{"gene":"N4BP1","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2007,"finding":"N4BP1 binds the WW2 domain of the E3 ubiquitin ligase Itch, competing with Itch substrates (p73α, c-Jun, p63) for the same binding site, thereby inhibiting Itch-mediated ubiquitylation of these substrates and Itch autoubiquitylation activity both in vitro and in vivo.","method":"Co-immunoprecipitation, in vitro and in vivo ubiquitylation assays, RNAi knockdown, N4BP1 knockout cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro ubiquitylation assay plus in vivo assay, domain-level mapping (WW2), RNAi and genetic KO, multiple orthogonal methods in a single rigorous study","pmids":["17592138"],"is_preprint":false},{"year":2010,"finding":"N4BP1 is a substrate for Nedd4-mediated polyubiquitylation and proteasomal degradation. SUMOylation of N4BP1 by SUMO1 abrogates its ubiquitylation, stabilizing it. Endogenous N4BP1 localizes predominantly to the nucleolus in primary cells, with a fraction at PML nuclear bodies; loss of the desumoylase SENP1 or proteasome inhibition causes accumulation of N4BP1 at PML nuclear bodies.","method":"Ubiquitylation assays, SUMOylation assays, immunofluorescence localization, subcellular fractionation, SENP1-mutant primary embryonic fibroblasts, proteasome inhibitor (MG132) treatment","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple orthogonal methods (in vivo ubiquitylation, SUMO conjugation, localization in genetic mutant cells), single lab","pmids":["20233849"],"is_preprint":false},{"year":2019,"finding":"N4BP1 harbors a PilT N-terminus (NYN)-like RNase domain and inhibits HIV-1 replication by binding to and degrading viral mRNA species. N4BP1 is cleaved at Arg509 by the paracaspase MALT1 following T cell activation, and this cleavage inactivates its HIV-1 restriction activity and facilitates reactivation of latent HIV-1 proviruses.","method":"RNA-binding and mRNA degradation assays, mutational analysis (Arg509), MALT1 knockout studies, primary T cell and macrophage infection models","journal":"Nature microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — mutational analysis of cleavage site, KO studies, primary cell functional assays, multiple orthogonal methods","pmids":["31133753"],"is_preprint":false},{"year":2019,"finding":"N4BP1 contains a novel ubiquitin-binding domain (CoCUN) in its C-terminal ~50 residues that recognizes the canonical hydrophobic patch of ubiquitin (Ile44) through a Phe-Pro motif, resembling CUE domain recognition but evolutionarily distinct. CoCUN does not bind NEDD8 (unlike the related CUBAN domain). Both CoCUN and CUBAN are polyubiquitinated in cells.","method":"NMR spectroscopy (15N chemical shift perturbation), mutagenesis, circular dichroism, protein sequence analysis","journal":"Biomolecules","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structural analysis with mutagenesis validation, single lab but multiple orthogonal biophysical methods","pmids":["31319543"],"is_preprint":false},{"year":2020,"finding":"N4BP1 is a suppressor of TLR1/2-, TLR7-, and TLR9-driven cytokine production (MyD88-dependent TLRs), but not TLR3 or TLR4 responses in wild-type macrophages. N4BP1 is cleaved and inactivated by caspase-8 downstream of TRIF signaling (TLR3, TLR4) or TNF/death receptor engagement, thereby licensing those pathways to produce higher cytokine levels. Caspase-8-deficient macrophages retain intact N4BP1, explaining their impaired cytokine responses to TLR3/4; co-deletion of N4BP1 in caspase-8-deficient macrophages largely rescues this defect.","method":"N4BP1 knockout mice, caspase-8-deficient macrophages, N4BP1/caspase-8 double-knockout genetic rescue, cytokine production assays, cleavage assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genetic rescue (double KO), multiple TLR stimuli, published in high-impact journal, multiple orthogonal approaches","pmids":["32971525"],"is_preprint":false},{"year":2021,"finding":"N4BP1 inhibits TLR-dependent NF-κB activation by directly interacting with the NEMO (IKKγ) COZI domain through its UBA-like and CUE-like domains, attenuating NEMO dimerization/oligomerization. Caspase-8, activated downstream of TRIF (TLR3/TLR4), cleaves N4BP1 distal to residues D424 and D490, abolishing its inhibitory effect on NF-κB.","method":"Co-immunoprecipitation, in vitro binding assays, domain deletion mapping, N4bp1-deficient mice, caspase-8 cleavage site mutagenesis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain-level mapping, in vitro and in vivo corroboration, cleavage site identification, N4BP1 KO mice with defined NF-κB phenotype","pmids":["33654074"],"is_preprint":false},{"year":2021,"finding":"In keratinocytes, N4BP1 binds JunB and FosB mRNAs and reduces their stability, thereby suppressing keratinocyte proliferation. In neutrophils, N4BP1 limits survival and tissue infiltration by targeting CXCL1, CCL20, and S100A8 mRNAs. N4BP1-deficient mice develop severe IMQ-induced psoriasis-like disease.","method":"RNA immunoprecipitation, transcriptome profiling, N4BP1 KO mice, primary keratinocyte proliferation assays, IMQ psoriasis model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — RNA immunoprecipitation linking N4BP1 to specific mRNA targets, KO mouse phenotype, but single lab","pmids":["33990547"],"is_preprint":false},{"year":2023,"finding":"N4BP1 negatively regulates Notch signaling by binding NICD (specifically via the NICD RAM domain) and promoting its ubiquitin-mediated proteasomal degradation after Notch1 S3 cleavage. The CoCUN domain Phe-Pro motif (residues 862/863) in N4BP1 is essential for NICD degradation. The E3 ligase Trim21 is required for N4BP1-regulated NICD degradation. In cortical neural progenitors, N4BP1 overexpression promotes differentiation while N4BP1 loss maintains stem-like properties.","method":"Protein binding assays, domain mutagenesis (Phe-Pro motif), Trim21 KO, overexpression and KO in neural progenitor cells, cortical development assays","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mutagenesis, KO of E3 ligase, in vivo progenitor cell phenotype, single lab","pmids":["37807845"],"is_preprint":false},{"year":2024,"finding":"N4BP1 acts as a dimerization-dependent linear ubiquitin reader through a novel ubiquitin-binding module named LUBIN, which positions two non-selective ubiquitin-binding domains to preferentially recognize linear (M1) ubiquitin chains. N4BP1 is recruited to the nascent TNFR1 signaling complex and limits NF-κB signaling duration in a LUBIN-dependent manner. N4BP1 deficiency accelerates TNFα-induced cell death by increasing complex II assembly. Under apoptotic conditions, caspase-8 cleaves N4BP1, leading to its rapid 26S proteasomal degradation.","method":"Structural modeling, mutagenesis, TNFR1 signaling complex immunoprecipitation, N4BP1 KO functional assays, ubiquitin-chain binding assays, caspase-8 cleavage assays","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mutagenesis plus complex recruitment, multiple functional readouts, single lab","pmids":["38643192"],"is_preprint":false},{"year":2024,"finding":"N4BP1 acts in concert with non-canonical IκB kinases (TBK1 and IKKε) and their adaptor TANK to limit the duration of canonical IKKα/β signaling after MyD88-dependent TLR activation. Deletion of TBK1/IKKε or TANK phenocopies