{"gene":"NOD1","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":1999,"finding":"NOD1 (CARD4) was identified as an Apaf-1/CED-4 family member containing an N-terminal CARD, a nucleotide-binding domain, and C-terminal leucine-rich repeats. It specifically activated caspase-9 and promoted caspase-9-induced apoptosis, requiring both the CARD and P-loop for function. NOD1 also induced NF-κB activation and physically bound RICK (a CARD-containing kinase) via homophilic CARD-CARD interaction; NOD1 mutants inhibited RICK-induced NF-κB but not TNF-α-induced NF-κB.","method":"Protein domain mapping, co-immunoprecipitation, dominant-negative mutant overexpression, caspase activity assays, NF-κB reporter assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (Co-IP, mutagenesis, enzymatic assays, reporter assays) in foundational study, replicated by subsequent work","pmids":["10329646"],"is_preprint":false},{"year":1999,"finding":"CARD4/NOD1 interacts with the serine-threonine kinase RICK and potently induces NF-κB activity through TRAF-6 and NIK signaling molecules. Coexpression of CARD4 also augmented caspase-9-induced apoptosis.","method":"Co-immunoprecipitation, NF-κB reporter gene assays, overexpression in cell lines","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP and reporter assays, independently replicated in multiple subsequent studies","pmids":["10224040"],"is_preprint":false},{"year":2000,"finding":"NOD1 confers responsiveness to bacterial lipopolysaccharides in a TLR4- and MyD88-independent manner in HEK293T cells expressing trace amounts of NOD1. The leucine-rich repeats of NOD1 were required for LPS-induced NF-κB activation. A LPS-binding activity was specifically co-immunopurified with NOD1 from cytosolic extracts.","method":"NF-κB reporter assays in HEK293T cells, co-immunopurification of LPS-binding activity, LRR deletion mutants","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-immunopurification and reporter assays, single lab; later studies refined the ligand to peptidoglycan muropeptides (iE-DAP) rather than LPS","pmids":["11058605"],"is_preprint":false},{"year":2001,"finding":"Upon infection with invasive Shigella flexneri, CARD4/NOD1 undergoes enhanced oligomerization, which is necessary for NF-κB induction. Dominant-negative CARD4 blocks both NF-κB and JNK activation by S. flexneri and microinjected LPS. S. flexneri infection triggers formation of a transient complex involving CARD4, RICK, and the IKK complex.","method":"Dominant-negative overexpression, NF-κB/JNK reporter assays, co-immunoprecipitation of CARD4-RICK-IKK complex, microinjection of LPS","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, DN mutants, microinjection, reporter assays) in a single rigorous study","pmids":["11463746"],"is_preprint":false},{"year":2002,"finding":"NOD1 binds to both RIP2 and caspase-1 via CARD-CARD interactions. NOD1 enhances pro-caspase-1 oligomerization and processing, and augments caspase-1-induced IL-1β secretion as well as LPS-induced IL-1β secretion in transfected and stably expressing cells.","method":"Co-immunoprecipitation, Western blot for caspase-1 processing, IL-1β ELISA, stable transfection","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and functional assays, single lab with two orthogonal readouts","pmids":["12459189"],"is_preprint":false},{"year":2006,"finding":"Nod1-dependent apoptosis is a caspase-8-mediated event that requires RIP2. In contrast, Nod1-dependent JNK activation and IL-8 production do not require caspase-8 but do require TAK1 and RIP2, placing TAK1 as a key downstream node in the Nod1 signaling pathway.","method":"Caspase-8 knockout/dominant-negative experiments, RIP2 and TAK1 knockdown/knockout, IL-8 ELISA, JNK kinase assays","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic knockouts and dominant-negatives with distinct pathway readouts, single lab but multiple orthogonal methods","pmids":["17186025"],"is_preprint":false},{"year":2007,"finding":"SGT1 positively regulates NOD1 activation. siRNA depletion of SGT1 prevented multiple NOD1-dependent cellular responses without affecting NOD1 protein stability or downstream signaling molecules. HSP90 depletion or pharmacological inhibition caused loss of NOD1 protein. Depletion of Chp1 (mammalian Rar1 orthologue) had no effect on NOD1-dependent activation.","method":"siRNA knockdown, pharmacological HSP90 inhibition, cell-based NOD1 activation assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — siRNA knockdown with multiple cellular readouts, genetic and pharmacological approaches, and negative control (Chp1)","pmids":["17420470"],"is_preprint":false},{"year":2007,"finding":"Nod1 stimulation in primary peritoneal mesothelial cells induces potent secretion of CXCL1 and CCL2 chemokines, as well as inducible NO synthase expression; these responses require RICK. In vivo intraperitoneal administration of a synthetic Nod1 agonist elicited chemokine production and neutrophil recruitment in wild-type but not RICK-deficient mice. Nod1/RICK-mediated chemokine responses cooperated with IFN-γ in NO production. Listeria-induced CXCL1 production was significantly reduced in Nod1- or RICK-deficient cells.","method":"Primary cell stimulation, cytokine ELISA, RICK-deficient mice (in vivo), Nod1-deficient cells, siNOS expression assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout (Nod1-/- and RICK-/-) in vitro and in vivo with specific pathway readouts","pmids":["17579072"],"is_preprint":false},{"year":2009,"finding":"NOD1 is required for H. pylori (cagPAI+)-induced p38 and ERK phosphorylation, as well as NF-κB and AP-1 activation in gastric epithelial cells. siNOD1-expressing cells showed significant reductions in p38 and ERK phosphorylation. NOD1-dependent JNK phosphorylation was observed in response to invasive Shigella but not H. pylori, indicating pathogen-specific responses.","method":"Stable siRNA knockdown of NOD1, kinase phosphorylation assays (p38, ERK, JNK), NF-κB and AP-1 reporter assays, pharmacological MAPK inhibitors","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — stable siRNA knockdown with multiple kinase and transcription factor readouts, pharmacological validation","pmids":["20007577"],"is_preprint":false},{"year":2009,"finding":"Rip2 kinase activity is required for its own protein stability; kinase-dead Rip2 mutants expressed in primary cells had severely compromised protein levels, and this instability could not be rescued by phospho-mimetic mutation. Loss of Rip2 kinase activity abrogated inflammatory cytokine production in response to Nod1 and Nod2 ligands both in vitro and in vivo.","method":"Retroviral expression of kinase-dead Rip2 mutants, Western blot for protein levels, cytokine production assays in primary cells and in vivo","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mutagenesis of kinase domain, in vitro and in vivo readouts, multiple mutants tested","pmids":["19473975"],"is_preprint":false},{"year":2011,"finding":"Purified recombinant NOD1 protein exists predominantly as a monomer; addition of the NOD1 ligand γ-Tri-DAP stimulates NOD1 protein oligomerization in vitro. NOD1 binds preferentially to ATP over ADP, AMP, or dATP (nucleotide binding specificity). Purified NOD1 protein directly binds purified pro-apoptotic protein Bid.","method":"Gel-sieve chromatography, fluorescence polarization assay for nucleotide binding, in vitro protein-protein binding with purified components","journal":"Journal of biotechnology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted in vitro with purified protein, multiple biochemical assays, single lab","pmids":["22062585"],"is_preprint":false},{"year":2011,"finding":"2-Aminobenzimidazole compounds (e.g., Nodinitib-1/ML130) selectively inhibit NOD1-induced NF-κB activity. Mechanistic studies indicate these small molecules cause conformational changes of NOD1 in vitro and alter NOD1 subcellular targeting in cells.","method":"High-throughput NF-κB reporter assay, multiple counter-screens, in vitro conformational change assays, subcellular localization imaging","journal":"Chemistry & biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — HTS with counter-screens and mechanistic follow-up, single lab","pmids":["21802003"],"is_preprint":false},{"year":2013,"finding":"PSMA7 (proteasome subunit alpha type-7) directly interacts with NOD1 and downregulates NOD1 expression in a proteasome-dependent manner. Overexpression of PSMA7 inhibits NOD1-mediated apoptosis and NF-κB activation; knockdown of PSMA7 enhances NOD1 activity.","method":"Yeast two-hybrid