADPH oxidase.\",\n      \"method\": \"NADPH dialdehyde affinity labeling of cytosol and purified recombinant p67phox; cell-free oxidase reconstitution rescue assay\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — chemical crosslinking combined with reconstitution activity assay\",\n      \"pmids\": [\"8770870\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"p67phox mediates translocation of p40phox and Rac1 from cytosol to membrane upon neutrophil stimulation; in p67phox-deficient CGD neutrophils, p40phox and Rac1 fail to translocate, whereas Rac2 and p47phox translocate normally.\",\n      \"method\": \"Subcellular fractionation and immunoblotting of stimulated normal and CGD (p67phox-deficient and p47phox-deficient) patient neutrophils\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function patient cells with multiple component tracking\",\n      \"pmids\": [\"8670049\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"A deletion of Lys58 in p67phox found in a CGD patient disrupts the interaction between p67phox and p21-Rac1, prevents translocation of p47phox and p67phox to the plasma membrane upon stimulation, and abolishes superoxide production — demonstrating that p67phox–Rac1 interaction is essential for oxidase complex assembly.\",\n      \"method\": \"CGD patient genetic analysis; co-immunoprecipitation of p67phox-Rac1; membrane translocation assay in patient neutrophils\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — naturally occurring disease mutation linked directly to disrupted protein–protein interaction and functional loss\",\n      \"pmids\": [\"8879195\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"p67phox is phosphorylated on serine residues upon neutrophil activation by fMLP and PMA; PKC phosphorylates p67phox at the same tryptic peptide site in vitro and in intact cells via a PKC-dependent pathway; an additional PKC-independent pathway also phosphorylates p67phox.\",\n      \"method\": \"32P-labeling of intact neutrophils; phosphoamino acid analysis; 2D tryptic peptide mapping; in vitro kinase assay with PKC and GST-p67phox\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro kinase assay plus intact-cell phosphopeptide mapping with inhibitors\",\n      \"pmids\": [\"9202043\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Rac1 effector region (residues 26–45) mutations increase Kd for p67phox binding, showing this region mediates direct Rac1–p67phox interaction; insert region mutations (residues 124–135) elevate EC50 for oxidase activity without affecting p67phox binding, suggesting the insert region interacts with another component (possibly cytochrome b558).\",\n      \"method\": \"Fluorescence (MANT-GTP) direct binding assay; steady-state kinetic NADPH oxidase reconstitution; site-directed Rac1 mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct binding measurements plus kinetic reconstitution with multiple mutants\",\n      \"pmids\": [\"9228059\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"An activation domain in p67phox (residues ~199–210, especially Val204) is essential for NADPH oxidase activity independently of p47phox or Rac binding; V204A mutation completely abolishes activity and acts as a dominant-negative by competing with wild-type p67phox for membrane translocation.\",\n      \"method\": \"C-terminal truncation series; single amino acid mutagenesis; cell-free oxidase reconstitution; dominant-negative competition assay; membrane translocation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — systematic mutagenesis with reconstitution and dominant-negative validation\",\n      \"pmids\": [\"9642219\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"C-terminal truncations of both p47phox and p67phox relieve autoinhibition, allowing amphiphile-independent NADPH oxidase activation in vitro; the C-terminal regions of both proteins function as negative regulatory elements, and the N-terminal SH3 domain of p47phox is normally masked by intramolecular interaction with its own C-terminus.\",\n      \"method\": \"Cell-free oxidase activation with truncated recombinant proteins; p22phox binding assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with multiple truncation mutants\",\n      \"pmids\": [\"9461621\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"The C-terminal SH3 domain of p67phox and the polyproline motif of p40phox constitute a PC motif-based interaction interface; mutagenesis of the PC motif in p40phox abolishes binding to p67phox.\",\n      \"method\": \"Yeast two-hybrid screening; in vitro binding with recombinant proteins; site-directed mutagenesis of PC motif\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — two orthogonal methods with mutagenesis\",\n      \"pmids\": [\"9490029\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Rac1 binds to p67phox at residues 170–199 (N-terminal region); deletion of the C-terminal SH3 domain or polyproline motif of p67phox increases Rac1 binding ~8-fold, revealing a cryptic Rac-binding site; PAK kinase phosphorylates p67phox adjacent to the Rac-binding site, and this phosphorylation is enhanced by C-terminal SH3 or polyproline deletion.