N4BP1 deficiency. Optimal suppression requires N4BP1's ability to bind polyubiquitin chains, as inactivating mutations in the ubiquitin-binding motif of N4BP1 increase TLR-induced cytokine production.","method":"N4BP1 KO and ubiquitin-binding mutant knock-in mice, TBK1/IKKε/TANK KO macrophages, cytokine production assays, genetic epistasis","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with multiple KOs, ubiquitin-binding domain point mutations in vivo, multiple orthogonal methods across mouse and cell models","pmids":["38697117"],"is_preprint":false},{"year":2024,"finding":"N4BP1 degrades its mRNA targets via their coding sequences (CDS) rather than the 3'-UTR. A critical 33-nt sequence (nt 289–322) near the 5' end of FosC CDS, containing a polyC motif, is required for N4BP1-mediated degradation. Both the KH domain and the NYN (YacP-like nuclease) domain are essential for N4BP1's mRNA-degrading activity. N4BP1-mediated mRNA turnover is independent of nonsense-mediated decay (NMD), as it is preserved in UPF1/UPF3A/UPF3B-deficient cells.","method":"Domain deletion mutagenesis, point mutations, reporter assays, CDS/3'UTR swap experiments, NMD-deficient cell lines (UPF1/UPF3A/UPF3B KO), RNA immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — domain mutagenesis defining catalytic requirement, multiple mRNA substrates tested, NMD-independent mechanism established using multiple KO cell lines, single lab","pmids":["39491646"],"is_preprint":false},{"year":2024,"finding":"Enteroviral 3C protease (3Cpro) from multiple human enteroviruses cleaves N4BP1 at residue Q816, destroying its ability to regulate TNFα-induced NF-κB signaling. Mouse N4BP1, which has a threonine at the P1' site, is resistant to human enteroviral 3Cpro, but rodent EMCV 3Cpro cleaves both human and mouse N4BP1 at species-specific sites.","method":"FIMO motif search, biochemical cleavage assays, cell biology experiments with 3Cpro expression, site mutagenesis (Q816), NF-κB signaling readouts","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cleavage site mutagenesis, endogenous N4BP1 cleavage during infection confirmed, functional consequence on NF-κB shown, single lab","pmids":["39655957"],"is_preprint":false},{"year":2025,"finding":"N4BP1 is a nucleocytoplasmic shuttling protein. Nuclear accumulation is induced by leptomycin B (blocking CRM1-mediated export). A nuclear localization signal (NLS) spanning residues 279–299 is required for nuclear import; its deletion abolishes nuclear import and fusion to GFP drives GFP into the nucleus. N4BP1 forms liquid-liquid phase separation aggregates in both cytoplasm and nucleus that colocalize with NEDD8-modified proteins (including cullin-1 and cullin-2), dependent on the CoCUN domain. Heat shock promotes N4BP1 aggregation, which confers cellular protection under stress.","method":"Leptomycin B treatment, NLS deletion and GFP fusion constructs, live imaging, 1,6-hexanediol phase separation assay, co-localization with neddylated proteins, heat shock experiments","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — NLS mutagenesis with GFP fusion rescue, pharmacological disruption of phase separation, co-localization with neddylated cullins, single lab","pmids":["40701250"],"is_preprint":false},{"year":2026,"finding":"N4BP1 suppresses IL-17 signaling by blocking translation of Act1 (TRAF3IP2) mRNA rather than affecting its stability. In N4BP1-deficient cells, Act1 mRNA is enriched in the translationally active polysome fraction, leading to elevated Act1 protein, hyperactivation of p38 MAPK, and increased downstream cytokines (CXCL1, CCL20, MMP9).","method":"Polysome profiling, mRNA stability assays, N4BP1 KO cells and mice, Act1 shRNA rescue experiments, p38 pharmacological inhibition, IMQ-induced skin model","journal":"Inflammation research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — polysome profiling directly demonstrates translational mechanism, KO rescue by Act1 silencing, in vivo corroboration, single lab","pmids":["41504891"],"is_preprint":false},{"year":2025,"finding":"N4BP1 is identified as a negative regulator of NF-κB that is targeted by poxvirus effector myxoma virus M3.1; N4BP1 inactivation by M3.1 unleashes an NF-κB ETI response, placing N4BP1 functionally alongside ZC3H12A and TBK1 as anti-viral NF-κB suppressors that can serve as pathogen sensors.","method":"Virulence factor expression screen in human monocytes, RNA-seq transcriptional response assays","journal":"bioRxiv (preprint)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single screen-based approach, indirect evidence that N4BP1 inactivation mediates NF-κB activation, no direct mechanistic dissection of N4BP1","pmids":["bio_10.1101_2025.03.04.641538"],"is_preprint":true}],"current_model":"N4BP1 is a multifunctional immune checkpoint protein that acts as an endoribonuclease (via its NYN/NYN-like RNase domain and KH domain) to degrade mRNA substrates through their coding sequences, and simultaneously suppresses NF-κB signaling by binding NEMO to block its oligomerization and by acting as a dimerization-dependent linear ubiquitin reader (LUBIN module) in the TNFR1 complex; N4BP1 is constitutively active against MyD88-dependent TLRs and TNF signaling but is inactivated by caspase-8-mediated cleavage (downstream of TRIF, death receptors, or MALT1) or by viral proteases (3Cpro), and its stability is regulated by Nedd4-mediated polyubiquitylation (opposed by SUMO1 conjugation), with nuclear–cytoplasmic shuttling governed by an NLS (residues 279–299) and phase-separation aggregate formation linked to neddylated cullins via its CoCUN ubiquitin-binding domain."},"narrative":{"mechanistic_narrative":"N4BP1 is a multifunctional immune-restriction protein that couples post-transcriptional silencing of inflammatory transcripts to direct suppression of NF-κB signaling, integrating its activity into checkpoints that limit antiviral and inflammatory output [PMID:32971525, PMID:38697117]. As an endoribonuclease, it uses both its KH domain and a PilT N-terminus (NYN/YacP-like) RNase domain to bind and degrade target mRNAs through their coding sequences—recognizing a polyC-containing element near the 5' CDS—in a manner independent of nonsense-mediated decay [PMID:31133753, PMID:39491646]; through this activity it restricts HIV-1 by degrading viral mRNA [PMID:31133753] and limits inflammation by destabilizing JunB, FosB, and chemokine transcripts in keratinocytes and neutrophils [PMID:33990547], while a parallel mode blocks translation of the IL-17 adaptor Act1 (TRAF3IP2) to restrain p38 MAPK–driven cytokine production [PMID:41504891]. In NF-κB control, N4BP1 is a constitutive suppressor of MyD88-dependent TLR (TLR1/2, TLR7, TLR9) and TNF responses [PMID:32971525]; it binds the NEMO COZI domain via UBA-like and CUE-like regions to block IKKγ oligomerization [PMID:33654074], reads linear (M1) polyubiquitin chains at the nascent TNFR1 complex through a dimerization-dependent LUBIN module to limit signaling duration and restrain complex II–driven cell death [PMID:38643192], and operates together with the non-canonical kinases TBK1/IKKε and the adaptor TANK in a polyubiquitin-binding-dependent manner [PMID:38697117]. N4BP1 is switched off by proteolysis: caspase-8 cleaves it downstream of TRIF, death receptors, and