{"gene":"HMGB1","run_date":"2026-06-10T01:55:22","timeline":{"discoveries":[{"year":1992,"finding":"HMGB1 (HMG1) HMG-box domain recognizes and binds four-way junction DNA structures (cross-shaped DNA with ~60° and ~120° angles between arms) in a structure-specific, sequence-independent manner, and binding to linear duplex DNA containing the SRY target sequence produces a sharp DNA bend.","method":"In vitro DNA binding assays with four-way junction DNA and linear duplex DNA; gel mobility shift and footprinting experiments","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro binding assay with defined DNA substrates, replicated across HMG1 and SRY HMG-box, demonstrating structure-specific recognition","pmids":["1425584"],"is_preprint":false},{"year":1979,"finding":"HMG1 unwinds the DNA double helix by local denaturation of base pairs; it has higher affinity for single-stranded than double-stranded DNA and introduces a net unwinding angle of ~22° per molecule. In absence of salt, HMG1 stabilizes DNA; in presence of salt, it destabilizes DNA.","method":"DNA melting absorption technique; competition unwinding experiments measuring topological winding numbers","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro biochemical assay with quantitative measurements of unwinding angle and DNA thermal stability, multiple orthogonal measurements","pmids":["226939"],"is_preprint":false},{"year":1994,"finding":"The acidic C-terminal tail of HMGB1 downregulates the DNA-binding and supercoiling activities of the tandem HMG-box domains (HMG3) in an ionic strength-dependent manner; removal of the tail increases DNA affinity and supercoiling activity. The tandem HMG-box domains can loop, compact, and change topology of DNA. The acidic tail directly modulates DNA-binding domain specificity.","method":"DNA supercoiling assays with topoisomerase I; electron microscopy of DNA-protein complexes; comparison of HMG1 vs. tryptic HMG3 (tail-deleted form)","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro assay with defined protein variants, electron microscopy, multiple orthogonal methods in one study","pmids":["8152909"],"is_preprint":false},{"year":1998,"finding":"HMGB1 (HMG-1) directly binds p53 protein in vitro and enhances p53 DNA-binding activity, including that of a constitutively active C-terminally deleted p53 (p53Δ30), by a mechanism distinct from other known p53 activators. HMG-1 promotes assembly of higher-order p53 nucleoprotein structures and stimulates p53-mediated transactivation in vivo.","method":"Biochemical purification from HeLa nuclear extracts; recombinant His-tagged HMG-1 in vitro binding and DNA-binding assays; transient transfection transactivation assays","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of direct protein-protein interaction and DNA-binding stimulation, supported by in vivo transactivation assay, single lab but multiple orthogonal methods","pmids":["9472015"],"is_preprint":false},{"year":1999,"finding":"HMG1 domain B binds cisplatin-modified DNA with high affinity (Kd ~60 nM) and induces a bend angle of 80–95° in the cisplatin-modified DNA, as determined by FRET distance measurements. Kinetic parameters: kon = 1.1 × 10⁹ M⁻¹s⁻¹, koff = 30 s⁻¹.","method":"Fluorescence resonance energy transfer (FRET) with fluorescein/rhodamine-labeled DNA probes; fluorescence titration; stopped-flow fluorescence spectroscopy","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with quantitative FRET structural measurement plus kinetic analysis, multiple orthogonal methods in one study","pmids":["10212205"],"is_preprint":false},{"year":2000,"finding":"HMGB1 stimulates DNA end joining (both cohesive-end and blunt-end ligation by T4 DNA ligase) by physically associating two DNA molecules via their ends. This activity requires the B domain of HMGB1, specifically the C-terminal flanking basic residues and conserved basic/hydrophobic residues within the HMG box.","method":"In vitro ligation assay with T4 DNA ligase; pull-down assays; electron microscopy and scanning force microscopy of DNA-protein complexes; domain deletion/mutation analysis","journal":"European journal of biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of ligation activity, domain mapping by mutagenesis, and direct visualization by EM/SFM in one study","pmids":["10866811"],"is_preprint":false},{"year":2007,"finding":"The acidic C-terminal tail of HMGB1 directly interacts with the N-terminal HMG-box domains (confirmed by NMR), and this intramolecular interaction can be competed more efficiently by four-way junction DNA than by linear dsDNA. Mutations in the N-terminal region that disrupt C-tail binding abolish HMGB1's ability to distinguish linear DNA from four-way junction DNA, defining a mechanism by which the acidic tail enhances domain specificity for structured DNAs.","method":"NMR; competitive DNA-binding assays; site-directed mutagenesis of the N-terminal region","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structural validation of intramolecular interaction combined with mutagenesis and functional competition assays","pmids":["17585880"],"is_preprint":false},{"year":2010,"finding":"Endogenous HMGB1 directly interacts with the autophagy protein Beclin1 and displaces Bcl-2 from Beclin1, thereby enhancing autophagic flux. Reactive oxygen species promote cytosolic translocation of HMGB1. Mutation of C106 (but not C23 or C45) promotes cytosolic HMGB1 localization and sustained autophagy. The intramolecular disulfide bond between C23 and C45 is required for HMGB1 binding to Beclin1 and sustaining autophagy.","method":"Co-immunoprecipitation; direct protein interaction assays; site-directed mutagenesis of cysteine residues; pharmacological inhibition (ethyl pyruvate); autophagy flux measurement; HMGB1 knockout/knockdown cells","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, cysteine mutagenesis with functional readout, genetic KO with phenotypic rescue, multiple orthogonal methods across multiple cell systems","pmids":["20819940"],"is_preprint":false},{"year":2012,"finding":"PKR (double-stranded RNA-dependent protein kinase) physically interacts with multiple inflammasome components (NLRP3, NLRP1, NLRC4, AIM2) and is required for inflammasome activation; PKR autophosphorylation in a cell-free reconstituted system with recombinant NLRP3, ASC, and pro-caspase-1 is sufficient to reconstitute inflammasome activity, leading to HMGB1 release. PKR deficiency severely impairs HMGB1 secretion in response to diverse inflammasome agonists.","method":"Genetic deletion and pharmacological inhibition of PKR; Co-IP of PKR with inflammasome components; cell-free reconstitution with recombinant proteins; in vivo peritonitis model measuring HMGB1 secretion","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — cell-free reconstitution plus genetic KO plus Co-IP across multiple agonists, replicated in vivo","pmids":["22801494"],"is_preprint":false},{"year":2012,"finding":"HMGB1 and p53 form a complex that reciprocally regulates autophagy and apoptosis. p53 knockout increases cytosolic HMGB1 expression and induces autophagy; HMGB1 knockout increases p53 cytoplasmic localization and decreases autophagy. p53 acts as a negative regulator of the HMGB1/Beclin1 complex. The HMGB1/p53 complex affects the cytoplasmic localization of the reciprocal binding partner.","method":"Co-immunoprecipitation; genetic knockout of p53 (HCT116) and HMGB1 (MEFs); subcellular fractionation; autophagy measurement","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and genetic KO in two cell systems, single lab","pmids":["22345153"],"is_preprint":false},{"year":2014,"finding":"PARP-1 regulates LPS-induced HMGB1 translocation and release from macrophages by two mechanisms: (1) PARylating HMGB1 to facilitate its subsequent acetylation, and (2) increasing the HAT/HDAC activity ratio to promote HMGB1 acetylation. PARylated HMGB1 remains nuclear, whereas acetylated HMGB1 localizes to the cytoplasm. Genetic or pharmacological inhibition of PARP-1 suppresses HMGB1 acetylation, nuclear-to-cytoplasm translocation, and extracellular release. ROS and ERK signaling upstream of PARP activation are also required.","method":"Genetic PARP-1 depletion and PARP inhibitors in bone marrow-derived macrophages; in vitro enzymatic PARylation reaction; HAT/HDAC activity assays; subcellular fractionation; immunofluorescence","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO plus pharmacological inhibition plus in vitro