{"gene":"NLRP4","run_date":"2026-04-29T11:37:57","timeline":{"discoveries":[{"year":2002,"finding":"NLRP4 (then called PAN2/PAAD-NACHT-containing protein 2) suppresses NF-κB activation induced by TNF-α and IL-1β signaling, acting downstream of TRAF2, TRAF6, RIP, IRAK2, and NIK as well as IKKα and IKKβ. Co-immunoprecipitation demonstrated direct association of NLRP4 with IKKα, and in vitro kinase assays showed NLRP4 inhibits IKKα/β activity.","method":"Co-immunoprecipitation, NF-κB reporter assays, EMSA, in vitro kinase assays, overexpression in HEK293 cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 — reciprocal Co-IP plus functional kinase assay in single study; multiple orthogonal methods but single lab","pmids":["12093792"],"is_preprint":false},{"year":2002,"finding":"NLRP4 (as PYPAF7) recruits to distinct cytoplasmic loci upon co-expression with ASC and synergistically activates NF-κB and caspase-1-dependent IL-1β processing. Pro-caspase-1 was identified as a binding partner of ASC in this pathway.","method":"Co-expression and co-localization in HEK cells, NF-κB reporter assays, caspase-1 activity assays, mammalian two-hybrid screen","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 — multiple functional readouts in single study, single lab; note later work shows NLRP4 does NOT interact with ASC, so this finding is contested","pmids":["12019269"],"is_preprint":false},{"year":2004,"finding":"The NACHT domain of NLRP4 (as PAN2) participates in heterotypic NACHT-domain interactions, co-immunoprecipitating with NACHT domains from CLAN, Nod1, Nod2, cryopyrin, NAC, and NAIP when co-expressed in HEK-293T cells, suggesting NLRP4 can form protein-interaction networks with other NLR family members.","method":"Co-immunoprecipitation in HEK-293T cells, NF-κB reporter assays","journal":"The Biochemical journal","confidence":"Low","confidence_rationale":"Tier 3 — single Co-IP experiment, single lab, overexpression system","pmids":["15107016"],"is_preprint":false},{"year":2007,"finding":"NLRP4, as a member of the NLR family, interacts with SGT1 and HSP90. Knockdown of SGT1 or chemical inhibition of HSP90 abrogated NLR-dependent inflammasome activity, placing SGT1 and HSP90 as conserved regulators of NLR function including NLRP4-containing complexes.","method":"siRNA knockdown, chemical inhibition (geldanamycin), co-immunoprecipitation, inflammasome activity assays","journal":"Nature immunology","confidence":"Medium","confidence_rationale":"Tier 2-3 — multiple NLR members tested including NLRP4, replicated across NLR family; specific NLRP4 interaction validated by Co-IP","pmids":["17435760"],"is_preprint":false},{"year":2011,"finding":"NLRP4 negatively regulates autophagy by binding Beclin1 through its PYD (NACHT domain region), with NLRP4 showing the strongest affinity among tested NLRs to the Beclin1 evolutionarily conserved domain. Upon Group A Streptococcus infection, NLRP4 is recruited to sub-plasma membrane phagosomes and transiently dissociates from Beclin1 to permit autophagic responses. NLRP4 also physically associates with the class C vacuolar protein-sorting (VPS) complex to negatively regulate autophagosome and endosome maturation.","method":"Co-immunoprecipitation, siRNA knockdown, immunofluorescence/confocal microscopy, bacterial infection assays, autophagic flux measurements","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (Co-IP, localization, functional KD phenotype), two distinct complexes identified (Beclin1 and VPS-C), replicated across bacterial infection models","pmids":["21209283"],"is_preprint":false},{"year":2012,"finding":"NLRP4 negatively regulates type I interferon signaling by recruiting the E3 ubiquitin ligase DTX4 to TBK1, promoting K48-linked polyubiquitination of TBK1 at Lys670 and its proteasomal degradation. Knockdown of either NLRP4 or DTX4 abolished K48-linked ubiquitination and degradation of TBK1, and enhanced TBK1 phosphorylation and IRF3 activation.","method":"Co-immunoprecipitation, siRNA knockdown, ubiquitination assays (K48-specific), immunoblot for TBK1 degradation and phospho-IRF3, dsRNA/dsDNA stimulation assays","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 1-2 — mechanistic ubiquitination site mapping (Lys670), K48-linkage specificity established, multiple orthogonal methods, high citation count indicating broad acceptance","pmids":["22388039"],"is_preprint":false},{"year":2012,"finding":"The crystal structure of the NLRP4 pyrin domain (PYD) was solved at 2.3 Å resolution, revealing a death domain superfamily fold with six α-helices. The NLRP4 PYD has unique structural features: an unusually long helix α3 and a distinct α2-α3 connecting loop compared to other PYDs. Despite a highly charged surface, NLRP4 PYD does not interact with the adaptor protein ASC, as demonstrated by co-immunoprecipitation, yeast two-hybrid, and NMR chemical shift perturbation analysis.","method":"X-ray crystallography (2.3 Å), co-immunoprecipitation, yeast two-hybrid, NMR chemical shift perturbation","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus three orthogonal binding assays; establishes both structure and negative interaction result with ASC","pmids":["22928810"],"is_preprint":false},{"year":2014,"finding":"Molecular dynamics simulations comparing NLRP4 PYD (as a representative native pyrin domain fold) with NLRP14 PYD revealed that intrinsic flexibility of NLRP pyrin domains is a key factor in their conformational dynamics, fold stability, and dimerization propensity. A charge relay system within NLRPs critically influences their conformational ensemble in solution.","method":"Molecular dynamics simulations, comparative structural analysis","journal":"Protein science","confidence":"Low","confidence_rationale":"Tier 4 — computational prediction only, no experimental validation of NLRP4-specific findings","pmids":["25403012"],"is_preprint":false},{"year":2015,"finding":"DYRK2 phosphorylates TBK1 at Ser527, and this phosphorylation is essential for the subsequent recruitment of NLRP4 and the E3 ubiquitin ligase DTX4 to TBK1, priming TBK1 for K48-linked ubiquitination and proteasomal degradation. This establishes DYRK2-mediated phosphorylation as an upstream event that gates NLRP4-DTX4-dependent TBK1 degradation.","method":"In vitro kinase assays, co-immunoprecipitation, ubiquitination assays, site-directed mutagenesis (Ser527), siRNA knockdown, interferon reporter assays","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro kinase assay plus mutagenesis defines phosphorylation site, Co-IP demonstrates NLRP4 recruitment dependence on Ser527 phosphorylation","pmids":["26407194"],"is_preprint":false},{"year":2016,"finding":"USP38 acts within the NLRP4 signalosome to negatively regulate type I IFN signaling. USP38 specifically cleaves K33-linked polyubiquitin chains from TBK1 at Lys670, removing them to allow subsequent K48-linked ubiquitination at the same site by DTX4 and TRIP (which are recruited via NLRP4). Knockout of USP38 increases K33-linked ubiquitination but abrogates K48-linked ubiquitination and TBK1 degradation.","method":"Co-immunoprecipitation, USP38 knockout (CRISPR), ubiquitination assays (K33- and K48-linkage specific), in vitro deubiquitination assays, interferon reporter assays, in vivo viral infection models","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro deubiquitination assay plus CRISPR KO plus linkage-specific ubiquitination analysis; mechanistically defines sequential ubiquitin editing at Lys670 within NLRP4 signalosome","pmids":["27692986"],"is_preprint":false},{"year":2017,"finding":"NLRP4 is recruited to Group A Streptococcus (GAS)-containing compartments and binds ARHGDIA (Rho GDP dissociation inhibitor α) to regulate Rho GTPase signaling, facilitating actin-mediated xenophagy. ARHGDIA Tyr156 phosphorylation acts as a regulatory gate for this interaction and for Rho-mediated autophagosome formation. Rho GTPases recruited via this NLRP4-ARHGDIA axis promote ATG9A recruitment to phagophores for autophagosome elongation.","method":"Co-immunoprecipitation, immunofluorescence microscopy, siRNA knockdown, bacterial infection assays (GAS), phosphorylation site mutagenesis (ARHGDIA Tyr156), autophagic flux assays","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 — Co-IP identifies novel binding partner, mutagenesis establishes regulatory phosphorylation site, functional assays link NLRP4-ARHGDIA to autophagosome