MALT1 to license TLR3/4 and death-receptor cytokine output and HIV reactivation [PMID:31133753, PMID:32971525, PMID:33654074], and enteroviral 3C protease cleaves it at Q816 to relieve NF-κB restriction [PMID:39655957]. The protein additionally binds the E3 ligase Itch to inhibit substrate ubiquitylation [PMID:17592138] and antagonizes Notch signaling by promoting Trim21-dependent degradation of NICD via its CoCUN domain [PMID:37807845]. Its abundance and localization are regulated by Nedd4-mediated polyubiquitylation opposed by SUMO1 conjugation [PMID:20233849], an NLS (residues 279–299) governing CRM1-dependent nucleocytoplasmic shuttling, and CoCUN-dependent phase separation with neddylated cullins under stress [PMID:40701250].","teleology":[{"year":2007,"claim":"Established the first molecular activity for N4BP1—as a competitive inhibitor of the E3 ligase Itch—answering whether it participates in ubiquitin pathway regulation.","evidence":"Co-IP, in vitro and in vivo ubiquitylation assays, RNAi and knockout cells","pmids":["17592138"],"confidence":"High","gaps":["Did not address RNase or NF-κB functions later attributed to N4BP1","Physiological context of Itch antagonism not defined"]},{"year":2010,"claim":"Defined how N4BP1 protein levels and subnuclear localization are controlled, showing a Nedd4-ubiquitylation/SUMO1 balance and nucleolar/PML body residence.","evidence":"Ubiquitylation and SUMOylation assays, immunofluorescence, SENP1-mutant fibroblasts, proteasome inhibition","pmids":["20233849"],"confidence":"High","gaps":["Functional consequence of nucleolar vs PML localization unresolved","Link between stability control and downstream signaling roles not established"]},{"year":2019,"claim":"Identified N4BP1 as an NYN-domain endoribonuclease that restricts HIV-1 and is inactivated by MALT1 cleavage, defining both an enzymatic activity and a switch-off mechanism.","evidence":"mRNA degradation assays, Arg509 mutagenesis, MALT1 KO, primary T cell/macrophage infection","pmids":["31133753"],"confidence":"High","gaps":["Substrate sequence determinants not yet defined","Mechanism of RNA target selection unknown"]},{"year":2019,"claim":"Structurally characterized the C-terminal CoCUN domain as a ubiquitin-binding module distinct from CUBAN, defining how N4BP1 reads ubiquitin.","evidence":"NMR chemical shift perturbation, mutagenesis, circular dichroism, sequence analysis","pmids":["31319543"],"confidence":"High","gaps":["In-cell function of CoCUN ubiquitin reading not yet linked to a pathway","Selectivity for chain linkage types not addressed"]},{"year":2020,"claim":"Placed N4BP1 as a constitutive suppressor of MyD88-dependent TLRs and TNF that is licensed off by caspase-8 cleavage, establishing it as an inflammatory checkpoint.","evidence":"N4BP1 KO mice, caspase-8-deficient macrophages, N4BP1/caspase-8 double-KO rescue, cytokine and cleavage assays","pmids":["32971525"],"confidence":"High","gaps":["Molecular mechanism by which N4BP1 suppresses signaling not defined here","Whether RNase activity mediates the cytokine suppression unaddressed"]},{"year":2021,"claim":"Provided a molecular mechanism for NF-κB suppression—direct binding to the NEMO COZI domain to block IKK oligomerization—and mapped the caspase-8 cleavage that abolishes it.","evidence":"Co-IP, in vitro binding, domain deletion mapping, N4bp1-deficient mice, cleavage site mutagenesis","pmids":["33654074"],"confidence":"High","gaps":["Relationship between NEMO binding and ubiquitin reading not resolved","Stoichiometry of NEMO inhibition unknown"]},{"year":2021,"claim":"Connected N4BP1 RNase activity to specific inflammatory mRNA targets and disease, showing destabilization of JunB/FosB and chemokine transcripts and a psoriasis-like phenotype in KO mice.","evidence":"RNA-IP, transcriptome profiling, N4BP1 KO mice, keratinocyte proliferation assays, IMQ psoriasis model","pmids":["33990547"],"confidence":"Medium","gaps":["Single lab","Direct demonstration that catalytic RNase activity drives each phenotype not shown"]},{"year":2023,"claim":"Extended N4BP1 function to Notch signaling, showing CoCUN-dependent, Trim21-mediated NICD degradation and a role in neural progenitor differentiation.","evidence":"Protein binding assays, Phe-Pro motif mutagenesis, Trim21 KO, progenitor overexpression/KO, cortical development assays","pmids":["37807845"],"confidence":"Medium","gaps":["Single lab","How CoCUN ubiquitin binding directs NICD to Trim21 mechanistically unclear"]},{"year":2024,"claim":"Defined the LUBIN module as a dimerization-dependent linear-ubiquitin reader that recruits N4BP1 to the TNFR1 complex to limit signaling duration and cell death.","evidence":"Structural modeling, mutagenesis, TNFR1 complex IP, KO functional assays, ubiquitin-chain binding, caspase-8 cleavage","pmids":["38643192"],"confidence":"Medium","gaps":["Single lab","High-resolution structure of LUBIN–M1 chain interaction not determined"]},{"year":2024,"claim":"Demonstrated genetic cooperation of N4BP1 with TBK1/IKKε and TANK in a ubiquitin-binding-dependent manner to terminate TLR-driven IKK signaling.","evidence":"N4BP1 KO and ubiquitin-binding mutant knock-in mice, TBK1/IKKε/TANK KO macrophages, cytokine assays, epistasis","pmids":["38697117"],"confidence":"High","gaps":["Biochemical nature of the N4BP1–TBK1/TANK complex not resolved","Whether the same module mediates both TLR and TNFR1 effects unaddressed"]},{"year":2024,"claim":"Resolved the RNA-degradation mechanism, showing CDS-targeted, polyC-element-dependent cleavage requiring KH and NYN domains and independent of NMD.","evidence":"Domain/point mutagenesis, reporter and CDS/3'UTR swap assays, UPF1/UPF3A/UPF3B KO cells, RNA-IP","pmids":["39491646"],"confidence":"High","gaps":["Full consensus of the targeted RNA element not defined","How CDS engagement is coordinated with translation unknown"]},{"year":2024,"claim":"Showed that enteroviral 3C protease cleaves N4BP1 at Q816 to relieve NF-κB restriction, identifying a viral evasion strategy and species-specific susceptibility.","evidence":"Motif search, cleavage assays, 3Cpro expression in cells, Q816 mutagenesis, NF-κB readouts","pmids":["39655957"],"confidence":"Medium","gaps":["Single lab","Whether cleavage affects RNase function in addition to NF-κB not tested"]},{"year":2025,"claim":"Characterized N4BP1 trafficking and biophysics, defining an NLS, CRM1-dependent export, and CoCUN-dependent phase separation with neddylated cullins that confers stress protection.","evidence":"Leptomycin B, NLS deletion/GFP fusion, live imaging, 1,6-hexanediol assay, co-localization with neddylated cullins, heat shock","pmids":["40701250"],"confidence":"Medium","gaps":["Single lab","Functional link between phase separation and RNase/NF-κB roles not established"]},{"year":2026,"claim":"Identified a translational-control mechanism, showing N4BP1 blocks Act1 (TRAF3IP2) mRNA translation to restrain IL-17/p38 signaling, distinct from mRNA destabilization.","evidence":"Polysome profiling, mRNA stability assays, N4BP1 KO cells/mice, Act1 shRNA rescue, p38 inhibition, IMQ skin model","pmids":["41504891"],"confidence":"Medium","gaps":["Single lab","How N4BP1 selects translational vs degradative fate for different mRNAs unknown"]},{"year":null,"claim":"It remains unresolved how N4BP1 integrates its distinct activities—CDS-directed RNA cleavage, translational repression, NEMO/linear-ubiquitin-based NF-κB suppression, and E3-ligase modulation—into a unified regulatory logic, and which activity dominates in a given cell type or stimulus.