{"gene":"DUOX2","run_date":"2026-04-28T17:46:03","timeline":{"discoveries":[{"year":1999,"finding":"DUOX2 (p138Tox) was purified from pig thyroid plasma membrane as a flavoprotein that constitutes the main component of the thyroid Ca2+-dependent NAD(P)H oxidase, generating H2O2 by transferring electrons from NAD(P)H to molecular oxygen; it contains two EF-hand motifs accounting for calcium-dependent activity and consensus FAD- and NADPH-binding sites.","method":"Protein purification from pig thyroid plasma membrane, microsequencing, cDNA cloning, domain analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — direct purification and biochemical characterization of enzymatic activity with structural domain identification","pmids":["10601291"],"is_preprint":false},{"year":2000,"finding":"DUOX2 (ThOX2) and DUOX1 (ThOX1) encode new NADPH oxidase family members that accumulate at the apical membrane of thyrocytes co-localized with thyroperoxidase, and their mRNA expression in dog thyroid is regulated by cAMP pathway activation.","method":"cDNA cloning, Western blot, immunolocalization, Northern blot, cAMP stimulation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — original cloning paper with functional characterization, subcellular localization, and regulatory studies; highly cited foundational work","pmids":["10806195"],"is_preprint":false},{"year":2002,"finding":"DUOX2 (ThOX2) proteins are N-glycosylated (apparent MW 180-190 kDa; drops to 160 kDa upon deglycosylation), and their enzymatic H2O2-generating activity requires thyroid-specific processing: the immature intracellular form (present in non-thyroid transfected cells) is enzymatically inactive; co-expression with thyroperoxidase or p22Phox does not rescue activity, indicating that additional thyroid-specific components are required for full processing and activity.","method":"Western blot, deglycosylation assays, heterologous transfection in non-thyroid cell lines, co-expression with thyroperoxidase and p22Phox, H2O2 generation assays, PLB-XCGD cell complementation","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods establishing maturation requirements and subcellular localization linked to enzymatic activity","pmids":["11822874"],"is_preprint":false},{"year":2002,"finding":"Biallelic (homozygous) inactivating mutations in DUOX2 (THOX2) result in complete disruption of thyroid hormone synthesis due to insufficient H2O2 production, causing severe permanent congenital hypothyroidism; monoallelic heterozygous truncating mutations cause partial H2O2 deficiency and milder transient congenital hypothyroidism.","method":"DNA sequencing of patient and family genomic DNA, genotype-phenotype correlation, perchlorate discharge test for iodide organification defect","journal":"The New England journal of medicine","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis by loss-of-function mutations with defined molecular and clinical phenotype; highly cited foundational study","pmids":["12110737"],"is_preprint":false},{"year":2001,"finding":"DUOX2 (LNOX2) protein localizes to the apical pole of thyrocytes and is a 164 kDa glycoprotein in which N-glycosylation accounts for at least 10-20 kDa of apparent molecular mass.","method":"Western blot with antipeptide antibodies, Northern blot across 23 human tissues, immunohistochemistry","journal":"The Journal of clinical endocrinology and metabolism","confidence":"Medium","confidence_rationale":"Tier 2-3 — direct localization experiments with glycoprotein characterization; single lab","pmids":["11443211"],"is_preprint":false},{"year":2004,"finding":"The human THOX2 gene spans 75 kb, is composed of 34 exons, is arranged in head-to-head configuration with THOX1 separated by ~16 kb, and its promoter lacks a TATA box and is not positively controlled by cAMP (unlike thyroglobulin or NIS gene promoters), yet displays significant transcriptional activity in differentiated thyroid cell lines.","method":"Gene structure determination, promoter transfection assays in thyroid cell lines, cAMP stimulation","journal":"Molecular and cellular endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 — functional promoter characterization with transfection assays; single lab","pmids":["15062544"],"is_preprint":false},{"year":2010,"finding":"THOX2 and DUOXA2 share a bidirectional promoter region with DUOXA2 containing a TATA box and THOX2 containing an Inr element; the THOX1-DUOXA1 promoter (but not THOX2-DUOXA2) contains functional Sp1 binding sites that drive bidirectional transcription.","method":"RLM-RACE transcription start site mapping, bidirectional reporter transfection assays in rat thyroid PCCl3 cells, gel shift/EMSA with Sp1, site-directed mutation of Sp1 sites","journal":"Molecular and cellular endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 — functional promoter characterization with EMSA and