formation","pmids":["29099277"],"is_preprint":false},{"year":2017,"finding":"NLRP4 negatively regulates both the TBK1/IRF3 and IKK/NF-κB signaling pathways in cardiac cells under fructose-induced injury. Overexpression of NLRP4 reduced pro-inflammatory cytokine release and TBK1 phosphorylation, while NLRP4 knockdown enhanced IKK/NF-κB activation, confirming NLRP4's role as a negative regulator of these inflammatory kinase cascades in cardiomyocytes.","method":"NLRP4 overexpression and siRNA knockdown, RT-qPCR, Western blotting for phospho-TBK1, phospho-IRF3, IKK activity, cytokine measurements, in vivo fructose-feeding mouse model","journal":"Biomedicine & pharmacotherapy","confidence":"Medium","confidence_rationale":"Tier 3 — gain- and loss-of-function with multiple signaling readouts but no direct biochemical interaction mapping; confirms known pathway roles in new cellular context","pmids":["28486191"],"is_preprint":false},{"year":2022,"finding":"NLRP4 knockdown in lung cancer cells enhanced type I IFN (IFN-α/β) production through the cGAS-STING-IRF3/IRF7 axis, placing NLRP4 as a negative regulator of cGAS-STING pathway signaling upstream of IRF3/IRF7 activation.","method":"NLRP4 siRNA knockdown, IFN-α/β ELISA, IRF3/IRF7 reporter assays, in vivo tumor models (Lewis lung cancer), flow cytometry for tumor-infiltrating CD8+ T cells","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2-3 — loss-of-function with defined pathway readouts (cGAS-STING-IRF3/7), in vitro and in vivo validation; pathway placement but no direct biochemical interaction with cGAS-STING components shown","pmids":["34927309"],"is_preprint":false},{"year":2024,"finding":"NLRP4 upregulation promotes olaparib resistance in pancreatic cancer by enhancing DNA repair capacity and inducing mitochondrial ROS production that drives autophagy. Co-IP mass spectrometry identified NLRP4 as involved in the DNA damage response and autophagy pathways. NLRP4-generated mitochondrial ROS promote autophagy without directly affecting DNA damage, and inhibition of mitochondrial ROS (MitoQ) or autophagy (chloroquine) sensitizes cells to olaparib.","method":"Co-IP mass spectrometry, RNA sequencing, NLRP4 overexpression/knockdown, mitochondrial ROS measurement, DNA damage assays, olaparib resistance assays in vitro and in vivo","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP MS identifies pathway membership, functional assays with specific inhibitors establish ROS-autophagy link; mechanism linking NLRP4 to mitochondrial ROS generation not fully defined","pmids":["39187531"],"is_preprint":false},{"year":2025,"finding":"NLRP4 overexpression suppresses PP2A (protein phosphatase 2A) activity and thereby activates the PI3K/Akt-NF-κB signaling axis in lung cancer, leading to chemokine reprogramming (CCL5 and CXCL2) that recruits TIGIT+TNFα+ NK cells and iNOS+ M1 macrophages. Co-IP and Western blot experiments validated NLRP4 interaction with components of this pathway.","method":"NLRP4 stable overexpression, co-immunoprecipitation, Western blotting, bulk RNA sequencing, proteomics, mass spectrometry, flow cytometry, multiplex immunofluorescence, subcutaneous tumor models in C57BL/6J mice","journal":"Biomarker research","confidence":"Medium","confidence_rationale":"Tier 2-3 — multiple omics approaches plus Co-IP validation; PP2A suppression by NLRP4 is novel mechanistic claim supported by functional rescue with PP2A inhibitor LB-100","pmids":["40087771"],"is_preprint":false}],"current_model":"NLRP4 is a cytosolic NLR family receptor that functions primarily as a negative regulator of innate immune signaling: it recruits the E3 ubiquitin ligase DTX4 (gated by DYRK2-mediated phosphorylation of TBK1 at Ser527) to promote K48-linked ubiquitination and proteasomal degradation of TBK1 at Lys670, with the deubiquitinase USP38 acting within this NLRP4 signalosome to edit K33-linked chains on TBK1 to permit subsequent K48-linked degradation; NLRP4 also suppresses IKK/NF-κB activity through direct association with IKKα, inhibits the cGAS-STING-IRF3/7 axis, negatively regulates autophagy by binding Beclin1 (via its PYD) and associating with the class C VPS complex, and during bacterial infection is recruited to pathogen-containing vacuoles where it binds ARHGDIA to direct Rho GTPase-actin dynamics for xenophagy; structurally, the NLRP4 PYD adopts a six-helix death domain fold with unique α3 features that prevent ASC interaction."},"narrative":{"teleology":[{"year":2002,"claim":"Initial characterization established that NLRP4 suppresses NF-κB signaling by directly binding IKKα and inhibiting IKK kinase activity, positioning it as a negative regulator of innate inflammatory signaling rather than a canonical inflammasome activator.","evidence":"Co-immunoprecipitation, NF-κB reporter assays, EMSA, and in vitro kinase assays in HEK293 cells","pmids":["12093792"],"confidence":"Medium","gaps":["Single-lab study without independent replication","Mechanism by which NLRP4 inhibits IKK kinase activity not defined","Endogenous expression context not examined"]},{"year":2002,"claim":"A parallel study reported that NLRP4 co-expression with ASC synergistically activated NF-κB and caspase-1-dependent IL-1β processing, suggesting inflammasome-like function; this was later contradicted by structural and biochemical evidence showing NLRP4 PYD does not interact with ASC.","evidence":"Co-expression in HEK cells, NF-κB reporter and caspase-1 activity assays","pmids":["12019269"],"confidence":"Medium","gaps":["ASC interaction was subsequently refuted by crystal structure and three orthogonal binding assays (PMID:22928810)","Overexpression artifacts not excluded"]},{"year":2011,"claim":"NLRP4 was shown to be a negative regulator of autophagy, binding Beclin 1 through its PYD with the highest affinity among NLRs tested and associating with the class C VPS complex to restrain autophagosome/endosome maturation, establishing a non-inflammasome function for the protein.","evidence":"Co-immunoprecipitation, siRNA knockdown, confocal microscopy, and bacterial infection assays with Group A Streptococcus","pmids":["21209283"],"confidence":"High","gaps":["Structural basis of Beclin 1–PYD interaction not resolved","Whether NLRP4 dissociation from Beclin 1 during infection is actively regulated remains unknown"]},{"year":2012,"claim":"The central mechanism of NLRP4-mediated type I IFN suppression was elucidated: NLRP4 recruits the E3 ligase DTX4 to TBK1, promoting site-specific K48-linked ubiquitination at Lys670 and proteasomal degradation, establishing a direct biochemical pathway for IFN attenuation.","evidence":"Co-immunoprecipitation, siRNA knockdown, K48-linkage-specific ubiquitination assays, TBK1 degradation immunoblots, dsRNA/dsDNA stimulation","pmids":["22388039"],"confidence":"High","gaps":["How NLRP4 is activated or derepressed to engage TBK1 was not defined","Physiological triggers for NLRP4–DTX4 complex assembly unclear"]},{"year":2012,"claim":"Crystal structure of NLRP4 PYD at 2.3 Å resolved its six-helix death-domain fold with a uniquely elongated α3 helix, and definitively demonstrated that it does not interact with ASC, clarifying why NLRP4 does not form a canonical inflammasome.","evidence":"X-ray crystallography, co-immunoprecipitation, yeast two-hybrid, NMR chemical shift perturbation","pmids":["22928810"],"confidence":"High","gaps":["No full-length NLRP4 structure available","Binding partners that do engage the PYD (beyond Beclin 1) not structurally mapped"]},{"year":2015,"claim":"DYRK2 was identified as the kinase that phosphorylates TBK1 at Ser527, establishing this modification as a prerequisite for NLRP4 and DTX4 recruitment, thereby defining the upstream gating event for the NLRP4-dependent TBK1 degradation pathway.","evidence":"In vitro kinase assays, Ser527 mutagenesis, co-immunoprecipitation, ubiquitination assays, interferon reporter assays","pmids":["26407194"],"confidence":"High","gaps":["Whether DYRK2 phosphorylation is the sole gating event or acts cooperatively with additional signals is unknown"]},{"year":2016,"claim":"USP38 was placed within the NLRP4 signalosome as a deubiquitinase that edits K33-linked ubiquitin chains from TBK1 Lys670 to permit subsequent K48-linked ubiquitination by DTX4/TRIP, establishing a sequential ubiquitin-editing mechanism for TBK1 turnover.","evidence":"USP38 CRISPR knockout, K33- and K48-linkage-specific ubiquitination assays, in vitro deubiquitination, in vivo viral infection