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No single-molecule or structural framework unifying the RNase and ubiquitin-reader modules","Determinants selecting between degradation, translational block, and signaling roles undefined","Crosstalk between localization/phase separation and effector functions unmapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[2,10]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[2,6,10]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,4,5,9]},{"term_id":"GO:0045182","term_label":"translation regulator activity","supporting_discovery_ids":[13]},{"term_id":"GO:0031386","term_label":"protein tag activity","supporting_discovery_ids":[3,8,9]}],"localization":[{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[1]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,12]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[12]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[4,5,9]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[5,8,9]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[2,10]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[8]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[2,11]}],"complexes":["TNFR1 signaling complex"],"partners":["ITCH","IKBKG","MALT1","CASP8","TRIM21","TBK1","IKBKE","TANK"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O75113","full_name":"NEDD4-binding protein 1","aliases":[],"length_aa":896,"mass_kda":100.4,"function":"Potent suppressor of cytokine production that acts as a regulator of innate immune signaling and inflammation. Acts as a key negative regulator of select cytokine and chemokine responses elicited by TRIF-independent Toll-like receptors (TLRs), thereby limiting inflammatory cytokine responses to minor insults. In response to more threatening pathogens, cleaved by CASP8 downstream of TLR3 or TLR4, leading to its inactivation, thereby allowing production of inflammatory cytokines (By similarity). Acts as a restriction factor against some viruses, such as HIV-1: restricts HIV-1 replication by binding to HIV-1 mRNAs and mediating their degradation via its ribonuclease activity (PubMed:31133753). Also acts as an inhibitor of the E3 ubiquitin-protein ligase ITCH: acts by interacting with the second WW domain of ITCH, leading to compete with ITCH's substrates and impairing ubiquitination of substrates (By similarity)","subcellular_location":"Cytoplasm, cytosol; Nucleus; Nucleus, nucleolus; Nucleus, PML body","url":"https://www.uniprot.org/uniprotkb/O75113/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/N4BP1","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/N4BP1","total_profiled":1310},"omim":[{"mim_id":"619140","title":"NEDD4-BINDING PROTEIN 3; N4BP3","url":"https://www.omim.org/entry/619140"},{"mim_id":"619139","title":"NEDD4-BINDING PROTEIN 2; N4BP2","url":"https://www.omim.org/entry/619139"},{"mim_id":"619138","title":"NEDD4-BINDING PROTEIN 1; N4BP1","url":"https://www.omim.org/entry/619138"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoli fibrillar center","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/N4BP1"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"O75113","domains":[{"cath_id":"3.30.1370.10","chopping":"2-75","consensus_level":"medium","plddt":86.1981,"start":2,"end":75},{"cath_id":"3.30.1370.10","chopping":"78-192","consensus_level":"medium","plddt":92.2952,"start":78,"end":192},{"cath_id":"3.40.50.11980","chopping":"615-772","consensus_level":"high","plddt":95.6813,"start":615,"end":772},{"cath_id":"-","chopping":"853-896","consensus_level":"high","plddt":90.113,"start":853,"end":896},{"cath_id":"1.10.8","chopping":"345-387","consensus_level":"medium","plddt":93.3181,"start":345,"end":387}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O75113","model_url":"https://alphafold.ebi.ac.uk/files/AF-O75113-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O75113-F1-predicted_aligned_error_v6.png","plddt_mean":66.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=N4BP1","jax_strain_url":"https://www.jax.org/strain/search?query=N4BP1"},"sequence":{"accession":"O75113","fasta_url":"https://rest.uniprot.org/uniprotkb/O75113.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O75113/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O75113"}},"corpus_meta":[{"pmid":"17592138","id":"PMC_17592138","title":"The Nedd4-binding partner 1 (N4BP1) protein is an inhibitor of the E3 ligase Itch.","date":"2007","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/17592138","citation_count":98,"is_preprint":false},{"pmid":"32971525","id":"PMC_32971525","title":"Integration of innate immune signalling by caspase-8 cleavage of N4BP1.","date":"2020","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/32971525","citation_count":93,"is_preprint":false},{"pmid":"31133753","id":"PMC_31133753","title":"N4BP1 restricts HIV-1 and its inactivation by MALT1 promotes viral reactivation.","date":"2019","source":"Nature microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/31133753","citation_count":63,"is_preprint":false},{"pmid":"33654074","id":"PMC_33654074","title":"N4BP1 negatively regulates NF-κB by binding and inhibiting NEMO oligomerization.","date":"2021","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/33654074","citation_count":41,"is_preprint":false},{"pmid":"20233849","id":"PMC_20233849","title":"N4BP1 is a newly identified nucleolar protein that undergoes SUMO-regulated polyubiquitylation and proteasomal turnover at promyelocytic leukemia nuclear bodies.","date":"2010","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/20233849","citation_count":36,"is_preprint":false},{"pmid":"28350078","id":"PMC_28350078","title":"miR-28-5p promotes the development and progression of ovarian cancer through inhibition of N4BP1.","date":"2017","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/28350078","citation_count":26,"is_preprint":false},{"pmid":"38697117","id":"PMC_38697117","title":"N4BP1 coordinates ubiquitin-dependent crosstalk within the IκB kinase family to limit Toll-like receptor signaling and inflammation.","date":"2024","source":"Immunity","url":"https://pubmed.ncbi.nlm.nih.gov/38697117","citation_count":23,"is_preprint":false},{"pmid":"33990547","id":"PMC_33990547","title":"The endoribonuclease N4BP1 prevents psoriasis by controlling both keratinocytes proliferation and neutrophil infiltration.","date":"2021","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/33990547","citation_count":22,"is_preprint":false},{"pmid":"31319543","id":"PMC_31319543","title":"CoCUN, a Novel Ubiquitin Binding Domain Identified in N4BP1.","date":"2019","source":"Biomolecules","url":"https://pubmed.ncbi.nlm.nih.gov/31319543","citation_count":16,"is_preprint":false},{"pmid":"38643192","id":"PMC_38643192","title":"N4BP1 functions as a dimerization-dependent linear ubiquitin reader which regulates TNF signalling.","date":"2024","source":"Cell death discovery","url":"https://pubmed.ncbi.nlm.nih.gov/38643192","citation_count":10,"is_preprint":false},{"pmid":"37807845","id":"PMC_37807845","title":"N4BP1 