mutagenesis; single lab","pmids":["20060878"],"is_preprint":false},{"year":2024,"finding":"An IL-17-DUOX2 axis controls gastrointestinal colonization by Candida albicans: IL-17 receptor signaling is required for upregulation of Duox2/Duoxa2 in intestinal epithelium in response to C. albicans (specifically requiring the yeast-hyphal transition and the hyphal toxin candidalysin); IL-17A addition to colonoids induces Duox2/Duoxa2 expression and H2O2 production; loss of intestinal DUOX2 function reduces fungal colonization at extended time points and increases hyphal proportion, with elevated IL-17A levels indicating cross-regulation.","method":"Germ-free mouse colonization with C. albicans, expression profiling, IL-17 receptor knockout mice, colonoid culture with IL-17A, intestinal DUOX2-deficient mice, fungal colonization quantification","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — multiple genetic models with defined cellular and molecular phenotypes; preprint not yet peer-reviewed","pmids":["bio_10.1101_2024.08.16.608271"],"is_preprint":true},{"year":2025,"finding":"Social disruption stress upregulates Duox2 and Duoxa2 expression in intestinal epithelial cells via β-adrenergic signaling; this upregulation is reversed by β-adrenergic receptor blockade (propranolol) but not by α2-adrenergic, CRH, or glucocorticoid inhibition; NADPH oxidase inhibition with apocynin mitigates stress-induced ROS production and colitis severity.","method":"Social disruption stress mouse model, pharmacological antagonists (propranolol, idazoxan, mifepristone, antalarmin), intestinal epithelial cell gene expression analysis, apocynin treatment, ROS/RNS measurement, colitis models (DSS and C. rodentium)","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — multiple pharmacological interventions with defined molecular readouts; preprint, not peer-reviewed","pmids":["bio_10.1101_2025.07.15.664961"],"is_preprint":true},{"year":2024,"finding":"Duox2 is required for sentinel goblet cell-mediated secretory responses in the developing neonatal colon; microbiota-dependent development of functional sentinel goblet cells depends on the NADPH/Dual oxidase family member Duox2.","method":"In vivo and ex vivo analyses of pre- and post-weaning colonic mucus barrier in neonatal mice, Duox2-deficient mouse model","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 — single preprint with loss-of-function phenotype but limited mechanistic detail","pmids":["bio_10.1101_2024.07.03.601781"],"is_preprint":true}],"current_model":"DUOX2 is an apical membrane-localized, Ca2+-dependent flavoprotein NADPH oxidase in thyrocytes that generates H2O2 (by transferring electrons from NAD(P)H to O2 via FAD) to serve as the oxidant for thyroperoxidase-catalyzed iodination of thyroglobulin during thyroid hormone synthesis; its enzymatic activity requires thyroid-specific maturation factors beyond thyroperoxidase and p22Phox, and in the intestine it is regulated by IL-17 and β-adrenergic signaling to produce reactive oxygen species that modulate microbial colonization and epithelial barrier function."},"narrative":{"teleology":[{"year":1999,"claim":"Identification of DUOX2 as the principal thyroid H₂O₂-generating enzyme resolved the long-standing question of which oxidase supplies the oxidant for thyroid hormone synthesis, establishing its flavoprotein nature and Ca²⁺-dependent regulation via EF-hand motifs.","evidence":"Purification from pig thyroid plasma membrane, microsequencing, cDNA cloning, and domain analysis","pmids":["10601291"],"confidence":"High","gaps":["Catalytic mechanism and stoichiometry of electron transfer not resolved","Whether DUOX2 produces superoxide or directly H₂O₂ not distinguished"]},{"year":2000,"claim":"Cloning of DUOX2 and DUOX1 as a paralog pair and demonstration of their apical thyrocyte colocalization with thyroperoxidase established the spatial framework for coupled H₂O₂ production and iodination at the thyroid apical surface.","evidence":"cDNA cloning, immunolocalization, Northern blot, and cAMP stimulation in dog thyroid","pmids":["10806195"],"confidence":"High","gaps":["Relative contributions of DUOX1 versus DUOX2 to thyroidal H₂O₂ not determined","Mechanism of apical membrane targeting unknown"]},{"year":2001,"claim":"Confirmation that DUOX2 is an N-glycosylated apical membrane glycoprotein refined understanding of its post-translational processing as a prerequisite for surface expression.","evidence":"Western blot with antipeptide antibodies, immunohistochemistry, and tissue expression profiling","pmids":["11443211"],"confidence":"Medium","gaps":["Identity of glycosylation sites and their functional necessity not mapped","Single-lab observation without independent replication"]},{"year":2002,"claim":"Demonstration that heterologously expressed DUOX2 is enzymatically inactive and that neither thyroperoxidase