enzymatic reconstitution plus subcellular fractionation, multiple orthogonal methods in one study","pmids":["25392528"],"is_preprint":false},{"year":2013,"finding":"The inflammatory activity of extracellular HMGB1 depends on its redox state: an intramolecular disulfide bond between C23 and C45 combined with a reduced C106 confers cytokine/TLR4-stimulating activity. Oxidation of C106 blocks stimulatory activity and promotes immune tolerance. Thus the chemoattractant and cytokine-inducing activities of HMGB1 are separable and depend on distinct cysteine redox states.","method":"Redox state characterization with defined recombinant HMGB1 isoforms; TLR4 activation assays; leukocyte recruitment assays","journal":"Antioxidants & redox signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple defined redox isoforms tested in functional assays, reviewed/replicated by multiple labs but primary evidence in single lab per experiment","pmids":["23373897"],"is_preprint":false},{"year":2012,"finding":"The chemoattractant activity of HMGB1 is entirely mediated through formation of a heterocomplex with the chemokine CXCL12, which acts on CXCR4 more potently than CXCL12 alone. Only the all-thiol (fully reduced) form of HMGB1 can form this heterocomplex with CXCL12. The disulfide HMGB1 (C23–C45 bond) activates TLR4 to induce cytokine release but cannot recruit leukocytes, demonstrating that chemoattractant and cytokine-inducing activities of HMGB1 are separable redox-dependent functions.","method":"Biochemical characterization of HMGB1-CXCL12 complex; receptor binding assays on CXCR4; leukocyte migration assays with distinct redox isoforms","journal":"Molecular immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — defined recombinant redox isoforms, receptor-specific functional assays, complex formation characterization, findings replicated across multiple reports","pmids":["23207101"],"is_preprint":false},{"year":2010,"finding":"HMGB1 acts as a cofactor in base excision repair (BER), inhibiting single-nucleotide BER and stimulating long-patch BER by modulating the activities of BER enzymes. HMGB1 was found cross-linked to a BER intermediate in cells, indicating direct participation in the BER pathway.","method":"In vitro BER assays; cross-linking of HMGB1 to BER intermediates; enzymatic activity measurements of BER enzymes in presence/absence of HMGB1","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro reconstituted BER assays with direct cross-linking evidence, single lab","pmids":["20123074"],"is_preprint":false},{"year":2016,"finding":"TLR5 is an HMGB1 receptor. HMGB1 binds TLR5 and initiates NF-κB signaling via MyD88-dependent signaling, resulting in proinflammatory cytokine production and pain hypersensitivity in vivo. The C-terminal tail region of HMGB1 is essential for its interaction with TLR5.","method":"Biophysical binding assays; in vitro NF-κB signaling assays with TLR5-expressing cells; in vivo allodynia model; C-terminal tail truncation analysis","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biophysical binding plus functional NF-κB signaling plus in vivo phenotype, domain mapping, single lab","pmids":["27760316"],"is_preprint":false},{"year":2019,"finding":"HMGB1 is released by ferroptotic cells in an autophagy-dependent manner. Genetic ablation (ATG5-/- or ATG7-/- cells) or pharmacological inhibition of autophagy blocks ferroptosis-induced HMGB1 release. Autophagy-mediated HDAC inhibition promotes HMGB1 acetylation leading to its release during ferroptosis. AGER (RAGE), but not TLR4, is required for HMGB1-mediated inflammation in macrophages in response to ferroptotic cells.","method":"Genetic KO of ATG5/ATG7; pharmacological autophagy inhibitors; HMGB1 release measurement; HDAC inhibition assays; receptor-specific macrophage stimulation assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple genetic KOs plus pharmacological inhibitors plus receptor specificity testing, single lab","pmids":["30686534"],"is_preprint":false},{"year":2020,"finding":"HMGB1 release after inflammasome activation in macrophages occurs only under conditions causing cell lysis (pyroptosis), not during inflammasome activation per se. When pyroptosis is prevented, HMGB1 is not released despite inflammasome activation and IL-1β secretion. Gasdermin D knockout mice secrete HMGB1 normally during endotoxemia (indirect/pyroptosis-independent mechanism in vivo), while IL-1β secretion is completely blocked.","method":"Genetic gasdermin D knockout macrophages; LPS stimulation; separation of pyroptosis from inflammasome activation; measurement of HMGB1 and IL-1β secretion in vitro and in vivo","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO in vitro and in vivo with well-controlled separation of pyroptosis from inflammasome activation, rigorous mechanistic dissection","pmids":["32917873"],"is_preprint":false},{"year":2022,"finding":"During cuproptosis, copper accumulation-induced ATP depletion activates AMPK, which phosphorylates HMGB1 to promote its extracellular release. Genetic or pharmacological inhibition of AMPK limits cuproptosis-associated HMGB1 release. Extracellular HMGB1 from cuproptotic cells signals through AGER (RAGE) to induce inflammatory cytokine production; HMGB1-deficient cuproptotic cells show greatly reduced AGER-dependent inflammatory signaling.","method":"RNAi knockdown of AMPK; pharmacological AMPK inhibition (dorsomorphin); HMGB1 phosphorylation analysis; AGER-dependent cytokine production assays; cuproptosis inducers","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and pharmacological inhibition of AMPK with HMGB1 phosphorylation readout and receptor-specific functional assays, single lab","pmids":["36211458"],"is_preprint":false},{"year":2011,"finding":"Intracellular HMGB1 negatively regulates efferocytosis (phagocytosis of apoptotic cells). HMGB1 knockdown in macrophages and fibroblasts increases phagocytosis of apoptotic cells, enhances Rac-1 activation and cytoskeletal rearrangement, and increases phosphorylation of ERK and FAK. HMGB1 physically interacts with Src kinase and inhibits Src-FAK interactions; in vitro, purified HMGB1 directly diminishes interactions between purified FAK and Src.","method":"HMGB1 siRNA knockdown; phagocytosis assays; Rac-1 activation assay; phosphorylation analysis; Src inhibition; in vitro pulldown of purified FAK and Src with/without HMGB1","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD phenotype confirmed by in vitro reconstitution of FAK-Src interaction, multiple methods, single lab","pmids":["21957148"],"is_preprint":false},{"year":2021,"finding":"Nociceptors directly release HMGB1 upon activation: transgenic nociceptors expressing channelrhodopsin-2 release HMGB1 in response to light stimulation. Neuron-specific HMGB1 silencing (Syn-Cre × HMGB1f/f mice) protects against cutaneous inflammation, allodynia after sciatic nerve injury, and joint inflammation in collagen antibody-induced arthritis, establishing a required role for nociceptor-derived HMGB1 in neurogenic inflammation.","method":"Optogenetic stimulation of channelrhodopsin-2-expressing nociceptors; conditional neuron-specific HMGB1 knockout (Syn-Cre/HMGB1fl/fl); sciatic nerve injury model; collagen antibody-induced arthritis model","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — optogenetic direct release demonstration plus conditional KO in two disease models with defined phenotypic readouts, multiple orthogonal approaches","pmids":["34385304"],"is_preprint":false},{"year":1994,"finding":"The mouse hmg1 gene contains five exons (first untranslated), with a promoter coinciding with a CpG island and lacking a TATA sequence, consistent with ubiquitous expression. No evidence for a membrane localization signal was found in the coding sequence.","method":"Gene isolation, sequencing, exon mapping; reporter gene expression assays; Southern blot analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct gene characterization with reporter assays, single lab","pmids":["7961836"],"is_preprint":false},{"year":2016,"finding":"C1q and HMGB1 cooperate via a complex involving RAGE and LAIR-1 to induce monocyte differentiation into anti-inflammatory M2-like macrophages, counterbalancing HMGB1's pro-inflammatory M1-inducing activity. This pathway depends on the relative levels of C1q and HMGB1.","method":"In vitro monocyte differentiation assays; receptor blocking experiments (RAGE and LAIR-1); co-stimulation with C1q and