mediates RAM domain-dependent notch signaling turnover during neocortical development.","date":"2023","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/37807845","citation_count":8,"is_preprint":false},{"pmid":"24220004","id":"PMC_24220004","title":"Identification of a novel N4BP1-like gene from grass carp (Ctenopharyngodon idella) in response to GCRV infection.","date":"2013","source":"Fish & shellfish immunology","url":"https://pubmed.ncbi.nlm.nih.gov/24220004","citation_count":8,"is_preprint":false},{"pmid":"39655957","id":"PMC_39655957","title":"Enteroviral 3C protease cleaves N4BP1 to impair the host inflammatory response.","date":"2024","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/39655957","citation_count":7,"is_preprint":false},{"pmid":"39491646","id":"PMC_39491646","title":"The NEDD4-binding protein N4BP1 degrades mRNA substrates through the coding sequence independent of nonsense-mediated decay.","date":"2024","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/39491646","citation_count":6,"is_preprint":false},{"pmid":"37579928","id":"PMC_37579928","title":"N4BP1 regulates keratinocytes development and plays protective role in burn- and adhesive-related skin injury via MMP9.","date":"2023","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/37579928","citation_count":4,"is_preprint":false},{"pmid":"40201312","id":"PMC_40201312","title":"Exosomal miR-92b-5p regulates N4BP1 to enhance PTEN mono-ubiquitination in doxorubicin-resistant AML.","date":"2025","source":"Cancer drug resistance (Alhambra, Calif.)","url":"https://pubmed.ncbi.nlm.nih.gov/40201312","citation_count":4,"is_preprint":false},{"pmid":"40312219","id":"PMC_40312219","title":"N4BP1 as a modulator of the NF-κB pathway.","date":"2025","source":"Cytokine & growth factor reviews","url":"https://pubmed.ncbi.nlm.nih.gov/40312219","citation_count":1,"is_preprint":false},{"pmid":"40701250","id":"PMC_40701250","title":"N4BP1 is a nucleocytoplasmic shuttling protein and recognizes aggregates of the ubiquitin-like protein NEDD8 to protect cells under heat shock.","date":"2025","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/40701250","citation_count":0,"is_preprint":false},{"pmid":"42052091","id":"PMC_42052091","title":"Altered expression of ADAR1, N4BP1, and PSME1 in PBMCs correlated with therapeutic outcomes in HBeAg-negative chronic hepatitis B patients treated with Peg-IFN-α.","date":"2026","source":"Frontiers in cellular and infection microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/42052091","citation_count":0,"is_preprint":false},{"pmid":"41513619","id":"PMC_41513619","title":"N4BP1 is essential for the development of oral cancer via controlling both cancer cells and immune microenvironment.","date":"2026","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/41513619","citation_count":0,"is_preprint":false},{"pmid":"41504891","id":"PMC_41504891","title":"N4BP1 acts as a potent negative regulator of IL-17 signaling by blocking the translation of Act1 mRNA.","date":"2026","source":"Inflammation research : official journal of the European Histamine Research Society ... [et al.]","url":"https://pubmed.ncbi.nlm.nih.gov/41504891","citation_count":0,"is_preprint":false},{"pmid":"38749393","id":"PMC_38749393","title":"Non-canonical IKKs side with N4BP1 against the family.","date":"2024","source":"Immunity","url":"https://pubmed.ncbi.nlm.nih.gov/38749393","citation_count":0,"is_preprint":false},{"pmid":"41611111","id":"PMC_41611111","title":"N4BP1 promotes the progression of acute glaucoma by promoting pyroptosis through the JAK2/STAT3 pathway.","date":"2026","source":"Experimental eye research","url":"https://pubmed.ncbi.nlm.nih.gov/41611111","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.12.23.630104","title":"Structural and functional characterization of the KHNYN extended-diKH domain for mediating ZAP antiviral activity","date":"2024-12-23","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.23.630104","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.03.04.641538","title":"Poxvirus attack of anti-viral defense pathways unleashes an effector-triggered NF-κB response","date":"2025-03-11","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.04.641538","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":15233,"output_tokens":4650,"usd":0.057724,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12655,"output_tokens":5026,"usd":0.094463,"stage2_stop_reason":"end_turn"},"total_usd":0.152187,"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\": 2007,\n      \"finding\": \"N4BP1 binds the WW2 domain of the E3 ubiquitin ligase Itch, competing with Itch substrates (p73α, c-Jun, p63) for the same binding site, thereby inhibiting Itch-mediated ubiquitylation of these substrates and Itch autoubiquitylation activity both in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation, in vitro and in vivo ubiquitylation assays, RNAi knockdown, N4BP1 knockout cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro ubiquitylation assay plus in vivo assay, domain-level mapping (WW2), RNAi and genetic KO, multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"17592138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"N4BP1 is a substrate for Nedd4-mediated polyubiquitylation and proteasomal degradation. SUMOylation of N4BP1 by SUMO1 abrogates its ubiquitylation, stabilizing it. Endogenous N4BP1 localizes predominantly to the nucleolus in primary cells, with a fraction at PML nuclear bodies; loss of the desumoylase SENP1 or proteasome inhibition causes accumulation of N4BP1 at PML nuclear bodies.\",\n      \"method\": \"Ubiquitylation assays, SUMOylation assays, immunofluorescence localization, subcellular fractionation, SENP1-mutant primary embryonic fibroblasts, proteasome inhibitor (MG132) treatment\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple orthogonal methods (in vivo ubiquitylation, SUMO conjugation, localization in genetic mutant cells), single lab\",\n      \"pmids\": [\"20233849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"N4BP1 harbors a PilT N-terminus (NYN)-like RNase domain and inhibits HIV-1 replication by binding to and degrading viral mRNA species. N4BP1 is cleaved at Arg509 by the paracaspase MALT1 following T cell activation, and this cleavage inactivates its HIV-1 restriction activity and facilitates reactivation of latent HIV-1 proviruses.\",\n      \"method\": \"RNA-binding and mRNA degradation assays, mutational analysis (Arg509), MALT1 knockout studies, primary T cell and macrophage infection models\",\n      \"journal\": \"Nature microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mutational analysis of cleavage site, KO studies, primary cell functional assays, multiple orthogonal methods\",\n      \"pmids\": [\"31133753\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"N4BP1 contains a novel ubiquitin-binding domain (CoCUN) in its C-terminal ~50 residues that recognizes the canonical hydrophobic patch of ubiquitin (Ile44) through a Phe-Pro motif, resembling CUE domain recognition but evolutionarily distinct. CoCUN does not bind NEDD8 (unlike the related CUBAN domain). Both CoCUN and CUBAN are polyubiquitinated in cells.