\",\n      \"method\": \"Pulldown binding assays with p67phox truncations/deletions; in vitro PAK kinase assay; NADPH oxidase inhibition assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — binding assays with multiple deletions, single lab\",\n      \"pmids\": [\"9624165\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The major phosphorylation site of p67phox is Thr233; mutagenesis of Thr233 to Ala abolishes in vitro phosphorylation; this site is phosphorylated by MAP kinase and cytosol in vitro and in stimulated neutrophils.\",\n      \"method\": \"Phosphopeptide mapping by cyanogen bromide digestion and HPLC-MS; Thr233Ala mutagenesis; in vitro kinase assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mass spectrometry site identification combined with mutagenesis confirmation\",\n      \"pmids\": [\"9931304\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Crystal structure of the N-terminal TPR domain of p67phox bound to Rac·GTP at 2.7 Å reveals a novel Rho effector interaction mode in which an insertion between TPR motifs mediates complex formation and GTPase specificity.\",\n      \"method\": \"X-ray crystallography of Rac·GTP–p67phox TPR domain complex\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure at atomic resolution\",\n      \"pmids\": [\"11090627\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Crystal structure of the active N-terminal fragment of p67phox (residues 1–213) at 1.8 Å resolution reveals four TPR motifs with the C-terminus folding back into a hydrophobic groove; an additional short C-terminal helix (residues 187–193) likely constitutes part of the activation domain; CGD mutations G78E and A128V map to this region, with A128V exhibiting temperature-sensitive activity.\",\n      \"method\": \"X-ray crystallography; temperature-shift cell-free oxidase reconstitution with CGD mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure combined with functional mutagenesis and disease variant characterization\",\n      \"pmids\": [\"11262407\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"In the resting cytosolic complex, p67phox acts as the central bridging molecule that connects p40phox and p47phox; the complex contains one copy of each protein forming a 1:1:1 heterotrimer with an extended non-globular shape; isothermal titration calorimetry shows p67phox is the primary binding partner of p47phox in the resting state.\",\n      \"method\": \"Gel filtration; isothermal titration calorimetry (ITC); analytical ultracentrifugation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple biophysical methods including ITC for thermodynamic characterization\",\n      \"pmids\": [\"11796733\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"A prenylated chimera of p67phox(1–212) fused to full-length Rac1, when loaded with GTP, activates NADPH oxidase in the absence of p47phox and amphiphile by spontaneously associating with membranes, demonstrating that Rac serves as a membrane-targeting carrier for p67phox and that intrachimeric p67phox–Rac interaction induces a conformational change in the p67phox activation domain.\",\n      \"method\": \"Cell-free NADPH oxidase reconstitution with prenylated chimeric proteins; membrane association assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted system with engineered chimeras plus prenylation, mechanistically dissects Rac function\",\n      \"pmids\": [\"11896062\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"ERK2 phosphorylates p67phox primarily in its N-terminal fragment (residues 1–243), while p38MAPK phosphorylates sites in the C-terminal fragment (residues 244–526); a C-terminal phosphorylation site is cryptic in the intact protein due to an intramolecular interaction with the N-terminal TPR-rich region; both kinases phosphorylate p67phox in fMLP- and PMA-stimulated neutrophils.\",\n      \"method\": \"In vitro kinase assays with ERK2 and p38MAPK; phosphopeptide mapping; truncation constructs; MEK1/2 and p38MAPK inhibitors in intact neutrophils\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro kinase assays with domain mapping confirmed in intact cells with inhibitors\",\n      \"pmids\": [\"12693948\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Rac has a dual role in NADPH oxidase assembly: (1) tethering p67phox to the membrane via Rac's C-terminal basic residues, and (2) inducing an activating conformational change in p67phox (via intrachimeric p67phox–Rac interaction); inactive Rac moiety chimeras can be rescued by exogenous Rac1-GTP but not by exogenous p67phox, demonstrating the two functions are separable.