HMGB1","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — receptor-specific blocking experiments with defined functional readout, single lab","pmids":["27683415"],"is_preprint":false},{"year":2018,"finding":"Extracellular HMGB1 can be internalized via RAGE-receptor-mediated endocytosis into the endolysosomal compartment while bound to other extracellular proinflammatory molecules. After endocytosis, HMGB1 destabilizes lysosomal membranes, facilitating access of HMGB1-bound partner molecules to endosomal and cytosolic pattern recognition receptors.","method":"RAGE-dependent endocytosis assays; lysosomal membrane destabilization assays; co-internalization experiments with HMGB1 and bound molecules","journal":"Seminars in immunology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — review paper summarizing mechanistic findings without detailed primary experimental description in abstract; single mechanism proposed","pmids":["29530410"],"is_preprint":false},{"year":2010,"finding":"HMGB1 acts as a cofactor in base excision repair, stimulating long-patch BER sub-pathway while inhibiting single-nucleotide BER, thereby directing repair toward long-patch BER, which can result in trinucleotide repeat instability.","method":"In vitro BER assays; cross-linking of HMGB1 to BER intermediates","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — reconstituted in vitro BER assay, cross-linking evidence, single lab","pmids":["20123074"],"is_preprint":false}],"current_model":"HMGB1 is a multifunctional nuclear protein that, inside the nucleus, acts as an architectural DNA-binding chromatin protein (binding distorted/structured DNA preferentially via its HMG-box domains, bending DNA, modulating transcription factor binding including p53 activation, and participating in base excision repair); in the cytoplasm, it promotes autophagy by directly binding Beclin1 and displacing Bcl-2 (requiring the C23–C45 disulfide bond) and is negatively regulated by its complex with p53; its secretion and extracellular release are tightly controlled by post-translational modifications (particularly acetylation driven by PARP-1/HAT/HDAC balance, and phosphorylation by AMPK), with active secretion requiring nuclear-to-cytoplasmic translocation, while passive release occurs during necrosis/lytic cell death; extracellularly, HMGB1's biological activity is determined by its cysteine redox state—all-thiol HMGB1 forms a heterocomplex with CXCL12 to act as a chemoattractant via CXCR4, disulfide (C23–C45) HMGB1 activates TLR4 (with MD-2) to induce cytokine release, and fully oxidized HMGB1 is inactive—while HMGB1 also signals via RAGE (mediating endocytosis), TLR2, TLR4, and TLR5 (via its C-terminal tail, triggering MyD88-dependent NF-κB activation), and PKR serves as an upstream activator of inflammasome-dependent HMGB1 release."},"narrative":{"mechanistic_narrative":"HMGB1 is a multifunctional protein that bridges nuclear chromatin architecture, DNA repair, and extracellular danger signaling, with its activity in each compartment governed by domain structure and post-translational/redox state [PMID:1425584, PMID:20819940, PMID:23207101]. In the nucleus it functions as an architectural DNA-binding protein: its tandem HMG-box domains recognize distorted and structured DNA (four-way junctions, cisplatin adducts) in a sequence-independent manner, bend and unwind the duplex, and can loop, compact, and supercoil DNA, while the acidic C-terminal tail folds back onto the boxes to tune their specificity for structured over linear DNA [PMID:1425584, PMID:226939, PMID:8152909, PMID:10212205, PMID:17585880]. Through these activities HMGB1 stimulates p53 DNA binding and transactivation by promoting higher-order nucleoprotein assembly [PMID:9472015], promotes DNA end joining by juxtaposing DNA ends [PMID:10866811], and acts as a cofactor in base excision repair, directing repair toward the long-patch sub-pathway [PMID:20123074]. In the cytoplasm HMGB1 promotes autophagy by binding Beclin1 and displacing Bcl-2 in a manner requiring the C23–C45 disulfide bond, an activity reciprocally restrained by complex formation with p53 [PMID:20819940, PMID:22345153]. HMGB1 release is actively controlled: PARP-1 drives PARylation and an increased HAT/HDAC ratio to acetylate HMGB1 and trigger nuclear-to-cytoplasmic translocation [PMID:25392528], AMPK phosphorylates HMGB1 during cuproptosis [PMID:36211458], and autophagy-dependent HDAC inhibition promotes its acetylation and release during ferroptosis [PMID:30686534], with PKR-dependent inflammasome activity required for secretion but pyroptotic membrane rupture governing actual release in macrophages [PMID:22801494, PMID:32917873]. Once extracellular, HMGB1's function is redox-determined: the all-thiol form pairs with CXCL12 to chemoattract leukocytes via CXCR4, the C23–C45 disulfide form activates TLR4 to induce cytokines, and full oxidation abolishes activity [PMID:23373897, PMID:23207101]; it additionally signals through TLR5 via its C-terminal tail to drive MyD88/NF-κB-dependent inflammation and pain [PMID:27760316], and through RAGE to mediate endocytosis, M2 macrophage polarization with C1q, and inflammation from regulated cell death [PMID:30686534, PMID:36211458, PMID:27683415, PMID:29530410]. Nociceptor-derived HMGB1 is required for neurogenic inflammation in vivo [PMID:34385304].","teleology":[{"year":1979,"claim":"Established that HMGB1 actively alters DNA topology rather than passively coating DNA, defining a biochemical basis for its architectural role.","evidence":"DNA melting and competition unwinding assays measuring unwinding angle and thermal stability in vitro","pmids":["226939"],"confidence":"High","gaps":["Did not define structural preference for distorted DNA","No domain-level dissection of the unwinding activity"]},{"year":1992,"claim":"Showed HMGB1's HMG-box recognizes DNA by structure rather than sequence and bends the duplex, explaining how it targets junctions and distorted sites.","evidence":"In vitro four-way junction and linear duplex binding, gel shift and footprinting","pmids":["1425584"],"confidence":"High","gaps":["Role of the acidic tail not addressed","Cellular consequences of structure-specific binding untested"]},{"year":1994,"claim":"Defined the acidic C-terminal tail as an autoregulatory element that downregulates and tunes the DNA-binding/supercoiling activity of the tandem boxes.","evidence":"Supercoiling assays, EM, and comparison of full-length vs tail-deleted HMG3","pmids":["8152909"],"confidence":"High","gaps":["Molecular mechanism of tail-box interaction not yet structurally resolved","Physiological setting of looping/compaction unclear"]},{"year":1994,"claim":"Characterized the gene structure and TATA-less CpG-island promoter, supporting ubiquitous expression with no encoded membrane-targeting signal.","evidence":"Gene isolation, exon mapping, reporter assays in mouse hmg1","pmids":["7961836"],"confidence":"Medium","gaps":["Regulation of expression in stress/inflammation not addressed","Secretion route left unexplained given lack of signal sequence"]},{"year":1998,"claim":"Linked HMGB1's DNA architecture to transcription by showing it directly binds p53 and stimulates its DNA binding and transactivation.","evidence":"Biochemical purification, recombinant in vitro binding, transactivation assays","pmids":["9472015"],"confidence":"High","gaps":["Genomic targets of HMGB1/p53 cooperation not mapped","In vivo relevance to p53 programs untested here"]},{"year":1999,"claim":"Quantified high-affinity binding and severe bending of cisplatin-damaged DNA, mechanistically connecting HMGB1 to recognition of damaged DNA.","evidence":"FRET distance measurement and stopped-flow kinetics with HMG1 domain B","pmids":["10212205"],"confidence":"High","gaps":["Downstream effect on repair or cisplatin sensitivity not assessed","Only domain B examined"]},{"year":2000,"claim":"Showed HMGB1 physically juxtaposes DNA ends to stimulate ligation, mapping the activity to the B domain and flanking basic residues.","evidence":"In vitro T4 ligase joining assays, EM/SFM, domain mutagenesis","pmids":["10866811"],"confidence":"High","gaps":["Relevance to cellular NHEJ pathway not established","Partner repair factors not identified"]},{"year":2010,"claim":"Defined HMGB1 as a base excision repair cofactor that biases repair toward the long-patch sub-pathway, with potential consequences for repeat instability.","evidence":"In vitro reconstituted BER assays and cross-linking to BER