{"gene":"NOSIP","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2001,"finding":"NOSIP (eNOS interacting protein) binds directly to the carboxyl-terminal region of the eNOS oxygenase domain, as identified by yeast two-hybrid and confirmed by coimmunoprecipitation in vitro and in vivo; this interaction is inhibited by a synthetic peptide of the caveolin-1 scaffolding domain.","method":"Yeast two-hybrid screen, coimmunoprecipitation, peptide competition","journal":"FASEB Journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP in vitro and in vivo, yeast two-hybrid, multiple orthogonal methods in a single rigorous study","pmids":["11149895"],"is_preprint":false},{"year":2001,"finding":"NOSIP overexpression promotes translocation of eNOS from plasma membrane caveolae to intracellular compartments, reducing NO production in CHO-eNOS cells; this was demonstrated by subcellular fractionation showing a shift of eNOS out of caveolin-rich membrane fractions and by immunofluorescence loss of plasma membrane eNOS staining.","method":"Subcellular fractionation, detergent solubility assay, immunofluorescence, NO production assay in CHO-eNOS cells overexpressing NOSIP","journal":"FASEB Journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (fractionation, immunofluorescence, NO assay) in a single rigorous study","pmids":["11149895"],"is_preprint":false},{"year":2005,"finding":"Endogenous NOSIP reduces eNOS enzymatic activity specifically in the G2 phase of the cell cycle by targeting eNOS to the actin cytoskeleton; knockdown of NOSIP by RNAi completely abolishes G2-specific cytoskeletal association and inhibition of eNOS.","method":"RNA interference (siRNA knockdown), cell cycle synchronization, cytoskeletal association assay, eNOS activity assay","journal":"Molecular and Cellular Biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — RNAi loss-of-function with specific cell-cycle-phase phenotype and cytoskeletal fractionation readout, multiple orthogonal methods","pmids":["16135813"],"is_preprint":false},{"year":2005,"finding":"NOSIP shuttles between the nucleus and cytoplasm; nuclear import depends on a bipartite nuclear localization sequence (NLS) that mediates interaction with importin alpha, and mutational destruction of the NLS abolishes both nuclear import and importin alpha interaction. Nuclear export is leptomycin B-insensitive, indicating a CRM1-independent export mechanism. Cytoplasmic accumulation in G2 is required for cytoskeletal targeting of eNOS.","method":"Mutational analysis of NLS, importin alpha interaction assay (Co-IP), leptomycin B treatment, immunofluorescence of subcellular localization across cell cycle phases","journal":"Molecular and Cellular Biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — site-directed mutagenesis of NLS combined with binding assay and pharmacological inhibition, multiple orthogonal methods in one study","pmids":["16135813"],"is_preprint":false},{"year":2014,"finding":"NOSIP functions as a ubiquitin E3 ligase that mediates monoubiquitination of the PP2A catalytic subunit; loss of NOSIP in knockout mice results in increased PP2A activity in craniofacial tissue. NOSIP was identified as a novel interaction partner of PP2A by mass spectrometry-based protein interaction screen.","method":"Ubiquitin E3 ligase assay (monoubiquitination), mass spectrometry-based protein interaction screen, NOSIP knockout mouse, PP2A activity assay in craniofacial tissue","journal":"PLoS ONE","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro ubiquitination assay, MS-based interactome, and in vivo KO with enzymatic activity readout in one study","pmids":["25546391"],"is_preprint":false},{"year":2014,"finding":"NOSIP knockout mice develop holoprosencephaly and craniofacial anomalies (cleft lip/palate, cyclopia, facial midline clefting), establishing NOSIP as a critical modulator of brain and craniofacial development.","method":"NOSIP knockout mouse generation and phenotypic analysis","journal":"PLoS ONE","confidence":"High","confidence_rationale":"Tier 2 / Moderate — defined loss-of-function phenotype in KO mouse with specific developmental readout","pmids":["25546391"],"is_preprint":false},{"year":2017,"finding":"NOSIP is required for neural stem/progenitor cell self-renewal and neurogenesis; Nosip knockout mouse embryos show reduced brain size, cortical thickness, and postmitotic neuron formation, with decreased proliferation and increased apoptosis. Nosip functions upstream of Rbp1 (a regulator of retinoic acid synthesis) in neurogenesis, as shown by proteome analysis of neurospheres and synergy/epistasis experiments in Xenopus.","method":"NOSIP knockout mouse neurosphere assay, mass spectrometry proteomics of neurospheres, Xenopus nosip morpholino knockdown with rescue by human NOSIP mRNA, Rbp1 synergy knockdown epistasis","journal":"Developmental Biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis in two vertebrate models, rescue experiment, MS proteomics, multiple orthogonal methods","pmids":["28663132"],"is_preprint":false},{"year":2002,"finding":"NOSIP specifically coimmunoprecipitates with eNOS (but not nNOS) from rat small intestine, confirming the in vivo interaction in non-vascular cells of the gastrointestinal tract.","method":"Coimmunoprecipitation from rat small intestine tissue lysate","journal":"Gastroenterology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single Co-IP from tissue, replicated finding from original 2001 paper but in a new tissue context","pmids":["12105859"],"is_preprint":false},{"year":2005,"finding":"NOSIP interacts with eNOS in rat trachea and lung, as demonstrated by coimmunoprecipitation from lung tissue, consistent with a role in regulating eNOS activity in airway and vascular cells.","method":"Coimmunoprecipitation from rat lung tissue, RT-PCR, immunohistochemistry","journal":"Journal of Histochemistry and Cytochemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single Co-IP from tissue; corroborates prior findings in a new tissue","pmids":["15684328"],"is_preprint":false},{"year":2021,"finding":"NOSIP interacts with nNOS during neutrophil differentiation; NOSIP expression is significantly decreased at the final stage of neutrophil differentiation, and this decrease correlates with augmented NO release, suggesting that NOSIP-nNOS interaction modulates NO generation during granulopoiesis.","method":"nNOS overexpression in K562 cells, nNOS inhibitor treatment in mice and human CD34+ HSPCs, NOSIP expression analysis during differentiation by Western blot/RT-PCR","journal":"Biochimica et Biophysica Acta – Molecular Cell Research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — correlative expression plus functional inhibitor experiments; direct NOSIP-nNOS interaction established by prior literature, but mechanistic link here is inferred from expression changes","pmids":["33771575"],"is_preprint":false},{"year":2018,"finding":"Nosip is required for eye development and anterior neural crest cell induction and migration in Xenopus; Nosip deficiency disrupts retinal lamination, dorso-ventral patterning, eye field induction markers (rax, pax6, otx2), cranial cartilage formation, and neural crest markers (twist, snai2, egr2), with foxc1 (a downstream factor of retinoic acid signalling) also affected, linking Nosip to retinoic acid signalling in anterior neural tissue.","method":"Xenopus nosip morpholino knockdown, marker gene expression analysis, mouse Nosip conditional analysis, immunohistochemistry","journal":"Developmental Dynamics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function in two model organisms with specific molecular marker readouts; single lab","pmids":["30055071"],"is_preprint":false}],"current_model":"NOSIP is a ubiquitin E3 ligase and nucleocytoplasmic shuttling protein that binds the oxygenase domain of eNOS (and nNOS), translocates eNOS from plasma membrane caveolae to the actin cytoskeleton in a cell cycle-dependent (G2-phase) manner to inhibit NO production, monoubiquitinates the PP2A catalytic subunit to modulate its activity, and is required for neural progenitor self-renewal and craniofacial development by acting upstream of Rbp1 in a retinoic acid signalling pathway."},"narrative":{"mechanistic_narrative":"NOSIP is a nucleocytoplasmic shuttling protein and ubiquitin E3 ligase that couples nitric oxide synthase regulation, phosphatase control, and vertebrate neurodevelopment [PMID:11149895, PMID:25546391, PMID:28663132]. It binds directly to the carboxyl-terminal region of the eNOS oxygenase domain, a contact antagonized by the caveolin-1 scaffolding domain, and upon overexpression drives eNOS out of plasma membrane caveolae into intracellular compartments to suppress NO production [PMID:11149895]. Endogenous NOSIP executes this regulation in a cell-cycle-restricted manner, targeting eNOS to the actin cytoskeleton specifically in G2 phase to inhibit its enzymatic activity [PMID:16135813]. Its localization is governed by a bipartite NLS that mediates importin-alpha-dependent nuclear import and a CRM1-independent export pathway, with G2-phase cytoplasmic accumulation being required for cytoskeletal targeting of eNOS [PMID:16135813]. Independently, NOSIP acts as an E3 ligase that monoubiquitinates the PP2A catalytic subunit, and its loss elevates PP2A activity in vivo [PMID:25546391]. NOSIP is essential for brain and craniofacial development: knockout mice develop holoprosencephaly and midline craniofacial defects, and NOSIP sustains neural progenitor self-renewal and neurogenesis by acting upstream of Rbp1 within a retinoic acid signalling pathway that also controls eye field induction and neural crest formation [PMID:25546391, PMID:28663132, PMID:30055071].","teleology":[{"year":2001,"claim":"Established the molecular