\",\n      \"method\": \"NMR spectroscopy (15N chemical shift perturbation), mutagenesis, circular dichroism, protein sequence analysis\",\n      \"journal\": \"Biomolecules\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structural analysis with mutagenesis validation, single lab but multiple orthogonal biophysical methods\",\n      \"pmids\": [\"31319543\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"N4BP1 is a suppressor of TLR1/2-, TLR7-, and TLR9-driven cytokine production (MyD88-dependent TLRs), but not TLR3 or TLR4 responses in wild-type macrophages. N4BP1 is cleaved and inactivated by caspase-8 downstream of TRIF signaling (TLR3, TLR4) or TNF/death receptor engagement, thereby licensing those pathways to produce higher cytokine levels. Caspase-8-deficient macrophages retain intact N4BP1, explaining their impaired cytokine responses to TLR3/4; co-deletion of N4BP1 in caspase-8-deficient macrophages largely rescues this defect.\",\n      \"method\": \"N4BP1 knockout mice, caspase-8-deficient macrophages, N4BP1/caspase-8 double-knockout genetic rescue, cytokine production assays, cleavage assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal genetic rescue (double KO), multiple TLR stimuli, published in high-impact journal, multiple orthogonal approaches\",\n      \"pmids\": [\"32971525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"N4BP1 inhibits TLR-dependent NF-κB activation by directly interacting with the NEMO (IKKγ) COZI domain through its UBA-like and CUE-like domains, attenuating NEMO dimerization/oligomerization. Caspase-8, activated downstream of TRIF (TLR3/TLR4), cleaves N4BP1 distal to residues D424 and D490, abolishing its inhibitory effect on NF-κB.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding assays, domain deletion mapping, N4bp1-deficient mice, caspase-8 cleavage site mutagenesis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain-level mapping, in vitro and in vivo corroboration, cleavage site identification, N4BP1 KO mice with defined NF-κB phenotype\",\n      \"pmids\": [\"33654074\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In keratinocytes, N4BP1 binds JunB and FosB mRNAs and reduces their stability, thereby suppressing keratinocyte proliferation. In neutrophils, N4BP1 limits survival and tissue infiltration by targeting CXCL1, CCL20, and S100A8 mRNAs. N4BP1-deficient mice develop severe IMQ-induced psoriasis-like disease.\",\n      \"method\": \"RNA immunoprecipitation, transcriptome profiling, N4BP1 KO mice, primary keratinocyte proliferation assays, IMQ psoriasis model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — RNA immunoprecipitation linking N4BP1 to specific mRNA targets, KO mouse phenotype, but single lab\",\n      \"pmids\": [\"33990547\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"N4BP1 negatively regulates Notch signaling by binding NICD (specifically via the NICD RAM domain) and promoting its ubiquitin-mediated proteasomal degradation after Notch1 S3 cleavage. The CoCUN domain Phe-Pro motif (residues 862/863) in N4BP1 is essential for NICD degradation. The E3 ligase Trim21 is required for N4BP1-regulated NICD degradation. In cortical neural progenitors, N4BP1 overexpression promotes differentiation while N4BP1 loss maintains stem-like properties.\",\n      \"method\": \"Protein binding assays, domain mutagenesis (Phe-Pro motif), Trim21 KO, overexpression and KO in neural progenitor cells, cortical development assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mutagenesis, KO of E3 ligase, in vivo progenitor cell phenotype, single lab\",\n      \"pmids\": [\"37807845\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"N4BP1 acts as a dimerization-dependent linear ubiquitin reader through a novel ubiquitin-binding module named LUBIN, which positions two non-selective ubiquitin-binding domains to preferentially recognize linear (M1) ubiquitin chains. N4BP1 is recruited to the nascent TNFR1 signaling complex and limits NF-κB signaling duration in a LUBIN-dependent manner. N4BP1 deficiency accelerates TNFα-induced cell death by increasing complex II assembly. Under apoptotic conditions, caspase-8 cleaves N4BP1, leading to its rapid 26S proteasomal degradation.\",\n      \"method\": \"Structural modeling, mutagenesis, TNFR1 signaling complex immunoprecipitation, N4BP1 KO functional assays, ubiquitin-chain binding assays, caspase-8 cleavage assays\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mutagenesis plus complex recruitment, multiple functional readouts, single lab\",\n      \"pmids\": [\"38643192\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"N4BP1 acts in concert with non-canonical IκB kinases (TBK1 and IKKε) and their adaptor TANK to limit the duration of canonical IKKα/β signaling after MyD88-dependent TLR activation. Deletion of TBK1/IKKε or TANK phenocopies N4BP1 deficiency. Optimal suppression requires N4BP1's ability to bind polyubiquitin chains, as inactivating mutations in the ubiquitin-binding motif of N4BP1 increase TLR-induced cytokine production.\",\n      \"method\": \"N4BP1 KO and ubiquitin-binding mutant knock-in mice, TBK1/IKKε/TANK KO macrophages, cytokine production assays, genetic epistasis\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with multiple KOs, ubiquitin-binding domain point mutations in vivo, multiple orthogonal methods across mouse and cell models\",\n      \"pmids\": [\"38697117\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"N4BP1 degrades its mRNA targets via their coding sequences (CDS) rather than the 3'-UTR. A critical 33-nt sequence (nt 289–322) near the 5' end of FosC CDS, containing a polyC motif, is required for N4BP1-mediated degradation. Both the KH domain and the NYN (YacP-like nuclease) domain are essential for N4BP1's mRNA-degrading activity. N4BP1-mediated mRNA turnover is independent of nonsense-mediated decay (NMD), as it is preserved in UPF1/UPF3A/UPF3B-deficient cells.\",\n      \"method\": \"Domain deletion mutagenesis, point mutations, reporter assays, CDS/3'UTR swap experiments, NMD-deficient cell lines (UPF1/UPF3A/UPF3B KO), RNA immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — domain mutagenesis defining catalytic requirement, multiple mRNA substrates tested, NMD-independent mechanism established using multiple KO cell lines, single lab\",\n      \"pmids\": [\"39491646\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Enteroviral 3C protease (3Cpro) from multiple human enteroviruses cleaves N4BP1 at residue Q816, destroying its ability to regulate TNFα-induced NF-κB signaling. Mouse N4BP1, which has a threonine at the P1' site, is resistant to human enteroviral 3Cpro, but rodent EMCV 3Cpro cleaves both human and mouse N4BP1 at species-specific sites.\",\n      \"method\": \"FIMO motif search, biochemical cleavage assays, cell biology experiments with 3Cpro expression, site mutagenesis (Q816), NF-κB signaling readouts\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cleavage site mutagenesis, endogenous N4BP1 cleavage during infection confirmed, functional consequence on NF-κB shown, single lab\",\n      \"pmids\": [\"39655957\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"N4BP1 is a nucleocytoplasmic shuttling protein. Nuclear accumulation is induced by leptomycin B (blocking CRM1-mediated export). A nuclear localization signal (NLS) spanning residues 279–299 is required for nuclear import; its deletion abolishes nuclear import and fusion to GFP drives GFP into the nucleus. N4BP1 forms liquid-liquid phase separation aggregates in both cytoplasm and nucleus that colocalize with NEDD8-modified proteins (including cullin-1 and cullin-2), dependent on the CoCUN domain. Heat shock promotes N4BP1 aggregation, which confers cellular protection under stress.