screening, co-immunoprecipitation, GST-pulldown, ubiquitin assay, siRNA knockdown, Western blot","journal":"Cellular physiology and biochemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — three orthogonal binding methods (Y2H, Co-IP, GST pulldown) plus functional readouts, single lab","pmids":["23839082"],"is_preprint":false},{"year":2016,"finding":"NOD1 and NOD2 are important mediators of ER-stress-induced inflammation. The ER stress inducers thapsigargin and DTT trigger IL-6 production in a NOD1/2-dependent fashion. Brucella abortus-induced inflammation via its type IV secretion effector VceC is TRAF2-, NOD1/2-, and RIP2-dependent; it can be reduced by ER stress inhibitor TUDCA or an IRE1α kinase inhibitor, placing NOD1/2 downstream of the IRE1α/TRAF2 ER stress signaling pathway.","method":"NOD1/2-deficient mouse and human cells, ER stress inducers, pharmacological inhibitors (TUDCA, IRE1α inhibitor), bacterial infection model, IL-6 cytokine measurement","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout cells plus pharmacological inhibition with multiple orthogonal stimuli and readouts, published in high-impact journal","pmids":["27007849"],"is_preprint":false},{"year":2019,"finding":"Nod1 and its downstream adapter Rip2 are required for insulin trafficking in pancreatic beta cells in a cell-autonomous manner. Upon recognizing cognate ligands, Nod1 and Rip2 localize to insulin vesicles and recruit Rab1a to direct insulin trafficking through the cytoplasm. Intestinal lysozyme liberates Nod1 ligands into circulation, enabling long-range gut-islet communication that modulates host glucose tolerance.","method":"Nod1-/- and Rip2-/- beta cells, subcellular fractionation and co-localization imaging, Rab1a recruitment assays, intestinal lysozyme manipulation, glucose tolerance tests in mice","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockouts with cell-autonomous functional readout, subcellular localization with functional consequence, in vivo validation","pmids":["31201384"],"is_preprint":false},{"year":2020,"finding":"NOD1 promotes antiviral signaling by binding viral RNA (poly(I:C)) and regulating the interaction of MDA5 and MAVS. NOD1 enhances MDA5-MAVS complex formation, increasing type I IFN transcription. NOD1 also targets TRAF3 to modulate TRAF3-MAVS complexes. Human NOD1-MDA5 and NOD1-TRAF3 interactions were confirmed by co-immunoprecipitation.","method":"Co-immunoprecipitation, RNA binding assays (poly(I:C)), NOD1 knockdown/overexpression, IFN reporter assays, zebrafish NOD1-/- survival experiments, human cell confirmation","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with functional readouts, cross-species validation, but primarily teleost model with human confirmation","pmids":["32169843"],"is_preprint":false},{"year":2021,"finding":"Systemic Nod1 activation promotes autophagy-dependent reprogramming of macrophages toward an alternative phenotype and induces expansion of myeloid-derived suppressor cells (MDSCs), maintaining their immunosuppressive potential via arginase-1 activity. Myeloid-intrinsic Nod1 expression sustains intra-tumoral arginase-1 levels to foster an immunosuppressive tumor microenvironment during colorectal cancer development.","method":"Nod1-/- mice, peritonitis model, autophagy inhibitors, bone marrow chimeras, arginase-1 activity assays, colorectal cancer tumor model","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic models (KO mice, chimeras), biochemical assays, in vivo cancer model with mechanistic readouts","pmids":["33503439"],"is_preprint":false},{"year":2018,"finding":"Endothelial NOD1 triggers VCAM-1 upregulation through the RIP2-NF-κB axis in an autocrine manner in response to peptidoglycan or oxidized LDLs, enhancing firm adhesion of monocytes and neutrophils to the vasculature in vivo. NOD1 genetic inactivation in Apoe-/- mice reduced atherosclerosis burden and monocyte/neutrophil accumulation in atheromata.","method":"NOD1-/- Apoe-/- mice, endothelial cell stimulation, VCAM-1 expression assays, leukocyte adhesion assays in vivo, RIP2-NF-κB pathway pharmacological inhibition","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic knockout model with in vitro mechanistic pathway dissection and multiple orthogonal readouts","pmids":["30496704"],"is_preprint":false},{"year":2015,"finding":"Selective NOD1 activation with C12-iE-DAP in mice induces cardiac dysfunction with decreased L-type Ca2+ current, reduced [Ca2+]i transients and Ca2+ load, decreased phospho-phospholamban, SERCA, and NCX expression, and increased RyR2 over-phosphorylation causing diastolic Ca2+ leak. These effects were lost in NOD1-/- mice. Co-treatment with NF-κB inhibitor BAY11-7082 prevented iE-DAP-induced cardiac dysfunction and Ca2+ handling impairment, placing NF-κB downstream of NOD1 in this pathway.","method":"NOD1-/- mice, selective NOD1 agonist C12-iE-DAP, echocardiography, patch-clamp electrophysiology, Ca2+ imaging, Western blot, NF-κB pharmacological inhibition","journal":"Cardiovascular research","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO controls, multiple electrophysiological and biochemical readouts, pharmacological pathway validation","pmids":["25824149"],"is_preprint":false},{"year":2007,"finding":"Nod1 activation in lung epithelial cells strongly activates C/EBPβ transcription factors in addition to modest NF-κB activation. This C/EBPβ activation appears independent of de novo protein synthesis, identifying a novel downstream transcriptional pathway from NOD1 in lung epithelium.","method":"Electrophoretic mobility shift assay (EMSA), reporter gene assays, protein synthesis inhibitor experiments in BEAS-2B cells","journal":"The European respiratory journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — EMSA and reporter assays with pharmacological controls, single lab, single cell type","pmids":["17666556"],"is_preprint":false},{"year":2014,"finding":"Bacterial peptidoglycan (PGN) stimulates lipolysis in adipocytes via NOD1 in a cell-autonomous manner. NOD1-activating PGN stimulated MAPK (ERK1/2), PKA, and NF-κB in 3T3-L1 adipocytes. NOD1-mediated lipolysis was completely suppressed by simultaneous inhibition of ERK1/2 and PKA, or by hormone-sensitive lipase (HSL) inhibition. WAT explants from NOD1-/- mice did not respond to NOD1-activating PGN.","method":"NOD1-/- mouse adipose tissue explants, 3T3-L1 adipocyte stimulation, kinase inhibitors (ERK1/2, PKA, JNK), HSL inhibitor, NF-κB inhibitor, lipolysis assays","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO tissue and pharmacological dissection with multiple pathway inhibitors, cell-autonomous mechanism established","pmids":["24828250"],"is_preprint":false},{"year":2019,"finding":"NOD1 inhibited proliferation of hepatocellular carcinoma (HCC) cells by directly interacting with proto-oncogene SRC and suppressing SRC activation, thereby inhibiting the SRC/MAPK axis. NOD1 overexpression induced cell cycle arrest at G1 phase and enhanced chemosensitivity to sorafenib and 5-FU in vitro and in vivo.","method":"Co-immunoprecipitation (NOD1-SRC interaction), overexpression, siRNA knockdown, cell cycle analysis, xenograft tumor model, Western blot for SRC/MAPK","journal":"Journal of molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP for direct interaction, functional KO/OE with mechanistic readouts, single lab","pmids":["31872284"],"is_preprint":false},{"year":2022,"finding":"METTL3 (m6A methyltransferase) negatively regulates NOD1 expression by promoting m6A-dependent mRNA degradation of NOD1 and RIPK2 transcripts via YTHDF1 and YTHDF2 m6A readers. METTL3 knockdown increased NOD1 and RIPK2 mRNA stability and expression, activating the NOD1 pathway and amplifying LPS-induced inflammatory responses in macrophages.","method":"METTL3 siRNA knockdown, YTHDF1/YTHDF2 silencing, mRNA stability assays, NOD1/RIPK2 expression by Western blot and qPCR, cytokine assays, NOD1 pathway-specific inhibitors","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple siRNA knockdowns with mRNA stability readout, pathway inhibitor confirmation, single lab","pmids":["35176453"],"is_preprint":false},{"year":2024,"finding":"BFT-1 (B. fragilis toxin) directly binds to NOD1 and stabilizes NOD1 protein. NOD1, highly expressed on ALDH+ breast cancer stem cells, cooperates with GAK to phosphorylate NUMB, promoting its lysosomal degradation and thereby activating the NOTCH1-HEY1 signaling pathway to increase breast cancer stem cells.","method":"Direct binding assays (BFT-1 to NOD1), protein stability assays, co-immunoprecipitation (NOD1-GAK), NUMB phosphorylation and lysosomal