models","pmids":["27692986"],"confidence":"High","gaps":["How the switch from K33 to K48 ubiquitin chains is temporally coordinated is not fully resolved","Whether additional DUBs participate in NLRP4 signalosome ubiquitin editing is unknown"]},{"year":2017,"claim":"NLRP4 was found to bind ARHGDIA at pathogen-containing vacuoles, directing Rho GTPase activation and actin dynamics for xenophagy; ARHGDIA Tyr156 phosphorylation gates this interaction, linking NLRP4 to cytoskeletal control during antibacterial autophagy.","evidence":"Co-immunoprecipitation, immunofluorescence, siRNA knockdown, ARHGDIA Y156 mutagenesis, GAS infection assays, autophagic flux assays","pmids":["29099277"],"confidence":"High","gaps":["Kinase responsible for ARHGDIA Tyr156 phosphorylation not identified","Whether NLRP4-ARHGDIA interaction operates beyond GAS infection is untested"]},{"year":2022,"claim":"NLRP4 was demonstrated to suppress the cGAS–STING–IRF3/IRF7 axis, broadening its role as a negative regulator of cytosolic nucleic acid sensing beyond TBK1 degradation alone.","evidence":"NLRP4 siRNA knockdown, IFN-α/β ELISA, IRF3/IRF7 reporter assays, Lewis lung cancer tumor models, flow cytometry for CD8+ T cells","pmids":["34927309"],"confidence":"Medium","gaps":["Direct biochemical interaction with cGAS or STING components not shown","Whether suppression occurs through TBK1 degradation or an independent mechanism is unresolved"]},{"year":2025,"claim":"A new axis was reported in which NLRP4 suppresses PP2A phosphatase activity to activate PI3K/Akt–NF-κB signaling, driving chemokine reprogramming that shapes the tumor immune microenvironment, revealing context-dependent pro-tumorigenic functions.","evidence":"NLRP4 stable overexpression, co-immunoprecipitation, proteomics, bulk RNA-seq, PP2A inhibitor rescue, subcutaneous tumor models","pmids":["40087771"],"confidence":"Medium","gaps":["Mechanism by which NLRP4 inhibits PP2A catalytic activity is undefined","Apparent contradiction with canonical NF-κB-suppressive role not reconciled","Single-study claim requiring independent replication"]},{"year":null,"claim":"Key unresolved questions include how NLRP4 is itself activated or regulated at the protein level, the full-length structure and oligomeric state, the mechanism linking NLRP4 to mitochondrial ROS and DNA repair, and reconciliation of its context-dependent pro- versus anti-inflammatory roles.","evidence":"","pmids":[],"confidence":"Low","gaps":["No full-length structure or cryo-EM model","Ligand or danger signal that activates NLRP4 is unknown","NLRP4 post-translational regulation poorly characterized","Context-dependent switching between NF-κB suppression and PP2A-mediated NF-κB activation unexplained"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,5,8,9,14]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[5,9,10]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,4,5]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[4,10]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,5,8,9,12]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[4,10,13]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,11,14]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[5,9]}],"complexes":["NLRP4-DTX4-TBK1 signalosome","NLRP4-Beclin1-class C VPS complex"],"partners":["TBK1","DTX4","IKBKA","BECN1","ARHGDIA","USP38","DYRK2","SGT1"],"other_free_text":[]},"mechanistic_narrative":"NLRP4 is a cytoplasmic NLR family member that functions predominantly as a negative regulator of innate immune signaling and autophagy. It suppresses type I interferon production by scaffolding a signalosome in which DYRK2-mediated phosphorylation of TBK1 at Ser527 enables NLRP4 to recruit the E3 ligase DTX4 for K48-linked ubiquitination and proteasomal degradation of TBK1 at Lys670, a process gated by USP38-dependent removal of competing K33-linked ubiquitin chains [PMID:22388039, PMID:26407194, PMID:27692986]. NLRP4 also inhibits NF-κB signaling through direct association with IKKα and suppression of IKK kinase activity, and negatively regulates the cGAS–STING–IRF3/7 axis [PMID:12093792, PMID:34927309]. In parallel, NLRP4 restrains autophagy by binding Beclin 1 via its PYD and associating with the class C VPS complex, yet upon bacterial infection it is recruited to pathogen-containing vacuoles where it engages ARHGDIA to redirect Rho GTPase–actin dynamics for xenophagy [PMID:21209283, PMID:29099277]."},"prefetch_data":{"uniprot":{"accession":"Q96MN2","full_name":"NACHT, LRR and PYD domains-containing protein 4","aliases":["Cancer/testis antigen 58","CT58","PAAD and NACHT-containing protein 2","PAN2","PYRIN and NACHT-containing protein 2","PYRIN-containing APAF1-like protein 4","PYPAF4","Ribonuclease inhibitor 2"],"length_aa":994,"mass_kda":113.4,"function":"May be involved in inflammation and recognition of cytosolic pathogen-associated molecular patterns (PAMPs) not intercepted by membrane-bound receptors. Acts as a negative regulator of the type I interferon signaling pathway by serving as an adapter to promote DTX4-mediated ubiquitination of activated TBK1, and its subsequent degradation. Suppresses NF-kappaB induction by the cytokines TNFA and IL1B, suggesting that it operates at a point of convergence in these two cytokine signaling pathways","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q96MN2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NLRP4","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"DDX6","stoichiometry":0.2},{"gene":"GSPT1","stoichiometry":0.2},{"gene":"PABPC4","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/NLRP4","total_profiled":1310},"omim":[{"mim_id":"616110","title":"DELTEX E3 UBIQUITIN LIGASE 4; DTX4","url":"https://www.omim.org/entry/616110"},{"mim_id":"609645","title":"NLR FAMILY, PYRIN DOMAIN-CONTAINING 4; NLRP4","url":"https://www.omim.org/entry/609645"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Mitochondria","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"lymphoid tissue","ntpm":4.7},{"tissue":"testis","ntpm":6.6}],"url":"https://www.proteinatlas.org/search/NLRP4"},"hgnc":{"alias_symbol":["PYPAF4","FLJ32126","PAN2","RNH2","CLR19.5","CT58"],"prev_symbol":["NALP4"]},"alphafold":{"accession":"Q96MN2","domains":[{"cath_id":"1.10.533.10","chopping":"2-94","consensus_level":"high","plddt":79.4889,"start":2,"end":94},{"cath_id":"3.40.50,3.40.50","chopping":"96-298","consensus_level":"high","plddt":85.8431,"start":96,"end":298},{"cath_id":"-","chopping":"370-409_421-554","consensus_level":"medium","plddt":89.6337,"start":370,"end":554},{"cath_id":"3.80.10.10","chopping":"825-994","consensus_level":"medium","plddt":93.1664,"start":825,"end":994},{"cath_id":"1.10.1900","chopping":"303-366","consensus_level":"medium","plddt":92.5761,"start":303,"end":366}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96MN2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96MN2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96MN2-F1-predicted_aligned_error_v6.png","plddt_mean":89.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NLRP4","jax_strain_url":"https://www.jax.org/strain/search?query=NLRP4"},"sequence":{"accession":"Q96MN2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96MN2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96MN2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96MN2"}},"corpus_meta":[{"pmid":"22388039","id":"PMC_22388039","title":"NLRP4 negatively regulates type I interferon signaling by targeting the kinase TBK1 for degradation via the ubiquitin ligase DTX4.","date":"2012","source":"Nature immunology","url":"https://pubmed.ncbi.nlm.nih.gov/22388039","citation_count":247,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"23337855","id":"PMC_23337855","title":"RNA decay machines: deadenylation by the Ccr4-not and Pan2-Pan3 complexes.","date":"2013","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/23337855","citation_count":193,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"8550599","id":"PMC_8550599","title":"The yeast Pan2 protein is required for poly(A)-binding protein-stimulated poly(A)-nuclease activity.","date":"1996","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8550599","citation_count":153,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"21209283","id":"PMC_21209283","title":"NLRP4 negatively regulates 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Molecular cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/12563287","citation_count":554,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15489334","id":"PMC_15489334","title":"The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).","date":"2004","source":"Genome research","url":"https://pubmed.ncbi.nlm.nih.gov/15489334","citation_count":438,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"26344197","id":"PMC_26344197","title":"Panorama of ancient metazoan macromolecular complexes.","date":"2015","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/26344197","citation_count":407,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"17435760","id":"PMC_17435760","title":"A crucial function of SGT1 and HSP90 in inflammasome activity links mammalian and plant innate immune responses.","date":"2007","source":"Nature immunology","url":"https://pubmed.ncbi.nlm.nih.gov/17435760","citation_count":356,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"12019269","id":"PMC_12019269","title":"PYPAF7, a novel PYRIN-containing Apaf1-like protein that regulates activation of NF-kappa B and caspase-1-dependent cytokine processing.