nor p22Phox rescues activity revealed a requirement for unidentified thyroid-specific maturation factor(s), later found to be the DUOXA proteins.","evidence":"Heterologous transfection in non-thyroid cells, co-expression with TPO and p22Phox, H₂O₂ generation assays","pmids":["11822874"],"confidence":"High","gaps":["Identity of the maturation factor not determined at this point","ER-to-plasma-membrane trafficking mechanism unresolved"]},{"year":2002,"claim":"Discovery that biallelic DUOX2 mutations cause permanent congenital hypothyroidism—while monoallelic mutations cause transient forms—provided the definitive genetic proof that DUOX2-generated H₂O₂ is non-redundant for thyroid hormone synthesis in humans.","evidence":"Patient genomic DNA sequencing, genotype-phenotype correlation, perchlorate discharge test","pmids":["12110737"],"confidence":"High","gaps":["Whether DUOX1 partially compensates in monoallelic cases not tested","Extrathyroidal consequences of DUOX2 loss not investigated"]},{"year":2004,"claim":"Characterization of the DUOX2 promoter as TATA-less and cAMP-unresponsive distinguished its transcriptional regulation from other thyroid-specific genes (thyroglobulin, NIS), indicating an independent regulatory logic.","evidence":"Gene structure determination, promoter-reporter transfection assays in thyroid cell lines, cAMP stimulation","pmids":["15062544"],"confidence":"Medium","gaps":["Transcription factors driving thyroid-specific DUOX2 expression not identified","In vivo promoter activity not validated"]},{"year":2010,"claim":"Mapping of the bidirectional DUOX2–DUOXA2 promoter, with an Inr element driving DUOX2 and a TATA box driving DUOXA2, established co-regulation of the oxidase and its maturation factor as a coordinated transcriptional unit.","evidence":"RLM-RACE, bidirectional reporter assays in PCCl3 cells, EMSA, Sp1-site mutagenesis","pmids":["20060878"],"confidence":"Medium","gaps":["Regulatory elements specific to intestinal DUOX2 expression not characterized","Whether bidirectional regulation ensures stoichiometric co-expression not tested"]},{"year":2024,"claim":"An IL-17–DUOX2 signaling axis was shown to control intestinal Candida albicans colonization, revealing DUOX2 as a key effector of mucosal antifungal defense downstream of candidalysin-triggered IL-17 signaling.","evidence":"Germ-free mouse colonization, IL-17 receptor knockout mice, colonoid culture with IL-17A, intestinal DUOX2-deficient mice (preprint)","pmids":["bio_10.1101_2024.08.16.608271"],"confidence":"Medium","gaps":["Not yet peer-reviewed","Whether DUOX2-derived H₂O₂ is directly fungicidal or signals through downstream pathways not resolved","Relevance to human intestinal candidiasis not established"]},{"year":null,"claim":"The structural basis for DUOX2 activation by calcium, the identity of all maturation cofactors required for ER exit, and the precise mechanism by which DUOX2-derived ROS modulate intestinal epithelial barrier function remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of full-length DUOX2 in active versus inactive states","Mechanism of DUOXA2-dependent ER-to-plasma-membrane trafficking not fully elucidated","Relative contributions of DUOX2 versus other NOX family members to intestinal ROS not quantified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[0,2]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,1,4]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,1,3]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[7]}],"complexes":[],"partners":["DUOXA2","TPO"],"other_free_text":[]},"mechanistic_narrative":"DUOX2 is a Ca²⁺-dependent, flavoprotein NADPH oxidase that generates hydrogen peroxide at the apical membrane of thyrocytes and intestinal epithelial cells, serving as the essential oxidant source for thyroperoxidase-catalyzed iodination of thyroglobulin during thyroid hormone biosynthesis and as a regulator of mucosal barrier function. The enzyme contains two EF-hand motifs conferring calcium dependence, plus FAD- and NADPH-binding domains that mediate electron transfer from NAD(P)H to molecular oxygen [PMID:10601291]. Full enzymatic activity requires thyroid-specific maturation factors beyond thyroperoxidase and p22Phox, as the immature, incompletely glycosylated intracellular form produced in heterologous cells is catalytically inactive [PMID:11822874]. Biallelic loss-of-function mutations in DUOX2 cause permanent congenital hypothyroidism due to defective H₂O₂ production and iodide organification, while monoallelic mutations produce transient hypothyroidism [PMID:12110737]."