{"gene":"PYCARD","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2002,"finding":"ASC functions as a caspase-1-activating adaptor by interacting with procaspase-1 via CARD-CARD interactions and inducing its oligomerization. The PYRIN domain acts as an oligomerization domain while the CARD domain is the effector domain; full-length ASC (but not isolated domains alone) activates procaspase-1 and processes pro-IL-1β in transfected cells.","method":"Co-IP, FKBP12-inducible oligomerization domain swap, transfection/overexpression in HEK cells, siRNA knockdown in THP-1 cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1/2 / Strong — direct in vitro and cellular reconstitution with domain-swap mutagenesis and loss-of-function, multiple orthogonal methods in a single study","pmids":["11967258"],"is_preprint":false},{"year":2001,"finding":"Pyrin (the MEFV gene product) interacts with ASC via its pyrin domain (exon 1) in a yeast two-hybrid assay and co-localizes with ASC in cytosolic specks in HeLa cells. Co-expression of pyrin with ASC increases speck formation and promotes survival of speck-positive cells, modulating ASC-induced apoptosis.","method":"Yeast two-hybrid, co-transfection/immunofluorescence co-localization, cell survival assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid and co-localization in transfected cells, two orthogonal methods, single lab","pmids":["11498534"],"is_preprint":false},{"year":2004,"finding":"ASC functions as a Bax adaptor in the p53-dependent mitochondrial apoptosis pathway. ASC directly interacts with Bax, co-localizes with Bax at mitochondria, induces cytochrome c release, loss of mitochondrial membrane potential, and activation of caspases-9, -2, and -3. Rapid apoptosis induction is absent in Bax-deficient cells, and siRNA knockdown of ASC inhibits Bax translocation to mitochondria after p53 or genotoxic insult.","method":"Co-IP, co-localization by fluorescence microscopy, cytochrome c release assay, mitochondrial membrane potential assay, Bax-deficient cell lines, siRNA knockdown","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, multiple orthogonal cellular assays, genetic rescue with Bax-null cells, and loss-of-function with siRNA; multiple independent validations in single study","pmids":["14730312"],"is_preprint":false},{"year":2009,"finding":"AIM2 (PYHIN family member) binds cytosolic dsDNA via its HIN200 domain, while its pyrin domain associates with ASC. This AIM2-ASC interaction activates both NF-κB and caspase-1 as a functional inflammasome. Knockdown of AIM2 abrogates caspase-1 activation in response to cytoplasmic dsDNA.","method":"Co-IP (PYD-PYD interaction), siRNA knockdown, reporter assay for NF-κB, caspase-1 activity assay, viral infection model","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, domain-mapping, siRNA knockdown with functional caspase-1 readout, replicated with multiple stimuli and viral infection model","pmids":["19158675"],"is_preprint":false},{"year":2013,"finding":"Procaspase-8 localizes to ASC inflammasome specks and binds directly to the pyrin domain of ASC. AIM2 and NLRP3 inflammasomes activate caspase-8 and caspase-1 leading to both apoptotic and pyroptotic cell death, with caspase-8 being the apical caspase in the ASC-dependent apoptotic pathway (with little requirement for caspase-9).","method":"Gene knockdown, fluorescence co-localization of procaspase-8 with ASC specks, direct binding assay (PYD-procaspase-8 interaction), cell death quantification with varying DNA concentrations","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding demonstrated, co-localization confirmed, gene knockdown with functional readout; single lab","pmids":["23645208"],"is_preprint":false},{"year":2014,"finding":"After pyroptosis, ASC specks accumulate in the extracellular space where they continue to promote IL-1β maturation. Phagocytosis of extracellular ASC specks by macrophages causes lysosomal damage and nucleation of soluble ASC, activating IL-1β in recipient cells (prionoid propagation). Autoantibodies to ASC specks develop in patients and mice with autoimmune pathologies.","method":"Cell imaging (confocal microscopy), cell-free IL-1β maturation assay with extracellular specks, phagocytosis assay with lysosomal damage readout, autoantibody detection in patient samples","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (cell-free assay, imaging, phagocytosis experiments), validated in human patient samples; single lab but comprehensive","pmids":["24952505"],"is_preprint":false},{"year":2014,"finding":"IKKα negatively regulates ASC-dependent inflammasomes by interacting with ASC in the nucleus of resting macrophages in an IKKα kinase-dependent manner. IKKi (IKK-related kinase) facilitates translocation of ASC from nucleus to perinuclear area during inflammasome activation; upon NLRP3 signal 2, PP2A is recruited and inhibits IKKα kinase activity, releasing ASC to participate in inflammasome assembly.","method":"Co-IP (ASC-IKKα interaction), subcellular fractionation/localization, kinase-dead mutant analysis, PP2A recruitment assay, macrophage inflammasome activation assays","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, subcellular fractionation, kinase-dead mutant, multiple mechanistic steps validated; single lab","pmids":["25266676"],"is_preprint":false},{"year":2015,"finding":"The structure of the mouse ASC filament was determined at atomic resolution by combining cryo-EM and solid-state NMR spectroscopy. ASC-PYD forms the rigid core of the filament while the CARD domain is highly mobile relative to this core. NMR identified specific PYD-PYD binding interfaces, and structure-based mutagenesis validated their functional relevance in macrophage inflammasome activation.","method":"Cryo-electron microscopy, solid-state NMR spectroscopy, structure-based site-directed mutagenesis, macrophage functional assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — atomic-resolution structure by two complementary methods (cryo-EM + NMR) plus mutagenesis validation in primary macrophages","pmids":["26464513"],"is_preprint":false},{"year":2016,"finding":"ASC speck assembly involves two steps: oligomerization of ASC-PYD into filaments, and cross-linking of these filaments by ASC-CARD. ASC mutants with non-functional CARD only assemble filaments but not specks and disrupt endogenous specks. ASC speck formation is required for efficient IL-1β processing but is dispensable for gasdermin-D cleavage and pyroptosis induction, indicating that ASC oligomerization serves as a signal amplification mechanism for caspase-1-dependent cytokine production.","method":"Systematic site-directed mutagenesis of ASC-PYD, ASC-CARD mutant expression in primary macrophages, IL-1β processing assay, gasdermin-D cleavage assay, pyroptosis assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic mutagenesis combined with functional dissection of IL-1β processing vs. gasdermin-D cleavage; multiple orthogonal readouts in primary macrophages","pmids":["27329339"],"is_preprint":false},{"year":2006,"finding":"ASC directs caspase-1 away from RIP2-mediated NF-κB activation toward caspase-1-mediated IL-1β processing. ASC interferes with the caspase-1/RIP2 interaction in a dose-dependent manner, and siRNA knockdown of ASC in THP-1 cells decreases caspase-1 activity while enhancing NF-κB signaling.","method":"Co-transfection with NF-κB reporter gene assay, Co-IP (caspase-1/RIP2 interaction), siRNA knockdown, caspase-1-knockout macrophages","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reporter assay, Co-IP, siRNA knockdown, and knockout macrophages; multiple methods in single