degradation assays, NOTCH1 pathway reporter, ALDH+ cell sorting","journal":"Protein & cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding and Co-IP with mechanistic downstream readouts, single lab","pmids":["38437016"],"is_preprint":false},{"year":2019,"finding":"NOD1 activation in adipocytes causes diacylglycerol (DAG) accumulation through lipolysis, which activates PKCδ. PKCδ then stimulates IRAK1/4, leading to NF-κB and JNK activation and proinflammatory cytokine expression. Pharmacological inhibition or siRNA-mediated knockdown of IRAK1/4 attenuated NOD1-mediated NF-κB, JNK activation, and cytokine production.","method":"NOD1 agonist stimulation of 3T3-L1 adipocytes, DAG measurement, PKCδ activity assays, IRAK1/4 siRNA and pharmacological inhibition, NF-κB/JNK assays, cytokine measurement","journal":"Biochimica et biophysica acta. Molecular basis of disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA and pharmacological dissection of pathway in adipocytes, single lab with multiple readouts","pmids":["30391544"],"is_preprint":false}],"current_model":"NOD1 (CARD4/NLRC1) is an intracellular pattern recognition receptor that, upon sensing meso-diaminopimelic acid-containing bacterial peptidoglycan muropeptides, undergoes ATP-dependent oligomerization and recruits the serine-threonine kinase RICK/RIP2 via homophilic CARD-CARD interaction; RIP2 then activates TAK1 and the IKK complex to drive NF-κB and MAPK (p38, ERK, JNK) signaling, inducing proinflammatory cytokine and chemokine production, while NOD1 can also promote caspase-9-dependent apoptosis, interact with MDA5/MAVS for antiviral signaling, localize to insulin vesicles to recruit Rab1a for insulin trafficking, engage SRC to suppress MAPK-driven proliferation, and be negatively regulated by PSMA7-mediated proteasomal degradation and METTL3-mediated m6A mRNA destabilization, with HSP90 and SGT1 required as chaperones for NOD1 stability and activation."},"narrative":{"mechanistic_narrative":"NOD1 (CARD4) is an intracellular pattern-recognition receptor of the Apaf-1/CED-4 family, comprising an N-terminal CARD, a central nucleotide-binding domain, and C-terminal leucine-rich repeats, that couples cytosolic sensing of bacterial products to inflammatory and cell-fate signaling [PMID:10329646]. Ligand engagement drives ATP-dependent oligomerization—purified NOD1 is a monomer that oligomerizes upon addition of the muropeptide ligand γ-Tri-DAP, and binds ATP preferentially over other nucleotides [PMID:22062585]; this oligomerization is enhanced during invasive bacterial infection and is required for downstream activation [PMID:11463746]. Activated NOD1 recruits the CARD-containing kinase RICK/RIP2 through homophilic CARD-CARD interaction [PMID:10329646, PMID:10224040], and RIP2 kinase activity is required both for its own stability and for downstream cytokine output [PMID:19473975]. Signaling proceeds via TAK1 to NF-κB and MAPK (p38, ERK, JNK) activation [PMID:17186025, PMID:20007577], engaging TRAF6/NIK and the IKK complex [PMID:10224040, PMID:11463746] and additional transcriptional effectors such as C/EBPβ [PMID:17666556], to induce proinflammatory cytokines and chemokines and neutrophil recruitment in vivo [PMID:17579072]. In parallel, NOD1 promotes programmed cell death, originally as a caspase-9 activator [PMID:10329646] and subsequently as a RIP2- and caspase-8-dependent apoptotic event [PMID:17186025]. Beyond infection, NOD1 transduces ER-stress-induced inflammation downstream of the IRE1α/TRAF2 axis [PMID:27007849] and operates cell-autonomously in metabolic and vascular tissues: it localizes to insulin vesicles to recruit Rab1a for insulin trafficking in pancreatic beta cells [PMID:31201384], drives lipolysis and inflammatory signaling in adipocytes via ERK/PKA/HSL and a DAG–PKCδ–IRAK1/4 cascade [PMID:24828250, PMID:30391544], and promotes endothelial VCAM-1 expression and atherosclerosis through the RIP2–NF-κB axis [PMID:30496704]. NOD1 activity is constrained by PSMA7-mediated proteasomal degradation [PMID:23839082] and METTL3-directed m6A destabilization of NOD1 and RIPK2 mRNA [PMID:35176453], while HSP90 and SGT1 act as chaperones required for NOD1 stability and activation [PMID:17420470].","teleology":[{"year":1999,"claim":"Established NOD1 as a CARD-bearing Apaf-1/CED-4 family member that links bacterial-sensing architecture to both NF-κB activation and caspase-9-dependent apoptosis, defining its dual signaling identity.","evidence":"Domain mapping, Co-IP, dominant-negative mutagenesis, caspase and NF-κB reporter assays","pmids":["10329646","10224040"],"confidence":"High","gaps":["Physiological ligand not yet defined","Endogenous oligomerization state untested"]},{"year":2000,"claim":"Implicated the leucine-rich repeats in conferring responsiveness to bacterial products independent of TLR4/MyD88, positioning NOD1 as a cytosolic sensor.","evidence":"NF-κB reporter assays and co-immunopurification of ligand-binding activity in HEK293T cells with LRR deletion mutants","pmids":["11058605"],"confidence":"Medium","gaps":["Ligand initially assigned to LPS; later refined to peptidoglycan muropeptides","Single lab, no direct structural binding data"]},{"year":2001,"claim":"Demonstrated that infection-triggered NOD1 oligomerization assembles a transient NOD1-RICK-IKK complex necessary for NF-κB and JNK activation, defining the signaling assembly.","evidence":"Shigella infection, dominant-negative overexpression, Co-IP, microinjection of LPS, reporter assays","pmids":["11463746"],"confidence":"High","gaps":["Stoichiometry of the complex unresolved","Kinetics of complex disassembly not defined"]},{"year":2002,"claim":"Extended NOD1 partner repertoire to caspase-1, linking it to pro-caspase-1 oligomerization and IL-1β secretion.","evidence":"Reciprocal Co-IP, caspase-1 processing Western blots, IL-1β ELISA in transfected/stable cells","pmids":["12459189"],"confidence":"Medium","gaps":["Relies on overexpression","Endogenous relevance to inflammasome assembly untested"]},{"year":2006,"claim":"Genetically separated NOD1 outputs: apoptosis is caspase-8- and RIP2-dependent, whereas JNK/IL-8 responses require TAK1 and RIP2, placing TAK1 as a central downstream node.","evidence":"Caspase-8 and RIP2/TAK1 knockout/dominant-negative, IL-8 ELISA, JNK kinase assays","pmids":["17186025"],"confidence":"High","gaps":["Branch-point selection between apoptosis and inflammation not mechanistically resolved"]},{"year":2007,"claim":"Identified HSP90 and SGT1 as chaperones required for NOD1 protein stability and activation respectively, establishing a folding-dependent regulatory layer.","evidence":"siRNA knockdown, pharmacological HSP90 inhibition, cellular NOD1 activation assays with Chp1 negative control","pmids":["17420470"],"confidence":"High","gaps":["Direct chaperone-NOD1 binding interface not mapped"]},{"year":2007,"claim":"Showed in vivo that NOD1/RICK drive chemokine (CXCL1, CCL2) production, iNOS expression, and neutrophil recruitment, demonstrating physiological inflammatory function and a novel C/EBPβ transcriptional arm.","evidence":"Nod1-/- and RICK-/- cells and mice, cytokine ELISA, EMSA and reporter assays in epithelial cells","pmids":["17579072","17666556"],"confidence":"High","gaps":["Mechanism of C/EBPβ activation independent of protein synthesis undefined"]},{"year":2009,"claim":"Established that RIP2 kinase activity is required for RIP2 stability and for NOD1/NOD2-driven cytokine production, identifying the kinase as a tractable signaling chokepoint.","evidence":"Retroviral kinase-dead RIP2 mutants, protein-level Western blots, cytokine assays in vitro and in vivo","pmids":["19473975"],"confidence":"High","gaps":["Relevant RIP2 autophosphorylation substrates not enumerated"]},{"year":2011,"claim":"Reconstituted NOD1 activation biochemically, showing ligand-induced oligomerization, ATP-binding specificity, and direct binding to pro-apoptotic Bid, plus chemical-probe modulation of conformation and localization.","evidence":"Gel-sieve chromatography, fluorescence polarization, purified protein binding; HTS small-molecule inhibitors with conformational and imaging assays","pmids":["22062585","21802003"],"confidence":"High","gaps":["No high-resolution structure of activated oligomer","Functional role of NOD1-Bid interaction in cells untested"]},{"year":2013,"claim":"Defined PSMA7 as a direct NOD1-interacting factor driving proteasomal turnover, establishing negative regulation of NOD1 abundance.","evidence":"Y2H, Co-IP, GST-pulldown, ubiquitin assay, siRNA with NF-κB/apoptosis readouts","pmids":["23839082"],"confidence":"High","gaps":["E3 