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12019269","citation_count":314,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"12387869","id":"PMC_12387869","title":"Functional screening of five PYPAF family members identifies PYPAF5 as a novel regulator of NF-kappaB and caspase-1.","date":"2002","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/12387869","citation_count":245,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"14703513","id":"PMC_14703513","title":"Identification of Ser-386 of interferon regulatory factor 3 as critical target for inducible phosphorylation that determines activation.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/14703513","citation_count":184,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15107016","id":"PMC_15107016","title":"Heterotypic interactions among NACHT domains: implications for regulation of innate immune responses.","date":"2004","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/15107016","citation_count":129,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"23376485","id":"PMC_23376485","title":"Proteomic analysis of podocyte exosome-enriched fraction from normal human urine.","date":"2013","source":"Journal of proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/23376485","citation_count":126,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"17420470","id":"PMC_17420470","title":"SGT1 is essential for Nod1 activation.","date":"2007","source":"Proceedings of the National Academy of Sciences of the United States of 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publication of the Protein Society","url":"https://pubmed.ncbi.nlm.nih.gov/25403012","citation_count":21,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"34257569","id":"PMC_34257569","title":"Somatic Mutation of NLRP Genes in Gastric and Colonic Cancers.","date":"2021","source":"Pathology oncology research : POR","url":"https://pubmed.ncbi.nlm.nih.gov/34257569","citation_count":9,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"40593736","id":"PMC_40593736","title":"Human protein interaction networks of ancestral and variant SARS-CoV-2 in organ-specific cells and bodily fluids.","date":"2025","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/40593736","citation_count":2,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16697,"output_tokens":2312,"usd":0.042385},"stage2":{"model":"claude-opus-4-6","input_tokens":5654,"output_tokens":2245,"usd":0.126592},"total_usd":0.427836,"stage1_batch_id":"msgbatch_012ZiiCUBfB62NCTQd4Bk1Jo","stage2_batch_id":"msgbatch_01Xuqq9aUHoQouS7UVGZNStZ","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":26288,"output_tokens":5688,"usd":0.082092},"round2_rules_fired":"R2","round2_stage2":{"model":"claude-opus-4-6","input_tokens":8289,"output_tokens":3056,"usd":0.176767}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2012,\n      \"finding\": \"NLRP4 recruits the E3 ubiquitin ligase DTX4 to TBK1, promoting K48-linked polyubiquitination of TBK1 at Lys670, leading to TBK1 proteasomal degradation and suppression of type I interferon signaling downstream of double-stranded RNA or DNA sensing.\",\n      \"method\": \"Co-immunoprecipitation, knockdown experiments, ubiquitination assays with K48-linkage specificity, phosphorylation analysis of TBK1 and IRF3\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, specific ubiquitin-linkage mapping, functional knockdown phenotype; highly cited foundational paper\",\n      \"pmids\": [\"22388039\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NLRP4 forms a signalosome with USP38 and DTX4/TRIP: USP38 removes K33-linked polyubiquitin from TBK1 at Lys670, enabling subsequent K48-linked ubiquitination by DTX4/TRIP (facilitated by NLRP4), thereby targeting active TBK1 for degradation and dampening type I IFN signaling.\",\n      \"method\": \"Co-immunoprecipitation, USP38 knockout/knockdown, in vitro deubiquitinase assays, linkage-specific ubiquitination assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro assay plus KO/KD with defined molecular mechanism, reciprocal Co-IP; replicated mechanistic node from prior NLRP4-DTX4 paper\",\n      \"pmids\": [\"27692986\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"NLRP4 interacts with Beclin1 through its PYD/NACHT domain and with the class C vacuolar protein-sorting (VPS-C) complex, negatively regulating autophagosome initiation and maturation; NLRP4 is transiently released from Beclin1 upon bacterial infection to permit autophagic bactericidal responses against Group A Streptococcus.\",\n      \"method\": \"Co-immunoprecipitation, RNA interference knockdown, autophagic flux assays, confocal localization during bacterial infection\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, RNAi loss-of-function with defined autophagy phenotype and bacterial clearance readout; well-cited\",\n      \"pmids\": [\"21209283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Crystal structure of the NLRP4 pyrin domain (PYD) at 2.3 Å resolution reveals a death-domain fold with six α-helices; unique helix α3 length and α2–α3 loop distinguish it from other PYDs. Functional experiments (co-immunoprecipitation, yeast two-hybrid, NMR chemical shift perturbation) demonstrate that NLRP4 PYD does not interact with the adaptor protein ASC, unlike other NLR PYDs.\",\n      \"method\": \"X-ray crystallography, co-immunoprecipitation, yeast two-hybrid, NMR chemical shift perturbation\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus three orthogonal binding assays confirming non-interaction with ASC\",\n      \"pmids\": [\"22928810\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NLRP4 binds ARHGDIA (Rho GDP dissociation inhibitor α) at GAS-containing autophagosome-like vacuoles (GcAVs) to regulate Rho GTPase signaling; ARHGDIA Tyr156 phosphorylation controls this interaction and is required for actin-mediated ATG9A recruitment to phagophores, facilitating xenophagy of Group A Streptococcus.\",\n      \"method\": \"Co-immunoprecipitation, confocal colocalization, knockdown, phosphorylation site mutation analysis\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP with phosphorylation mutant, defined subcellular localization with functional autophagy readout; single lab\",\n      \"pmids\": [\"29099277\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NLRP4 negatively regulates the IKK/NF-κB signaling pathway through direct interaction with IKK kinase, in addition to suppressing TBK1/IRF3 signaling, thereby attenuating pro-inflammatory cytokine production in cardiac cells.\",\n      \"method\": \"Overexpression and knockdown with western blotting, RT-qPCR for cytokine outputs\",\n      \"journal\": \"Biomedicine & pharmacotherapy\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, indirect functional readouts without direct binding or mechanistic reconstitution of IKK interaction\",\n      \"pmids\": [\"28486191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Knockdown of NLRP4 in lung cancer cells enhances IFN-α/β production through the cGAS-STING-IRF3/IRF7 axis, demonstrating that NLRP4 negatively regulates innate DNA-sensing signaling upstream of IRF3/IRF7 activation.