lab","pmids":["16585594"],"is_preprint":false},{"year":2011,"finding":"ASC/PYCARD regulates MAPK (ERK) phosphorylation in an inflammasome-independent manner by suppressing the dual-specificity phosphatase DUSP10/MKP5. ASC also regulates chemokine induction independently of caspase-1 and IL-1β. MAPK activation by pathogen was abrogated in Asc-/- but not Nlrp3-/-, Nlrc4-/-, or Casp1-/- macrophages.","method":"ASC shRNA knockdown, microarray/network analysis, MAPK phosphorylation assay, comparison of Asc-/-, Nlrp3-/-, Nlrc4-/-, Casp1-/- macrophages","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with multiple knockout macrophage lines plus shRNA knockdown, microarray network analysis, and direct phosphorylation readout across multiple stimuli","pmids":["21487011"],"is_preprint":false},{"year":2015,"finding":"ASC filaments nucleate procaspase-8 death effector domain (DED) filaments in vitro and in vivo. Interaction between ASC-PYD and procaspase-8 tandem DEDs requires both DEDs and represents a heterotypic interaction between death fold superfamily domains. ASC PYD mutagenesis showed that procaspase-8 interaction surfaces overlap with those required for ASC self-association.","method":"In vitro filament nucleation assay, in vivo co-localization, ASC-PYD mutagenesis, co-IP of ASC and procaspase-8","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis, confirmed in vivo; single lab but multiple orthogonal approaches","pmids":["26468282"],"is_preprint":false},{"year":2016,"finding":"ASC-PYD self-associates and binds NLRP3-PYD through equivalent protein regions (located at opposite sides of the protein), with higher affinity for NLRP3-PYD than for itself. NLRP3-PYD coexists as monomer and large oligomeric species in solution. These binding interfaces allow multimeric complex formation consistent with ASC-PYD fibril assemblies.","method":"Solution NMR spectroscopy, analytical ultracentrifugation, structural modeling","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — NMR structure and binding characterization with orthogonal biophysical method (AUC); single lab","pmids":["27432880"],"is_preprint":false},{"year":2017,"finding":"The deubiquitinating enzyme USP50 binds ASC and deubiquitinates K63-linked polyubiquitin chains on ASC. USP50 knockdown in THP-1 cells and BMDMs significantly decreases procaspase-1 cleavage, IL-1β/IL-18 secretion, and ASC speck formation/oligomerization upon NLRP3 stimuli.","method":"Co-IP (USP50-ASC binding), deubiquitination assay (K63-linked Ub), siRNA knockdown of USP50, caspase-1 cleavage assay, IL-1β/IL-18 ELISA, ASC speck formation assay","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, deubiquitination assay, and loss-of-function knockdown with multiple functional readouts; single lab","pmids":["28094437"],"is_preprint":false},{"year":2019,"finding":"Tyrosine dephosphorylation of ASC is required for NLRP3 and AIM2 inflammasome activation. Site-directed mutagenesis identified tyrosine residues Y60 and Y137 of ASC as critical for inflammasome assembly and function (ASC nucleation, caspase-1 activation, IL-1β/IL-18 processing, and pyroptosis).","method":"Site-directed mutagenesis of ASC tyrosine residues (Y60, Y137), pharmacological PTPase inhibition (phenylarsine oxide), caspase-1 activation assay, IL-1β/IL-18 release assay, pyroptosis assay","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-directed mutagenesis combined with pharmacological inhibition and functional readouts; single lab","pmids":["31333677"],"is_preprint":false},{"year":2021,"finding":"The E3 ubiquitin ligase Peli1 conjugates K63-linked ubiquitin chains specifically to lysine 55 of ASC, which facilitates ASC/NLRP3 interaction and ASC oligomerization, thereby promoting NLRP3 inflammasome activation. Peli1 deficiency impairs NLRP3-induced (but not AIM2, NLRP1, or NLRC4) caspase-1 activation and IL-1β maturation.","method":"Co-IP (ASC-Peli1 interaction), ubiquitination assay (K63-specific, lysine-55 mapping by mutagenesis), Peli1 knockout macrophages and mice, IL-1β secretion assay, peritonitis mouse model","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — site-specific ubiquitination mapping by mutagenesis, Peli1 KO cells and in vivo mouse model, inflammasome-selectivity analysis across multiple sensors","pmids":["34706239"],"is_preprint":false},{"year":2021,"finding":"cAbl kinase phosphorylates ASC at tyrosine 146 (Y146), and this phosphorylation is required for ASC oligomerization and inflammasome activation. CRISPR/Cas9 deletion of cABL in THP-1 cells inhibits inflammasome function and reduces phosphorylated ASC release after LPS/ATP stimulation. Complementation of ASC-knockout cells with Y146A-mutant ASC abrogates inflammasome activation and ASC oligomerization.","method":"CRISPR/Cas9 cABL knockout, site-directed mutagenesis (Y146A), ASC phosphorylation assay, IL-18/IL-1β release assay, ASC oligomerization assay","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1 / Strong — CRISPR KO combined with Y146A mutagenesis and functional complementation; multiple orthogonal readouts in single study","pmids":["33568399"],"is_preprint":false},{"year":2022,"finding":"USP3 is a deubiquitinating enzyme that directly binds ASC, removes K48-linked ubiquitin chains, and stabilizes ASC protein by blocking proteasomal degradation. USP3 promotes inflammasome activation, confirmed in mouse models of alum-induced peritonitis, F. novicida infection, and flagellin-induced pneumonia.","method":"Co-IP (USP3-ASC), deubiquitination assay (K48-linked Ub), proteasome inhibitor experiments, USP3-deficient mice models (peritonitis, F. novicida, flagellin pneumonia)","journal":"Cellular & molecular immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, K48-Ub-specific deubiquitination assay, proteasomal degradation rescue, and three independent in vivo mouse models","pmids":["36050480"],"is_preprint":false},{"year":2023,"finding":"ZBTB16 (a nuclear zinc-finger/BTB domain protein) promotes SUMOylation of ASC, which controls ASC function in inflammasome assembly. Ablation of ZBTB16 in a mouse model of Muckle-Wells syndrome reduces acute inflammatory pathogenesis driven by constitutively hyperactive inflammasome.","method":"SUMOylation assay (ASC-SUMO modification), ZBTB16 knockout in Muckle-Wells mouse model, inflammasome activation assays","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — SUMOylation assay and in vivo genetic model; single lab, mechanistic detail of exact SUMO site not specified in abstract","pmids":["38123560"],"is_preprint":false},{"year":2023,"finding":"Low intracellular K+ concentration causes a structural change in ASC oligomers that makes the ASC CARD domain more accessible for recruitment of the pro-caspase-1 CARD domain, thereby enhancing caspase-1 activation. This structural change in ASC is independent of NLRP3.","method":"ASC oligomerization assays under varying K+ conditions, domain accessibility experiments, caspase-1 recruitment assays","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic dissection of K+-dependent structural change in ASC oligomers with functional caspase-1 recruitment readout; single lab","pmids":["37402211"],"is_preprint":false},{"year":2023,"finding":"Cryo-electron tomography of NLRP3-activated cells reveals that ASC puncta are composed of branched filaments with a tubular core formed by the PYD. Ribosomes and Golgi-like/endosomal vesicles permeate the ASC filament network. Mitochondria are not associated with ASC filaments but show outer-membrane discontinuities consistent with gasdermin D pores.","method":"Correlative cryo-light microscopy and cryo-electron tomography of NLRP3-activated cells","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-ET structural determination at near-native conditions in activated cells; single lab but direct structural data with organelle co-localization","pmids":["37945612"],"is_preprint":false},{"year":2017,"finding":"In live zebrafish, CRISPR/Cas9-tagging of endogenous ASC showed strong expression in skin epithelia. Toxic stimulus triggered speck formation followed by rapid pyroptosis in keratinocytes, and macrophages engulfed and digested speck-containing pyroptotic debris. 