ligase mediating NOD1 ubiquitination not identified"]},{"year":2016,"claim":"Placed NOD1/NOD2 downstream of the IRE1α/TRAF2 ER-stress pathway, connecting unfolded-protein-response signaling to RIP2-dependent inflammation.","evidence":"NOD1/2-deficient mouse and human cells, ER stress inducers, TUDCA/IRE1α inhibitors, Brucella infection, IL-6 measurement","pmids":["27007849"],"confidence":"High","gaps":["Whether ER stress generates an endogenous NOD1 ligand or activates it ligand-independently is unresolved"]},{"year":2015,"claim":"Demonstrated tissue-level consequences of NOD1 activation in the heart, with NF-κB-dependent impairment of Ca2+ handling and contractile function.","evidence":"NOD1-/- mice, C12-iE-DAP agonist, echocardiography, patch-clamp, Ca2+ imaging, NF-κB inhibition","pmids":["25824149"],"confidence":"High","gaps":["Cell type within myocardium mediating the effect not isolated"]},{"year":2018,"claim":"Established endothelial NOD1 as a driver of leukocyte adhesion and atherosclerosis via the RIP2-NF-κB-VCAM-1 axis, responsive to peptidoglycan and oxidized LDL.","evidence":"NOD1-/-Apoe-/- mice, endothelial stimulation, VCAM-1 and adhesion assays, RIP2-NF-κB pharmacological inhibition","pmids":["30496704"],"confidence":"High","gaps":["Direct sensing of oxLDL versus indirect activation not distinguished"]},{"year":2019,"claim":"Revealed cell-autonomous metabolic functions: NOD1/RIP2 localize to insulin vesicles to recruit Rab1a for insulin trafficking, and NOD1 inhibits hepatocellular carcinoma proliferation by binding and suppressing SRC.","evidence":"Nod1-/-/Rip2-/- beta cells with fractionation/colocalization and glucose tolerance tests; Co-IP, cell-cycle analysis and xenografts for SRC suppression","pmids":["31201384","31872284"],"confidence":"High","gaps":["How vesicle localization is targeted versus plasma-membrane sensing is unclear","SRC interaction surface not mapped"]},{"year":2020,"claim":"Expanded NOD1 to antiviral sensing, where it binds viral RNA and promotes MDA5-MAVS and modulates TRAF3-MAVS complex formation to boost type I IFN.","evidence":"Co-IP, poly(I:C) binding, IFN reporters, zebrafish NOD1-/- survival, human cell confirmation","pmids":["32169843"],"confidence":"Medium","gaps":["Primarily teleost model","Direct RNA-binding region of NOD1 undefined"]},{"year":2021,"claim":"Defined NOD1 in myeloid cells as a driver of immunosuppression, sustaining arginase-1 and MDSC expansion through autophagy-dependent macrophage reprogramming in colorectal cancer.","evidence":"Nod1-/- mice, bone-marrow chimeras, autophagy inhibitors, arginase-1 assays, colorectal cancer model","pmids":["33503439"],"confidence":"High","gaps":["Endogenous ligand sustaining systemic NOD1 activation in tumors not defined"]},{"year":2022,"claim":"Identified METTL3-directed m6A modification as a post-transcriptional brake on NOD1 and RIPK2 expression via YTHDF1/2-mediated mRNA decay.","evidence":"METTL3 and YTHDF1/2 siRNA, mRNA stability assays, NOD1/RIPK2 expression and cytokine readouts in macrophages","pmids":["35176453"],"confidence":"Medium","gaps":["Specific m6A sites on NOD1 transcript not mapped","Single lab"]},{"year":2024,"claim":"Linked NOD1 stabilization by the bacterial toxin BFT-1 to a kinase-scaffolding role with GAK in phosphorylating NUMB, activating NOTCH1-HEY1 signaling to expand breast cancer stem cells.","evidence":"Direct BFT-1 binding and stability assays, NOD1-GAK Co-IP, NUMB phosphorylation/degradation, NOTCH reporter, ALDH+ sorting","pmids":["38437016"],"confidence":"Medium","gaps":["Whether NOD1 itself has kinase activity versus scaffolds GAK is unresolved","Single lab"]},{"year":null,"claim":"How a single sensor selects among its divergent outputs—inflammation, apoptosis, antiviral IFN, vesicular trafficking, and tumor-context signaling—as a function of ligand, tissue, and partner availability remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of the activated NOD1 oligomer in any output context","Endogenous ligands driving non-infectious (metabolic, vascular, tumor) functions undefined","Determinants of subcellular targeting versus cytosolic signaling unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[10]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,4]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,3,7]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[15]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,23]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,3]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[14]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[17]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[3,7,13]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,5,8]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[0,5]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[13]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[16]}],"complexes":["NOD1-RICK/RIP2-IKK signaling complex"],"partners":["RIPK2","CASP1","TRAF6","PSMA7","SRC","GAK","MDA5","TRAF3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y239","full_name":"Nucleotide-binding oligomerization domain-containing protein 1","aliases":["Caspase recruitment domain-containing protein 4"],"length_aa":953,"mass_kda":107.7,"function":"Pattern recognition receptor (PRR) that detects bacterial peptidoglycan fragments and other danger signals and thus participates in both innate and adaptive immune responses (PubMed:11058605, PubMed:12791997, PubMed:12796777, PubMed:15044951, PubMed:16172124, PubMed:19043560, PubMed:22672233, PubMed:27099311). Specifically recognizes and binds gamma-D-glutamyl-meso-diaminopimelic acid (iE-DAP), a dipeptide present in peptidoglycan of Gram-negative bacteria (PubMed:12791997, PubMed:12796777, PubMed:12871942, PubMed:16172124, PubMed:16211083). Preferentially binds iE-DAP in tripeptide-containing muropeptides (MurNAc-TriDAP or TriDAP) (PubMed:16211083). Ligand binding triggers oligomerization that facilitates the binding and subsequent activation of the proximal adapter receptor-interacting RIPK2 (PubMed:12791997, PubMed:12796777, PubMed:17054981). Following recruitment, RIPK2 undergoes 'Met-1'- (linear) and 'Lys-63'-linked polyubiquitination by E3 ubiquitin-protein ligases XIAP, BIRC2, BIRC3 and the LUBAC complex, becoming a scaffolding protein for downstream effectors, triggering activation of the NF-kappa-B and MAP kinases signaling (PubMed:10880512, PubMed:12791997, PubMed:19043560). This in turn leads to the transcriptional activation of hundreds of genes involved in immune response (PubMed:10880512, PubMed:19043560). Also acts as a regulator of antiviral response elicited by dsRNA and the expression of RLR pathway members by targeting IFIH1 and TRAF3 to modulate the formation of IFIH1-MAVS and TRAF3-MAVS complexes leading to increased transcription of type I IFNs (PubMed:32169843). Also acts as a regulator of autophagy via its interaction with ATG16L1, possibly by recruiting ATG16L1 at the site of bacterial entry (By similarity). Besides recognizing pathogens, also involved in the endoplasmic reticulum stress response: acts by sensing and binding to the cytosolic metabolite sphingosine-1-phosphate generated in response to endoplasmic reticulum stress, initiating an inflammation process that leads to activation of the NF-kappa-B and MAP kinases signaling (PubMed:27007849, PubMed:33942347). In addition, plays a role in insulin trafficking in beta cells in a cell-autonomous manner (By similarity). Mechanistically, upon recognizing cognate ligands, NOD1 and RIPK2 localize to insulin vesicles where they recruit RAB1A to direct insulin trafficking through the cytoplasm (By similarity) In contrast to isoform 1, does not efficiently recognize and bind gamma-D-glutamyl-meso-diaminopimelic acid (iE-DAP) ligand","subcellular_location":"Cell membrane; Apical cell membrane; Basolateral cell membrane; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9Y239/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NOD1","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/NOD1","total_profiled":1310},"omim":[{"mim_id":"615929","title":"ANKYRIN REPEAT DOMAIN-CONTAINING PROTEIN 17; ANKRD17","url":"https://www.omim.org/entry/615929"},{"mim_id":"614061","title":"OLFACTOMEDIN 4; OLFM4","url":"https://www.omim.org/entry/614061"},{"mim_id":"613635","title":"EUKARYOTIC TRANSLATION INITIATION FACTOR 2-ALPHA KINASE 1; EIF2AK1","url":"https://www.omim.org/entry/613635"},{"mim_id":"609986","title":"CASPASE RECRUITMENT DOMAIN-CONTAINING PROTEIN 6; CARD6","url":"https://www.omim.org/entry/609986"},{"mim_id":"609650","title":"NLR FAMILY, PYRIN DOMAIN-CONTAINING 6; NLRP6","url":"https://www.omim.org/entry/609650"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Mitochondria","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NOD1"},"hgnc":{"alias_symbol":["NLRC1","CLR7.1"],"prev_symbol":["CARD4"]},"alphafold":{"accession":"Q9Y239","domains":[{"cath_id":"1.10.533.10","chopping":"18-123","consensus_level":"high","plddt":87.4321,"start":18,"end":123},{"cath_id":"3.40.50.300","chopping":"129-350","consensus_level":"high","plddt":84.6897,"start":129,"end":350},{"cath_id":"-","chopping":"366-406","consensus_level":"medium","plddt":79.5,"start":366,"end":406},{"cath_id":"-","chopping":"418-434_450-502_512-546_582-600","consensus_level":"medium","plddt":85.1572,"start":418,"end":600}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y239","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y239-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y239-F1-predicted_aligned_error_v6.png","plddt_mean":85.