\",\n      \"method\": \"Leptomycin B treatment, NLS deletion and GFP fusion constructs, live imaging, 1,6-hexanediol phase separation assay, co-localization with neddylated proteins, heat shock experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — NLS mutagenesis with GFP fusion rescue, pharmacological disruption of phase separation, co-localization with neddylated cullins, single lab\",\n      \"pmids\": [\"40701250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"N4BP1 suppresses IL-17 signaling by blocking translation of Act1 (TRAF3IP2) mRNA rather than affecting its stability. In N4BP1-deficient cells, Act1 mRNA is enriched in the translationally active polysome fraction, leading to elevated Act1 protein, hyperactivation of p38 MAPK, and increased downstream cytokines (CXCL1, CCL20, MMP9).\",\n      \"method\": \"Polysome profiling, mRNA stability assays, N4BP1 KO cells and mice, Act1 shRNA rescue experiments, p38 pharmacological inhibition, IMQ-induced skin model\",\n      \"journal\": \"Inflammation research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — polysome profiling directly demonstrates translational mechanism, KO rescue by Act1 silencing, in vivo corroboration, single lab\",\n      \"pmids\": [\"41504891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"N4BP1 is identified as a negative regulator of NF-κB that is targeted by poxvirus effector myxoma virus M3.1; N4BP1 inactivation by M3.1 unleashes an NF-κB ETI response, placing N4BP1 functionally alongside ZC3H12A and TBK1 as anti-viral NF-κB suppressors that can serve as pathogen sensors.\",\n      \"method\": \"Virulence factor expression screen in human monocytes, RNA-seq transcriptional response assays\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single screen-based approach, indirect evidence that N4BP1 inactivation mediates NF-κB activation, no direct mechanistic dissection of N4BP1\",\n      \"pmids\": [\"bio_10.1101_2025.03.04.641538\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"N4BP1 is a multifunctional immune checkpoint protein that acts as an endoribonuclease (via its NYN/NYN-like RNase domain and KH domain) to degrade mRNA substrates through their coding sequences, and simultaneously suppresses NF-κB signaling by binding NEMO to block its oligomerization and by acting as a dimerization-dependent linear ubiquitin reader (LUBIN module) in the TNFR1 complex; N4BP1 is constitutively active against MyD88-dependent TLRs and TNF signaling but is inactivated by caspase-8-mediated cleavage (downstream of TRIF, death receptors, or MALT1) or by viral proteases (3Cpro), and its stability is regulated by Nedd4-mediated polyubiquitylation (opposed by SUMO1 conjugation), with nuclear–cytoplasmic shuttling governed by an NLS (residues 279–299) and phase-separation aggregate formation linked to neddylated cullins via its CoCUN ubiquitin-binding domain.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"N4BP1 is a multifunctional immune-restriction protein that couples post-transcriptional silencing of inflammatory transcripts to direct suppression of NF-\\u03baB signaling, integrating its activity into checkpoints that limit antiviral and inflammatory output [#4, #9]. As an endoribonuclease, it uses both its KH domain and a PilT N-terminus (NYN/YacP-like) RNase domain to bind and degrade target mRNAs through their coding sequences\\u2014recognizing a polyC-containing element near the 5' CDS\\u2014in a manner independent of nonsense-mediated decay [#2, #10]; through this activity it restricts HIV-1 by degrading viral mRNA [#2] and limits inflammation by destabilizing JunB, FosB, and chemokine transcripts in keratinocytes and neutrophils [#6], while a parallel mode blocks translation of the IL-17 adaptor Act1 (TRAF3IP2) to restrain p38 MAPK\\u2013driven cytokine production [#13]. In NF-\\u03baB control, N4BP1 is a constitutive suppressor of MyD88-dependent TLR (TLR1/2, TLR7, TLR9) and TNF responses [#4]; it binds the NEMO COZI domain via UBA-like and CUE-like regions to block IKK\\u03b3 oligomerization [#5], reads linear (M1) polyubiquitin chains at the nascent TNFR1 complex through a dimerization-dependent LUBIN module to limit signaling duration and restrain complex II\\u2013driven cell death [#8], and operates together with the non-canonical kinases TBK1/IKK\\u03b5 and the adaptor TANK in a polyubiquitin-binding-dependent manner [#9]. N4BP1 is switched off by proteolysis: caspase-8 cleaves it downstream of TRIF, death receptors, and MALT1 to license TLR3/4 and death-receptor cytokine output and HIV reactivation [#2, #4, #5], and enteroviral 3C protease cleaves it at Q816 to relieve NF-\\u03baB restriction [#11]. The protein additionally binds the E3 ligase Itch to inhibit substrate ubiquitylation [#0] and antagonizes Notch signaling by promoting Trim21-dependent degradation of NICD via its CoCUN domain [#7]. Its abundance and localization are regulated by Nedd4-mediated polyubiquitylation opposed by SUMO1 conjugation [#1], an NLS (residues 279\\u2013299) governing CRM1-dependent nucleocytoplasmic shuttling, and CoCUN-dependent phase separation with neddylated cullins under stress [#12].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established the first molecular activity for N4BP1\\u2014as a competitive inhibitor of the E3 ligase Itch\\u2014answering whether it participates in ubiquitin pathway regulation.\",\n      \"evidence\": \"Co-IP, in vitro and in vivo ubiquitylation assays, RNAi and knockout cells\",\n      \"pmids\": [\"17592138\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address RNase or NF-\\u03baB functions later attributed to N4BP1\", \"Physiological context of Itch antagonism not defined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined how N4BP1 protein levels and subnuclear localization are controlled, showing a Nedd4-ubiquitylation/SUMO1 balance and nucleolar/PML body residence.\",\n      \"evidence\": \"Ubiquitylation and SUMOylation assays, immunofluorescence, SENP1-mutant fibroblasts, proteasome inhibition\",\n      \"pmids\": [\"20233849\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of nucleolar vs PML localization unresolved\", \"Link between stability control and downstream signaling roles not established\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified N4BP1 as an NYN-domain endoribonuclease that restricts HIV-1 and is inactivated by MALT1 cleavage, defining both an enzymatic activity and a switch-off mechanism.\",\n      \"evidence\": \"mRNA degradation assays, Arg509 mutagenesis, MALT1 KO, primary T cell/macrophage infection\",\n      \"pmids\": [\"31133753\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrate sequence determinants not yet defined\", \"Mechanism of RNA target selection unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Structurally characterized the C-terminal CoCUN domain as a ubiquitin-binding module distinct from CUBAN, defining how N4BP1 reads ubiquitin.