},"prefetch_data":{"uniprot":{"accession":"Q9NRD8","full_name":"Dual oxidase 2","aliases":["Large NOX 2","Long NOX 2","NADH/NADPH thyroid oxidase p138-tox","NADPH oxidase/peroxidase DUOX2","NADPH thyroid oxidase 2","Thyroid oxidase 2","p138 thyroid oxidase"],"length_aa":1548,"mass_kda":175.4,"function":"Generates hydrogen peroxide which is required for the activity of thyroid peroxidase/TPO and lactoperoxidase/LPO (PubMed:15972824). Plays a role in thyroid hormone synthesis. Also required for lactoperoxidase-mediated antimicrobial defense at the surface of mucosa (PubMed:12824283). Synthesizes NAADP from its reduced NAADPH form which promotes Ca(2+) signaling during T cell activation (PubMed:34784249). May have its own peroxidase activity through its N-terminal peroxidase-like domain","subcellular_location":"Apical cell membrane; Cell junction","url":"https://www.uniprot.org/uniprotkb/Q9NRD8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/DUOX2","classification":"Not Classified","n_dependent_lines":18,"n_total_lines":1208,"dependency_fraction":0.014900662251655629},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/DUOX2","total_profiled":1310},"omim":[{"mim_id":"617792","title":"THIOREDOXIN DOMAIN-CONTAINING PROTEIN 11; TXNDC11","url":"https://www.omim.org/entry/617792"},{"mim_id":"612772","title":"DUAL OXIDASE MATURATION FACTOR 2; DUOXA2","url":"https://www.omim.org/entry/612772"},{"mim_id":"612771","title":"DUAL OXIDASE MATURATION FACTOR 1; DUOXA1","url":"https://www.omim.org/entry/612771"},{"mim_id":"609893","title":"HYPOTHYROIDISM, CONGENITAL, NONGOITROUS, 3; CHNG3","url":"https://www.omim.org/entry/609893"},{"mim_id":"607200","title":"THYROID DYSHORMONOGENESIS 6; TDH6","url":"https://www.omim.org/entry/607200"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"gallbladder","ntpm":273.1},{"tissue":"thyroid gland","ntpm":70.8},{"tissue":"urinary bladder","ntpm":86.8}],"url":"https://www.proteinatlas.org/search/DUOX2"},"hgnc":{"alias_symbol":["P138-TOX","P138(TOX)","THOX2","LNOX2"],"prev_symbol":[]},"alphafold":{"accession":"Q9NRD8","domains":[{"cath_id":"1.10.640.10","chopping":"56-555","consensus_level":"medium","plddt":93.6057,"start":56,"end":555},{"cath_id":"2.30.29.140","chopping":"643-753","consensus_level":"high","plddt":81.1881,"start":643,"end":753},{"cath_id":"1.10.238","chopping":"762-854","consensus_level":"medium","plddt":73.1728,"start":762,"end":854},{"cath_id":"1.10.238.10","chopping":"856-937","consensus_level":"medium","plddt":68.437,"start":856,"end":937},{"cath_id":"-","chopping":"1068-1267","consensus_level":"high","plddt":91.7651,"start":1068,"end":1267},{"cath_id":"2.40.30.10","chopping":"1268-1371","consensus_level":"medium","plddt":83.7622,"start":1268,"end":1371},{"cath_id":"3.40.50.80","chopping":"1372-1548","consensus_level":"medium","plddt":85.4995,"start":1372,"end":1548}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NRD8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NRD8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NRD8-F1-predicted_aligned_error_v6.png","plddt_mean":83.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DUOX2","jax_strain_url":"https://www.jax.org/strain/search?query=DUOX2"},"sequence":{"accession":"Q9NRD8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NRD8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NRD8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NRD8"}},"corpus_meta":[{"pmid":"10806195","id":"PMC_10806195","title":"Cloning of two human thyroid cDNAs encoding new members of the NADPH oxidase family.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10806195","citation_count":479,"is_preprint":false},{"pmid":"12110737","id":"PMC_12110737","title":"Inactivating mutations in the gene for thyroid oxidase 2 (THOX2) and congenital hypothyroidism.","date":"2002","source":"The New England journal of medicine","url":"https://pubmed.ncbi.nlm.nih.gov/12110737","citation_count":364,"is_preprint":false},{"pmid":"10601291","id":"PMC_10601291","title":"Purification of a novel flavoprotein involved in the thyroid NADPH oxidase. Cloning of the porcine and human cdnas.","date":"1999","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10601291","citation_count":356,"is_preprint":false},{"pmid":"15863666","id":"PMC_15863666","title":"Genetics of congenital hypothyroidism.","date":"2005","source":"Journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/15863666","citation_count":263,"is_preprint":false},{"pmid":"11822874","id":"PMC_11822874","title":"Characterization of ThOX proteins as components of the thyroid H(2)O(2)-generating system.","date":"2002","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/11822874","citation_count":147,"is_preprint":false},{"pmid":"11443211","id":"PMC_11443211","title":"Expression of reduced nicotinamide adenine dinucleotide phosphate oxidase (ThoX, LNOX, Duox) genes and proteins in human thyroid tissues.","date":"2001","source":"The Journal of clinical endocrinology and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/11443211","citation_count":93,"is_preprint":false},{"pmid":"15062544","id":"PMC_15062544","title":"Structural and functional