\",\n      \"method\": \"Cell-free NADPH oxidase reconstitution with p67phox(1–212)–Rac1 chimeras bearing site-directed mutations in activation domain, Rac effector region, and C-terminal basic residues\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — systematic chimeric mutagenesis with reconstitution, complementation tests\",\n      \"pmids\": [\"14761978\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"PKCdelta forms a complex with p67phox in activated monocytes and directly phosphorylates p67phox in vitro; rottlerin (PKCdelta inhibitor) and PKCdelta antisense both block p67phox phosphorylation and reduce superoxide production, identifying PKCdelta as a regulator of p67phox in human monocytes.\",\n      \"method\": \"Co-immunoprecipitation of PKCdelta–p67phox; in vitro kinase assay with recombinant PKCdelta; PKCdelta antisense knockdown; selective inhibitor studies; superoxide assay\",\n      \"journal\": \"Journal of leukocyte biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — co-IP, in vitro kinase assay, KD with defined phenotype\",\n      \"pmids\": [\"15591124\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"S100A8 (but not S100A9) interacts with p67phox and Rac in vitro; S100A8/A9 promotes NADPH oxidase activation by transferring arachidonic acid to the complex; S100A9 knockdown in NB4 cells and S100A9-/- mouse neutrophils show impaired oxidase activity, confirming functional relevance.\",\n      \"method\": \"Protein-protein interaction studies (pulldown); cell-free oxidase assay with S100A8/A9 mutant unable to bind arachidonic acid; S100A9 antibody neutralization; S100A9-/- mouse neutrophils; S100A9 siRNA in NB4 cells\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including KO mice and in vitro functional assays\",\n      \"pmids\": [\"15642721\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"p67phox depends on p40phox (via PX domain-phosphatidylinositol 3-phosphate interaction) or p47phox (via PX domain-plasma membrane interaction) for membrane targeting; GFP-p67phox expressed alone does not translocate to membranes; mutations disrupting p67phox binding to p40phox or p47phox abolish p67phox translocation to their respective membrane compartments.\",\n      \"method\": \"Live-cell GFP imaging of translocation; mutations in PX and PB1 domains; co-expression in COS-7 cells\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — live imaging with mutagenesis in intact cells\",\n      \"pmids\": [\"17122360\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PKC phosphorylates gp91phox/NOX2 in stimulated neutrophils; this phosphorylation enhances diaphorase activity of the NOX2 flavoprotein domain and increases its binding to Rac2, p67phox, and p47phox, indicating a novel mechanism of NADPH oxidase regulation through phosphorylation-enhanced assembly.\",\n      \"method\": \"32P-labeling of intact neutrophils; in vitro PKC kinase assay on recombinant NOX2 domain; 2D tryptic peptide mapping; co-IP pulldown after phosphorylation; diaphorase activity assay\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro kinase assay with binding and activity readouts confirmed in intact cells\",\n      \"pmids\": [\"19028840\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"SAXS analysis shows p67phox in solution is a very elongated multidomain protein with semi-flexible linkers, adopting a beads-on-a-string arrangement with evidence of intramolecular interactions that constrain the overall conformation; this architecture underlies its ability to interact simultaneously with all other NADPH oxidase components.\",\n      \"method\": \"Small-angle X-ray scattering (SAXS) of full-length p67phox\",\n      \"journal\": \"Journal of structural biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structural method with quantitative solution-state data\",\n      \"pmids\": [\"19723583\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"A naturally occurring NCF2 missense mutation H389Q in the PB1 domain reduces binding efficiency to the Vav1 guanine nucleotide exchange factor and causes a ~2-fold decrease in ROS production stimulated via FcγR-elicited (Vav-dependent) pathway, linking this SLE-risk variant to a specific loss of Vav1-dependent oxidase activation.\",\n      \"method\": \"Computational modeling of NCF2–Vav1 interaction; site-directed mutagenesis at position 389; FcγR-stimulated ROS production assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis with functional ROS assay, supported by structural modeling\",\n      \"pmids\": [\"22203994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"A rare NCF2 missense variant found in patients with very early onset IBD reduces binding of p67phox to RAC2, providing a direct mechanistic link between partial oxidase impairment and intestinal inflammation.\",\n      \"method\": \"Direct sequencing; functional binding assay of variant p67phox–RAC2 interaction\",\n      \"journal\": \"Gut\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — binding assay for variant, single lab, limited mechanistic depth\",\n      \"pmids\": [\"21900546\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"NCF2/p67phox is a direct transcriptional target of p53; p53 binds to the NCF2 promoter in vivo; NCF2 knockdown by siRNA reduces ROS production and stimulates cell death, indicating p53-driven NCF2 expression generates ROS that promote cell survival against apoptosis.