3D ultrastructural reconstruction (CLEM) revealed compact networks of highly intercrossed filaments in in vivo specks. Full-length ASC (not PYD or CARD alone) is required to form a single compact speck and rapidly induce cell death.","method":"CRISPR/Cas9 endogenous tagging in zebrafish, live imaging, correlative light and electron microscopy (CLEM), 3D ultrastructural reconstruction","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — endogenous tagging + live in vivo imaging + CLEM ultrastructural reconstruction; orthogonal domain-deletion functional analysis","pmids":["28701426"],"is_preprint":false},{"year":2022,"finding":"The ASC alternative splicing isoform ASCb (differing from ASC only in the linker length between PYD and CARD) self-associates more slowly and into less compact macrostructures than ASC, with a reduced tendency to form densely packed filaments. This kinetic/structural difference, attributable solely to linker length, explains ASCb's reduced ability to activate the inflammasome compared to ASC.","method":"Real-time NMR, dynamic light scattering (DLS), size-exclusion chromatography, transmission electron microscopy, molecular docking with procaspase-1 CARD","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal biophysical methods (NMR + DLS + SEC + TEM + docking) characterizing isoform-specific self-assembly differences","pmids":["35007535"],"is_preprint":false},{"year":2021,"finding":"A SNP in the 3' UTR of the mouse Pycard gene (DBA/2 vs. AKR alleles) regulates ASC mRNA stability without altering transcription rate, leading to higher Pycard mRNA and ASC protein levels in DBA/2 macrophages and increased inflammasome speck formation. CRISPR/Cas9 editing of the DBA/2 3'UTR SNP to the AKR allele reduced Pycard expression and inflammasome activity, confirming a post-transcriptional regulatory mechanism.","method":"QTL mapping, mRNA stability assay, ASC protein quantification, inflammasome speck formation, CRISPR/Cas9 single-base editing of 3'UTR SNP","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — CRISPR single-base editing validation of mRNA stability mechanism; multiple orthogonal methods confirming post-transcriptional regulation","pmids":["34197316"],"is_preprint":false},{"year":2010,"finding":"PYCARD/ASC, but not NLRP3 or caspase-1, is required for granuloma formation and host protection during chronic Mycobacterium tuberculosis infection in vivo, revealing an inflammasome-independent role for ASC. Pycard-/- mice showed abrupt decrease in survival with defective granuloma formation, whereas Nlrp3-/- and Casp1-/- mice showed no difference from wild-type.","method":"Pycard-/-, Nlrp3-/-, and Casp1-/- mouse aerosol infection model, survival analysis, bacterial burden measurement, histological granuloma assessment, macrophage IL-1β secretion assay","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with three separate knockouts in vivo, defining an inflammasome-independent function of ASC in granuloma formation","pmids":["20808838"],"is_preprint":false},{"year":2020,"finding":"ASC stabilizes HIF-1α protein in oral squamous cell carcinoma cells under normoxia by interacting with HIF-1α in both cytoplasm and nucleus. ASC interaction decreases HIF-1α hydroxylation (HIF-1α-OH) and ubiquitination without affecting VHL or PHD2 levels, thereby promoting HIF-1α-dependent transcription of genes driving cell migration and invasion.","method":"Co-IP (ASC-HIF-1α), subcellular fractionation, HIF-1α hydroxylation and ubiquitination assays, transcriptomic analysis (RNA-seq), HIF-1α synthesis inhibitor treatment, migration/invasion assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with mechanistic follow-up (hydroxylation/ubiquitination assays), subcellular localization, and functional rescue experiments; single lab","pmids":["32883954"],"is_preprint":false},{"year":2007,"finding":"Pyrin-only protein 2 (POP2) co-localizes with ASC in perinuclear specks and disrupts ASC:CLR (CIAS1/NALP3) interactions, inhibiting speck formation by NLRP3. POP2 also modulates NF-κB activity through changes in nuclear import or distribution.","method":"Co-transfection/immunofluorescence co-localization, NF-κB reporter assay, speck formation assay with NLRP3 co-expression","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-localization and reporter assays, two orthogonal readouts; single lab, no direct binding assay for POP2-ASC","pmids":["17339483"],"is_preprint":false},{"year":2018,"finding":"NLRC4 activates an ASC- and caspase-8-dependent apoptotic pathway that is distinct from caspase-1-mediated pyroptosis. A genome-wide CRISPR/Cas9 screen and gene-targeted mouse validation confirmed that ASC and caspase-8 are required for the NLRC4-dependent alternative apoptotic program. Caspase-1 catalytic dead knock-in BMDMs confirm that caspase-1 does not compete with ASC for NLRC4 interactions.","method":"Genome-wide CRISPR/Cas9 screen, gene-targeted (caspase-8, ASC knockout) mice, caspase-1 catalytic dead knock-in mice, cell death characterization","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — unbiased genome-wide screen with multiple genetic validations in gene-targeted mice; caspase-1 knock-in provides mechanistic dissection","pmids":["29491424"],"is_preprint":false},{"year":2016,"finding":"T cell-intrinsic ASC-NLRP3-caspase-8 inflammasome in TH17 cells drives IL-1β production upon ATP stimulation of the T cell antigen receptor, promoting autocrine TH17 cell survival during experimental autoimmune encephalomyelitis (EAE).","method":"T cell-specific ASC knockout, caspase-8 activation assay in T cells, IL-1β production assay, ATP stimulation of TCR-activated TH17 and TH1 cells, EAE mouse model","journal":"Nature immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — T cell-intrinsic ASC KO with functional caspase-8 and IL-1β readouts plus in vivo EAE model; single lab","pmids":["26998763"],"is_preprint":false},{"year":2023,"finding":"Pristimerin (PM) promotes K63-linked ubiquitination of ASC at K158/161 by inhibiting the deubiquitinase USP50 (through covalent binding at Cys53), which serves as a recognition signal for SQSTM1/p62-mediated selective autophagic degradation of the AIM2-ASC complex, thereby suppressing AIM2 inflammasome activation.","method":"Ubiquitination assay (K63-specific, K158/161 mapping), DARTS assay (PM binding to USP50 at Cys53), co-IP (AIM2-ASC), autophagy flux assay, SQSTM1/p62 siRNA, tendinopathy mouse model","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 1 / Strong — site-specific ubiquitination mapping, drug-target binding (DARTS), receptor-ligand delineation, and in vivo model; multiple orthogonal methods","pmids":["37647255"],"is_preprint":false},{"year":2018,"finding":"Zebrafish ASC (zASC) forms speck and filament structures when overexpressed in vitro and in vivo. Crystal structures of the N-terminal PYD and C-terminal CARD of zASC were determined. Structure-guided mutagenesis revealed functional relevance of the PYD hydrophilic surface. The fish caspase-1 homolog Caspy (not Caspy2) interacts with zASC via PYD-PYD interactions, differing from the mammalian CARD-CARD