characterization of the two human ThOX/Duox genes and their 5'-flanking regions.","date":"2004","source":"Molecular and cellular endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/15062544","citation_count":64,"is_preprint":false},{"pmid":"17121535","id":"PMC_17121535","title":"Congenital hypothyroidism caused by new mutations in the thyroid oxidase 2 (THOX2) gene.","date":"2006","source":"Clinical endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/17121535","citation_count":45,"is_preprint":false},{"pmid":"14751036","id":"PMC_14751036","title":"Thyroperoxidase gene mutations in congenital goitrous hypothyroidism with total and partial iodide organification defect.","date":"2003","source":"Thyroid : official journal of the American Thyroid Association","url":"https://pubmed.ncbi.nlm.nih.gov/14751036","citation_count":44,"is_preprint":false},{"pmid":"21835056","id":"PMC_21835056","title":"Molecules important for thyroid hormone synthesis and action - known facts and future perspectives.","date":"2011","source":"Thyroid research","url":"https://pubmed.ncbi.nlm.nih.gov/21835056","citation_count":39,"is_preprint":false},{"pmid":"20060878","id":"PMC_20060878","title":"Delimitation and functional characterization of the bidirectional THOX-DUOXA promoter regions in thyrocytes.","date":"2010","source":"Molecular and cellular endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/20060878","citation_count":24,"is_preprint":false},{"pmid":"15611819","id":"PMC_15611819","title":"[Genetic aspects in congenital hypothyrodism].","date":"2004","source":"Arquivos brasileiros de endocrinologia e metabologia","url":"https://pubmed.ncbi.nlm.nih.gov/15611819","citation_count":3,"is_preprint":false},{"pmid":"32207419","id":"PMC_32207419","title":"Insilico study of genes involved in Congenital Hypothyroidism.","date":"2020","source":"JPMA. The Journal of the Pakistan Medical Association","url":"https://pubmed.ncbi.nlm.nih.gov/32207419","citation_count":1,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.07.15.664961","title":"Social stress worsens colitis through β-adrenergic–driven oxidative stress in intestinal mucosal compartments","date":"2025-07-18","source":"bioRxiv","url":"https://doi.org/10.1101/2025.07.15.664961","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.08.16.608271","title":"An IL-17-DUOX2 axis controls gastrointestinal colonization by<i>Candida albicans</i>","date":"2024-08-19","source":"bioRxiv","url":"https://doi.org/10.1101/2024.08.16.608271","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.07.03.601781","title":"Neonatal microbiota colonization drives maturation of primary and secondary goblet cell mediated protection in the pre-weaning colon","date":"2024-07-05","source":"bioRxiv","url":"https://doi.org/10.1101/2024.07.03.601781","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.08.27.25333852","title":"Ultrarare Variants in DNA Damage Repair and Mitochondrial Genes in Pediatric Acute-Onset Neuropsychiatric Syndrome and Acute Behavioral Regression in Neurodevelopmental Disorders","date":"2025-08-27","source":"bioRxiv","url":"https://doi.org/10.1101/2025.08.27.25333852","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.09.08.674949","title":"TNF signaling drives expansion of Reg4+ epithelial cells in colitis","date":"2025-09-09","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.08.674949","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.12.04.626838","title":"Multi-omics analysis of the mechanism of alfalfa and wheat-induced rumen flatulence in Xizang sheep","date":"2024-12-04","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.04.626838","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.07.23.24310848","title":"Inflammatory Bowel Disease in patients with Primary Sclerosing Cholangitis: a distinct form of colitis","date":"2024-07-23","source":"bioRxiv","url":"https://doi.org/10.1101/2024.07.23.24310848","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12342,"output_tokens":2616,"usd":0.038133},"stage2":{"model":"claude-opus-4-6","input_tokens":5938,"output_tokens":2194,"usd":0.12681},"total_usd":0.164943,"stage1_batch_id":"msgbatch_0118RScg3WGaGkaZRtWZbtce","stage2_batch_id":"msgbatch_01Cpfzvtbj42aqp5oYZ5Te2s","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"DUOX2 (p138Tox) was purified from pig thyroid plasma membrane as a flavoprotein that constitutes the main component of the thyroid Ca2+-dependent NAD(P)H oxidase, generating H2O2 by transferring electrons from NAD(P)H to molecular oxygen; it contains two EF-hand motifs accounting for calcium-dependent activity and consensus FAD- and NADPH-binding sites.