\",\n      \"method\": \"Microarray identification; luciferase reporter assay; chromatin immunoprecipitation (ChIP); siRNA knockdown with ROS and apoptosis readouts\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — ChIP plus reporter assay plus siRNA KD with functional phenotype\",\n      \"pmids\": [\"23187810\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"IFN-γ-induced transcription of NCF2 (p67phox) requires cooperative recruitment of PU.1, IRF1, and ICSBP to a specific NCF2 cis element; this transcription factor complex then recruits the coactivator CBP (CREB-binding protein) to activate NCF2 transcription.\",\n      \"method\": \"Promoter deletion and mutation analysis; gel shift assays; reporter gene assays; co-IP of transcription factor complex with CBP\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — promoter mapping with mutagenesis and co-IP of activating complex\",\n      \"pmids\": [\"10570299\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TLR4/TLR2 signaling activates an IRAK–ERK pathway that drives ERK–p67phox interaction, p67phox translocation to membrane, and p67phox–Nox2 interaction, resulting in ROS generation that mediates IL-1β transcription and processing; disruption at any node (IRAK, ERK, p67phox, or Nox2 siRNA) blocks IL-1β production.\",\n      \"method\": \"Co-IP of ERK–p67phox and p67phox–Nox2 complexes; siRNA knockdown of Nox2; pharmacological inhibitors; membrane translocation assay; IL-1β reporter and ELISA\",\n      \"journal\": \"Cellular & molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — multiple co-IPs and siRNA data but pathway assigned primarily in a cell line\",\n      \"pmids\": [\"26320741\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"M. tuberculosis PPE2 protein directly interacts with p67phox via its Trp236 residue; this interaction prevents p67phox and p47phox translocation to the membrane and reduces NADPH oxidase activity; Trp236Ala PPE2 mutant loses both p67phox binding and inhibition of ROS production.\",\n      \"method\": \"Co-IP of PPE2–p67phox; site-directed mutagenesis (Trp236Ala); membrane translocation assay; ROS quantification in macrophages; bacterial survival assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus mutagenesis plus translocation and activity phenotype\",\n      \"pmids\": [\"31375544\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"p67phox translocation to forming phagosomes is deficient in neutrophils lacking p47phox, demonstrating that p47phox is required for stable phagosomal recruitment of p67phox; in X-linked CGD neutrophils lacking flavocytochrome b558, both p47phox and p67phox accumulate transiently but are shed from nascent phagosomes once particles are internalized, showing flavocytochrome b558 is required for stable retention of p67phox at the phagosome.\",\n      \"method\": \"Synchronized phagocytosis assay with immunofluorescence microscopy; immunoblotting of phagosome fractions from CGD patient neutrophils\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct imaging with defined genetic loss-of-function cells\",\n      \"pmids\": [\"14623873\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NCF2-encoded p67phox is an essential cytosolic activator of the phagocyte NADPH oxidase that serves as the central scaffold of the resting p40phox–p47phox–p67phox heterotrimer; upon cell stimulation it is phosphorylated on multiple serine/threonine sites by PKC, ERK2, p38MAPK, and PKCδ, then translocated to the plasma membrane/phagosome via p47phox (plasma membrane) or p40phox (early endosomes) as carrier proteins, where its N-terminal TPR domain binds GTP-loaded Rac2 (Kd ~60 nM) to induce a conformational change in its activation domain (Val204-containing region) that directly stimulates electron transfer from NADPH to the FAD center of flavocytochrome b558, while its two SH3 domains contribute to stable membrane association and its PB1 domain mediates interaction with Vav1.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"NCF2 encodes p67phox, an essential cytosolic activator of the phagocyte NADPH oxidase (NOX2) that functions as the central scaffold of the resting p40phox–p47phox–p67phox heterotrimer and as the direct catalytic activator of electron transfer from NADPH to the FAD center of flavocytochrome b558 [PMID:1512217, PMID:7896790]. Its N-terminal TPR domain binds GTP-loaded Rac2 (preferentially over Rac1), inducing a conformational change in the activation domain (centered on Val204) that stimulates oxidase catalysis, while its C-terminal SH3 domain anchors the trimer via interaction with p47phox and its PB1 domain mediates interaction with p40phox and the guanine nucleotide exchange factor Vav1 [PMID:11090627, PMID:9642219, PMID:7890694, PMID:22203994]. p67phox lacks intrinsic membrane-targeting capacity and depends on p47phox for plasma-membrane/phagosomal recruitment and on p40phox for endosomal recruitment; upon stimulation, it is phosphorylated by PKC, ERK2, p38 MAPK, and PKCδ on multiple serine/threonine residues including Thr233, modulating oxidase assembly and activity [PMID:17122360, PMID:9202043, PMID:12693948, PMID:15591124, PMID:9931304]. Loss-of-function mutations in NCF2 cause autosomal recessive chronic granulomatous disease (CGD), and coding variants that impair Rac2 or Vav1 binding are linked to very early onset inflammatory bowel disease and systemic lupus erythematosus susceptibility [PMID:8879195, PMID:21900546, PMID:22203994].