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NOD1","jax_strain_url":"https://www.jax.org/strain/search?query=NOD1"},"sequence":{"accession":"Q9Y239","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y239.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y239/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y239"}},"corpus_meta":[{"pmid":"11087742","id":"PMC_11087742","title":"Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF-kappaB.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11087742","citation_count":1124,"is_preprint":false},{"pmid":"25526305","id":"PMC_25526305","title":"NOD1 and NOD2: signaling, host defense, and inflammatory disease.","date":"2014","source":"Immunity","url":"https://pubmed.ncbi.nlm.nih.gov/25526305","citation_count":649,"is_preprint":false},{"pmid":"16493424","id":"PMC_16493424","title":"Signalling pathways and molecular interactions of NOD1 and NOD2.","date":"2006","source":"Nature reviews. 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identified as an Apaf-1/CED-4 family member containing an N-terminal CARD, a nucleotide-binding domain, and C-terminal leucine-rich repeats. It specifically activated caspase-9 and promoted caspase-9-induced apoptosis, requiring both the CARD and P-loop for function. NOD1 also induced NF-κB activation and physically bound RICK (a CARD-containing kinase) via homophilic CARD-CARD interaction; NOD1 mutants inhibited RICK-induced NF-κB but not TNF-α-induced NF-κB.\",\n      \"method\": \"Protein domain mapping, co-immunoprecipitation, dominant-negative mutant overexpression, caspase activity assays, NF-κB reporter assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (Co-IP, mutagenesis, enzymatic assays, reporter assays) in foundational study, replicated by subsequent work\",\n      \"pmids\": [\"10329646\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"CARD4/NOD1 interacts with the serine-threonine kinase RICK and potently induces NF-κB activity through TRAF-6 and NIK signaling molecules. Coexpression of CARD4 also augmented caspase-9-induced apoptosis.\",\n      \"method\": \"Co-immunoprecipitation, NF-κB reporter gene assays, overexpression in cell lines\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP and reporter assays, independently replicated in multiple subsequent studies\",\n      \"pmids\": [\"10224040\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"NOD1 confers responsiveness to bacterial lipopolysaccharides in a TLR4- and MyD88-independent manner in HEK293T cells expressing trace amounts of NOD1. The leucine-rich repeats of NOD1 were required for LPS-induced NF-κB activation. A LPS-binding activity was specifically co-immunopurified with NOD1 from cytosolic extracts.\",\n      \"method\": \"NF-κB reporter assays in HEK293T cells, co-immunopurification of LPS-binding activity, LRR deletion mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-immunopurification and reporter assays, single lab; later studies refined the ligand to peptidoglycan muropeptides (iE-DAP) rather than LPS\",\n      \"pmids\": [\"11058605\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Upon infection with invasive Shigella flexneri, CARD4/NOD1 undergoes enhanced oligomerization, which is necessary for NF-κB induction. Dominant-negative CARD4 blocks both NF-κB and JNK activation by S. flexneri and microinjected LPS. S. flexneri infection triggers formation of a transient complex involving CARD4, RICK, and the IKK complex.\",\n      \"method\": \"Dominant-negative overexpression, NF-κB/JNK reporter assays, co-immunoprecipitation of CARD4-RICK-IKK complex, microinjection of LPS\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, DN mutants, microinjection, reporter assays) in a single rigorous study\",\n      \"pmids\": [\"11463746\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"NOD1 binds to both RIP2 and caspase-1 via CARD-CARD interactions. NOD1 enhances pro-caspase-1 oligomerization and processing, and augments caspase-1-induced IL-1β secretion as well as LPS-induced IL-1β secretion in transfected and stably expressing cells.\",\n      \"method\": \"Co-immunoprecipitation, Western blot for caspase-1 processing, IL-1β ELISA, stable transfection\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and functional assays, single lab with two orthogonal readouts\",\n      \"pmids\": [\"12459189\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Nod1-dependent apoptosis is a caspase-8-mediated event that requires RIP2. In contrast, Nod1-dependent JNK activation and IL-8 production do not require caspase-8 but do require TAK1 and RIP2, placing TAK1 as a key downstream node in the Nod1 signaling pathway.\",\n      \"method\": \"Caspase-8 knockout/dominant-negative experiments, RIP2 and TAK1 knockdown/knockout, IL-8 ELISA, JNK kinase assays\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic knockouts and dominant-negatives with distinct pathway readouts, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"17186025\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SGT1 positively regulates NOD1 activation. siRNA depletion of SGT1 prevented multiple NOD1-dependent cellular responses without affecting NOD1 protein stability or downstream signaling molecules. HSP90 depletion or pharmacological inhibition caused loss of NOD1 protein. Depletion of Chp1 (mammalian Rar1 orthologue) had no effect on NOD1-dependent activation.\",\n      \"method\": \"siRNA knockdown, pharmacological HSP90 inhibition, cell-based NOD1 activation assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — siRNA knockdown with multiple cellular readouts, genetic and pharmacological approaches, and negative control (Chp1)\",\n      \"pmids\": [\"17420470\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Nod1 stimulation in primary peritoneal mesothelial cells induces potent secretion of CXCL1 and CCL2 chemokines, as well as inducible NO synthase expression; these responses require RICK. In vivo intraperitoneal administration of a synthetic Nod1 agonist elicited chemokine production and neutrophil recruitment in wild-type but not RICK-deficient mice. Nod1/RICK-mediated chemokine responses cooperated with IFN-γ in NO production. Listeria-induced CXCL1 production was significantly reduced in Nod1- or RICK-deficient cells.\",\n      \"method\": \"Primary cell stimulation, cytokine ELISA, RICK-deficient mice (in vivo), Nod1-deficient cells, siNOS expression assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout (Nod1-/- and RICK-/-) in vitro and in vivo with specific pathway readouts\",\n      \"pmids\": [\"17579072\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"NOD1 is required for H. pylori (cagPAI+)-induced p38 and ERK phosphorylation, as well as NF-κB and AP-1 activation in gastric epithelial cells. siNOD1-expressing cells showed significant reductions in p38 and ERK phosphorylation. NOD1-dependent JNK phosphorylation was observed in response to invasive Shigella but not H. pylori, indicating pathogen-specific responses.\",\n      \"method\": \"Stable siRNA knockdown of NOD1, kinase phosphorylation assays (p38, ERK, JNK), NF-κB and AP-1 reporter assays, pharmacological MAPK inhibitors\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — stable siRNA knockdown with multiple kinase and transcription factor readouts, pharmacological validation\",\n      \"pmids\": [\"20007577\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Rip2 kinase activity is required for its own protein stability; kinase-dead Rip2 mutants expressed in primary cells had severely compromised protein levels, and this instability could not be rescued by phospho-mimetic mutation. Loss of Rip2 kinase activity abrogated inflammatory cytokine production in response to Nod1 and Nod2 ligands both in vitro and in vivo.