identity of NOSIP as a direct eNOS partner and defined where it binds, answering whether a dedicated protein regulates eNOS independently of caveolin.","evidence":"Yeast two-hybrid screen with reciprocal Co-IP in vitro and in vivo, plus caveolin-1 scaffolding-domain peptide competition","pmids":["11149895"],"confidence":"High","gaps":["Did not define whether binding alters eNOS catalysis directly or only via relocalization","No structural model of the NOSIP-eNOS interface"]},{"year":2001,"claim":"Showed that NOSIP functionally suppresses NO output by physically displacing eNOS from caveolae, linking binding to a downstream signalling consequence.","evidence":"Subcellular fractionation, detergent solubility, immunofluorescence, and NO assays in CHO-eNOS cells overexpressing NOSIP","pmids":["11149895"],"confidence":"High","gaps":["Overexpression context may not reflect endogenous stoichiometry","Destination compartment of displaced eNOS not resolved at this stage"]},{"year":2005,"claim":"Demonstrated that endogenous NOSIP regulates eNOS in a cell-cycle-specific window, revealing physiological timing and the actin cytoskeleton as the relocalization target.","evidence":"siRNA knockdown with cell-cycle synchronization, cytoskeletal association assay, and eNOS activity assay","pmids":["16135813"],"confidence":"High","gaps":["Trigger that activates NOSIP-mediated targeting specifically in G2 not identified","Functional purpose of G2-restricted NO suppression unclear"]},{"year":2005,"claim":"Explained the localization logic by defining NOSIP shuttling, showing nuclear import requires a bipartite NLS and importin alpha while export is CRM1-independent, and that cytoplasmic accumulation enables eNOS targeting.","evidence":"NLS mutagenesis, importin-alpha Co-IP, leptomycin B treatment, and cell-cycle immunofluorescence","pmids":["16135813"],"confidence":"High","gaps":["Identity of the CRM1-independent export receptor unknown","Nuclear function of NOSIP not defined"]},{"year":2002,"claim":"Extended the eNOS interaction beyond vascular cells, confirming physiological relevance in gastrointestinal tissue.","evidence":"Co-IP from rat small intestine lysate","pmids":["12105859"],"confidence":"Medium","gaps":["Single Co-IP without functional readout in this tissue","No reciprocal pulldown reported"]},{"year":2005,"claim":"Broadened the eNOS interaction to airway and lung tissue, supporting a general role in eNOS regulation across organs.","evidence":"Co-IP from rat lung, RT-PCR, immunohistochemistry","pmids":["15684328"],"confidence":"Medium","gaps":["Functional consequence for lung NO signalling not tested","Single-tissue correlative localization"]},{"year":2014,"claim":"Reframed NOSIP as an enzyme, establishing it as a ubiquitin E3 ligase that monoubiquitinates the PP2A catalytic subunit and restrains PP2A activity in vivo.","evidence":"In vitro monoubiquitination assay, MS-based interactome, and PP2A activity assay in NOSIP knockout craniofacial tissue","pmids":["25546391"],"confidence":"High","gaps":["Catalytic domain/RING or HECT identity of NOSIP not specified","Functional outcome of PP2A monoubiquitination on substrate specificity unresolved"]},{"year":2014,"claim":"Connected NOSIP loss to a defined developmental disease phenotype, establishing it as a modulator of brain and craniofacial morphogenesis.","evidence":"NOSIP knockout mouse generation and phenotypic analysis","pmids":["25546391"],"confidence":"High","gaps":["Whether the phenotype derives from PP2A, eNOS, or another pathway not disentangled","Human disease correlate not established in this study"]},{"year":2017,"claim":"Placed NOSIP in a defined neurogenic pathway, showing it sustains neural progenitor self-renewal and acts genetically upstream of Rbp1 in retinoic acid signalling.","evidence":"Knockout mouse neurosphere assay, MS proteomics, and Xenopus morpholino knockdown with human NOSIP rescue and Rbp1 epistasis","pmids":["28663132"],"confidence":"High","gaps":["Direct biochemical link between NOSIP and Rbp1 not established","Whether E3 ligase activity is required for neurogenic function untested"]},{"year":2018,"claim":"Extended the developmental role to eye and neural crest induction, reinforcing the link to retinoic acid signalling via foxc1.","evidence":"Xenopus morpholino knockdown with marker gene analysis, mouse conditional analysis, immunohistochemistry","pmids":["30055071"],"confidence":"Medium","gaps":["Mechanistic step connecting NOSIP to retinoic acid output not defined","Single-lab loss-of-function data"]},{"year":2021,"claim":"Implicated NOSIP in NO regulation during granulopoiesis, where its downregulation tracks with increased NO release via nNOS.","evidence":"nNOS overexpression in K562 cells, nNOS inhibitor treatment in mice and human CD34+ HSPCs, NOSIP expression analysis during differentiation","pmids":["33771575"],"confidence":"Medium","gaps":["Direct