recruitment mechanism.","method":"Crystal structure determination (X-ray crystallography), structure-guided mutagenesis, co-IP (zASC-Caspy PYD-PYD interaction), in vitro and in vivo speck formation assays","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional mutagenesis validation and direct binding assays; ortholog with evolutionary insight into mammalian mechanism","pmids":["29791979"],"is_preprint":false},{"year":2008,"finding":"PYCARD expression is induced in hematopoietic KG1a cells by contact-dependent stroma-derived signals (co-culture with stromal cell line HS5), and suppression of PYCARD by miRNA inhibits stroma-dependent TNF-α-induced apoptosis and caspase-3 activation.","method":"Gene expression profiling (microarray), miRNA knockdown of PYCARD, caspase-3 activation assay, TNF-α-induced apoptosis assay in co-culture","journal":"Blood","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single method (miRNA KD) with functional readout; correlative expression data with single mechanistic follow-up","pmids":["18945969"],"is_preprint":false}],"current_model":"PYCARD/ASC is a bipartite adaptor protein (PYD + CARD) that acts as the central scaffold of inflammasome complexes: upon danger signal detection by sensors such as NLRP3, AIM2, or NLRC4, ASC-PYD polymerizes into filaments (cross-linked via CARD domains) into a micron-sized 'speck' that recruits and activates procaspase-1 via CARD-CARD interactions and procaspase-8 via PYD-DED interactions, leading to IL-1β/IL-18 maturation and pyroptotic or apoptotic cell death; ASC activity is tightly regulated by post-translational modifications including K63-linked ubiquitination (added by Peli1, removed by USP50; required for NLRP3/ASC interaction), K48-linked ubiquitination (removed by USP3 to prevent proteasomal degradation), tyrosine phosphorylation at Y60/Y137 and Y146 (by cAbl kinase; required for speck formation), and SUMOylation (promoted by ZBTB16; modulates assembly); subcellular translocation of ASC from nucleus to perinuclear area is regulated by IKKi/IKKα; extracellular ASC specks released after pyroptosis continue to process IL-1β and propagate inflammasome activation in neighboring cells in a prionoid manner; independently of inflammasome function, ASC regulates MAPK/ERK signaling via DUSP10, serves as a Bax adaptor in the p53-dependent mitochondrial apoptosis pathway, and is required for granuloma formation during chronic mycobacterial infection."},"narrative":{"mechanistic_narrative":"PYCARD/ASC is a bipartite (PYD + CARD) adaptor that serves as the central scaffold of inflammasome complexes, transducing danger-sensor activation into caspase activation and inflammatory cell death [PMID:11967258]. Cytosolic sensors engage ASC through homotypic PYD–PYD contacts—NLRP3-PYD binds ASC-PYD with high affinity [PMID:27432880] and AIM2 couples cytosolic dsDNA detection to ASC [PMID:19158675]—nucleating ASC into micron-scale specks. Assembly is a two-tier process: ASC-PYD polymerizes into a rigid tubular filament core while the mobile CARD cross-links these filaments into the compact speck [PMID:26464513, PMID:27329339, PMID:37945612], a higher-order architecture resolved at atomic and near-native resolution and recapitulated in vivo as intercrossed filament networks [PMID:28701426]. Speck formation amplifies caspase-1-dependent IL-1β maturation but is dispensable for gasdermin-D cleavage and pyroptosis, defining ASC oligomerization as a signal-amplification device [PMID:27329339]; full-length ASC, not isolated domains, is required for both speck formation and rapid death [PMID:11967258, PMID:28701426]. ASC recruits effectors via two distinct death-fold interfaces—procaspase-1 through CARD–CARD contacts, gated by K+-dependent CARD accessibility [PMID:11967258, PMID:37402211], and procaspase-8 through ASC-PYD nucleation of caspase-8 tandem-DED filaments—driving an apoptotic arm including NLRC4- and TH17-associated programs [PMID:23645208, PMID:26468282, PMID:29491424, PMID:26998763]. ASC function is tuned by extensive post-translational control: K63-linked ubiquitination (added by Peli1 at K55, removed by USP50) promotes ASC/NLRP3 interaction and oligomerization [PMID:28094437, PMID:34706239], USP3 removes K48 chains to block proteasomal degradation [PMID:36050480], tyrosine status (cAbl phosphorylation at Y146; requisite dephosphorylation at Y60/Y137) governs nucleation [PMID:31333677, PMID:33568399], SUMOylation via ZBTB16 modulates assembly [PMID:38123560], and IKKα/IKKi control nuclear-to-perinuclear translocation [PMID:25266676]. Beyond canonical inflammasome signaling, ASC drives MAPK/ERK activation by suppressing DUSP10 [PMID:21487011], acts as a Bax adaptor in p53-dependent mitochondrial apoptosis [PMID:14730312], stabilizes HIF-1α to promote tumor cell invasion [PMID:32883954], and is selectively required for granuloma formation during chronic mycobacterial infection independently of NLRP3 and caspase-1 [PMID:20808838]. Released extracellular ASC specks propagate inflammation in a prionoid manner by nucleating soluble ASC in recipient macrophages [PMID:24952505].","teleology":[{"year":2001,"claim":"Established that ASC engages an upstream pyrin-domain sensor (MEFV/pyrin) and that this interaction modulates ASC-driven speck formation and cell fate, first placing ASC in a PYD-based signaling module.","evidence":"Yeast two-hybrid, co-transfection co-localization, and survival assay in HeLa cells","pmids":["11498534"],"confidence":"Medium","gaps":["No structural basis for the pyrin–ASC interface","Endogenous physiological context not tested"]},{"year":2002,"claim":"Defined ASC's bipartite logic—PYD as oligomerization domain, CARD as effector—showing full-length ASC oligomerizes procaspase-1 via CARD–CARD contacts to process pro-IL-1β, answering how an adaptor converts sensing into caspase activation.","evidence":"Co-IP, FKBP-inducible oligomerization domain swap, transfection and THP-1 siRNA","pmids":["11967258"],"confidence":"High","gaps":["Did not resolve filament/speck ultrastructure","Sensor specificity upstream not addressed"]},{"year":2004,"claim":"Revealed an inflammasome-independent role for ASC as a Bax adaptor in p53-dependent mitochondrial apoptosis, expanding ASC function beyond IL-1β maturation.","evidence":"Reciprocal Co-IP, cytochrome c release, Bax-null cells, siRNA in cell lines","pmids":["14730312"],"confidence":"High","gaps":["Domain requirement for Bax interaction not mapped","Relationship to ASC speck formation unclear"]},{"year":2006,"claim":"Showed ASC routes caspase-1 toward IL-1β processing and away from RIP2/NF-κB signaling, establishing ASC as a switch between inflammatory outputs.","evidence":"NF-κB reporter, Co-IP, siRNA, caspase-1-knockout macrophages","pmids":["16585594"],"confidence":"Medium","gaps":["Mechanism of caspase-1 partitioning between partners not structurally defined"]},{"year":2009,"claim":"Identified AIM2 as a cytosolic dsDNA sensor that recruits ASC via PYD–PYD to assemble a functional caspase-1 inflammasome, broadening the repertoire of ASC-coupled sensors.","evidence":"Co-IP domain mapping, siRNA, NF-κB reporter, caspase-1 assay, viral infection","pmids":["19158675"],"confidence":"High","gaps":["Stoichiometry of AIM2–ASC assembly not resolved"]},{"year":2011,"claim":"Demonstrated an inflammasome-independent ASC function in MAPK/ERK signaling through suppression of DUSP10, using genetic epistasis to separate it from NLRP3/NLRC4/caspase-1.","evidence":"shRNA, microarray network analysis, MAPK phosphorylation, multiple knockout macrophages","pmids":["21487011"],"confidence":"High","gaps":["Direct ASC–DUSP10 interaction not