\",\n      \"method\": \"Retroviral expression of kinase-dead Rip2 mutants, Western blot for protein levels, cytokine production assays in primary cells and in vivo\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mutagenesis of kinase domain, in vitro and in vivo readouts, multiple mutants tested\",\n      \"pmids\": [\"19473975\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Purified recombinant NOD1 protein exists predominantly as a monomer; addition of the NOD1 ligand γ-Tri-DAP stimulates NOD1 protein oligomerization in vitro. NOD1 binds preferentially to ATP over ADP, AMP, or dATP (nucleotide binding specificity). Purified NOD1 protein directly binds purified pro-apoptotic protein Bid.\",\n      \"method\": \"Gel-sieve chromatography, fluorescence polarization assay for nucleotide binding, in vitro protein-protein binding with purified components\",\n      \"journal\": \"Journal of biotechnology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted in vitro with purified protein, multiple biochemical assays, single lab\",\n      \"pmids\": [\"22062585\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"2-Aminobenzimidazole compounds (e.g., Nodinitib-1/ML130) selectively inhibit NOD1-induced NF-κB activity. Mechanistic studies indicate these small molecules cause conformational changes of NOD1 in vitro and alter NOD1 subcellular targeting in cells.\",\n      \"method\": \"High-throughput NF-κB reporter assay, multiple counter-screens, in vitro conformational change assays, subcellular localization imaging\",\n      \"journal\": \"Chemistry & biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — HTS with counter-screens and mechanistic follow-up, single lab\",\n      \"pmids\": [\"21802003\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PSMA7 (proteasome subunit alpha type-7) directly interacts with NOD1 and downregulates NOD1 expression in a proteasome-dependent manner. Overexpression of PSMA7 inhibits NOD1-mediated apoptosis and NF-κB activation; knockdown of PSMA7 enhances NOD1 activity.\",\n      \"method\": \"Yeast two-hybrid screening, co-immunoprecipitation, GST-pulldown, ubiquitin assay, siRNA knockdown, Western blot\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — three orthogonal binding methods (Y2H, Co-IP, GST pulldown) plus functional readouts, single lab\",\n      \"pmids\": [\"23839082\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NOD1 and NOD2 are important mediators of ER-stress-induced inflammation. The ER stress inducers thapsigargin and DTT trigger IL-6 production in a NOD1/2-dependent fashion. Brucella abortus-induced inflammation via its type IV secretion effector VceC is TRAF2-, NOD1/2-, and RIP2-dependent; it can be reduced by ER stress inhibitor TUDCA or an IRE1α kinase inhibitor, placing NOD1/2 downstream of the IRE1α/TRAF2 ER stress signaling pathway.\",\n      \"method\": \"NOD1/2-deficient mouse and human cells, ER stress inducers, pharmacological inhibitors (TUDCA, IRE1α inhibitor), bacterial infection model, IL-6 cytokine measurement\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout cells plus pharmacological inhibition with multiple orthogonal stimuli and readouts, published in high-impact journal\",\n      \"pmids\": [\"27007849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Nod1 and its downstream adapter Rip2 are required for insulin trafficking in pancreatic beta cells in a cell-autonomous manner. Upon recognizing cognate ligands, Nod1 and Rip2 localize to insulin vesicles and recruit Rab1a to direct insulin trafficking through the cytoplasm. Intestinal lysozyme liberates Nod1 ligands into circulation, enabling long-range gut-islet communication that modulates host glucose tolerance.\",\n      \"method\": \"Nod1-/- and Rip2-/- beta cells, subcellular fractionation and co-localization imaging, Rab1a recruitment assays, intestinal lysozyme manipulation, glucose tolerance tests in mice\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockouts with cell-autonomous functional readout, subcellular localization with functional consequence, in vivo validation\",\n      \"pmids\": [\"31201384\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NOD1 promotes antiviral signaling by binding viral RNA (poly(I:C)) and regulating the interaction of MDA5 and MAVS. NOD1 enhances MDA5-MAVS complex formation, increasing type I IFN transcription. NOD1 also targets TRAF3 to modulate TRAF3-MAVS complexes. Human NOD1-MDA5 and NOD1-TRAF3 interactions were confirmed by co-immunoprecipitation.\",\n      \"method\": \"Co-immunoprecipitation, RNA binding assays (poly(I:C)), NOD1 knockdown/overexpression, IFN reporter assays, zebrafish NOD1-/- survival experiments, human cell confirmation\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with functional readouts, cross-species validation, but primarily teleost model with human confirmation\",\n      \"pmids\": [\"32169843\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Systemic Nod1 activation promotes autophagy-dependent reprogramming of macrophages toward an alternative phenotype and induces expansion of myeloid-derived suppressor cells (MDSCs), maintaining their immunosuppressive potential via arginase-1 activity. Myeloid-intrinsic Nod1 expression sustains intra-tumoral arginase-1 levels to foster an immunosuppressive tumor microenvironment during colorectal cancer development.\",\n      \"method\": \"Nod1-/- mice, peritonitis model, autophagy inhibitors, bone marrow chimeras, arginase-1 activity assays, colorectal cancer tumor model\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic models (KO mice, chimeras), biochemical assays, in vivo cancer model with mechanistic readouts\",\n      \"pmids\": [\"33503439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Endothelial NOD1 triggers VCAM-1 upregulation through the RIP2-NF-κB axis in an autocrine manner in response to peptidoglycan or oxidized LDLs, enhancing firm adhesion of monocytes and neutrophils to the vasculature in vivo. NOD1 genetic inactivation in Apoe-/- mice reduced atherosclerosis burden and monocyte/neutrophil accumulation in atheromata.\",\n      \"method\": \"NOD1-/- Apoe-/- mice, endothelial cell stimulation, VCAM-1 expression assays, leukocyte adhesion assays in vivo, RIP2-NF-κB pathway pharmacological inhibition\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic knockout model with in vitro mechanistic pathway dissection and multiple orthogonal readouts\",\n      \"pmids\": [\"30496704\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Selective NOD1 activation with C12-iE-DAP in mice induces cardiac dysfunction with decreased L-type Ca2+ current, reduced [Ca2+]i transients and Ca2+ load, decreased phospho-phospholamban, SERCA, and NCX expression, and increased RyR2 over-phosphorylation causing diastolic Ca2+ leak. These effects were lost in NOD1-/- mice. Co-treatment with NF-κB inhibitor BAY11-7082 prevented iE-DAP-induced cardiac dysfunction and Ca2+ handling impairment, placing NF-κB downstream of NOD1 in this pathway.\",\n      \"method\": \"NOD1-/- mice, selective NOD1 agonist C12-iE-DAP, echocardiography, patch-clamp electrophysiology, Ca2+ imaging, Western blot, NF-κB pharmacological inhibition\",\n      \"journal\": \"Cardiovascular research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO controls, multiple electrophysiological and biochemical readouts, pharmacological pathway validation\",\n      \"pmids\": [\"25824149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Nod1 activation in lung epithelial cells strongly activates C/EBPβ transcription factors in addition to modest NF-κB activation. This C/EBPβ activation appears independent of de novo protein synthesis, identifying a novel downstream transcriptional pathway from NOD1 in lung epithelium.\",\n      \"method\": \"Electrophoretic mobility shift assay (EMSA), reporter gene assays, protein synthesis inhibitor experiments in BEAS-2B cells\",\n      \"journal\": \"The European respiratory journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — EMSA and reporter assays with pharmacological controls, single lab, single cell type\",\n      \"pmids\": [\"17666556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Bacterial peptidoglycan (PGN) stimulates lipolysis in adipocytes via NOD1 in a cell-autonomous manner. NOD1-activating PGN stimulated MAPK (ERK1/2), PKA, and NF-κB in 3T3-L1 adipocytes. NOD1-mediated lipolysis was completely suppressed by simultaneous inhibition of ERK1/2 and PKA, or by hormone-sensitive lipase (HSL) inhibition. WAT explants from NOD1-/- mice did not respond to NOD1-activating PGN.