\",\n      \"method\": \"Protein purification from pig thyroid plasma membrane, microsequencing, cDNA cloning, domain analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct purification and biochemical characterization of enzymatic activity with structural domain identification\",\n      \"pmids\": [\"10601291\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"DUOX2 (ThOX2) and DUOX1 (ThOX1) encode new NADPH oxidase family members that accumulate at the apical membrane of thyrocytes co-localized with thyroperoxidase, and their mRNA expression in dog thyroid is regulated by cAMP pathway activation.\",\n      \"method\": \"cDNA cloning, Western blot, immunolocalization, Northern blot, cAMP stimulation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — original cloning paper with functional characterization, subcellular localization, and regulatory studies; highly cited foundational work\",\n      \"pmids\": [\"10806195\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"DUOX2 (ThOX2) proteins are N-glycosylated (apparent MW 180-190 kDa; drops to 160 kDa upon deglycosylation), and their enzymatic H2O2-generating activity requires thyroid-specific processing: the immature intracellular form (present in non-thyroid transfected cells) is enzymatically inactive; co-expression with thyroperoxidase or p22Phox does not rescue activity, indicating that additional thyroid-specific components are required for full processing and activity.\",\n      \"method\": \"Western blot, deglycosylation assays, heterologous transfection in non-thyroid cell lines, co-expression with thyroperoxidase and p22Phox, H2O2 generation assays, PLB-XCGD cell complementation\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods establishing maturation requirements and subcellular localization linked to enzymatic activity\",\n      \"pmids\": [\"11822874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Biallelic (homozygous) inactivating mutations in DUOX2 (THOX2) result in complete disruption of thyroid hormone synthesis due to insufficient H2O2 production, causing severe permanent congenital hypothyroidism; monoallelic heterozygous truncating mutations cause partial H2O2 deficiency and milder transient congenital hypothyroidism.\",\n      \"method\": \"DNA sequencing of patient and family genomic DNA, genotype-phenotype correlation, perchlorate discharge test for iodide organification defect\",\n      \"journal\": \"The New England journal of medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis by loss-of-function mutations with defined molecular and clinical phenotype; highly cited foundational study\",\n      \"pmids\": [\"12110737\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"DUOX2 (LNOX2) protein localizes to the apical pole of thyrocytes and is a 164 kDa glycoprotein in which N-glycosylation accounts for at least 10-20 kDa of apparent molecular mass.\",\n      \"method\": \"Western blot with antipeptide antibodies, Northern blot across 23 human tissues, immunohistochemistry\",\n      \"journal\": \"The Journal of clinical endocrinology and metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — direct localization experiments with glycoprotein characterization; single lab\",\n      \"pmids\": [\"11443211\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The human THOX2 gene spans 75 kb, is composed of 34 exons, is arranged in head-to-head configuration with THOX1 separated by ~16 kb, and its promoter lacks a TATA box and is not positively controlled by cAMP (unlike thyroglobulin or NIS gene promoters), yet displays significant transcriptional activity in differentiated thyroid cell lines.\",\n      \"method\": \"Gene structure determination, promoter transfection assays in thyroid cell lines, cAMP stimulation\",\n      \"journal\": \"Molecular and cellular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional promoter characterization with transfection assays; single lab\",\n      \"pmids\": [\"15062544\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"THOX2 and DUOXA2 share a bidirectional promoter region with DUOXA2 containing a TATA box and THOX2 containing an Inr element; the THOX1-DUOXA1 promoter (but not THOX2-DUOXA2) contains functional Sp1 binding sites that drive bidirectional transcription.\",\n      \"method\": \"RLM-RACE transcription start site mapping, bidirectional reporter transfection assays in rat thyroid PCCl3 cells, gel shift/EMSA with Sp1, site-directed mutation of Sp1 sites\",\n      \"journal\": \"Molecular and cellular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional promoter characterization with EMSA and mutagenesis; single lab\",\n      \"pmids\": [\"20060878\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"An IL-17-DUOX2 axis controls gastrointestinal colonization by Candida albicans: IL-17 receptor signaling is required for upregulation of Duox2/Duoxa2 in intestinal epithelium in response to C. albicans (specifically requiring the yeast-hyphal transition and the hyphal toxin candidalysin); IL-17A addition to colonoids induces Duox2/Duoxa2 expression and H2O2 production; loss of intestinal DUOX2 function reduces fungal colonization at extended time points and increases hyphal proportion, with elevated IL-17A levels indicating cross-regulation.