intermediates","pmids":["20123074"],"confidence":"Medium","gaps":["Single lab, in vitro reconstitution","BER enzyme contacts not structurally defined","In vivo repair phenotype not shown"]},{"year":2010,"claim":"Established the cytoplasmic autophagy function of HMGB1, showing it binds Beclin1, displaces Bcl-2, and requires the C23–C45 disulfide bond, with ROS driving cytosolic translocation.","evidence":"Reciprocal Co-IP, cysteine mutagenesis, autophagy flux in KO/KD cells","pmids":["20819940"],"confidence":"High","gaps":["Structural basis of Beclin1 binding not resolved","Redox sensing mechanism of translocation unclear"]},{"year":2011,"claim":"Revealed an intracellular brake on efferocytosis by showing HMGB1 inhibits Src-FAK interaction and dampens Rac1-driven phagocytic cytoskeletal signaling.","evidence":"siRNA knockdown phenotypes plus in vitro pulldown of purified FAK/Src with HMGB1","pmids":["21957148"],"confidence":"Medium","gaps":["Single lab","Binding site on Src not mapped","In vivo efferocytosis relevance untested"]},{"year":2012,"claim":"Placed PKR upstream of inflammasome-dependent HMGB1 secretion, reconstituting inflammasome activity in a cell-free system.","evidence":"PKR KO/inhibition, Co-IP with inflammasome components, cell-free reconstitution, in vivo peritonitis","pmids":["22801494"],"confidence":"High","gaps":["Direct PKR substrates within the inflammasome not defined","Link to membrane permeabilization not addressed"]},{"year":2012,"claim":"Demonstrated that HMGB1's chemoattractant activity is mediated by an all-thiol HMGB1–CXCL12 heterocomplex acting on CXCR4, distinct from its disulfide-dependent TLR4 cytokine activity.","evidence":"Complex characterization, CXCR4 binding and leukocyte migration with defined redox isoforms","pmids":["23207101"],"confidence":"High","gaps":["Structure of the HMGB1–CXCL12 complex not resolved","In vivo redox dynamics not quantified"]},{"year":2012,"claim":"Showed the HMGB1–p53 complex reciprocally regulates autophagy and apoptosis by controlling each partner's cytoplasmic localization, with p53 restraining the HMGB1/Beclin1 axis.","evidence":"Reciprocal Co-IP and p53/HMGB1 KO with subcellular fractionation and autophagy readout","pmids":["22345153"],"confidence":"Medium","gaps":["Single lab","Direct competition with Beclin1 not biochemically isolated"]},{"year":2013,"claim":"Refined the redox code by showing the C23–C45 disulfide plus reduced C106 confers TLR4/cytokine activity while C106 oxidation enforces tolerance, separating chemoattractant and cytokine functions.","evidence":"Defined recombinant redox isoforms in TLR4 activation and leukocyte recruitment assays","pmids":["23373897"],"confidence":"Medium","gaps":["Per-experiment evidence from single lab","In vivo redox transition kinetics not measured"]},{"year":2016,"claim":"Identified TLR5 as an HMGB1 receptor driving MyD88/NF-κB inflammation and pain, mapping the interaction to the C-terminal tail.","evidence":"Biophysical binding, NF-κB assays in TLR5 cells, in vivo allodynia, C-terminal truncation","pmids":["27760316"],"confidence":"Medium","gaps":["Single lab","Redox-state dependence of TLR5 binding not tested"]},{"year":2016,"claim":"Showed HMGB1 can drive anti-inflammatory M2 polarization when cooperating with C1q via RAGE and LAIR-1, balancing its pro-inflammatory M1 activity.","evidence":"Monocyte differentiation with C1q/HMGB1 co-stimulation and RAGE/LAIR-1 blocking","pmids":["27683415"],"confidence":"Medium","gaps":["Single lab","Molecular composition of the C1q/HMGB1/RAGE/LAIR-1 complex not resolved"]},{"year":2018,"claim":"Proposed that RAGE-mediated endocytosis delivers HMGB1 and its bound cargo to the endolysosome, where HMGB1 destabilizes membranes to reach cytosolic pattern recognition receptors.","evidence":"Review summarizing RAGE endocytosis and lysosomal destabilization assays","pmids":["29530410"],"confidence":"Low","gaps":["Review-level, lacking detailed primary experimental description","Membrane destabilization mechanism not biochemically defined"]},{"year":2019,"claim":"Connected ferroptotic HMGB1 release to autophagy and HDAC inhibition-driven acetylation, with RAGE rather than TLR4 mediating downstream inflammation.","evidence":"ATG5/ATG7 KO, autophagy inhibitors, HDAC assays, receptor-specific macrophage stimulation","pmids":["30686534"],"confidence":"Medium","gaps":["Single lab","Mechanism linking autophagy to HDAC activity not resolved"]},{"year":2020,"claim":"Dissected secretion from inflammasome activation, showing macrophage HMGB1 release requires pyroptotic lysis while in vivo release can proceed independently of gasdermin D.","evidence":"Gasdermin D KO macrophages and mice separating pyroptosis from inflammasome activation","pmids":["32917873"],"confidence":"High","gaps":["Pyroptosis-independent in vivo release route not identified","Cell types responsible in vivo not defined"]},{"year":2021,"claim":"Established a required role for nociceptor-derived HMGB1 in neurogenic inflammation by direct optogenetic release and neuron-specific knockout.","evidence":"Channelrhodopsin-2 nociceptor stimulation and Syn-Cre HMGB1 conditional KO in nerve injury and arthritis models","pmids":["34385304"],"confidence":"High","gaps":["Receptor mediating neuronal HMGB1 effects not defined here","Redox form of neuron-released HMGB1 unknown"]},{"year":2022,"claim":"Added a phosphorylation-based release route, showing AMPK phosphorylates HMGB1 during cuproptosis to drive RAGE-dependent inflammation.","evidence":"AMPK knockdown/inhibition, HMGB1 phosphorylation analysis, AGER-dependent cytokine assays","pmids":["36211458"],"confidence":"Medium","gaps":["Single lab","AMPK phosphorylation site on HMGB1 not mapped"]},{"year":null,"claim":"How the nuclear, autophagic, and extracellular roles of HMGB1 are integrated in vivo, and how its post-translational/redox states are coordinately switched within a given cellular context, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified structural model linking redox state, modifications, and receptor choice","Pyroptosis-independent in vivo release mechanism unidentified","Quantitative control of nuclear retention vs secretion in physiological settings unclear"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0,1,2,4,5,6]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[18]},{"term_id":"GO:0140110","term_label":"transcription regulator 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Attenuates Diabetic Nephropathy in Mice.","date":"2018","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/29844451","citation_count":55,"is_preprint":false},{"pmid":"31215389","id":"PMC_31215389","title":"High Mobility Group Box-1 (HMGB1): A Potential Target in Therapeutics.","date":"2019","source":"Current drug targets","url":"https://pubmed.ncbi.nlm.nih.gov/31215389","citation_count":49,"is_preprint":false},{"pmid":"17948903","id":"PMC_17948903","title":"Is HMGB1 an osteocyte alarmin?","date":"2008","source":"Journal of cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17948903","citation_count":49,"is_preprint":false},{"pmid":"22033335","id":"PMC_22033335","title":"HMGB1, an alarmin promoting HIV dissemination and latency in dendritic cells.","date":"2011","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/22033335","citation_count":48,"is_preprint":false},{"pmid":"10866811","id":"PMC_10866811","title":"HMG1 protein 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research","url":"https://pubmed.ncbi.nlm.nih.gov/35820449","citation_count":35,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":41757,"output_tokens":6786,"usd":0.113531,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":15683,"output_tokens":6010,"usd":0.114333,"stage2_stop_reason":"end_turn"},"total_usd":0.227864,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1992,\n      \"finding\": \"HMGB1 (HMG1) HMG-box domain recognizes and binds four-way junction DNA structures (cross-shaped DNA with ~60° and ~120° angles between arms) in a structure-specific, sequence-independent manner, and binding to linear duplex DNA containing the SRY target sequence produces a sharp DNA bend.