demonstrated","Domain requirement unknown"]},{"year":2014,"claim":"Discovered that released extracellular ASC specks propagate inflammation in a prionoid fashion and act as autoantigens, redefining the speck as an intercellular danger signal.","evidence":"Confocal imaging, cell-free IL-1β maturation, phagocytosis/lysosomal damage assays, patient autoantibody detection","pmids":["24952505"],"confidence":"High","gaps":["Determinants of speck stability in the extracellular milieu unclear"]},{"year":2014,"claim":"Defined spatial regulation of ASC by IKKα/IKKi-controlled nuclear-to-perinuclear translocation with PP2A relief, explaining how resting macrophages restrain inflammasome assembly.","evidence":"Co-IP, subcellular fractionation, kinase-dead mutants, PP2A recruitment, macrophage assays","pmids":["25266676"],"confidence":"Medium","gaps":["Phosphosites on ASC mediating retention not mapped","Single lab"]},{"year":2015,"claim":"Solved the ASC filament architecture—rigid PYD core with mobile CARD—and validated PYD–PYD interfaces functionally, providing the structural basis for speck assembly.","evidence":"Cryo-EM, solid-state NMR, structure-based mutagenesis in macrophages (mouse)","pmids":["26464513"],"confidence":"High","gaps":["CARD cross-linking geometry not resolved at the same resolution"]},{"year":2015,"claim":"Showed ASC-PYD nucleates procaspase-8 tandem-DED filaments via a heterotypic death-fold interaction overlapping the self-association surface, mechanistically coupling ASC to the apoptotic caspase-8 arm.","evidence":"In vitro filament nucleation, in vivo co-localization, ASC-PYD mutagenesis, Co-IP","pmids":["26468282"],"confidence":"High","gaps":["Competition between caspase-8 and self-association in cells not quantified"]},{"year":2016,"claim":"Dissected speck assembly into PYD filament formation followed by CARD cross-linking and showed speck formation amplifies IL-1β processing but is dispensable for gasdermin-D cleavage/pyroptosis, defining ASC oligomerization as a signal amplifier.","evidence":"Systematic PYD/CARD mutagenesis in primary macrophages, IL-1β, gasdermin-D, pyroptosis readouts","pmids":["27329339"],"confidence":"High","gaps":["Why pyroptosis bypasses speck requirement not fully explained"]},{"year":2016,"claim":"Quantified NLRP3-PYD/ASC-PYD binding showing higher affinity for the sensor than for self-association, clarifying how NLRP3 seeds ASC fibril nucleation.","evidence":"Solution NMR, analytical ultracentrifugation, structural modeling","pmids":["27432880"],"confidence":"Medium","gaps":["In-cell relevance of measured affinities not tested"]},{"year":2016,"claim":"Identified a T cell-intrinsic ASC-NLRP3-caspase-8 inflammasome that sustains TH17 survival via autocrine IL-1β, extending ASC function to adaptive immunity in autoimmunity.","evidence":"T cell-specific ASC KO, caspase-8/IL-1β assays, TCR/ATP stimulation, EAE model","pmids":["26998763"],"confidence":"Medium","gaps":["Caspase-8 vs caspase-1 dependence in T cells not fully separated","Single lab"]},{"year":2017,"claim":"Imaged endogenous ASC speck assembly, pyroptosis, and clearance in live zebrafish and resolved in vivo speck ultrastructure as intercrossed filament networks, validating filament-based assembly under native conditions.","evidence":"CRISPR endogenous tagging, live imaging, CLEM, 3D reconstruction (zebrafish)","pmids":["28701426"],"confidence":"High","gaps":["Quantitative kinetics of human speck assembly not directly transferable"]},{"year":2017,"claim":"Identified USP50 as a K63-deubiquitinase of ASC required for NLRP3-driven caspase-1 cleavage and speck formation, establishing ubiquitin editing of ASC as a checkpoint.","evidence":"Co-IP, K63-DUB assay, USP50 siRNA, caspase-1/IL-1β/speck readouts in THP-1 and BMDM","pmids":["28094437"],"confidence":"Medium","gaps":["Ubiquitination site on ASC not mapped in this study","Apparent context-dependent role versus later degradation findings"]},{"year":2018,"claim":"Used an unbiased CRISPR screen and gene-targeted mice to show NLRC4 drives an ASC- and caspase-8-dependent apoptotic program distinct from caspase-1 pyroptosis, formalizing ASC's apoptotic arm.","evidence":"Genome-wide CRISPR screen, ASC/caspase-8 KO mice, caspase-1 catalytic-dead knock-in","pmids":["29491424"],"confidence":"High","gaps":["Molecular trigger choosing apoptosis vs pyroptosis not defined"]},{"year":2018,"claim":"Determined crystal structures of zebrafish ASC PYD and CARD and revealed a PYD–PYD caspase recruitment mode in fish, providing evolutionary contrast to mammalian CARD–CARD recruitment.","evidence":"X-ray crystallography, structure-guided mutagenesis, Co-IP of zASC–Caspy (zebrafish)","pmids":["29791979"],"confidence":"High","gaps":["Mammalian PYD surface usage not directly compared functionally here"]},{"year":2020,"claim":"Revealed an inflammasome-independent ASC function stabilizing HIF-1α by reducing its hydroxylation and ubiquitination, linking ASC to tumor cell migration and invasion.","evidence":"Co-IP, fractionation, HIF-1α hydroxylation/ubiquitination assays, RNA-seq, invasion assays in OSCC","pmids":["32883954"],"confidence":"Medium","gaps":["Direct enzymatic mechanism by which ASC blocks hydroxylation unclear","Single lab/tumor context"]},{"year":2010,"claim":"Demonstrated via genetic epistasis that ASC, but not NLRP3 or caspase-1, is required for granuloma formation and host protection in chronic M. tuberculosis infection, cementing an inflammasome-independent in vivo role.","evidence":"Aerosol infection of Pycard-/-, Nlrp3-/-, Casp1-/- mice; survival, burden, histology","pmids":["20808838"],"confidence":"High","gaps":["Molecular effector pathway downstream of ASC in granuloma formation unknown"]},{"year":2021,"claim":"Showed cAbl phosphorylates ASC at Y146 to enable oligomerization and inflammasome activation, defining a positive tyrosine-phosphorylation input to assembly.","evidence":"CRISPR cABL KO, Y146A complementation, phospho/oligomerization/IL-1β assays in THP-1","pmids":["33568399"],"confidence":"High","gaps":["Reconciliation with required dephosphorylation at other tyrosines not addressed here"]},{"year":2021,"claim":"Identified Peli1-mediated K63 ubiquitination of ASC at K55 as a selective promoter of NLRP3 (but not AIM2/NLRP1/NLRC4) inflammasome activation, mapping a sensor-specific ubiquitin switch.","evidence":"Co-IP, K63/K55-mapping mutagenesis, Peli1 KO cells and mice, peritonitis model","pmids":["34706239"],"confidence":"High","gaps":["Why the modification is NLRP3-selective not mechanistically explained"]},{"year":2021,"claim":"Showed a 3'UTR SNP controls Pycard mRNA stability and ASC protein dosage to set inflammasome activity, establishing post-transcriptional regulation of ASC abundance.","evidence":"QTL mapping, mRNA stability, CRISPR single-base 3'UTR editing in mouse macrophages","pmids":["34197316"],"confidence":"High","gaps":["trans-acting factor binding the SNP region not identified"]},{"year":2022,"claim":"Identified USP3 as a K48-deubiquitinase that stabilizes ASC against proteasomal degradation, defining abundance control as a determinant of inflammasome output in vivo.","evidence":"Co-IP, K48-DUB assay, proteasome inhibition, USP3-deficient mouse models","pmids":["36050480"],"confidence":"High","gaps":["E3 ligase opposing USP3 on K48 chains not identified"]},{"year":2022,"claim":"Showed the ASCb splice isoform, differing only in PYD–CARD linker length, self-assembles more slowly into less compact structures, explaining isoform-specific reduction in inflammasome activity.","evidence":"Real-time NMR, DLS, SEC, TEM, docking with procaspase-1 CARD","pmids":["35007535"],"confidence":"High","gaps":["Physiological