NOSIP-nNOS interaction not demonstrated in this study; inferred from expression changes","Causality between NOSIP decrease and NO release not established by loss-of-function"]},{"year":null,"claim":"How NOSIP's two molecular activities — eNOS relocalization and PP2A monoubiquitination — mechanistically converge to drive retinoic-acid-dependent neurodevelopment remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No defined catalytic domain or full substrate repertoire for the E3 ligase activity","Unknown whether NO regulation and PP2A ubiquitination operate in the same developmental pathway","Mechanism connecting NOSIP to Rbp1/retinoic acid signalling not biochemically defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[4]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[4]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[3]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[3]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[2]}],"complexes":[],"partners":["NOS3","NOS1","PPP2CA","KPNA"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y314","full_name":"Nitric oxide synthase-interacting protein","aliases":["E3 ubiquitin-protein ligase NOSIP","RING-type E3 ubiquitin transferase NOSIP","eNOS-interacting protein"],"length_aa":301,"mass_kda":33.2,"function":"E3 ubiquitin-protein ligase that is essential for proper development of the forebrain, the eye, and the face. Catalyzes monoubiquitination of serine/threonine-protein phosphatase 2A (PP2A) catalytic subunit PPP2CA/PPP2CB (By similarity). Negatively regulates nitric oxide production by inducing NOS1 and NOS3 translocation to actin cytoskeleton and inhibiting their enzymatic activity (PubMed:11149895, PubMed:15548660, PubMed:16135813)","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9Y314/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NOSIP","classification":"Not Classified","n_dependent_lines":85,"n_total_lines":1208,"dependency_fraction":0.07036423841059603},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CAPZB","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/NOSIP","total_profiled":1310},"omim":[{"mim_id":"616759","title":"NITRIC OXIDE SYNTHASE-INTERACTING PROTEIN; NOSIP","url":"https://www.omim.org/entry/616759"},{"mim_id":"176916","title":"PROTEIN PHOSPHATASE 2, CATALYTIC SUBUNIT, BETA ISOFORM; PPP2CB","url":"https://www.omim.org/entry/176916"},{"mim_id":"176915","title":"PROTEIN PHOSPHATASE 2, CATALYTIC SUBUNIT, ALPHA ISOFORM; PPP2CA","url":"https://www.omim.org/entry/176915"},{"mim_id":"163729","title":"NITRIC OXIDE SYNTHASE 3; NOS3","url":"https://www.omim.org/entry/163729"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NOSIP"},"hgnc":{"alias_symbol":["CGI-25"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y314","domains":[{"cath_id":"3.30.40.10","chopping":"32-112_179-281","consensus_level":"medium","plddt":90.4257,"start":32,"end":281}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y314","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y314-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y314-F1-predicted_aligned_error_v6.png","plddt_mean":81.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NOSIP","jax_strain_url":"https://www.jax.org/strain/search?query=NOSIP"},"sequence":{"accession":"Q9Y314","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y314.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y314/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y314"}},"corpus_meta":[{"pmid":"11149895","id":"PMC_11149895","title":"NOSIP, a novel modulator of endothelial nitric oxide synthase activity.","date":"2001","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/11149895","citation_count":146,"is_preprint":false},{"pmid":"16135813","id":"PMC_16135813","title":"Cell cycle-regulated inactivation of endothelial NO synthase through NOSIP-dependent targeting to the cytoskeleton.","date":"2005","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/16135813","citation_count":34,"is_preprint":false},{"pmid":"12105859","id":"PMC_12105859","title":"Distribution of the novel eNOS-interacting protein NOSIP in the liver, pancreas, and gastrointestinal tract of the rat.","date":"2002","source":"Gastroenterology","url":"https://pubmed.ncbi.nlm.nih.gov/12105859","citation_count":30,"is_preprint":false},{"pmid":"15684328","id":"PMC_15684328","title":"NOSIP and its interacting protein, eNOS, in the rat trachea and lung.","date":"2005","source":"The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society","url":"https://pubmed.ncbi.nlm.nih.gov/15684328","citation_count":23,"is_preprint":false},{"pmid":"25546391","id":"PMC_25546391","title":"The ubiquitin E3 ligase NOSIP modulates protein phosphatase 2A activity in craniofacial development.