\",\n  \"teleology\": [\n    {\n      \"year\": 1992,\n      \"claim\": \"Establishing that p67phox is an indispensable component for reconstituting NADPH oxidase activity resolved the question of whether all four cytosolic/membrane factors are individually required, positioning p67phox as a direct catalytic activator rather than an accessory factor.\",\n      \"evidence\": \"Cell-free reconstitution with purified recombinant p67phox, p47phox, Rac1, and cytochrome b-245\",\n      \"pmids\": [\"1512217\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of p67phox activation of electron transfer unknown\", \"Which domain(s) of p67phox are catalytically essential not yet defined\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Identification of p67phox as the direct effector of Rac GTPase answered which oxidase subunit reads the Rac activation signal, establishing the Rac–p67phox axis as the trigger for oxidase assembly.\",\n      \"evidence\": \"Co-immunoprecipitation with GTP-dependence and Rac effector-site mutagenesis; two-hybrid confirmation with Rac2 preference\",\n      \"pmids\": [\"8036496\", \"8550629\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Rac–p67phox recognition unknown\", \"Whether Rac2 selectivity matters in vivo not tested\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Demonstrating that p67phox translocation to membranes requires p47phox, while p67phox itself carries p40phox and Rac1 to the membrane, defined a hierarchical assembly pathway in which p67phox acts as the central scaffold bridging upstream carriers and downstream effectors.\",\n      \"evidence\": \"Subcellular fractionation of stimulated normal and CGD (p47phox-deficient and p67phox-deficient) neutrophils\",\n      \"pmids\": [\"8120032\", \"8670049\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether p40phox provides an alternative membrane-targeting route not yet explored\", \"Phagosomal versus plasma membrane targeting not distinguished\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Mapping the domain architecture of the p67phox-centered trimer — SH3 domains required for membrane association and activity, C-terminal SH3 binding p47phox, inter-SH3 region binding p40phox — established p67phox as the central bridging molecule of the resting cytosolic complex.\",\n      \"evidence\": \"SH3 deletion mutagenesis in reconstituted CGD B-cells; two-hybrid and in vitro binding with domain constructs; co-IP of native p40phox–p67phox complex\",\n      \"pmids\": [\"8206939\", \"7890694\", \"8147882\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and shape of native complex not determined\", \"Whether SH3 domains contact membrane targets directly unknown\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Showing that p67phox is specifically required for NADPH→FAD electron transfer while p47phox is additionally needed for FAD→heme transfer dissected the catalytic roles of individual cytosolic subunits for the first time.\",\n      \"evidence\": \"Cell-free oxidase reconstitution with CGD patient cytosol selectively depleted of each subunit; spectrophotometric electron-flow assays\",\n      \"pmids\": [\"7896790\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether p67phox directly contacts the FAD-binding site of gp91phox unknown\", \"Structural basis for activation domain function not defined\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Discovery of an NADPH-binding site on p67phox itself suggested a substrate-channeling or regulatory role beyond simple protein–protein scaffolding, adding catalytic complexity to the activation mechanism.\",\n      \"evidence\": \"NADPH dialdehyde affinity labeling of purified recombinant p67phox; competition with NADPH; cell-free rescue assay\",\n      \"pmids\": [\"8770870\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological significance of p67phox NADPH binding versus gp91phox NADPH binding not resolved\", \"No structural data for NADPH-binding site\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"A CGD patient mutation (ΔLys58) that disrupts p67phox–Rac1 interaction and abolishes superoxide production provided the first human genetic proof that the Rac–p67phox interaction is essential for oxidase function in vivo.