\",\n      \"method\": \"NLRP4 knockdown with cytokine measurement, pathway inhibitor analysis, in vivo mouse tumor models\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KD with defined pathway placement (cGAS-STING-IRF3/7) and in vivo validation; consistent with prior TBK1 mechanism\",\n      \"pmids\": [\"34927309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NLRP4 promotes olaparib resistance in pancreatic cancer by enhancing DNA repair capacity and by generating mitochondrial ROS that drive autophagy; Co-IP/MS analysis confirmed NLRP4 involvement in DNA damage response and autophagy pathways.\",\n      \"method\": \"Co-IP mass spectrometry, RNA sequencing, ROS measurement, MitoQ and chloroquine inhibition, in vitro and in vivo olaparib sensitivity assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP MS plus pharmacological dissection; single lab with multiple orthogonal assays\",\n      \"pmids\": [\"39187531\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NLRP4 suppresses PP2A phosphatase activity, thereby activating the PI3K/Akt-NF-κB axis; this drives CCL5 and CXCL2 chemokine reprogramming that recruits TIGIT+TNFα+ NK cells and iNOS+ M1 macrophages in lung cancer.\",\n      \"method\": \"Co-immunoprecipitation, western blot, pharmacological PP2A inhibition (LB-100), co-culture transwell assays, bulk RNA-seq and proteomics\",\n      \"journal\": \"Biomarker research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP and indirect functional readouts; single lab, novel claim lacking in vitro reconstitution of PP2A inhibition\",\n      \"pmids\": [\"40087771\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NLRP4 is a cytosolic NLR-family receptor that acts as a negative regulator of innate immune signaling: it recruits the E3 ubiquitin ligase DTX4 (and cooperates with the deubiquitinase USP38) to catalyze K48-linked polyubiquitination and proteasomal degradation of TBK1 at Lys670, thereby suppressing type I interferon induction downstream of nucleic acid sensing; it also binds Beclin1 and the VPS-C complex to restrain autophagosome formation and maturation, and its unique PYD domain (structurally resolved at 2.3 Å) does not interact with the canonical inflammasome adaptor ASC, distinguishing NLRP4 from inflammasome-forming NLRs.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries only from KEEP papers.\n\n**Classification:**\n\nKEEP (canonical NLRP4 protein):\n- PMID:22388039 — NLRP4 regulates TBK1 degradation via DTX4\n- PMID:27692986 — USP38/NLRP4 signalosome regulates TBK1 ubiquitination\n- PMID:21209283 — NLRP4 regulates autophagy via Beclin1\n- PMID:22928810 — Crystal structure of NLRP4 pyrin domain\n- PMID:29099277 — NLRP4 directs Rho-actin signaling in xenophagy\n- PMID:28486191 — NLRP4 regulates IKK/NF-κB and TBK1/IRF3 in cardiac cells\n- PMID:39187531 — NLRP4 in DNA damage response and autophagy in pancreatic cancer\n- PMID:34927309 — NLRP4 knockdown enhances cGAS-STING-IRF3/7 signaling\n- PMID:40087771 — NLRP4 suppresses PP2A, activates PI3K/Akt-NF-κB\n- PMID:26407194 — DYRK2 phosphorylates TBK1 Ser527, recruiting NLRP4/DTX4\n- PMID:12093792 — PAN2 (=NLRP4 alias) suppresses NF-κB, associates with IKK\n- PMID:15107016 — NACHT domain heterotypic interactions including PAN2 (NLRP4)\n- PMID:12563287 — NALPs family overview (includes NLRP4 context)\n- PMID:12019269 — PYPAF7 (=NLRP4) activates NF-κB/caspase-1 with ASC\n- PMID:12387869 — PYPAF screening; NLRP4 (PYPAF3/PYPAF4) context\n- PMID:17435760 — SGT1/HSP90 interact with NLRs including NLRP4\n- PMID:25403012 — MD simulations of NLRP4 pyrin domain conformational dynamics\n- PMID:24871464 — Not NLRP4 specifically (NLRP2P pseudogene) — EXCLUDE\n- PMID:40395525 — NLRP4 in olaparib resistance (companion commentary) — KEEP\n\nEXCLUDE (alias collisions — PAN2 as deadenylase, maize PAN2, yeast Pan2-Pan3, etc.):\n- PMID:23337855, 8550599, 23932717, 31104843, 23175742, 24872509, 23398456, 31110294, 24450649, 24880344, 22402495, 12435598, 11953437, 24578508, 37936339, 34280615, 35304602, 12182447, 40491293, 41556236, 41275497, 41714623, bio_10.1101_2025.09.27.678968 — all about Pan2-Pan3 deadenylase complex or maize PAN2\n\nEXCLUDE from curated list:\n- PMID:12477932, 14702039, 21873635, 15489334, 26344197, 17420470, 23376485, 19074885, 20403135, 14703513, 34257569, 40593736 — not mechanistically about NLRP4\n\nNote on PMID:12019269 (PYPAF7): This paper describes PYPAF7 which maps to chromosome 19q13.4 — this is actually NLRP4's genomic locus. PYPAF7 is an alias for NLRP4. KEEP.\n\nNote on PMID:12387869: PYPAF3 = NLRP3, PYPAF4 = NLRP4? Need to check carefully. The paper screens PYPAF2/3/4/5/6 — PYPAF4 could be NLRP4, but the paper shows PYPAF4 \"failed to colocalize with ASC and activate NF-κB.\" This is relevant but minimal mechanistic detail for NLRP4 specifically. I'll include as low confidence.\n\nNote on PMID:12093792: PAN2 here = \"PAAD and NACHT-containing protein 2\" which is an older alias for NLRP4. KEEP.\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2002,\n      \"finding\": \"NLRP4 (then called PAN2/PAAD-NACHT-containing protein 2) suppresses NF-κB activation induced by TNF-α and IL-1β signaling, acting downstream of TRAF2, TRAF6, RIP, IRAK2, and NIK as well as IKKα and IKKβ. Co-immunoprecipitation demonstrated direct association of NLRP4 with IKKα, and in vitro kinase assays showed NLRP4 inhibits IKKα/β activity.\",\n      \"method\": \"Co-immunoprecipitation, NF-κB reporter assays, EMSA, in vitro kinase assays, overexpression in HEK293 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — reciprocal Co-IP plus functional kinase assay in single study; multiple orthogonal methods but single lab\",\n      \"pmids\": [\"12093792\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"NLRP4 (as PYPAF7) recruits to distinct cytoplasmic loci upon co-expression with ASC and synergistically activates NF-κB and caspase-1-dependent IL-1β processing. Pro-caspase-1 was identified as a binding partner of ASC in this pathway.\",\n      \"method\": \"Co-expression and co-localization in HEK cells, NF-κB reporter assays, caspase-1 activity assays, mammalian two-hybrid screen\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — multiple functional readouts in single study, single lab; note later work shows NLRP4 does NOT interact with ASC, so this finding is contested\",\n      \"pmids\": [\"12019269\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The NACHT domain of NLRP4 (as PAN2) participates in heterotypic NACHT-domain interactions, co-immunoprecipitating with NACHT domains from CLAN, Nod1, Nod2, cryopyrin, NAC, and NAIP when co-expressed in HEK-293T cells, suggesting NLRP4 can form protein-interaction networks with other NLR family members.\",\n      \"method\": \"Co-immunoprecipitation in HEK-293T cells, NF-κB reporter assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP experiment, single lab, overexpression system\",\n      \"pmids\": [\"15107016\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"NLRP4, as a member of the NLR family, interacts with SGT1 and HSP90. Knockdown of SGT1 or chemical inhibition of HSP90 abrogated NLR-dependent inflammasome activity, placing SGT1 and HSP90 as conserved regulators of NLR function including NLRP4-containing complexes.\",\n      \"method\": \"siRNA knockdown, chemical inhibition (geldanamycin), co-immunoprecipitation, inflammasome activity assays\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — multiple NLR members tested including NLRP4, replicated across NLR family; specific NLRP4 interaction validated by Co-IP\",\n      \"pmids\": [\"17435760\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"NLRP4 negatively regulates autophagy by binding Beclin1 through its PYD (NACHT domain region), with NLRP4 showing the strongest affinity among tested NLRs to the Beclin1 evolutionarily conserved domain. Upon Group A Streptococcus infection, NLRP4 is recruited to sub-plasma membrane phagosomes and transiently dissociates from Beclin1 to permit autophagic responses. NLRP4 also physically associates with the class C vacuolar protein-sorting (VPS) complex to negatively regulate autophagosome and endosome maturation.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, immunofluorescence/confocal microscopy, bacterial infection assays, autophagic flux measurements\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (Co-IP, localization, functional KD phenotype), two distinct complexes identified (Beclin1 and VPS-C), replicated across bacterial infection models\",\n      \"pmids\": [\"21209283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"NLRP4 negatively regulates type I interferon signaling by recruiting the E3 ubiquitin ligase DTX4 to TBK1, promoting K48-linked polyubiquitination of TBK1 at Lys670 and its proteasomal degradation. Knockdown of either NLRP4 or DTX4 abolished K48-linked ubiquitination and degradation of TBK1, and enhanced TBK1 phosphorylation and IRF3 activation.