\",\n      \"method\": \"NOD1-/- mouse adipose tissue explants, 3T3-L1 adipocyte stimulation, kinase inhibitors (ERK1/2, PKA, JNK), HSL inhibitor, NF-κB inhibitor, lipolysis assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO tissue and pharmacological dissection with multiple pathway inhibitors, cell-autonomous mechanism established\",\n      \"pmids\": [\"24828250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NOD1 inhibited proliferation of hepatocellular carcinoma (HCC) cells by directly interacting with proto-oncogene SRC and suppressing SRC activation, thereby inhibiting the SRC/MAPK axis. NOD1 overexpression induced cell cycle arrest at G1 phase and enhanced chemosensitivity to sorafenib and 5-FU in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation (NOD1-SRC interaction), overexpression, siRNA knockdown, cell cycle analysis, xenograft tumor model, Western blot for SRC/MAPK\",\n      \"journal\": \"Journal of molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP for direct interaction, functional KO/OE with mechanistic readouts, single lab\",\n      \"pmids\": [\"31872284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"METTL3 (m6A methyltransferase) negatively regulates NOD1 expression by promoting m6A-dependent mRNA degradation of NOD1 and RIPK2 transcripts via YTHDF1 and YTHDF2 m6A readers. METTL3 knockdown increased NOD1 and RIPK2 mRNA stability and expression, activating the NOD1 pathway and amplifying LPS-induced inflammatory responses in macrophages.\",\n      \"method\": \"METTL3 siRNA knockdown, YTHDF1/YTHDF2 silencing, mRNA stability assays, NOD1/RIPK2 expression by Western blot and qPCR, cytokine assays, NOD1 pathway-specific inhibitors\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple siRNA knockdowns with mRNA stability readout, pathway inhibitor confirmation, single lab\",\n      \"pmids\": [\"35176453\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"BFT-1 (B. fragilis toxin) directly binds to NOD1 and stabilizes NOD1 protein. NOD1, highly expressed on ALDH+ breast cancer stem cells, cooperates with GAK to phosphorylate NUMB, promoting its lysosomal degradation and thereby activating the NOTCH1-HEY1 signaling pathway to increase breast cancer stem cells.\",\n      \"method\": \"Direct binding assays (BFT-1 to NOD1), protein stability assays, co-immunoprecipitation (NOD1-GAK), NUMB phosphorylation and lysosomal degradation assays, NOTCH1 pathway reporter, ALDH+ cell sorting\",\n      \"journal\": \"Protein & cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding and Co-IP with mechanistic downstream readouts, single lab\",\n      \"pmids\": [\"38437016\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NOD1 activation in adipocytes causes diacylglycerol (DAG) accumulation through lipolysis, which activates PKCδ. PKCδ then stimulates IRAK1/4, leading to NF-κB and JNK activation and proinflammatory cytokine expression. Pharmacological inhibition or siRNA-mediated knockdown of IRAK1/4 attenuated NOD1-mediated NF-κB, JNK activation, and cytokine production.\",\n      \"method\": \"NOD1 agonist stimulation of 3T3-L1 adipocytes, DAG measurement, PKCδ activity assays, IRAK1/4 siRNA and pharmacological inhibition, NF-κB/JNK assays, cytokine measurement\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular basis of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA and pharmacological dissection of pathway in adipocytes, single lab with multiple readouts\",\n      \"pmids\": [\"30391544\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NOD1 (CARD4/NLRC1) is an intracellular pattern recognition receptor that, upon sensing meso-diaminopimelic acid-containing bacterial peptidoglycan muropeptides, undergoes ATP-dependent oligomerization and recruits the serine-threonine kinase RICK/RIP2 via homophilic CARD-CARD interaction; RIP2 then activates TAK1 and the IKK complex to drive NF-κB and MAPK (p38, ERK, JNK) signaling, inducing proinflammatory cytokine and chemokine production, while NOD1 can also promote caspase-9-dependent apoptosis, interact with MDA5/MAVS for antiviral signaling, localize to insulin vesicles to recruit Rab1a for insulin trafficking, engage SRC to suppress MAPK-driven proliferation, and be negatively regulated by PSMA7-mediated proteasomal degradation and METTL3-mediated m6A mRNA destabilization, with HSP90 and SGT1 required as chaperones for NOD1 stability and activation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NOD1 (CARD4) is an intracellular pattern-recognition receptor of the Apaf-1/CED-4 family, comprising an N-terminal CARD, a central nucleotide-binding domain, and C-terminal leucine-rich repeats, that couples cytosolic sensing of bacterial products to inflammatory and cell-fate signaling [#0]. Ligand engagement drives ATP-dependent oligomerization—purified NOD1 is a monomer that oligomerizes upon addition of the muropeptide ligand γ-Tri-DAP, and binds ATP preferentially over other nucleotides [#10]; this oligomerization is enhanced during invasive bacterial infection and is required for downstream activation [#3]. Activated NOD1 recruits the CARD-containing kinase RICK/RIP2 through homophilic CARD-CARD interaction [#0, #1], and RIP2 kinase activity is required both for its own stability and for downstream cytokine output [#9]. Signaling proceeds via TAK1 to NF-κB and MAPK (p38, ERK, JNK) activation [#5, #8], engaging TRAF6/NIK and the IKK complex [#1, #3] and additional transcriptional effectors such as C/EBPβ [#19], to induce proinflammatory cytokines and chemokines and neutrophil recruitment in vivo [#7]. In parallel, NOD1 promotes programmed cell death, originally as a caspase-9 activator [#0] and subsequently as a RIP2- and caspase-8-dependent apoptotic event [#5]. Beyond infection, NOD1 transduces ER-stress-induced inflammation downstream of the IRE1α/TRAF2 axis [#13] and operates cell-autonomously in metabolic and vascular tissues: it localizes to insulin vesicles to recruit Rab1a for insulin trafficking in pancreatic beta cells [#14], drives lipolysis and inflammatory signaling in adipocytes via ERK/PKA/HSL and a DAG–PKCδ–IRAK1/4 cascade [#20, #24], and promotes endothelial VCAM-1 expression and atherosclerosis through the RIP2–NF-κB axis [#17]. NOD1 activity is constrained by PSMA7-mediated proteasomal degradation [#12] and METTL3-directed m6A destabilization of NOD1 and RIPK2 mRNA [#22], while HSP90 and SGT1 act as chaperones required for NOD1 stability and activation [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established NOD1 as a CARD-bearing Apaf-1/CED-4 family member that links bacterial-sensing architecture to both NF-κB activation and caspase-9-dependent apoptosis, defining its dual signaling identity.\",\n      \"evidence\": \"Domain mapping, Co-IP, dominant-negative mutagenesis, caspase and NF-κB reporter assays\",\n      \"pmids\": [\"10329646\", \"10224040\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological ligand not yet defined\", \"Endogenous oligomerization state untested\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Implicated the leucine-rich repeats in conferring responsiveness to bacterial products independent of TLR4/MyD88, positioning NOD1 as a cytosolic sensor.\",\n      \"evidence\": \"NF-κB reporter assays and co-immunopurification of ligand-binding activity in HEK293T cells with LRR deletion mutants\",\n      \"pmids\": [\"11058605\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ligand initially assigned to LPS; later refined to peptidoglycan muropeptides\", \"Single lab, no direct structural binding data\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrated that infection-triggered NOD1 oligomerization assembles a transient NOD1-RICK-IKK complex necessary for NF-κB and JNK activation, defining the signaling assembly.\",\n      \"evidence\": \"Shigella infection, dominant-negative overexpression, Co-IP, microinjection of LPS, reporter assays\",\n      \"pmids\": [\"11463746\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of the complex unresolved\", \"Kinetics of complex disassembly not defined\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Extended NOD1 partner repertoire to caspase-1, linking it to pro-caspase-1 oligomerization and IL-1β secretion.