\",\n      \"method\": \"In vitro DNA binding assays with four-way junction DNA and linear duplex DNA; gel mobility shift and footprinting experiments\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct in vitro binding assay with defined DNA substrates, replicated across HMG1 and SRY HMG-box, demonstrating structure-specific recognition\",\n      \"pmids\": [\"1425584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1979,\n      \"finding\": \"HMG1 unwinds the DNA double helix by local denaturation of base pairs; it has higher affinity for single-stranded than double-stranded DNA and introduces a net unwinding angle of ~22° per molecule. In absence of salt, HMG1 stabilizes DNA; in presence of salt, it destabilizes DNA.\",\n      \"method\": \"DNA melting absorption technique; competition unwinding experiments measuring topological winding numbers\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro biochemical assay with quantitative measurements of unwinding angle and DNA thermal stability, multiple orthogonal measurements\",\n      \"pmids\": [\"226939\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"The acidic C-terminal tail of HMGB1 downregulates the DNA-binding and supercoiling activities of the tandem HMG-box domains (HMG3) in an ionic strength-dependent manner; removal of the tail increases DNA affinity and supercoiling activity. The tandem HMG-box domains can loop, compact, and change topology of DNA. The acidic tail directly modulates DNA-binding domain specificity.\",\n      \"method\": \"DNA supercoiling assays with topoisomerase I; electron microscopy of DNA-protein complexes; comparison of HMG1 vs. tryptic HMG3 (tail-deleted form)\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro assay with defined protein variants, electron microscopy, multiple orthogonal methods in one study\",\n      \"pmids\": [\"8152909\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"HMGB1 (HMG-1) directly binds p53 protein in vitro and enhances p53 DNA-binding activity, including that of a constitutively active C-terminally deleted p53 (p53Δ30), by a mechanism distinct from other known p53 activators. HMG-1 promotes assembly of higher-order p53 nucleoprotein structures and stimulates p53-mediated transactivation in vivo.\",\n      \"method\": \"Biochemical purification from HeLa nuclear extracts; recombinant His-tagged HMG-1 in vitro binding and DNA-binding assays; transient transfection transactivation assays\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of direct protein-protein interaction and DNA-binding stimulation, supported by in vivo transactivation assay, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"9472015\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"HMG1 domain B binds cisplatin-modified DNA with high affinity (Kd ~60 nM) and induces a bend angle of 80–95° in the cisplatin-modified DNA, as determined by FRET distance measurements. Kinetic parameters: kon = 1.1 × 10⁹ M⁻¹s⁻¹, koff = 30 s⁻¹.\",\n      \"method\": \"Fluorescence resonance energy transfer (FRET) with fluorescein/rhodamine-labeled DNA probes; fluorescence titration; stopped-flow fluorescence spectroscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with quantitative FRET structural measurement plus kinetic analysis, multiple orthogonal methods in one study\",\n      \"pmids\": [\"10212205\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"HMGB1 stimulates DNA end joining (both cohesive-end and blunt-end ligation by T4 DNA ligase) by physically associating two DNA molecules via their ends. This activity requires the B domain of HMGB1, specifically the C-terminal flanking basic residues and conserved basic/hydrophobic residues within the HMG box.\",\n      \"method\": \"In vitro ligation assay with T4 DNA ligase; pull-down assays; electron microscopy and scanning force microscopy of DNA-protein complexes; domain deletion/mutation analysis\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of ligation activity, domain mapping by mutagenesis, and direct visualization by EM/SFM in one study\",\n      \"pmids\": [\"10866811\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The acidic C-terminal tail of HMGB1 directly interacts with the N-terminal HMG-box domains (confirmed by NMR), and this intramolecular interaction can be competed more efficiently by four-way junction DNA than by linear dsDNA. Mutations in the N-terminal region that disrupt C-tail binding abolish HMGB1's ability to distinguish linear DNA from four-way junction DNA, defining a mechanism by which the acidic tail enhances domain specificity for structured DNAs.\",\n      \"method\": \"NMR; competitive DNA-binding assays; site-directed mutagenesis of the N-terminal region\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structural validation of intramolecular interaction combined with mutagenesis and functional competition assays\",\n      \"pmids\": [\"17585880\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Endogenous HMGB1 directly interacts with the autophagy protein Beclin1 and displaces Bcl-2 from Beclin1, thereby enhancing autophagic flux. Reactive oxygen species promote cytosolic translocation of HMGB1. Mutation of C106 (but not C23 or C45) promotes cytosolic HMGB1 localization and sustained autophagy. The intramolecular disulfide bond between C23 and C45 is required for HMGB1 binding to Beclin1 and sustaining autophagy.\",\n      \"method\": \"Co-immunoprecipitation; direct protein interaction assays; site-directed mutagenesis of cysteine residues; pharmacological inhibition (ethyl pyruvate); autophagy flux measurement; HMGB1 knockout/knockdown cells\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, cysteine mutagenesis with functional readout, genetic KO with phenotypic rescue, multiple orthogonal methods across multiple cell systems\",\n      \"pmids\": [\"20819940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"PKR (double-stranded RNA-dependent protein kinase) physically interacts with multiple inflammasome components (NLRP3, NLRP1, NLRC4, AIM2) and is required for inflammasome activation; PKR autophosphorylation in a cell-free reconstituted system with recombinant NLRP3, ASC, and pro-caspase-1 is sufficient to reconstitute inflammasome activity, leading to HMGB1 release. PKR deficiency severely impairs HMGB1 secretion in response to diverse inflammasome agonists.\",\n      \"method\": \"Genetic deletion and pharmacological inhibition of PKR; Co-IP of PKR with inflammasome components; cell-free reconstitution with recombinant proteins; in vivo peritonitis model measuring HMGB1 secretion\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cell-free reconstitution plus genetic KO plus Co-IP across multiple agonists, replicated in vivo\",\n      \"pmids\": [\"22801494\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"HMGB1 and p53 form a complex that reciprocally regulates autophagy and apoptosis. p53 knockout increases cytosolic HMGB1 expression and induces autophagy; HMGB1 knockout increases p53 cytoplasmic localization and decreases autophagy. p53 acts as a negative regulator of the HMGB1/Beclin1 complex. The HMGB1/p53 complex affects the cytoplasmic localization of the reciprocal binding partner.\",\n      \"method\": \"Co-immunoprecipitation; genetic knockout of p53 (HCT116) and HMGB1 (MEFs); subcellular fractionation; autophagy measurement\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and genetic KO in two cell systems, single lab\",\n      \"pmids\": [\"22345153\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PARP-1 regulates LPS-induced HMGB1 translocation and release from macrophages by two mechanisms: (1) PARylating HMGB1 to facilitate its subsequent acetylation, and (2) increasing the HAT/HDAC activity ratio to promote HMGB1 acetylation. PARylated HMGB1 remains nuclear, whereas acetylated HMGB1 localizes to the cytoplasm. Genetic or pharmacological inhibition of PARP-1 suppresses HMGB1 acetylation, nuclear-to-cytoplasm translocation, and extracellular release. ROS and ERK signaling upstream of PARP activation are also required.\",\n      \"method\": \"Genetic PARP-1 depletion and PARP inhibitors in bone marrow-derived macrophages; in vitro enzymatic PARylation reaction; HAT/HDAC activity assays; subcellular fractionation; immunofluorescence\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO plus pharmacological inhibition plus in vitro enzymatic reconstitution plus subcellular fractionation, multiple orthogonal methods in one study\",\n      \"pmids\": [\"25392528\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The inflammatory activity of extracellular HMGB1 depends on its redox state: an intramolecular disulfide bond between C23 and C45 combined with a reduced C106 confers cytokine/TLR4-stimulating activity. Oxidation of C106 blocks stimulatory activity and promotes immune tolerance. Thus the chemoattractant and cytokine-inducing activities of HMGB1 are separable and depend on distinct cysteine redox states.