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, ubiquitination assays (K48-specific), immunoblot for TBK1 degradation and phospho-IRF3, dsRNA/dsDNA stimulation assays\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mechanistic ubiquitination site mapping (Lys670), K48-linkage specificity established, multiple orthogonal methods, high citation count indicating broad acceptance\",\n      \"pmids\": [\"22388039\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The crystal structure of the NLRP4 pyrin domain (PYD) was solved at 2.3 Å resolution, revealing a death domain superfamily fold with six α-helices. The NLRP4 PYD has unique structural features: an unusually long helix α3 and a distinct α2-α3 connecting loop compared to other PYDs. Despite a highly charged surface, NLRP4 PYD does not interact with the adaptor protein ASC, as demonstrated by co-immunoprecipitation, yeast two-hybrid, and NMR chemical shift perturbation analysis.\",\n      \"method\": \"X-ray crystallography (2.3 Å), co-immunoprecipitation, yeast two-hybrid, NMR chemical shift perturbation\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus three orthogonal binding assays; establishes both structure and negative interaction result with ASC\",\n      \"pmids\": [\"22928810\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Molecular dynamics simulations comparing NLRP4 PYD (as a representative native pyrin domain fold) with NLRP14 PYD revealed that intrinsic flexibility of NLRP pyrin domains is a key factor in their conformational dynamics, fold stability, and dimerization propensity. A charge relay system within NLRPs critically influences their conformational ensemble in solution.\",\n      \"method\": \"Molecular dynamics simulations, comparative structural analysis\",\n      \"journal\": \"Protein science\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 — computational prediction only, no experimental validation of NLRP4-specific findings\",\n      \"pmids\": [\"25403012\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"DYRK2 phosphorylates TBK1 at Ser527, and this phosphorylation is essential for the subsequent recruitment of NLRP4 and the E3 ubiquitin ligase DTX4 to TBK1, priming TBK1 for K48-linked ubiquitination and proteasomal degradation. This establishes DYRK2-mediated phosphorylation as an upstream event that gates NLRP4-DTX4-dependent TBK1 degradation.\",\n      \"method\": \"In vitro kinase assays, co-immunoprecipitation, ubiquitination assays, site-directed mutagenesis (Ser527), siRNA knockdown, interferon reporter assays\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro kinase assay plus mutagenesis defines phosphorylation site, Co-IP demonstrates NLRP4 recruitment dependence on Ser527 phosphorylation\",\n      \"pmids\": [\"26407194\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"USP38 acts within the NLRP4 signalosome to negatively regulate type I IFN signaling. USP38 specifically cleaves K33-linked polyubiquitin chains from TBK1 at Lys670, removing them to allow subsequent K48-linked ubiquitination at the same site by DTX4 and TRIP (which are recruited via NLRP4). Knockout of USP38 increases K33-linked ubiquitination but abrogates K48-linked ubiquitination and TBK1 degradation.\",\n      \"method\": \"Co-immunoprecipitation, USP38 knockout (CRISPR), ubiquitination assays (K33- and K48-linkage specific), in vitro deubiquitination assays, interferon reporter assays, in vivo viral infection models\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro deubiquitination assay plus CRISPR KO plus linkage-specific ubiquitination analysis; mechanistically defines sequential ubiquitin editing at Lys670 within NLRP4 signalosome\",\n      \"pmids\": [\"27692986\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NLRP4 is recruited to Group A Streptococcus (GAS)-containing compartments and binds ARHGDIA (Rho GDP dissociation inhibitor α) to regulate Rho GTPase signaling, facilitating actin-mediated xenophagy. ARHGDIA Tyr156 phosphorylation acts as a regulatory gate for this interaction and for Rho-mediated autophagosome formation. Rho GTPases recruited via this NLRP4-ARHGDIA axis promote ATG9A recruitment to phagophores for autophagosome elongation.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence microscopy, siRNA knockdown, bacterial infection assays (GAS), phosphorylation site mutagenesis (ARHGDIA Tyr156), autophagic flux assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP identifies novel binding partner, mutagenesis establishes regulatory phosphorylation site, functional assays link NLRP4-ARHGDIA to autophagosome formation\",\n      \"pmids\": [\"29099277\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NLRP4 negatively regulates both the TBK1/IRF3 and IKK/NF-κB signaling pathways in cardiac cells under fructose-induced injury. Overexpression of NLRP4 reduced pro-inflammatory cytokine release and TBK1 phosphorylation, while NLRP4 knockdown enhanced IKK/NF-κB activation, confirming NLRP4's role as a negative regulator of these inflammatory kinase cascades in cardiomyocytes.\",\n      \"method\": \"NLRP4 overexpression and siRNA knockdown, RT-qPCR, Western blotting for phospho-TBK1, phospho-IRF3, IKK activity, cytokine measurements, in vivo fructose-feeding mouse model\",\n      \"journal\": \"Biomedicine & pharmacotherapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — gain- and loss-of-function with multiple signaling readouts but no direct biochemical interaction mapping; confirms known pathway roles in new cellular context\",\n      \"pmids\": [\"28486191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NLRP4 knockdown in lung cancer cells enhanced type I IFN (IFN-α/β) production through the cGAS-STING-IRF3/IRF7 axis, placing NLRP4 as a negative regulator of cGAS-STING pathway signaling upstream of IRF3/IRF7 activation.\",\n      \"method\": \"NLRP4 siRNA knockdown, IFN-α/β ELISA, IRF3/IRF7 reporter assays, in vivo tumor models (Lewis lung cancer), flow cytometry for tumor-infiltrating CD8+ T cells\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — loss-of-function with defined pathway readouts (cGAS-STING-IRF3/7), in vitro and in vivo validation; pathway placement but no direct biochemical interaction with cGAS-STING components shown\",\n      \"pmids\": [\"34927309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NLRP4 upregulation promotes olaparib resistance in pancreatic cancer by enhancing DNA repair capacity and inducing mitochondrial ROS production that drives autophagy. Co-IP mass spectrometry identified NLRP4 as involved in the DNA damage response and autophagy pathways. NLRP4-generated mitochondrial ROS promote autophagy without directly affecting DNA damage, and inhibition of mitochondrial ROS (MitoQ) or autophagy (chloroquine) sensitizes cells to olaparib.\",\n      \"method\": \"Co-IP mass spectrometry, RNA sequencing, NLRP4 overexpression/knockdown, mitochondrial ROS measurement, DNA damage assays, olaparib resistance assays in vitro and in vivo\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP MS identifies pathway membership, functional assays with specific inhibitors establish ROS-autophagy link; mechanism linking NLRP4 to mitochondrial ROS generation not fully defined\",\n      \"pmids\": [\"39187531\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NLRP4 overexpression suppresses PP2A (protein phosphatase 2A) activity and thereby activates the PI3K/Akt-NF-κB signaling axis in lung cancer, leading to chemokine reprogramming (CCL5 and CXCL2) that recruits TIGIT+TNFα+ NK cells and iNOS+ M1 macrophages. Co-IP and Western blot experiments validated NLRP4 interaction with components of this pathway.