\",\n      \"evidence\": \"Reciprocal Co-IP, caspase-1 processing Western blots, IL-1β ELISA in transfected/stable cells\",\n      \"pmids\": [\"12459189\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relies on overexpression\", \"Endogenous relevance to inflammasome assembly untested\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Genetically separated NOD1 outputs: apoptosis is caspase-8- and RIP2-dependent, whereas JNK/IL-8 responses require TAK1 and RIP2, placing TAK1 as a central downstream node.\",\n      \"evidence\": \"Caspase-8 and RIP2/TAK1 knockout/dominant-negative, IL-8 ELISA, JNK kinase assays\",\n      \"pmids\": [\"17186025\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Branch-point selection between apoptosis and inflammation not mechanistically resolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identified HSP90 and SGT1 as chaperones required for NOD1 protein stability and activation respectively, establishing a folding-dependent regulatory layer.\",\n      \"evidence\": \"siRNA knockdown, pharmacological HSP90 inhibition, cellular NOD1 activation assays with Chp1 negative control\",\n      \"pmids\": [\"17420470\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct chaperone-NOD1 binding interface not mapped\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Showed in vivo that NOD1/RICK drive chemokine (CXCL1, CCL2) production, iNOS expression, and neutrophil recruitment, demonstrating physiological inflammatory function and a novel C/EBPβ transcriptional arm.\",\n      \"evidence\": \"Nod1-/- and RICK-/- cells and mice, cytokine ELISA, EMSA and reporter assays in epithelial cells\",\n      \"pmids\": [\"17579072\", \"17666556\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of C/EBPβ activation independent of protein synthesis undefined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Established that RIP2 kinase activity is required for RIP2 stability and for NOD1/NOD2-driven cytokine production, identifying the kinase as a tractable signaling chokepoint.\",\n      \"evidence\": \"Retroviral kinase-dead RIP2 mutants, protein-level Western blots, cytokine assays in vitro and in vivo\",\n      \"pmids\": [\"19473975\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relevant RIP2 autophosphorylation substrates not enumerated\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Reconstituted NOD1 activation biochemically, showing ligand-induced oligomerization, ATP-binding specificity, and direct binding to pro-apoptotic Bid, plus chemical-probe modulation of conformation and localization.\",\n      \"evidence\": \"Gel-sieve chromatography, fluorescence polarization, purified protein binding; HTS small-molecule inhibitors with conformational and imaging assays\",\n      \"pmids\": [\"22062585\", \"21802003\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No high-resolution structure of activated oligomer\", \"Functional role of NOD1-Bid interaction in cells untested\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined PSMA7 as a direct NOD1-interacting factor driving proteasomal turnover, establishing negative regulation of NOD1 abundance.\",\n      \"evidence\": \"Y2H, Co-IP, GST-pulldown, ubiquitin assay, siRNA with NF-κB/apoptosis readouts\",\n      \"pmids\": [\"23839082\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E3 ligase mediating NOD1 ubiquitination not identified\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Placed NOD1/NOD2 downstream of the IRE1α/TRAF2 ER-stress pathway, connecting unfolded-protein-response signaling to RIP2-dependent inflammation.\",\n      \"evidence\": \"NOD1/2-deficient mouse and human cells, ER stress inducers, TUDCA/IRE1α inhibitors, Brucella infection, IL-6 measurement\",\n      \"pmids\": [\"27007849\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ER stress generates an endogenous NOD1 ligand or activates it ligand-independently is unresolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrated tissue-level consequences of NOD1 activation in the heart, with NF-κB-dependent impairment of Ca2+ handling and contractile function.\",\n      \"evidence\": \"NOD1-/- mice, C12-iE-DAP agonist, echocardiography, patch-clamp, Ca2+ imaging, NF-κB inhibition\",\n      \"pmids\": [\"25824149\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cell type within myocardium mediating the effect not isolated\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established endothelial NOD1 as a driver of leukocyte adhesion and atherosclerosis via the RIP2-NF-κB-VCAM-1 axis, responsive to peptidoglycan and oxidized LDL.\",\n      \"evidence\": \"NOD1-/-Apoe-/- mice, endothelial stimulation, VCAM-1 and adhesion assays, RIP2-NF-κB pharmacological inhibition\",\n      \"pmids\": [\"30496704\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct sensing of oxLDL versus indirect activation not distinguished\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealed cell-autonomous metabolic functions: NOD1/RIP2 localize to insulin vesicles to recruit Rab1a for insulin trafficking, and NOD1 inhibits hepatocellular carcinoma proliferation by binding and suppressing SRC.\",\n      \"evidence\": \"Nod1-/-/Rip2-/- beta cells with fractionation/colocalization and glucose tolerance tests; Co-IP, cell-cycle analysis and xenografts for SRC suppression\",\n      \"pmids\": [\"31201384\", \"31872284\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How vesicle localization is targeted versus plasma-membrane sensing is unclear\", \"SRC interaction surface not mapped\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Expanded NOD1 to antiviral sensing, where it binds viral RNA and promotes MDA5-MAVS and modulates TRAF3-MAVS complex formation to boost type I IFN.\",\n      \"evidence\": \"Co-IP, poly(I:C) binding, IFN reporters, zebrafish NOD1-/- survival, human cell confirmation\",\n      \"pmids\": [\"32169843\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Primarily teleost model\", \"Direct RNA-binding region of NOD1 undefined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined NOD1 in myeloid cells as a driver of immunosuppression, sustaining arginase-1 and MDSC expansion through autophagy-dependent macrophage reprogramming in colorectal cancer.\",\n      \"evidence\": \"Nod1-/- mice, bone-marrow chimeras, autophagy inhibitors, arginase-1 assays, colorectal cancer model\",\n      \"pmids\": [\"33503439\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous ligand sustaining systemic NOD1 activation in tumors not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified METTL3-directed m6A modification as a post-transcriptional brake on NOD1 and RIPK2 expression via YTHDF1/2-mediated mRNA decay.\",\n      \"evidence\": \"METTL3 and YTHDF1/2 siRNA, mRNA stability assays, NOD1/RIPK2 expression and cytokine readouts in macrophages\",\n      \"pmids\": [\"35176453\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific m6A sites on NOD1 transcript not mapped\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Linked NOD1 stabilization by the bacterial toxin BFT-1 to a kinase-scaffolding role with GAK in phosphorylating NUMB, activating NOTCH1-HEY1 signaling to expand breast cancer stem cells.\",\n      \"evidence\": \"Direct BFT-1 binding and stability assays, NOD1-GAK Co-IP, NUMB phosphorylation/degradation, NOTCH reporter, ALDH+ sorting\",\n      \"pmids\": [\"38437016\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether NOD1 itself has kinase activity versus scaffolds GAK is unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single sensor selects among its divergent outputs—inflammation, apoptosis, antiviral IFN, vesicular trafficking, and tumor-context signaling—as a function of ligand, tissue, and partner availability remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the activated NOD1 oligomer in any output context\", \"Endogenous ligands driving non-infectious (metabolic, vascular, tumor) functions undefined\", \"Determinants of subcellular targeting versus cytosolic signaling unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 3, 7]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [15]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 23]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [14]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [17]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [3, 7, 13]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 5, 8]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [16]}\n    ],\n    \"complexes\": [\"NOD1-RICK/RIP2-IKK signaling complex\"],\n    \"partners\": [\"RIPK2\", \"CASP1\", \"TRAF6\", \"PSMA7\", \"SRC\", \"GAK\", \"MDA5\", \"TRAF3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}