\",\n      \"method\": \"Redox state characterization with defined recombinant HMGB1 isoforms; TLR4 activation assays; leukocyte recruitment assays\",\n      \"journal\": \"Antioxidants & redox signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple defined redox isoforms tested in functional assays, reviewed/replicated by multiple labs but primary evidence in single lab per experiment\",\n      \"pmids\": [\"23373897\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The chemoattractant activity of HMGB1 is entirely mediated through formation of a heterocomplex with the chemokine CXCL12, which acts on CXCR4 more potently than CXCL12 alone. Only the all-thiol (fully reduced) form of HMGB1 can form this heterocomplex with CXCL12. The disulfide HMGB1 (C23–C45 bond) activates TLR4 to induce cytokine release but cannot recruit leukocytes, demonstrating that chemoattractant and cytokine-inducing activities of HMGB1 are separable redox-dependent functions.\",\n      \"method\": \"Biochemical characterization of HMGB1-CXCL12 complex; receptor binding assays on CXCR4; leukocyte migration assays with distinct redox isoforms\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — defined recombinant redox isoforms, receptor-specific functional assays, complex formation characterization, findings replicated across multiple reports\",\n      \"pmids\": [\"23207101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"HMGB1 acts as a cofactor in base excision repair (BER), inhibiting single-nucleotide BER and stimulating long-patch BER by modulating the activities of BER enzymes. HMGB1 was found cross-linked to a BER intermediate in cells, indicating direct participation in the BER pathway.\",\n      \"method\": \"In vitro BER assays; cross-linking of HMGB1 to BER intermediates; enzymatic activity measurements of BER enzymes in presence/absence of HMGB1\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro reconstituted BER assays with direct cross-linking evidence, single lab\",\n      \"pmids\": [\"20123074\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TLR5 is an HMGB1 receptor. HMGB1 binds TLR5 and initiates NF-κB signaling via MyD88-dependent signaling, resulting in proinflammatory cytokine production and pain hypersensitivity in vivo. The C-terminal tail region of HMGB1 is essential for its interaction with TLR5.\",\n      \"method\": \"Biophysical binding assays; in vitro NF-κB signaling assays with TLR5-expressing cells; in vivo allodynia model; C-terminal tail truncation analysis\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biophysical binding plus functional NF-κB signaling plus in vivo phenotype, domain mapping, single lab\",\n      \"pmids\": [\"27760316\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HMGB1 is released by ferroptotic cells in an autophagy-dependent manner. Genetic ablation (ATG5-/- or ATG7-/- cells) or pharmacological inhibition of autophagy blocks ferroptosis-induced HMGB1 release. Autophagy-mediated HDAC inhibition promotes HMGB1 acetylation leading to its release during ferroptosis. AGER (RAGE), but not TLR4, is required for HMGB1-mediated inflammation in macrophages in response to ferroptotic cells.\",\n      \"method\": \"Genetic KO of ATG5/ATG7; pharmacological autophagy inhibitors; HMGB1 release measurement; HDAC inhibition assays; receptor-specific macrophage stimulation assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genetic KOs plus pharmacological inhibitors plus receptor specificity testing, single lab\",\n      \"pmids\": [\"30686534\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"HMGB1 release after inflammasome activation in macrophages occurs only under conditions causing cell lysis (pyroptosis), not during inflammasome activation per se. When pyroptosis is prevented, HMGB1 is not released despite inflammasome activation and IL-1β secretion. Gasdermin D knockout mice secrete HMGB1 normally during endotoxemia (indirect/pyroptosis-independent mechanism in vivo), while IL-1β secretion is completely blocked.\",\n      \"method\": \"Genetic gasdermin D knockout macrophages; LPS stimulation; separation of pyroptosis from inflammasome activation; measurement of HMGB1 and IL-1β secretion in vitro and in vivo\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO in vitro and in vivo with well-controlled separation of pyroptosis from inflammasome activation, rigorous mechanistic dissection\",\n      \"pmids\": [\"32917873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"During cuproptosis, copper accumulation-induced ATP depletion activates AMPK, which phosphorylates HMGB1 to promote its extracellular release. Genetic or pharmacological inhibition of AMPK limits cuproptosis-associated HMGB1 release. Extracellular HMGB1 from cuproptotic cells signals through AGER (RAGE) to induce inflammatory cytokine production; HMGB1-deficient cuproptotic cells show greatly reduced AGER-dependent inflammatory signaling.\",\n      \"method\": \"RNAi knockdown of AMPK; pharmacological AMPK inhibition (dorsomorphin); HMGB1 phosphorylation analysis; AGER-dependent cytokine production assays; cuproptosis inducers\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and pharmacological inhibition of AMPK with HMGB1 phosphorylation readout and receptor-specific functional assays, single lab\",\n      \"pmids\": [\"36211458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Intracellular HMGB1 negatively regulates efferocytosis (phagocytosis of apoptotic cells). HMGB1 knockdown in macrophages and fibroblasts increases phagocytosis of apoptotic cells, enhances Rac-1 activation and cytoskeletal rearrangement, and increases phosphorylation of ERK and FAK. HMGB1 physically interacts with Src kinase and inhibits Src-FAK interactions; in vitro, purified HMGB1 directly diminishes interactions between purified FAK and Src.\",\n      \"method\": \"HMGB1 siRNA knockdown; phagocytosis assays; Rac-1 activation assay; phosphorylation analysis; Src inhibition; in vitro pulldown of purified FAK and Src with/without HMGB1\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD phenotype confirmed by in vitro reconstitution of FAK-Src interaction, multiple methods, single lab\",\n      \"pmids\": [\"21957148\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Nociceptors directly release HMGB1 upon activation: transgenic nociceptors expressing channelrhodopsin-2 release HMGB1 in response to light stimulation. Neuron-specific HMGB1 silencing (Syn-Cre × HMGB1f/f mice) protects against cutaneous inflammation, allodynia after sciatic nerve injury, and joint inflammation in collagen antibody-induced arthritis, establishing a required role for nociceptor-derived HMGB1 in neurogenic inflammation.\",\n      \"method\": \"Optogenetic stimulation of channelrhodopsin-2-expressing nociceptors; conditional neuron-specific HMGB1 knockout (Syn-Cre/HMGB1fl/fl); sciatic nerve injury model; collagen antibody-induced arthritis model\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — optogenetic direct release demonstration plus conditional KO in two disease models with defined phenotypic readouts, multiple orthogonal approaches\",\n      \"pmids\": [\"34385304\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"The mouse hmg1 gene contains five exons (first untranslated), with a promoter coinciding with a CpG island and lacking a TATA sequence, consistent with ubiquitous expression. No evidence for a membrane localization signal was found in the coding sequence.\",\n      \"method\": \"Gene isolation, sequencing, exon mapping; reporter gene expression assays; Southern blot analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct gene characterization with reporter assays, single lab\",\n      \"pmids\": [\"7961836\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"C1q and HMGB1 cooperate via a complex involving RAGE and LAIR-1 to induce monocyte differentiation into anti-inflammatory M2-like macrophages, counterbalancing HMGB1's pro-inflammatory M1-inducing activity. This pathway depends on the relative levels of C1q and HMGB1.