\",\n      \"evidence\": \"Patient genetic analysis; co-IP of mutant p67phox with Rac1; membrane translocation and oxidase activity in patient neutrophils\",\n      \"pmids\": [\"8879195\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full spectrum of CGD-causing NCF2 mutations not catalogued\", \"Whether partial loss of Rac binding causes intermediate phenotypes unknown\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Identification of PKC-dependent and PKC-independent phosphorylation of p67phox on serine residues upon neutrophil activation opened the question of how post-translational modifications regulate oxidase assembly dynamics.\",\n      \"evidence\": \"32P-labeling of intact neutrophils; 2D tryptic peptide mapping; in vitro PKC kinase assay\",\n      \"pmids\": [\"9202043\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific phosphorylation sites not yet mapped\", \"Functional consequences of phosphorylation on oxidase activity not tested\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Identification of an activation domain (residues ~199–210, Val204 critical) that is essential for catalysis independently of Rac or p47phox binding defined the minimal catalytic surface on p67phox and provided a dominant-negative tool.\",\n      \"evidence\": \"Systematic truncation and single amino acid mutagenesis; cell-free oxidase reconstitution; dominant-negative competition assay\",\n      \"pmids\": [\"9642219\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Val204 region contacts flavocytochrome b558 structurally unknown\", \"Whether activation domain undergoes conformational change upon Rac binding not shown\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Mapping Thr233 as the major phosphorylation site and identifying MAP kinase as the responsible kinase provided a specific molecular handle for studying phospho-regulation of p67phox.\",\n      \"evidence\": \"Phosphopeptide mapping by CNBr digestion/HPLC-MS; Thr233Ala mutagenesis; in vitro kinase assay\",\n      \"pmids\": [\"9931304\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of Thr233 phosphorylation on oxidase activation not determined\", \"Additional phosphorylation sites not yet mapped\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"The crystal structure of the p67phox TPR domain–Rac·GTP complex at 2.7 Å revealed a novel Rho effector recognition mode mediated by an insertion between TPR motifs, explaining GTPase specificity and providing a structural basis for CGD mutations in this region.\",\n      \"evidence\": \"X-ray crystallography of Rac·GTP–p67phox(1–203) complex\",\n      \"pmids\": [\"11090627\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length p67phox structure not available\", \"How Rac binding transmits conformational change to the activation domain structurally unresolved\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"The 1.8 Å structure of the active N-terminal fragment revealed the four-TPR fold, mapped CGD mutations G78E and A128V to the hydrophobic core, and identified a C-terminal helix (187–193) as part of the activation domain, connecting structure to disease-causing loss of function.\",\n      \"evidence\": \"X-ray crystallography; temperature-shift cell-free oxidase assay with CGD mutants\",\n      \"pmids\": [\"11262407\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of activation domain in contact with flavocytochrome b558 not determined\", \"No structure of full-length p67phox\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Biophysical characterization of the 1:1:1 p40phox–p67phox–p47phox heterotrimer with p67phox as the central bridge, combined with chimeric Rac–p67phox demonstrating Rac's dual role as membrane anchor and conformational activator, provided an integrated model of oxidase assembly.\",\n      \"evidence\": \"ITC, gel filtration, analytical ultracentrifugation for trimer; cell-free reconstitution with prenylated p67phox–Rac1 chimeras\",\n      \"pmids\": [\"11796733\", \"11896062\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of complete ternary cytosolic complex not solved\", \"Kinetics of assembly in intact cells not measured\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Demonstration that ERK2 phosphorylates the N-terminal region and p38MAPK the C-terminal region of p67phox, with a cryptic C-terminal site unmasked by domain rearrangement, revealed compartmentalized phospho-regulation linked to intramolecular conformational control.\",\n      \"evidence\": \"In vitro kinase assays with truncation constructs; MEK1/2 and p38MAPK inhibitors in intact neutrophils\",\n      \"pmids\": [\"12693948\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of C-terminal phosphorylation sites not mapped\", \"Whether phosphorylation at these sites regulates oxidase activity directly not tested\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Showing that p47phox is required for stable phagosomal recruitment of p67phox and that flavocytochrome b558 is needed for retention of both subunits on nascent phagosomes extended the assembly hierarchy to the phagosome compartment.