\",\n      \"evidence\": \"NMR chemical shift perturbation, mutagenesis, circular dichroism, sequence analysis\",\n      \"pmids\": [\"31319543\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In-cell function of CoCUN ubiquitin reading not yet linked to a pathway\", \"Selectivity for chain linkage types not addressed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Placed N4BP1 as a constitutive suppressor of MyD88-dependent TLRs and TNF that is licensed off by caspase-8 cleavage, establishing it as an inflammatory checkpoint.\",\n      \"evidence\": \"N4BP1 KO mice, caspase-8-deficient macrophages, N4BP1/caspase-8 double-KO rescue, cytokine and cleavage assays\",\n      \"pmids\": [\"32971525\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism by which N4BP1 suppresses signaling not defined here\", \"Whether RNase activity mediates the cytokine suppression unaddressed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Provided a molecular mechanism for NF-\\u03baB suppression\\u2014direct binding to the NEMO COZI domain to block IKK oligomerization\\u2014and mapped the caspase-8 cleavage that abolishes it.\",\n      \"evidence\": \"Co-IP, in vitro binding, domain deletion mapping, N4bp1-deficient mice, cleavage site mutagenesis\",\n      \"pmids\": [\"33654074\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relationship between NEMO binding and ubiquitin reading not resolved\", \"Stoichiometry of NEMO inhibition unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Connected N4BP1 RNase activity to specific inflammatory mRNA targets and disease, showing destabilization of JunB/FosB and chemokine transcripts and a psoriasis-like phenotype in KO mice.\",\n      \"evidence\": \"RNA-IP, transcriptome profiling, N4BP1 KO mice, keratinocyte proliferation assays, IMQ psoriasis model\",\n      \"pmids\": [\"33990547\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Direct demonstration that catalytic RNase activity drives each phenotype not shown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Extended N4BP1 function to Notch signaling, showing CoCUN-dependent, Trim21-mediated NICD degradation and a role in neural progenitor differentiation.\",\n      \"evidence\": \"Protein binding assays, Phe-Pro motif mutagenesis, Trim21 KO, progenitor overexpression/KO, cortical development assays\",\n      \"pmids\": [\"37807845\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"How CoCUN ubiquitin binding directs NICD to Trim21 mechanistically unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined the LUBIN module as a dimerization-dependent linear-ubiquitin reader that recruits N4BP1 to the TNFR1 complex to limit signaling duration and cell death.\",\n      \"evidence\": \"Structural modeling, mutagenesis, TNFR1 complex IP, KO functional assays, ubiquitin-chain binding, caspase-8 cleavage\",\n      \"pmids\": [\"38643192\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"High-resolution structure of LUBIN\\u2013M1 chain interaction not determined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated genetic cooperation of N4BP1 with TBK1/IKK\\u03b5 and TANK in a ubiquitin-binding-dependent manner to terminate TLR-driven IKK signaling.\",\n      \"evidence\": \"N4BP1 KO and ubiquitin-binding mutant knock-in mice, TBK1/IKK\\u03b5/TANK KO macrophages, cytokine assays, epistasis\",\n      \"pmids\": [\"38697117\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biochemical nature of the N4BP1\\u2013TBK1/TANK complex not resolved\", \"Whether the same module mediates both TLR and TNFR1 effects unaddressed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Resolved the RNA-degradation mechanism, showing CDS-targeted, polyC-element-dependent cleavage requiring KH and NYN domains and independent of NMD.\",\n      \"evidence\": \"Domain/point mutagenesis, reporter and CDS/3'UTR swap assays, UPF1/UPF3A/UPF3B KO cells, RNA-IP\",\n      \"pmids\": [\"39491646\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full consensus of the targeted RNA element not defined\", \"How CDS engagement is coordinated with translation unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed that enteroviral 3C protease cleaves N4BP1 at Q816 to relieve NF-\\u03baB restriction, identifying a viral evasion strategy and species-specific susceptibility.\",\n      \"evidence\": \"Motif search, cleavage assays, 3Cpro expression in cells, Q816 mutagenesis, NF-\\u03baB readouts\",\n      \"pmids\": [\"39655957\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Whether cleavage affects RNase function in addition to NF-\\u03baB not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Characterized N4BP1 trafficking and biophysics, defining an NLS, CRM1-dependent export, and CoCUN-dependent phase separation with neddylated cullins that confers stress protection.\",\n      \"evidence\": \"Leptomycin B, NLS deletion/GFP fusion, live imaging, 1,6-hexanediol assay, co-localization with neddylated cullins, heat shock\",\n      \"pmids\": [\"40701250\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Functional link between phase separation and RNase/NF-\\u03baB roles not established\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identified a translational-control mechanism, showing N4BP1 blocks Act1 (TRAF3IP2) mRNA translation to restrain IL-17/p38 signaling, distinct from mRNA destabilization.\",\n      \"evidence\": \"Polysome profiling, mRNA stability assays, N4BP1 KO cells/mice, Act1 shRNA rescue, p38 inhibition, IMQ skin model\",\n      \"pmids\": [\"41504891\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"How N4BP1 selects translational vs degradative fate for different mRNAs unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how N4BP1 integrates its distinct activities\\u2014CDS-directed RNA cleavage, translational repression, NEMO/linear-ubiquitin-based NF-\\u03baB suppression, and E3-ligase modulation\\u2014into a unified regulatory logic, and which activity dominates in a given cell type or stimulus.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No single-molecule or structural framework unifying the RNase and ubiquitin-reader modules\", \"Determinants selecting between degradation, translational block, and signaling roles undefined\", \"Crosstalk between localization/phase separation and effector functions unmapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [2, 10]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [2, 6, 10]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 4, 5, 9]},\n      {\"term_id\": \"GO:0045182\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"GO:0031386\", \"supporting_discovery_ids\": [3, 8, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 12]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [12]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [4, 5, 9]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [5, 8, 9]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [2, 10]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [2, 11]}\n    ],\n    \"complexes\": [\n      \"TNFR1 signaling complex\"\n    ],\n    \"partners\": [\n      \"ITCH\",\n      \"IKBKG\",\n      \"MALT1\",\n      \"CASP8\",\n      \"TRIM21\",\n      \"TBK1\",\n      \"IKBKE\",\n      \"TANK\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}