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/25546391","citation_count":16,"is_preprint":false},{"pmid":"33771575","id":"PMC_33771575","title":"nNOS induction and NOSIP interaction impact granulopoiesis and neutrophil differentiation by modulating nitric oxide generation.","date":"2021","source":"Biochimica et biophysica acta. Molecular cell research","url":"https://pubmed.ncbi.nlm.nih.gov/33771575","citation_count":14,"is_preprint":false},{"pmid":"12962906","id":"PMC_12962906","title":"Spinal upregulation of the nitric oxide synthase-interacting protein NOSIP in a rat model of inflammatory pain.","date":"2003","source":"Neuroscience letters","url":"https://pubmed.ncbi.nlm.nih.gov/12962906","citation_count":14,"is_preprint":false},{"pmid":"28663132","id":"PMC_28663132","title":"Developmental neurogenesis in mouse and Xenopus is impaired in the absence of Nosip.","date":"2017","source":"Developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/28663132","citation_count":10,"is_preprint":false},{"pmid":"23135205","id":"PMC_23135205","title":"Increased expression of nitric oxide synthase interacting protein (NOSIP) following traumatic spinal cord injury in rats.","date":"2012","source":"Journal of molecular histology","url":"https://pubmed.ncbi.nlm.nih.gov/23135205","citation_count":9,"is_preprint":false},{"pmid":"28196663","id":"PMC_28196663","title":"NOS-interacting protein (NOSIP) is increased in the colon of patients with Hirschsprungs's disease.","date":"2017","source":"Journal of pediatric surgery","url":"https://pubmed.ncbi.nlm.nih.gov/28196663","citation_count":9,"is_preprint":false},{"pmid":"37529099","id":"PMC_37529099","title":"Nosip is a potential therapeutic target in hepatocellular carcinoma cells.","date":"2023","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/37529099","citation_count":6,"is_preprint":false},{"pmid":"30536263","id":"PMC_30536263","title":"Expression of Nitric Oxide Synthase Interacting Protein (NOSIP) is Decreased in the Pulmonary Vasculature of Nitrofen-Induced Congenital Diaphragmatic Hernia.","date":"2018","source":"European journal of pediatric surgery : official journal of Austrian Association of Pediatric Surgery ... [et al] = Zeitschrift fur Kinderchirurgie","url":"https://pubmed.ncbi.nlm.nih.gov/30536263","citation_count":5,"is_preprint":false},{"pmid":"30055071","id":"PMC_30055071","title":"Nosip functions during vertebrate eye and cranial cartilage development.","date":"2018","source":"Developmental dynamics : an official publication of the American Association of Anatomists","url":"https://pubmed.ncbi.nlm.nih.gov/30055071","citation_count":5,"is_preprint":false},{"pmid":"42164384","id":"PMC_42164384","title":"Tea polyphenols suppress the malignant progression of renal cancer cells by targeting Nosip.","date":"2026","source":"Translational andrology and urology","url":"https://pubmed.ncbi.nlm.nih.gov/42164384","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8955,"output_tokens":2656,"usd":0.033353,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9971,"output_tokens":3465,"usd":0.06824,"stage2_stop_reason":"end_turn"},"total_usd":0.101593,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"NOSIP (eNOS interacting protein) binds directly to the carboxyl-terminal region of the eNOS oxygenase domain, as identified by yeast two-hybrid and confirmed by coimmunoprecipitation in vitro and in vivo; this interaction is inhibited by a synthetic peptide of the caveolin-1 scaffolding domain.\",\n      \"method\": \"Yeast two-hybrid screen, coimmunoprecipitation, peptide competition\",\n      \"journal\": \"FASEB Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP in vitro and in vivo, yeast two-hybrid, multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"11149895\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"NOSIP overexpression promotes translocation of eNOS from plasma membrane caveolae to intracellular compartments, reducing NO production in CHO-eNOS cells; this was demonstrated by subcellular fractionation showing a shift of eNOS out of caveolin-rich membrane fractions and by immunofluorescence loss of plasma membrane eNOS staining.\",\n      \"method\": \"Subcellular fractionation, detergent solubility assay, immunofluorescence, NO production assay in CHO-eNOS cells overexpressing NOSIP\",\n      \"journal\": \"FASEB Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (fractionation, immunofluorescence, NO assay) in a single rigorous study\",\n      \"pmids\": [\"11149895\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Endogenous NOSIP reduces eNOS enzymatic activity specifically in the G2 phase of the cell cycle by targeting eNOS to the actin cytoskeleton; knockdown of NOSIP by RNAi completely abolishes G2-specific cytoskeletal association and inhibition of eNOS.