abundance and regulation of ASCb in tissues not quantified"]},{"year":2023,"claim":"Resolved ASC puncta in NLRP3-activated cells as branched PYD-cored filaments permeated by ribosomes and vesicles, providing near-native cellular context for speck architecture.","evidence":"Correlative cryo-light microscopy and cryo-electron tomography","pmids":["37945612"],"confidence":"High","gaps":["Functional consequence of organelle entrapment in the filament network unknown"]},{"year":2023,"claim":"Showed low intracellular K+ structurally exposes the ASC CARD to enhance procaspase-1 recruitment independently of NLRP3, linking the canonical K+-efflux trigger directly to ASC conformation.","evidence":"ASC oligomerization under varying K+, CARD accessibility and caspase-1 recruitment assays","pmids":["37402211"],"confidence":"Medium","gaps":["Structural model of the K+-sensitive conformational change not solved","Single lab"]},{"year":2023,"claim":"Showed ZBTB16-promoted SUMOylation of ASC controls inflammasome assembly, and its ablation mitigates Muckle-Wells pathology, adding SUMO as a regulatory modification.","evidence":"SUMOylation assay, ZBTB16 KO in Muckle-Wells mouse model","pmids":["38123560"],"confidence":"Medium","gaps":["Exact SUMO site on ASC not defined","Direct vs indirect ZBTB16 effect unclear"]},{"year":2023,"claim":"Demonstrated that K63 ubiquitination of ASC at K158/161 (driven pharmacologically via USP50 inhibition) targets the AIM2-ASC complex for SQSTM1/p62-mediated selective autophagy, defining an autophagic route for inflammasome termination.","evidence":"Site-specific ubiquitination, DARTS drug-target binding, Co-IP, autophagy flux, p62 siRNA, tendinopathy model","pmids":["37647255"],"confidence":"High","gaps":["Physiological (non-pharmacological) trigger of this pathway not defined"]},{"year":2008,"claim":"Linked stroma-induced PYCARD expression to TNF-α-dependent apoptosis in hematopoietic cells, an early correlative hint of microenvironment-regulated ASC function.","evidence":"Expression profiling, miRNA knockdown, caspase-3/apoptosis assays in co-culture","pmids":["18945969"],"confidence":"Low","gaps":["Single knockdown method with correlative expression data; mechanism not established","No direct ASC interaction partner identified"]},{"year":null,"claim":"How the many competing post-translational inputs (K63/K48 ubiquitination, tyrosine phosphorylation/dephosphorylation, SUMOylation) are integrated in time and space to license a single speck per cell, and how inflammasome-independent ASC functions are mechanistically partitioned from the canonical pathway, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model coupling modification state to assembly kinetics","Structural basis of CARD cross-linking in the speck incomplete","Mechanistic effectors of inflammasome-independent roles (granuloma, HIF-1α, MAPK) undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,2,4,11]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[7,8,20,21]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[9,10,25]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,6,21]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[6,25]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[2]},{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[5]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,3,8,15]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[2,4,27]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[19,10]}],"complexes":["NLRP3 inflammasome","AIM2 inflammasome","NLRC4 inflammasome","ASC speck/filament"],"partners":["NLRP3","AIM2","CASP1","CASP8","BAX","MEFV","PELI1","USP3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9ULZ3","full_name":"Apoptosis-associated speck-like protein containing a CARD","aliases":["Caspase recruitment domain-containing protein 5","PYD and CARD domain-containing protein","Target of methylation-induced silencing 1"],"length_aa":195,"mass_kda":21.6,"function":"Functions as a key mediator in apoptosis and inflammation (PubMed:11103777, PubMed:12646168, PubMed:15030775, PubMed:17349957, PubMed:17599095, PubMed:19158675, PubMed:19158676, PubMed:19234215, PubMed:19494289, PubMed:21487011, PubMed:24630722, PubMed:25847972, PubMed:30674671, PubMed:34678144, PubMed:36050480). Promotes caspase-mediated apoptosis involving predominantly caspase-8 and also caspase-9 in a probable cell type-specific manner (PubMed:11103777, PubMed:12646168). Involved in activation of the mitochondrial apoptotic pathway, promotes caspase-8-dependent proteolytic maturation of BID independently of FADD in certain cell types and also mediates mitochondrial translocation of BAX and activates BAX-dependent apoptosis coupled to activation of caspase-9, -2 and -3 (PubMed:14730312, PubMed:16964285). Involved in innate immune response by acting as an integral adapter in the assembly of various inflammasomes (NLRP1, NLRP2, NLRP3, NLRP6, AIM2 and probably IFI16) which recruit and activate caspase-1 leading to processing and secretion of pro-inflammatory cytokines (PubMed:15030775, PubMed:16982856, PubMed:17349957, PubMed:17599095, PubMed:19158675, PubMed:19158676, PubMed:19234215, PubMed:21487011, PubMed:23530044, PubMed:24630722, PubMed:25847972, PubMed:29440442, PubMed:30674671, PubMed:33980849, PubMed:34678144, PubMed:34706239). Caspase-1-dependent inflammation leads to macrophage pyroptosis, a form of cell death (PubMed:24630722). The function as activating adapter in different types of inflammasomes is mediated by the pyrin and CARD domains and their homotypic interactions (PubMed:14499617, PubMed:19234215, PubMed:24630722). Clustered PYCARD nucleates the formation of caspase-1 filaments through the interaction of their respective CARD domains, acting as a platform for of caspase-1 polymerization (PubMed:24630722). In the NLRP1 and NLRC4 inflammasomes seems not be required but facilitates the processing of procaspase-1 (PubMed:17349957). In cooperation with NOD2 involved in an inflammasome activated by bacterial muramyl dipeptide leading to caspase-1 activation (PubMed:16964285). May be involved in RIGI-triggered pro-inflammatory responses and inflammasome activation (PubMed:19915568). In collaboration with AIM2 which detects cytosolic double-stranded DNA may also be involved in a caspase-1-independent cell death that involves caspase-8 (PubMed:19158675, PubMed:19158676). In adaptive immunity may be involved in maturation of dendritic cells to stimulate T-cell immunity and in cytoskeletal rearrangements coupled to chemotaxis and antigen uptake may be involved in post-transcriptional regulation of the guanine nucleotide exchange factor DOCK2; the latter function is proposed to involve the nuclear form (PubMed:22732093). Also involved in transcriptional activation of cytokines and chemokines independent of the inflammasome; this function may involve AP-1, NF-kappa-B, MAPK and caspase-8 signaling pathways (PubMed:12486103, PubMed:16585594). For regulation of NF-kappa-B activating and inhibiting functions have been reported (PubMed:12486103). Modulates NF-kappa-B induction at the level of the IKK complex by inhibiting kinase activity of CHUK and IKBK (PubMed:12486103, PubMed:16585594). Proposed to compete with RIPK2 for association with CASP1 thereby down-regulating CASP1-mediated RIPK2-dependent NF-kappa-B activation and activating interleukin-1 beta processing (PubMed:16585594). Modulates host resistance to DNA virus infection, probably by inducing the cleavage of and inactivating CGAS in presence of cytoplasmic double-stranded DNA (PubMed:28314590) May have a regulating effect on the function as inflammasome