\",\n      \"method\": \"Germ-free mouse colonization with C. albicans, expression profiling, IL-17 receptor knockout mice, colonoid culture with IL-17A, intestinal DUOX2-deficient mice, fungal colonization quantification\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic models with defined cellular and molecular phenotypes; preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2024.08.16.608271\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Social disruption stress upregulates Duox2 and Duoxa2 expression in intestinal epithelial cells via β-adrenergic signaling; this upregulation is reversed by β-adrenergic receptor blockade (propranolol) but not by α2-adrenergic, CRH, or glucocorticoid inhibition; NADPH oxidase inhibition with apocynin mitigates stress-induced ROS production and colitis severity.\",\n      \"method\": \"Social disruption stress mouse model, pharmacological antagonists (propranolol, idazoxan, mifepristone, antalarmin), intestinal epithelial cell gene expression analysis, apocynin treatment, ROS/RNS measurement, colitis models (DSS and C. rodentium)\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple pharmacological interventions with defined molecular readouts; preprint, not peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.07.15.664961\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Duox2 is required for sentinel goblet cell-mediated secretory responses in the developing neonatal colon; microbiota-dependent development of functional sentinel goblet cells depends on the NADPH/Dual oxidase family member Duox2.\",\n      \"method\": \"In vivo and ex vivo analyses of pre- and post-weaning colonic mucus barrier in neonatal mice, Duox2-deficient mouse model\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single preprint with loss-of-function phenotype but limited mechanistic detail\",\n      \"pmids\": [\"bio_10.1101_2024.07.03.601781\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"DUOX2 is an apical membrane-localized, Ca2+-dependent flavoprotein NADPH oxidase in thyrocytes that generates H2O2 (by transferring electrons from NAD(P)H to O2 via FAD) to serve as the oxidant for thyroperoxidase-catalyzed iodination of thyroglobulin during thyroid hormone synthesis; its enzymatic activity requires thyroid-specific maturation factors beyond thyroperoxidase and p22Phox, and in the intestine it is regulated by IL-17 and β-adrenergic signaling to produce reactive oxygen species that modulate microbial colonization and epithelial barrier function.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"DUOX2 is a Ca²⁺-dependent, flavoprotein NADPH oxidase that generates hydrogen peroxide at the apical membrane of thyrocytes and intestinal epithelial cells, serving as the essential oxidant source for thyroperoxidase-catalyzed iodination of thyroglobulin during thyroid hormone biosynthesis and as a regulator of mucosal barrier function. The enzyme contains two EF-hand motifs conferring calcium dependence, plus FAD- and NADPH-binding domains that mediate electron transfer from NAD(P)H to molecular oxygen [PMID:10601291]. Full enzymatic activity requires thyroid-specific maturation factors beyond thyroperoxidase and p22Phox, as the immature, incompletely glycosylated intracellular form produced in heterologous cells is catalytically inactive [PMID:11822874]. Biallelic loss-of-function mutations in DUOX2 cause permanent congenital hypothyroidism due to defective H₂O₂ production and iodide organification, while monoallelic mutations produce transient hypothyroidism [PMID:12110737].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Identification of DUOX2 as the principal thyroid H₂O₂-generating enzyme resolved the long-standing question of which oxidase supplies the oxidant for thyroid hormone synthesis, establishing its flavoprotein nature and Ca²⁺-dependent regulation via EF-hand motifs.\",\n      \"evidence\": \"Purification from pig thyroid plasma membrane, microsequencing, cDNA cloning, and domain analysis\",\n      \"pmids\": [\"10601291\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Catalytic mechanism and stoichiometry of electron transfer not resolved\",\n        \"Whether DUOX2 produces superoxide or directly H₂O₂ not distinguished\"\n      ]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Cloning of DUOX2 and DUOX1 as a paralog pair and demonstration of their apical thyrocyte colocalization with thyroperoxidase established the spatial framework for coupled H₂O₂ production and iodination at the thyroid apical surface.