\",\n      \"method\": \"NLRP4 stable overexpression, co-immunoprecipitation, Western blotting, bulk RNA sequencing, proteomics, mass spectrometry, flow cytometry, multiplex immunofluorescence, subcutaneous tumor models in C57BL/6J mice\",\n      \"journal\": \"Biomarker research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — multiple omics approaches plus Co-IP validation; PP2A suppression by NLRP4 is novel mechanistic claim supported by functional rescue with PP2A inhibitor LB-100\",\n      \"pmids\": [\"40087771\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NLRP4 is a cytosolic NLR family receptor that functions primarily as a negative regulator of innate immune signaling: it recruits the E3 ubiquitin ligase DTX4 (gated by DYRK2-mediated phosphorylation of TBK1 at Ser527) to promote K48-linked ubiquitination and proteasomal degradation of TBK1 at Lys670, with the deubiquitinase USP38 acting within this NLRP4 signalosome to edit K33-linked chains on TBK1 to permit subsequent K48-linked degradation; NLRP4 also suppresses IKK/NF-κB activity through direct association with IKKα, inhibits the cGAS-STING-IRF3/7 axis, negatively regulates autophagy by binding Beclin1 (via its PYD) and associating with the class C VPS complex, and during bacterial infection is recruited to pathogen-containing vacuoles where it binds ARHGDIA to direct Rho GTPase-actin dynamics for xenophagy; structurally, the NLRP4 PYD adopts a six-helix death domain fold with unique α3 features that prevent ASC interaction.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"NLRP4 is a cytosolic NLR-family protein that functions as a negative regulator of innate immune signaling and autophagy. It recruits the E3 ubiquitin ligase DTX4 to TBK1, promoting K48-linked polyubiquitination at Lys670 and proteasomal degradation of TBK1, thereby suppressing type I interferon production downstream of cytosolic nucleic acid sensors such as cGAS-STING; the deubiquitinase USP38 cooperates by first removing K33-linked polyubiquitin from the same site to license K48-linked ubiquitination [PMID:22388039, PMID:27692986, PMID:34927309]. NLRP4 also restrains autophagy by binding Beclin1 via its PYD/NACHT domains and interacting with the VPS-C complex, limiting autophagosome initiation and maturation until bacterial infection transiently releases this inhibitory interaction [PMID:21209283]. The crystal structure of the NLRP4 pyrin domain reveals an atypical α2–α3 loop that precludes interaction with the inflammasome adaptor ASC, distinguishing NLRP4 from canonical inflammasome-forming NLRs [PMID:22928810].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"The first mechanistic role for NLRP4 was established as a tonic suppressor of autophagy, answering how an NLR could restrain, rather than activate, an innate defense pathway.\",\n      \"evidence\": \"Co-IP, RNAi knockdown, autophagic flux assays, and Group A Streptococcus clearance in human cells\",\n      \"pmids\": [\"21209283\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How NLRP4 release from Beclin1 is triggered upon infection is undefined\",\n        \"No structural basis for the NLRP4–Beclin1 interaction\",\n        \"Whether VPS-C interaction is direct or bridged through Beclin1 is unresolved\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"NLRP4 was shown to recruit DTX4 to TBK1 for K48-linked ubiquitination at Lys670, establishing the first mechanism by which an NLR drives proteasomal degradation of a kinase to suppress type I IFN.\",\n      \"evidence\": \"Reciprocal Co-IP, K48-linkage-specific ubiquitination assays, TBK1/IRF3 phosphorylation analysis upon NLRP4 knockdown\",\n      \"pmids\": [\"22388039\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The domain of NLRP4 mediating DTX4 recruitment was not mapped\",\n        \"Whether NLRP4 is catalytically involved or purely scaffolding is unknown\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"The 2.3 Å crystal structure of the NLRP4 PYD revealed why NLRP4 cannot form a canonical inflammasome — its unique α2–α3 loop abolishes ASC binding, resolving a longstanding question about NLR subfamily diversification.\",\n      \"evidence\": \"X-ray crystallography, yeast two-hybrid, Co-IP, and NMR chemical shift perturbation for ASC interaction\",\n      \"pmids\": [\"22928810\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No structure of full-length NLRP4 or NLRP4–DTX4/Beclin1 complexes\",\n        \"What the NLRP4 PYD does bind (if anything) remains unknown\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Discovery of the NLRP4–USP38–DTX4 signalosome explained how K33-to-K48 ubiquitin chain editing at TBK1 Lys670 controls kinase turnover, revealing a two-step ubiquitin code for innate immune signal termination.\",\n      \"evidence\": \"USP38 KO/KD, in vitro deubiquitinase assays, linkage-specific ubiquitination mapping\",\n      \"pmids\": [\"27692986\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Stoichiometry and assembly order of the ternary complex are unresolved\",\n        \"Whether other NLRPs participate in analogous ubiquitin-editing platforms is untested\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"NLRP4 was linked to Rho GTPase regulation through ARHGDIA binding at autophagosome-like vacuoles, extending its autophagy role to actin-dependent ATG9A recruitment during xenophagy.\",\n      \"evidence\": \"Co-IP, confocal colocalization, phosphorylation site mutants, Group A Streptococcus xenophagy assays\",\n      \"pmids\": [\"29099277\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct binding between NLRP4 and ARHGDIA has not been reconstituted in vitro\",\n        \"Whether this pathway operates outside bacterial infection is unknown\",\n        \"Single-lab finding\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"NLRP4 knockdown in lung cancer cells enhanced IFN-α/β through the cGAS-STING-IRF3/IRF7 axis, confirming that NLRP4 suppresses DNA-sensing innate immunity in a disease-relevant context.\",\n      \"evidence\": \"NLRP4 KD, pathway inhibitor epistasis, cytokine measurement, in vivo tumor models\",\n      \"pmids\": [\"34927309\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether the effect is entirely via TBK1 degradation or involves additional nodes is not delineated\",\n        \"Mechanism by which tumors upregulate NLRP4 is undefined\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"NLRP4 was found to promote olaparib resistance in pancreatic cancer by enhancing DNA repair and driving mitochondrial ROS-dependent autophagy, broadening its functional scope to DNA damage responses.\",\n      \"evidence\": \"Co-IP mass spectrometry, RNA-seq, ROS and autophagy inhibitor experiments, in vivo drug sensitivity assays\",\n      \"pmids\": [\"39187531\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Specific DNA repair proteins engaged by NLRP4 are not identified\",\n        \"Causal link between NLRP4 and mitochondrial ROS generation is correlative\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the full-length NLRP4 structure, the mechanism by which NLRP4 is activated or de-repressed, whether its autophagy and IFN-suppressive roles are coordinated or independent, and identification of physiological ligands or danger signals sensed by the NACHT/LRR domains.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No full-length structure or cryo-EM model exists\",\n        \"Endogenous activation signal or ligand for NLRP4 is unknown\",\n        \"Relationship between autophagy suppression and TBK1 degradation functions has not been dissected\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 2, 6]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 1, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 1, 6]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [2, 4, 7]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"complexes\": [\n      \"NLRP4-DTX4-USP38 signalosome\"\n    ],\n    \"partners\": [\n      \"DTX4\",\n      \"TBK1\",\n      \"USP38\",\n      \"BECN1\",\n      \"ARHGDIA\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"NLRP4 is a cytoplasmic NLR family member that functions predominantly as a negative regulator of innate immune signaling and autophagy. It suppresses type I interferon production by scaffolding a signalosome in which DYRK2-mediated phosphorylation of TBK1 at Ser527 enables NLRP4 to recruit the E3 ligase DTX4 for K48-linked ubiquitination and proteasomal degradation of TBK1 at Lys670, a process gated by USP38-dependent removal of competing K33-linked ubiquitin chains [PMID:22388039, PMID:26407194, PMID:27692986]. NLRP4 also inhibits NF-κB signaling through direct association with IKKα and suppression of IKK kinase activity, and negatively regulates the cGAS–STING–IRF3/7 axis [PMID:12093792, PMID:34927309]. In parallel, NLRP4 restrains autophagy by binding Beclin 1 via its PYD and associating with the class C VPS complex, yet upon bacterial infection it is recruited to pathogen-containing vacuoles where it engages ARHGDIA to redirect Rho GTPase–actin dynamics for xenophagy [PMID:21209283, PMID:29099277].