adapter Seems to inhibit inflammasome-mediated maturation of interleukin-1 beta","subcellular_location":"Golgi apparatus membrane","url":"https://www.uniprot.org/uniprotkb/Q9ULZ3/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PYCARD","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/PYCARD","total_profiled":1310},"omim":[{"mim_id":"620563","title":"UBIQUITIN-SPECIFIC PEPTIDASE 50; USP50","url":"https://www.omim.org/entry/620563"},{"mim_id":"620331","title":"HATIPOGLU IMMUNODEFICIENCY SYNDROME; HATIS","url":"https://www.omim.org/entry/620331"},{"mim_id":"618424","title":"ARMADILLO REPEAT-CONTAINING PROTEIN 2; ARMC2","url":"https://www.omim.org/entry/618424"},{"mim_id":"615701","title":"PYRIN DOMAIN-CONTAINING PROTEIN 2; PYDC2","url":"https://www.omim.org/entry/615701"},{"mim_id":"615700","title":"PYRIN DOMAIN-CONTAINING PROTEIN 1; PYDC1","url":"https://www.omim.org/entry/615700"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nucleoli","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"lymphoid tissue","ntpm":104.6},{"tissue":"skin 1","ntpm":111.0}],"url":"https://www.proteinatlas.org/search/PYCARD"},"hgnc":{"alias_symbol":["TMS-1","CARD5","ASC"],"prev_symbol":[]},"alphafold":{"accession":"Q9ULZ3","domains":[{"cath_id":"1.10.533.10","chopping":"2-90","consensus_level":"high","plddt":80.6694,"start":2,"end":90},{"cath_id":"1.10.533.10","chopping":"113-193","consensus_level":"high","plddt":69.7488,"start":113,"end":193}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9ULZ3","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9ULZ3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9ULZ3-F1-predicted_aligned_error_v6.png","plddt_mean":72.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PYCARD","jax_strain_url":"https://www.jax.org/strain/search?query=PYCARD"},"sequence":{"accession":"Q9ULZ3","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9ULZ3.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9ULZ3/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9ULZ3"}},"corpus_meta":[{"pmid":"19158675","id":"PMC_19158675","title":"AIM2 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induces caspase-1-dependent macrophage pyroptosis through the NLRP3-ASC inflammasome.","date":"2019","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/31316052","citation_count":249,"is_preprint":false},{"pmid":"11498534","id":"PMC_11498534","title":"Interaction between pyrin and the apoptotic speck protein (ASC) modulates ASC-induced apoptosis.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11498534","citation_count":238,"is_preprint":false},{"pmid":"14730312","id":"PMC_14730312","title":"ASC is a Bax adaptor and regulates the p53-Bax mitochondrial apoptosis pathway.","date":"2004","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/14730312","citation_count":204,"is_preprint":false},{"pmid":"25080478","id":"PMC_25080478","title":"ASC-1, PAT2, and P2RX5 are cell surface markers for white, beige, and brown adipocytes.","date":"2014","source":"Science translational 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requires PYCARD/ASC but not NLRP3 or caspase-1.","date":"2010","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/20808838","citation_count":132,"is_preprint":false},{"pmid":"26464513","id":"PMC_26464513","title":"Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy.","date":"2015","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/26464513","citation_count":132,"is_preprint":false},{"pmid":"20368281","id":"PMC_20368281","title":"Cutting edge: critical role for PYCARD/ASC in the development of experimental autoimmune encephalomyelitis.","date":"2010","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/20368281","citation_count":128,"is_preprint":false},{"pmid":"29491424","id":"PMC_29491424","title":"ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages.","date":"2018","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/29491424","citation_count":121,"is_preprint":false},{"pmid":"20200803","id":"PMC_20200803","title":"Exercise effects on methylation of ASC gene.","date":"2010","source":"International journal of sports medicine","url":"https://pubmed.ncbi.nlm.nih.gov/20200803","citation_count":115,"is_preprint":false},{"pmid":"31086376","id":"PMC_31086376","title":"A long noncoding RNA distributed in both nucleus and cytoplasm operates in the PYCARD-regulated apoptosis by coordinating the epigenetic and translational regulation.","date":"2019","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/31086376","citation_count":103,"is_preprint":false},{"pmid":"16585594","id":"PMC_16585594","title":"ASC directs NF-kappaB activation by regulating receptor interacting protein-2 (RIP2) caspase-1 interactions.","date":"2006","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/16585594","citation_count":102,"is_preprint":false},{"pmid":"25266676","id":"PMC_25266676","title":"IKKα negatively regulates ASC-dependent inflammasome activation.","date":"2014","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/25266676","citation_count":97,"is_preprint":false},{"pmid":"34345247","id":"PMC_34345247","title":"Squamous intraepithelial lesions (SIL: LSIL, HSIL, ASCUS, ASC-H, LSIL-H) of Uterine Cervix and Bethesda System.","date":"2021","source":"CytoJournal","url":"https://pubmed.ncbi.nlm.nih.gov/34345247","citation_count":93,"is_preprint":false},{"pmid":"29247990","id":"PMC_29247990","title":"The intra- and extracellular functions of ASC specks.","date":"2018","source":"Immunological reviews","url":"https://pubmed.ncbi.nlm.nih.gov/29247990","citation_count":88,"is_preprint":false},{"pmid":"17339483","id":"PMC_17339483","title":"Pyrin-only protein 2 modulates NF-kappaB and disrupts ASC:CLR 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The PYRIN domain acts as an oligomerization domain while the CARD domain is the effector domain; full-length ASC (but not isolated domains alone) activates procaspase-1 and processes pro-IL-1β in transfected cells.\",\n      \"method\": \"Co-IP, FKBP12-inducible oligomerization domain swap, transfection/overexpression in HEK cells, siRNA knockdown in THP-1 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 / Strong — direct in vitro and cellular reconstitution with domain-swap mutagenesis and loss-of-function, multiple orthogonal methods in a single study\",\n      \"pmids\": [\"11967258\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Pyrin (the MEFV gene product) interacts with ASC via its pyrin domain (exon 1) in a yeast two-hybrid assay and co-localizes with ASC in cytosolic specks in HeLa cells. Co-expression of pyrin with ASC increases speck formation and promotes survival of speck-positive cells, modulating ASC-induced apoptosis.\",\n      \"method\": \"Yeast two-hybrid, co-transfection/immunofluorescence co-localization, cell survival assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid and co-localization in transfected cells, two orthogonal methods, single lab\",\n      \"pmids\": [\"11498534\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ASC functions as a Bax adaptor in the p53-dependent mitochondrial apoptosis pathway. ASC directly interacts with Bax, co-localizes with Bax at mitochondria, induces cytochrome c release, loss of mitochondrial membrane potential, and activation of caspases-9, -2, and -3. Rapid apoptosis induction is absent in Bax-deficient cells, and siRNA knockdown of ASC inhibits Bax translocation to mitochondria after p53 or genotoxic insult.