\",\n      \"method\": \"RNA interference (siRNA knockdown), cell cycle synchronization, cytoskeletal association assay, eNOS activity assay\",\n      \"journal\": \"Molecular and Cellular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — RNAi loss-of-function with specific cell-cycle-phase phenotype and cytoskeletal fractionation readout, multiple orthogonal methods\",\n      \"pmids\": [\"16135813\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"NOSIP shuttles between the nucleus and cytoplasm; nuclear import depends on a bipartite nuclear localization sequence (NLS) that mediates interaction with importin alpha, and mutational destruction of the NLS abolishes both nuclear import and importin alpha interaction. Nuclear export is leptomycin B-insensitive, indicating a CRM1-independent export mechanism. Cytoplasmic accumulation in G2 is required for cytoskeletal targeting of eNOS.\",\n      \"method\": \"Mutational analysis of NLS, importin alpha interaction assay (Co-IP), leptomycin B treatment, immunofluorescence of subcellular localization across cell cycle phases\",\n      \"journal\": \"Molecular and Cellular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — site-directed mutagenesis of NLS combined with binding assay and pharmacological inhibition, multiple orthogonal methods in one study\",\n      \"pmids\": [\"16135813\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NOSIP functions as a ubiquitin E3 ligase that mediates monoubiquitination of the PP2A catalytic subunit; loss of NOSIP in knockout mice results in increased PP2A activity in craniofacial tissue. NOSIP was identified as a novel interaction partner of PP2A by mass spectrometry-based protein interaction screen.\",\n      \"method\": \"Ubiquitin E3 ligase assay (monoubiquitination), mass spectrometry-based protein interaction screen, NOSIP knockout mouse, PP2A activity assay in craniofacial tissue\",\n      \"journal\": \"PLoS ONE\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro ubiquitination assay, MS-based interactome, and in vivo KO with enzymatic activity readout in one study\",\n      \"pmids\": [\"25546391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NOSIP knockout mice develop holoprosencephaly and craniofacial anomalies (cleft lip/palate, cyclopia, facial midline clefting), establishing NOSIP as a critical modulator of brain and craniofacial development.\",\n      \"method\": \"NOSIP knockout mouse generation and phenotypic analysis\",\n      \"journal\": \"PLoS ONE\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined loss-of-function phenotype in KO mouse with specific developmental readout\",\n      \"pmids\": [\"25546391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NOSIP is required for neural stem/progenitor cell self-renewal and neurogenesis; Nosip knockout mouse embryos show reduced brain size, cortical thickness, and postmitotic neuron formation, with decreased proliferation and increased apoptosis. Nosip functions upstream of Rbp1 (a regulator of retinoic acid synthesis) in neurogenesis, as shown by proteome analysis of neurospheres and synergy/epistasis experiments in Xenopus.\",\n      \"method\": \"NOSIP knockout mouse neurosphere assay, mass spectrometry proteomics of neurospheres, Xenopus nosip morpholino knockdown with rescue by human NOSIP mRNA, Rbp1 synergy knockdown epistasis\",\n      \"journal\": \"Developmental Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis in two vertebrate models, rescue experiment, MS proteomics, multiple orthogonal methods\",\n      \"pmids\": [\"28663132\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"NOSIP specifically coimmunoprecipitates with eNOS (but not nNOS) from rat small intestine, confirming the in vivo interaction in non-vascular cells of the gastrointestinal tract.\",\n      \"method\": \"Coimmunoprecipitation from rat small intestine tissue lysate\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single Co-IP from tissue, replicated finding from original 2001 paper but in a new tissue context\",\n      \"pmids\": [\"12105859\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"NOSIP interacts with eNOS in rat trachea and lung, as demonstrated by coimmunoprecipitation from lung tissue, consistent with a role in regulating eNOS activity in airway and vascular cells.\",\n      \"method\": \"Coimmunoprecipitation from rat lung tissue, RT-PCR, immunohistochemistry\",\n      \"journal\": \"Journal of Histochemistry and Cytochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single Co-IP from tissue; corroborates prior findings in a new tissue\",\n      \"pmids\": [\"15684328\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NOSIP interacts with nNOS during neutrophil differentiation; NOSIP expression is significantly decreased at the final stage of neutrophil differentiation, and this decrease correlates with augmented NO release, suggesting that NOSIP-nNOS interaction modulates NO generation during granulopoiesis.