\",\n      \"method\": \"In vitro monocyte differentiation assays; receptor blocking experiments (RAGE and LAIR-1); co-stimulation with C1q and HMGB1\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — receptor-specific blocking experiments with defined functional readout, single lab\",\n      \"pmids\": [\"27683415\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Extracellular HMGB1 can be internalized via RAGE-receptor-mediated endocytosis into the endolysosomal compartment while bound to other extracellular proinflammatory molecules. After endocytosis, HMGB1 destabilizes lysosomal membranes, facilitating access of HMGB1-bound partner molecules to endosomal and cytosolic pattern recognition receptors.\",\n      \"method\": \"RAGE-dependent endocytosis assays; lysosomal membrane destabilization assays; co-internalization experiments with HMGB1 and bound molecules\",\n      \"journal\": \"Seminars in immunology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — review paper summarizing mechanistic findings without detailed primary experimental description in abstract; single mechanism proposed\",\n      \"pmids\": [\"29530410\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"HMGB1 acts as a cofactor in base excision repair, stimulating long-patch BER sub-pathway while inhibiting single-nucleotide BER, thereby directing repair toward long-patch BER, which can result in trinucleotide repeat instability.\",\n      \"method\": \"In vitro BER assays; cross-linking of HMGB1 to BER intermediates\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted in vitro BER assay, cross-linking evidence, single lab\",\n      \"pmids\": [\"20123074\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HMGB1 is a multifunctional nuclear protein that, inside the nucleus, acts as an architectural DNA-binding chromatin protein (binding distorted/structured DNA preferentially via its HMG-box domains, bending DNA, modulating transcription factor binding including p53 activation, and participating in base excision repair); in the cytoplasm, it promotes autophagy by directly binding Beclin1 and displacing Bcl-2 (requiring the C23–C45 disulfide bond) and is negatively regulated by its complex with p53; its secretion and extracellular release are tightly controlled by post-translational modifications (particularly acetylation driven by PARP-1/HAT/HDAC balance, and phosphorylation by AMPK), with active secretion requiring nuclear-to-cytoplasmic translocation, while passive release occurs during necrosis/lytic cell death; extracellularly, HMGB1's biological activity is determined by its cysteine redox state—all-thiol HMGB1 forms a heterocomplex with CXCL12 to act as a chemoattractant via CXCR4, disulfide (C23–C45) HMGB1 activates TLR4 (with MD-2) to induce cytokine release, and fully oxidized HMGB1 is inactive—while HMGB1 also signals via RAGE (mediating endocytosis), TLR2, TLR4, and TLR5 (via its C-terminal tail, triggering MyD88-dependent NF-κB activation), and PKR serves as an upstream activator of inflammasome-dependent HMGB1 release.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"HMGB1 is a multifunctional protein that bridges nuclear chromatin architecture, DNA repair, and extracellular danger signaling, with its activity in each compartment governed by domain structure and post-translational/redox state [#0, #7, #12]. In the nucleus it functions as an architectural DNA-binding protein: its tandem HMG-box domains recognize distorted and structured DNA (four-way junctions, cisplatin adducts) in a sequence-independent manner, bend and unwind the duplex, and can loop, compact, and supercoil DNA, while the acidic C-terminal tail folds back onto the boxes to tune their specificity for structured over linear DNA [#0, #1, #2, #4, #6]. Through these activities HMGB1 stimulates p53 DNA binding and transactivation by promoting higher-order nucleoprotein assembly [#3], promotes DNA end joining by juxtaposing DNA ends [#5], and acts as a cofactor in base excision repair, directing repair toward the long-patch sub-pathway [#13, #23]. In the cytoplasm HMGB1 promotes autophagy by binding Beclin1 and displacing Bcl-2 in a manner requiring the C23\\u2013C45 disulfide bond, an activity reciprocally restrained by complex formation with p53 [#7, #9]. HMGB1 release is actively controlled: PARP-1 drives PARylation and an increased HAT/HDAC ratio to acetylate HMGB1 and trigger nuclear-to-cytoplasmic translocation [#10], AMPK phosphorylates HMGB1 during cuproptosis [#17], and autophagy-dependent HDAC inhibition promotes its acetylation and release during ferroptosis [#15], with PKR-dependent inflammasome activity required for secretion but pyroptotic membrane rupture governing actual release in macrophages [#8, #16]. Once extracellular, HMGB1's function is redox-determined: the all-thiol form pairs with CXCL12 to chemoattract leukocytes via CXCR4, the C23\\u2013C45 disulfide form activates TLR4 to induce cytokines, and full oxidation abolishes activity [#11, #12]; it additionally signals through TLR5 via its C-terminal tail to drive MyD88/NF-\\u03baB-dependent inflammation and pain [#14], and through RAGE to mediate endocytosis, M2 macrophage polarization with C1q, and inflammation from regulated cell death [#15, #17, #21, #22]. Nociceptor-derived HMGB1 is required for neurogenic inflammation in vivo [#19].\",\n  \"teleology\": [\n    {\n      \"year\": 1979,\n      \"claim\": \"Established that HMGB1 actively alters DNA topology rather than passively coating DNA, defining a biochemical basis for its architectural role.\",\n      \"evidence\": \"DNA melting and competition unwinding assays measuring unwinding angle and thermal stability in vitro\",\n      \"pmids\": [\"226939\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define structural preference for distorted DNA\", \"No domain-level dissection of the unwinding activity\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Showed HMGB1's HMG-box recognizes DNA by structure rather than sequence and bends the duplex, explaining how it targets junctions and distorted sites.\",\n      \"evidence\": \"In vitro four-way junction and linear duplex binding, gel shift and footprinting\",\n      \"pmids\": [\"1425584\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Role of the acidic tail not addressed\", \"Cellular consequences of structure-specific binding untested\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Defined the acidic C-terminal tail as an autoregulatory element that downregulates and tunes the DNA-binding/supercoiling activity of the tandem boxes.\",\n      \"evidence\": \"Supercoiling assays, EM, and comparison of full-length vs tail-deleted HMG3\",\n      \"pmids\": [\"8152909\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism of tail-box interaction not yet structurally resolved\", \"Physiological setting of looping/compaction unclear\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Characterized the gene structure and TATA-less CpG-island promoter, supporting ubiquitous expression with no encoded membrane-targeting signal.\",\n      \"evidence\": \"Gene isolation, exon mapping, reporter assays in mouse hmg1\",\n      \"pmids\": [\"7961836\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Regulation of expression in stress/inflammation not addressed\", \"Secretion route left unexplained given lack of signal sequence\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Linked HMGB1's DNA architecture to transcription by showing it directly binds p53 and stimulates its DNA binding and transactivation.\",\n      \"evidence\": \"Biochemical purification, recombinant in vitro binding, transactivation assays\",\n      \"pmids\": [\"9472015\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Genomic targets of HMGB1/p53 cooperation not mapped\", \"In vivo relevance to p53 programs untested here\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Quantified high-affinity binding and severe bending of cisplatin-damaged DNA, mechanistically connecting HMGB1 to recognition of damaged DNA.\",\n      \"evidence\": \"FRET distance measurement and stopped-flow kinetics with HMG1 domain B\",\n      \"pmids\": [\"10212205\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream effect on repair or cisplatin sensitivity not assessed\", \"Only domain B examined\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Showed HMGB1 physically juxtaposes DNA ends to stimulate ligation, mapping the activity to the B domain and flanking basic residues.\",\n      \"evidence\": \"In vitro T4 ligase joining assays, EM/SFM, domain mutagenesis\",\n      \"pmids\": [\"10866811\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relevance to cellular NHEJ pathway not established\", \"Partner repair factors not identified\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined HMGB1 as a base excision repair cofactor that biases repair toward the long-patch sub-pathway, with potential consequences for repeat instability.