\",\n      \"evidence\": \"Synchronized phagocytosis with immunofluorescence in CGD patient neutrophils\",\n      \"pmids\": [\"14623873\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Role of p40phox in phagosomal targeting of p67phox not assessed here\", \"Whether retention mechanism involves direct p67phox–cytochrome b558 interaction unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identification of PKCδ as a direct kinase for p67phox in monocytes, with functional consequences for superoxide production, expanded the kinase network regulating p67phox beyond conventional PKC and MAPKs.\",\n      \"evidence\": \"Co-IP of PKCδ–p67phox; in vitro kinase assay; PKCδ antisense knockdown; rottlerin inhibitor; superoxide assay\",\n      \"pmids\": [\"15591124\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"PKCδ phosphorylation sites on p67phox not identified\", \"Whether PKCδ regulates p67phox translocation or catalytic activation not distinguished\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Live-cell imaging established that p67phox has no intrinsic membrane-targeting capacity and reaches the plasma membrane via p47phox and endosomes via p40phox, resolving the question of how p67phox accesses distinct membrane compartments.\",\n      \"evidence\": \"GFP-p67phox live imaging with PX and PB1 domain mutations in COS-7 cells\",\n      \"pmids\": [\"17122360\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether p40phox-mediated endosomal route is essential for antimicrobial ROS not tested in phagocytes\", \"Contribution of Rac-mediated membrane tethering in intact cells not assessed\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Linking the NCF2 PB1-domain variant H389Q to reduced Vav1 binding and impaired FcγR-stimulated ROS production connected a lupus-risk allele to a specific defect in the Vav1-dependent arm of oxidase activation.\",\n      \"evidence\": \"Site-directed mutagenesis at position 389; computational modeling; FcγR-stimulated ROS assay\",\n      \"pmids\": [\"22203994\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance in patient cells not demonstrated\", \"Whether other PB1-domain partners are affected not tested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Demonstrating that p53 directly activates NCF2 transcription revealed a link between tumor-suppressor signaling and oxidase-derived ROS, with NCF2 knockdown reducing ROS and enhancing apoptosis.\",\n      \"evidence\": \"ChIP of p53 on NCF2 promoter; luciferase reporter; siRNA knockdown with ROS and apoptosis readouts\",\n      \"pmids\": [\"23187810\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether p53-driven NCF2 expression is relevant in phagocytes or primarily in tumor cells not clarified\", \"Downstream ROS targets mediating survival not identified\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identification of M. tuberculosis PPE2 as a direct p67phox-binding virulence factor that blocks oxidase assembly exemplified pathogen subversion of the Rac–p67phox activation axis.\",\n      \"evidence\": \"Co-IP of PPE2–p67phox; Trp236Ala mutagenesis abolishing binding; membrane translocation and ROS assays in macrophages\",\n      \"pmids\": [\"31375544\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which domain of p67phox is targeted by PPE2 not mapped\", \"In vivo significance during TB infection not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A full-length structure of p67phox in complex with flavocytochrome b558 is lacking, leaving the precise contact surface through which the activation domain stimulates FAD reduction unresolved; the functional consequences of individual phosphorylation events and the integrated regulatory logic of multi-site phosphorylation remain to be determined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No full-length p67phox structure or p67phox–cytochrome b558 complex structure available\", \"Functional roles of individual phosphorylation sites (beyond Thr233) on oxidase kinetics not characterized\", \"How intramolecular conformational changes couple Rac binding to activation domain rearrangement structurally unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 6, 22, 23]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [6, 15, 25]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 5, 22]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 28, 37]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [28]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 6, 12, 27, 36, 37]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 25, 31, 35]}\n    ],\n    \"complexes\": [\n      \"NADPH oxidase (NOX2 complex)\",\n      \"p40phox–p67phox–p47phox cytosolic heterotrimer\"\n    ],\n    \"partners\": [\n      \"NCF1\",\n      \"NCF4\",\n      \"RAC2\",\n      \"RAC1\",\n      \"CYBB\",\n      \"VAV1\",\n      \"PRKCQ\",\n      \"S100A8\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}