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Initial characterization established that NLRP4 suppresses NF-κB signaling by directly binding IKKα and inhibiting IKK kinase activity, positioning it as a negative regulator of innate inflammatory signaling rather than a canonical inflammasome activator.\",\n      \"evidence\": \"Co-immunoprecipitation, NF-κB reporter assays, EMSA, and in vitro kinase assays in HEK293 cells\",\n      \"pmids\": [\"12093792\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study without independent replication\", \"Mechanism by which NLRP4 inhibits IKK kinase activity not defined\", \"Endogenous expression context not examined\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"A parallel study reported that NLRP4 co-expression with ASC synergistically activated NF-κB and caspase-1-dependent IL-1β processing, suggesting inflammasome-like function; this was later contradicted by structural and biochemical evidence showing NLRP4 PYD does not interact with ASC.\",\n      \"evidence\": \"Co-expression in HEK cells, NF-κB reporter and caspase-1 activity assays\",\n      \"pmids\": [\"12019269\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"ASC interaction was subsequently refuted by crystal structure and three orthogonal binding assays (PMID:22928810)\", \"Overexpression artifacts not excluded\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"NLRP4 was shown to be a negative regulator of autophagy, binding Beclin 1 through its PYD with the highest affinity among NLRs tested and associating with the class C VPS complex to restrain autophagosome/endosome maturation, establishing a non-inflammasome function for the protein.\",\n      \"evidence\": \"Co-immunoprecipitation, siRNA knockdown, confocal microscopy, and bacterial infection assays with Group A Streptococcus\",\n      \"pmids\": [\"21209283\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Beclin 1–PYD interaction not resolved\", \"Whether NLRP4 dissociation from Beclin 1 during infection is actively regulated remains unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"The central mechanism of NLRP4-mediated type I IFN suppression was elucidated: NLRP4 recruits the E3 ligase DTX4 to TBK1, promoting site-specific K48-linked ubiquitination at Lys670 and proteasomal degradation, establishing a direct biochemical pathway for IFN attenuation.\",\n      \"evidence\": \"Co-immunoprecipitation, siRNA knockdown, K48-linkage-specific ubiquitination assays, TBK1 degradation immunoblots, dsRNA/dsDNA stimulation\",\n      \"pmids\": [\"22388039\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How NLRP4 is activated or derepressed to engage TBK1 was not defined\", \"Physiological triggers for NLRP4–DTX4 complex assembly unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Crystal structure of NLRP4 PYD at 2.3 Å resolved its six-helix death-domain fold with a uniquely elongated α3 helix, and definitively demonstrated that it does not interact with ASC, clarifying why NLRP4 does not form a canonical inflammasome.\",\n      \"evidence\": \"X-ray crystallography, co-immunoprecipitation, yeast two-hybrid, NMR chemical shift perturbation\",\n      \"pmids\": [\"22928810\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No full-length NLRP4 structure available\", \"Binding partners that do engage the PYD (beyond Beclin 1) not structurally mapped\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"DYRK2 was identified as the kinase that phosphorylates TBK1 at Ser527, establishing this modification as a prerequisite for NLRP4 and DTX4 recruitment, thereby defining the upstream gating event for the NLRP4-dependent TBK1 degradation pathway.\",\n      \"evidence\": \"In vitro kinase assays, Ser527 mutagenesis, co-immunoprecipitation, ubiquitination assays, interferon reporter assays\",\n      \"pmids\": [\"26407194\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether DYRK2 phosphorylation is the sole gating event or acts cooperatively with additional signals is unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"USP38 was placed within the NLRP4 signalosome as a deubiquitinase that edits K33-linked ubiquitin chains from TBK1 Lys670 to permit subsequent K48-linked ubiquitination by DTX4/TRIP, establishing a sequential ubiquitin-editing mechanism for TBK1 turnover.\",\n      \"evidence\": \"USP38 CRISPR knockout, K33- and K48-linkage-specific ubiquitination assays, in vitro deubiquitination, in vivo viral infection models\",\n      \"pmids\": [\"27692986\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the switch from K33 to K48 ubiquitin chains is temporally coordinated is not fully resolved\", \"Whether additional DUBs participate in NLRP4 signalosome ubiquitin editing is unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"NLRP4 was found to bind ARHGDIA at pathogen-containing vacuoles, directing Rho GTPase activation and actin dynamics for xenophagy; ARHGDIA Tyr156 phosphorylation gates this interaction, linking NLRP4 to cytoskeletal control during antibacterial autophagy.\",\n      \"evidence\": \"Co-immunoprecipitation, immunofluorescence, siRNA knockdown, ARHGDIA Y156 mutagenesis, GAS infection assays, autophagic flux assays\",\n      \"pmids\": [\"29099277\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase responsible for ARHGDIA Tyr156 phosphorylation not identified\", \"Whether NLRP4-ARHGDIA interaction operates beyond GAS infection is untested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"NLRP4 was demonstrated to suppress the cGAS–STING–IRF3/IRF7 axis, broadening its role as a negative regulator of cytosolic nucleic acid sensing beyond TBK1 degradation alone.\",\n      \"evidence\": \"NLRP4 siRNA knockdown, IFN-α/β ELISA, IRF3/IRF7 reporter assays, Lewis lung cancer tumor models, flow cytometry for CD8+ T cells\",\n      \"pmids\": [\"34927309\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical interaction with cGAS or STING components not shown\", \"Whether suppression occurs through TBK1 degradation or an independent mechanism is unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"A new axis was reported in which NLRP4 suppresses PP2A phosphatase activity to activate PI3K/Akt–NF-κB signaling, driving chemokine reprogramming that shapes the tumor immune microenvironment, revealing context-dependent pro-tumorigenic functions.\",\n      \"evidence\": \"NLRP4 stable overexpression, co-immunoprecipitation, proteomics, bulk RNA-seq, PP2A inhibitor rescue, subcutaneous tumor models\",\n      \"pmids\": [\"40087771\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which NLRP4 inhibits PP2A catalytic activity is undefined\", \"Apparent contradiction with canonical NF-κB-suppressive role not reconciled\", \"Single-study claim requiring independent replication\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include how NLRP4 is itself activated or regulated at the protein level, the full-length structure and oligomeric state, the mechanism linking NLRP4 to mitochondrial ROS and DNA repair, and reconciliation of its context-dependent pro- versus anti-inflammatory roles.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No full-length structure or cryo-EM model\", \"Ligand or danger signal that activates NLRP4 is unknown\", \"NLRP4 post-translational regulation poorly characterized\", \"Context-dependent switching between NF-κB suppression and PP2A-mediated NF-κB activation unexplained\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 5, 8, 9, 14]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [5, 9, 10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 4, 5]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [4, 10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 5, 8, 9, 12]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [4, 10, 13]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 11, 14]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [5, 9]}\n    ],\n    \"complexes\": [\n      \"NLRP4-DTX4-TBK1 signalosome\",\n      \"NLRP4-Beclin1-class C VPS complex\"\n    ],\n    \"partners\": [\n      \"TBK1\",\n      \"DTX4\",\n      \"IKBKA\",\n      \"BECN1\",\n      \"ARHGDIA\",\n      \"USP38\",\n      \"DYRK2\",\n      \"SGT1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}