\",\n      \"evidence\": \"cDNA cloning, immunolocalization, Northern blot, and cAMP stimulation in dog thyroid\",\n      \"pmids\": [\"10806195\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Relative contributions of DUOX1 versus DUOX2 to thyroidal H₂O₂ not determined\",\n        \"Mechanism of apical membrane targeting unknown\"\n      ]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Confirmation that DUOX2 is an N-glycosylated apical membrane glycoprotein refined understanding of its post-translational processing as a prerequisite for surface expression.\",\n      \"evidence\": \"Western blot with antipeptide antibodies, immunohistochemistry, and tissue expression profiling\",\n      \"pmids\": [\"11443211\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Identity of glycosylation sites and their functional necessity not mapped\",\n        \"Single-lab observation without independent replication\"\n      ]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Demonstration that heterologously expressed DUOX2 is enzymatically inactive and that neither thyroperoxidase nor p22Phox rescues activity revealed a requirement for unidentified thyroid-specific maturation factor(s), later found to be the DUOXA proteins.\",\n      \"evidence\": \"Heterologous transfection in non-thyroid cells, co-expression with TPO and p22Phox, H₂O₂ generation assays\",\n      \"pmids\": [\"11822874\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Identity of the maturation factor not determined at this point\",\n        \"ER-to-plasma-membrane trafficking mechanism unresolved\"\n      ]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Discovery that biallelic DUOX2 mutations cause permanent congenital hypothyroidism—while monoallelic mutations cause transient forms—provided the definitive genetic proof that DUOX2-generated H₂O₂ is non-redundant for thyroid hormone synthesis in humans.\",\n      \"evidence\": \"Patient genomic DNA sequencing, genotype-phenotype correlation, perchlorate discharge test\",\n      \"pmids\": [\"12110737\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether DUOX1 partially compensates in monoallelic cases not tested\",\n        \"Extrathyroidal consequences of DUOX2 loss not investigated\"\n      ]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Characterization of the DUOX2 promoter as TATA-less and cAMP-unresponsive distinguished its transcriptional regulation from other thyroid-specific genes (thyroglobulin, NIS), indicating an independent regulatory logic.\",\n      \"evidence\": \"Gene structure determination, promoter-reporter transfection assays in thyroid cell lines, cAMP stimulation\",\n      \"pmids\": [\"15062544\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Transcription factors driving thyroid-specific DUOX2 expression not identified\",\n        \"In vivo promoter activity not validated\"\n      ]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Mapping of the bidirectional DUOX2–DUOXA2 promoter, with an Inr element driving DUOX2 and a TATA box driving DUOXA2, established co-regulation of the oxidase and its maturation factor as a coordinated transcriptional unit.\",\n      \"evidence\": \"RLM-RACE, bidirectional reporter assays in PCCl3 cells, EMSA, Sp1-site mutagenesis\",\n      \"pmids\": [\"20060878\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Regulatory elements specific to intestinal DUOX2 expression not characterized\",\n        \"Whether bidirectional regulation ensures stoichiometric co-expression not tested\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"An IL-17–DUOX2 signaling axis was shown to control intestinal Candida albicans colonization, revealing DUOX2 as a key effector of mucosal antifungal defense downstream of candidalysin-triggered IL-17 signaling.\",\n      \"evidence\": \"Germ-free mouse colonization, IL-17 receptor knockout mice, colonoid culture with IL-17A, intestinal DUOX2-deficient mice (preprint)\",\n      \"pmids\": [\"bio_10.1101_2024.08.16.608271\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Not yet peer-reviewed\",\n        \"Whether DUOX2-derived H₂O₂ is directly fungicidal or signals through downstream pathways not resolved\",\n        \"Relevance to human intestinal candidiasis not established\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The structural basis for DUOX2 activation by calcium, the identity of all maturation cofactors required for ER exit, and the precise mechanism by which DUOX2-derived ROS modulate intestinal epithelial barrier function remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No high-resolution structure of full-length DUOX2 in active versus inactive states\",\n        \"Mechanism of DUOXA2-dependent ER-to-plasma-membrane trafficking not fully elucidated\",\n        \"Relative contributions of DUOX2 versus other NOX family members to intestinal ROS not quantified\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"DUOXA2\",\n      \"TPO\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}