\",\n      \"method\": \"nNOS overexpression in K562 cells, nNOS inhibitor treatment in mice and human CD34+ HSPCs, NOSIP expression analysis during differentiation by Western blot/RT-PCR\",\n      \"journal\": \"Biochimica et Biophysica Acta – Molecular Cell Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — correlative expression plus functional inhibitor experiments; direct NOSIP-nNOS interaction established by prior literature, but mechanistic link here is inferred from expression changes\",\n      \"pmids\": [\"33771575\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Nosip is required for eye development and anterior neural crest cell induction and migration in Xenopus; Nosip deficiency disrupts retinal lamination, dorso-ventral patterning, eye field induction markers (rax, pax6, otx2), cranial cartilage formation, and neural crest markers (twist, snai2, egr2), with foxc1 (a downstream factor of retinoic acid signalling) also affected, linking Nosip to retinoic acid signalling in anterior neural tissue.\",\n      \"method\": \"Xenopus nosip morpholino knockdown, marker gene expression analysis, mouse Nosip conditional analysis, immunohistochemistry\",\n      \"journal\": \"Developmental Dynamics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function in two model organisms with specific molecular marker readouts; single lab\",\n      \"pmids\": [\"30055071\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NOSIP is a ubiquitin E3 ligase and nucleocytoplasmic shuttling protein that binds the oxygenase domain of eNOS (and nNOS), translocates eNOS from plasma membrane caveolae to the actin cytoskeleton in a cell cycle-dependent (G2-phase) manner to inhibit NO production, monoubiquitinates the PP2A catalytic subunit to modulate its activity, and is required for neural progenitor self-renewal and craniofacial development by acting upstream of Rbp1 in a retinoic acid signalling pathway.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NOSIP is a nucleocytoplasmic shuttling protein and ubiquitin E3 ligase that couples nitric oxide synthase regulation, phosphatase control, and vertebrate neurodevelopment [#0, #4, #6]. It binds directly to the carboxyl-terminal region of the eNOS oxygenase domain, a contact antagonized by the caveolin-1 scaffolding domain, and upon overexpression drives eNOS out of plasma membrane caveolae into intracellular compartments to suppress NO production [#0, #1]. Endogenous NOSIP executes this regulation in a cell-cycle-restricted manner, targeting eNOS to the actin cytoskeleton specifically in G2 phase to inhibit its enzymatic activity [#2]. Its localization is governed by a bipartite NLS that mediates importin-alpha-dependent nuclear import and a CRM1-independent export pathway, with G2-phase cytoplasmic accumulation being required for cytoskeletal targeting of eNOS [#3]. Independently, NOSIP acts as an E3 ligase that monoubiquitinates the PP2A catalytic subunit, and its loss elevates PP2A activity in vivo [#4]. NOSIP is essential for brain and craniofacial development: knockout mice develop holoprosencephaly and midline craniofacial defects, and NOSIP sustains neural progenitor self-renewal and neurogenesis by acting upstream of Rbp1 within a retinoic acid signalling pathway that also controls eye field induction and neural crest formation [#5, #6, #10].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established the molecular identity of NOSIP as a direct eNOS partner and defined where it binds, answering whether a dedicated protein regulates eNOS independently of caveolin.\",\n      \"evidence\": \"Yeast two-hybrid screen with reciprocal Co-IP in vitro and in vivo, plus caveolin-1 scaffolding-domain peptide competition\",\n      \"pmids\": [\"11149895\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define whether binding alters eNOS catalysis directly or only via relocalization\", \"No structural model of the NOSIP-eNOS interface\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Showed that NOSIP functionally suppresses NO output by physically displacing eNOS from caveolae, linking binding to a downstream signalling consequence.\",\n      \"evidence\": \"Subcellular fractionation, detergent solubility, immunofluorescence, and NO assays in CHO-eNOS cells overexpressing NOSIP\",\n      \"pmids\": [\"11149895\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Overexpression context may not reflect endogenous stoichiometry\", \"Destination compartment of displaced eNOS not resolved at this stage\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Demonstrated that endogenous NOSIP regulates eNOS in a cell-cycle-specific window, revealing physiological timing and the actin cytoskeleton as the relocalization target.