\",\n      \"evidence\": \"In vitro reconstituted BER assays and cross-linking to BER intermediates\",\n      \"pmids\": [\"20123074\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, in vitro reconstitution\", \"BER enzyme contacts not structurally defined\", \"In vivo repair phenotype not shown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Established the cytoplasmic autophagy function of HMGB1, showing it binds Beclin1, displaces Bcl-2, and requires the C23\\u2013C45 disulfide bond, with ROS driving cytosolic translocation.\",\n      \"evidence\": \"Reciprocal Co-IP, cysteine mutagenesis, autophagy flux in KO/KD cells\",\n      \"pmids\": [\"20819940\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Beclin1 binding not resolved\", \"Redox sensing mechanism of translocation unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Revealed an intracellular brake on efferocytosis by showing HMGB1 inhibits Src-FAK interaction and dampens Rac1-driven phagocytic cytoskeletal signaling.\",\n      \"evidence\": \"siRNA knockdown phenotypes plus in vitro pulldown of purified FAK/Src with HMGB1\",\n      \"pmids\": [\"21957148\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Binding site on Src not mapped\", \"In vivo efferocytosis relevance untested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Placed PKR upstream of inflammasome-dependent HMGB1 secretion, reconstituting inflammasome activity in a cell-free system.\",\n      \"evidence\": \"PKR KO/inhibition, Co-IP with inflammasome components, cell-free reconstitution, in vivo peritonitis\",\n      \"pmids\": [\"22801494\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct PKR substrates within the inflammasome not defined\", \"Link to membrane permeabilization not addressed\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Demonstrated that HMGB1's chemoattractant activity is mediated by an all-thiol HMGB1\\u2013CXCL12 heterocomplex acting on CXCR4, distinct from its disulfide-dependent TLR4 cytokine activity.\",\n      \"evidence\": \"Complex characterization, CXCR4 binding and leukocyte migration with defined redox isoforms\",\n      \"pmids\": [\"23207101\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of the HMGB1\\u2013CXCL12 complex not resolved\", \"In vivo redox dynamics not quantified\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showed the HMGB1\\u2013p53 complex reciprocally regulates autophagy and apoptosis by controlling each partner's cytoplasmic localization, with p53 restraining the HMGB1/Beclin1 axis.\",\n      \"evidence\": \"Reciprocal Co-IP and p53/HMGB1 KO with subcellular fractionation and autophagy readout\",\n      \"pmids\": [\"22345153\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Direct competition with Beclin1 not biochemically isolated\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Refined the redox code by showing the C23\\u2013C45 disulfide plus reduced C106 confers TLR4/cytokine activity while C106 oxidation enforces tolerance, separating chemoattractant and cytokine functions.\",\n      \"evidence\": \"Defined recombinant redox isoforms in TLR4 activation and leukocyte recruitment assays\",\n      \"pmids\": [\"23373897\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Per-experiment evidence from single lab\", \"In vivo redox transition kinetics not measured\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified TLR5 as an HMGB1 receptor driving MyD88/NF-\\u03baB inflammation and pain, mapping the interaction to the C-terminal tail.\",\n      \"evidence\": \"Biophysical binding, NF-\\u03baB assays in TLR5 cells, in vivo allodynia, C-terminal truncation\",\n      \"pmids\": [\"27760316\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Redox-state dependence of TLR5 binding not tested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed HMGB1 can drive anti-inflammatory M2 polarization when cooperating with C1q via RAGE and LAIR-1, balancing its pro-inflammatory M1 activity.\",\n      \"evidence\": \"Monocyte differentiation with C1q/HMGB1 co-stimulation and RAGE/LAIR-1 blocking\",\n      \"pmids\": [\"27683415\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Molecular composition of the C1q/HMGB1/RAGE/LAIR-1 complex not resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Proposed that RAGE-mediated endocytosis delivers HMGB1 and its bound cargo to the endolysosome, where HMGB1 destabilizes membranes to reach cytosolic pattern recognition receptors.\",\n      \"evidence\": \"Review summarizing RAGE endocytosis and lysosomal destabilization assays\",\n      \"pmids\": [\"29530410\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Review-level, lacking detailed primary experimental description\", \"Membrane destabilization mechanism not biochemically defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected ferroptotic HMGB1 release to autophagy and HDAC inhibition-driven acetylation, with RAGE rather than TLR4 mediating downstream inflammation.\",\n      \"evidence\": \"ATG5/ATG7 KO, autophagy inhibitors, HDAC assays, receptor-specific macrophage stimulation\",\n      \"pmids\": [\"30686534\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Mechanism linking autophagy to HDAC activity not resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Dissected secretion from inflammasome activation, showing macrophage HMGB1 release requires pyroptotic lysis while in vivo release can proceed independently of gasdermin D.\",\n      \"evidence\": \"Gasdermin D KO macrophages and mice separating pyroptosis from inflammasome activation\",\n      \"pmids\": [\"32917873\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Pyroptosis-independent in vivo release route not identified\", \"Cell types responsible in vivo not defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established a required role for nociceptor-derived HMGB1 in neurogenic inflammation by direct optogenetic release and neuron-specific knockout.\",\n      \"evidence\": \"Channelrhodopsin-2 nociceptor stimulation and Syn-Cre HMGB1 conditional KO in nerve injury and arthritis models\",\n      \"pmids\": [\"34385304\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor mediating neuronal HMGB1 effects not defined here\", \"Redox form of neuron-released HMGB1 unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Added a phosphorylation-based release route, showing AMPK phosphorylates HMGB1 during cuproptosis to drive RAGE-dependent inflammation.\",\n      \"evidence\": \"AMPK knockdown/inhibition, HMGB1 phosphorylation analysis, AGER-dependent cytokine assays\",\n      \"pmids\": [\"36211458\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"AMPK phosphorylation site on HMGB1 not mapped\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the nuclear, autophagic, and extracellular roles of HMGB1 are integrated in vivo, and how its post-translational/redox states are coordinately switched within a given cellular context, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified structural model linking redox state, modifications, and receptor choice\", \"Pyroptosis-independent in vivo release mechanism unidentified\", \"Quantitative control of nuclear retention vs secretion in physiological settings unclear\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0, 1, 2, 4, 5, 6]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [18]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 7, 9]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [12, 14]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [11, 12, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 3, 10]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [7, 9, 10]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [11, 12, 16, 19]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [22]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [8, 11, 12, 14, 16, 21]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [7, 9, 15]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [13, 23]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [15, 16, 17]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [12, 14, 18]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"TP53\", \"BECN1\", \"CXCL12\", \"TLR4\", \"TLR5\", \"AGER\", \"C1q\", \"SRC\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}