\",\n      \"evidence\": \"siRNA knockdown with cell-cycle synchronization, cytoskeletal association assay, and eNOS activity assay\",\n      \"pmids\": [\"16135813\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trigger that activates NOSIP-mediated targeting specifically in G2 not identified\", \"Functional purpose of G2-restricted NO suppression unclear\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Explained the localization logic by defining NOSIP shuttling, showing nuclear import requires a bipartite NLS and importin alpha while export is CRM1-independent, and that cytoplasmic accumulation enables eNOS targeting.\",\n      \"evidence\": \"NLS mutagenesis, importin-alpha Co-IP, leptomycin B treatment, and cell-cycle immunofluorescence\",\n      \"pmids\": [\"16135813\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the CRM1-independent export receptor unknown\", \"Nuclear function of NOSIP not defined\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Extended the eNOS interaction beyond vascular cells, confirming physiological relevance in gastrointestinal tissue.\",\n      \"evidence\": \"Co-IP from rat small intestine lysate\",\n      \"pmids\": [\"12105859\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Co-IP without functional readout in this tissue\", \"No reciprocal pulldown reported\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Broadened the eNOS interaction to airway and lung tissue, supporting a general role in eNOS regulation across organs.\",\n      \"evidence\": \"Co-IP from rat lung, RT-PCR, immunohistochemistry\",\n      \"pmids\": [\"15684328\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence for lung NO signalling not tested\", \"Single-tissue correlative localization\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Reframed NOSIP as an enzyme, establishing it as a ubiquitin E3 ligase that monoubiquitinates the PP2A catalytic subunit and restrains PP2A activity in vivo.\",\n      \"evidence\": \"In vitro monoubiquitination assay, MS-based interactome, and PP2A activity assay in NOSIP knockout craniofacial tissue\",\n      \"pmids\": [\"25546391\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic domain/RING or HECT identity of NOSIP not specified\", \"Functional outcome of PP2A monoubiquitination on substrate specificity unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Connected NOSIP loss to a defined developmental disease phenotype, establishing it as a modulator of brain and craniofacial morphogenesis.\",\n      \"evidence\": \"NOSIP knockout mouse generation and phenotypic analysis\",\n      \"pmids\": [\"25546391\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the phenotype derives from PP2A, eNOS, or another pathway not disentangled\", \"Human disease correlate not established in this study\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Placed NOSIP in a defined neurogenic pathway, showing it sustains neural progenitor self-renewal and acts genetically upstream of Rbp1 in retinoic acid signalling.\",\n      \"evidence\": \"Knockout mouse neurosphere assay, MS proteomics, and Xenopus morpholino knockdown with human NOSIP rescue and Rbp1 epistasis\",\n      \"pmids\": [\"28663132\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct biochemical link between NOSIP and Rbp1 not established\", \"Whether E3 ligase activity is required for neurogenic function untested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Extended the developmental role to eye and neural crest induction, reinforcing the link to retinoic acid signalling via foxc1.\",\n      \"evidence\": \"Xenopus morpholino knockdown with marker gene analysis, mouse conditional analysis, immunohistochemistry\",\n      \"pmids\": [\"30055071\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic step connecting NOSIP to retinoic acid output not defined\", \"Single-lab loss-of-function data\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Implicated NOSIP in NO regulation during granulopoiesis, where its downregulation tracks with increased NO release via nNOS.\",\n      \"evidence\": \"nNOS overexpression in K562 cells, nNOS inhibitor treatment in mice and human CD34+ HSPCs, NOSIP expression analysis during differentiation\",\n      \"pmids\": [\"33771575\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct NOSIP-nNOS interaction not demonstrated in this study; inferred from expression changes\", \"Causality between NOSIP decrease and NO release not established by loss-of-function\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How NOSIP's two molecular activities — eNOS relocalization and PP2A monoubiquitination — mechanistically converge to drive retinoic-acid-dependent neurodevelopment remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No defined catalytic domain or full substrate repertoire for the E3 ligase activity\", \"Unknown whether NO regulation and PP2A ubiquitination operate in the same developmental pathway\", \"Mechanism connecting NOSIP to Rbp1/retinoic acid signalling not biochemically defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"NOS3\", \"NOS1\", \"PPP2CA\", \"KPNA\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}