\",\n      \"method\": \"Co-IP, co-localization by fluorescence microscopy, cytochrome c release assay, mitochondrial membrane potential assay, Bax-deficient cell lines, siRNA knockdown\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, multiple orthogonal cellular assays, genetic rescue with Bax-null cells, and loss-of-function with siRNA; multiple independent validations in single study\",\n      \"pmids\": [\"14730312\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"AIM2 (PYHIN family member) binds cytosolic dsDNA via its HIN200 domain, while its pyrin domain associates with ASC. This AIM2-ASC interaction activates both NF-κB and caspase-1 as a functional inflammasome. Knockdown of AIM2 abrogates caspase-1 activation in response to cytoplasmic dsDNA.\",\n      \"method\": \"Co-IP (PYD-PYD interaction), siRNA knockdown, reporter assay for NF-κB, caspase-1 activity assay, viral infection model\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, domain-mapping, siRNA knockdown with functional caspase-1 readout, replicated with multiple stimuli and viral infection model\",\n      \"pmids\": [\"19158675\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Procaspase-8 localizes to ASC inflammasome specks and binds directly to the pyrin domain of ASC. AIM2 and NLRP3 inflammasomes activate caspase-8 and caspase-1 leading to both apoptotic and pyroptotic cell death, with caspase-8 being the apical caspase in the ASC-dependent apoptotic pathway (with little requirement for caspase-9).\",\n      \"method\": \"Gene knockdown, fluorescence co-localization of procaspase-8 with ASC specks, direct binding assay (PYD-procaspase-8 interaction), cell death quantification with varying DNA concentrations\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding demonstrated, co-localization confirmed, gene knockdown with functional readout; single lab\",\n      \"pmids\": [\"23645208\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"After pyroptosis, ASC specks accumulate in the extracellular space where they continue to promote IL-1β maturation. Phagocytosis of extracellular ASC specks by macrophages causes lysosomal damage and nucleation of soluble ASC, activating IL-1β in recipient cells (prionoid propagation). Autoantibodies to ASC specks develop in patients and mice with autoimmune pathologies.\",\n      \"method\": \"Cell imaging (confocal microscopy), cell-free IL-1β maturation assay with extracellular specks, phagocytosis assay with lysosomal damage readout, autoantibody detection in patient samples\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (cell-free assay, imaging, phagocytosis experiments), validated in human patient samples; single lab but comprehensive\",\n      \"pmids\": [\"24952505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"IKKα negatively regulates ASC-dependent inflammasomes by interacting with ASC in the nucleus of resting macrophages in an IKKα kinase-dependent manner. IKKi (IKK-related kinase) facilitates translocation of ASC from nucleus to perinuclear area during inflammasome activation; upon NLRP3 signal 2, PP2A is recruited and inhibits IKKα kinase activity, releasing ASC to participate in inflammasome assembly.\",\n      \"method\": \"Co-IP (ASC-IKKα interaction), subcellular fractionation/localization, kinase-dead mutant analysis, PP2A recruitment assay, macrophage inflammasome activation assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, subcellular fractionation, kinase-dead mutant, multiple mechanistic steps validated; single lab\",\n      \"pmids\": [\"25266676\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The structure of the mouse ASC filament was determined at atomic resolution by combining cryo-EM and solid-state NMR spectroscopy. ASC-PYD forms the rigid core of the filament while the CARD domain is highly mobile relative to this core. NMR identified specific PYD-PYD binding interfaces, and structure-based mutagenesis validated their functional relevance in macrophage inflammasome activation.\",\n      \"method\": \"Cryo-electron microscopy, solid-state NMR spectroscopy, structure-based site-directed mutagenesis, macrophage functional assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — atomic-resolution structure by two complementary methods (cryo-EM + NMR) plus mutagenesis validation in primary macrophages\",\n      \"pmids\": [\"26464513\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ASC speck assembly involves two steps: oligomerization of ASC-PYD into filaments, and cross-linking of these filaments by ASC-CARD. ASC mutants with non-functional CARD only assemble filaments but not specks and disrupt endogenous specks. ASC speck formation is required for efficient IL-1β processing but is dispensable for gasdermin-D cleavage and pyroptosis induction, indicating that ASC oligomerization serves as a signal amplification mechanism for caspase-1-dependent cytokine production.\",\n      \"method\": \"Systematic site-directed mutagenesis of ASC-PYD, ASC-CARD mutant expression in primary macrophages, IL-1β processing assay, gasdermin-D cleavage assay, pyroptosis assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic mutagenesis combined with functional dissection of IL-1β processing vs. gasdermin-D cleavage; multiple orthogonal readouts in primary macrophages\",\n      \"pmids\": [\"27329339\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ASC directs caspase-1 away from RIP2-mediated NF-κB activation toward caspase-1-mediated IL-1β processing. ASC interferes with the caspase-1/RIP2 interaction in a dose-dependent manner, and siRNA knockdown of ASC in THP-1 cells decreases caspase-1 activity while enhancing NF-κB signaling.\",\n      \"method\": \"Co-transfection with NF-κB reporter gene assay, Co-IP (caspase-1/RIP2 interaction), siRNA knockdown, caspase-1-knockout macrophages\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reporter assay, Co-IP, siRNA knockdown, and knockout macrophages; multiple methods in single lab\",\n      \"pmids\": [\"16585594\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"ASC/PYCARD regulates MAPK (ERK) phosphorylation in an inflammasome-independent manner by suppressing the dual-specificity phosphatase DUSP10/MKP5. ASC also regulates chemokine induction independently of caspase-1 and IL-1β. MAPK activation by pathogen was abrogated in Asc-/- but not Nlrp3-/-, Nlrc4-/-, or Casp1-/- macrophages.\",\n      \"method\": \"ASC shRNA knockdown, microarray/network analysis, MAPK phosphorylation assay, comparison of Asc-/-, Nlrp3-/-, Nlrc4-/-, Casp1-/- macrophages\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with multiple knockout macrophage lines plus shRNA knockdown, microarray network analysis, and direct phosphorylation readout across multiple stimuli\",\n      \"pmids\": [\"21487011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ASC filaments nucleate procaspase-8 death effector domain (DED) filaments in vitro and in vivo. Interaction between ASC-PYD and procaspase-8 tandem DEDs requires both DEDs and represents a heterotypic interaction between death fold superfamily domains. ASC PYD mutagenesis showed that procaspase-8 interaction surfaces overlap with those required for ASC self-association.\",\n      \"method\": \"In vitro filament nucleation assay, in vivo co-localization, ASC-PYD mutagenesis, co-IP of ASC and procaspase-8\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis, confirmed in vivo; single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"26468282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ASC-PYD self-associates and binds NLRP3-PYD through equivalent protein regions (located at opposite sides of the protein), with higher affinity for NLRP3-PYD than for itself. NLRP3-PYD coexists as monomer and large oligomeric species in solution. These binding interfaces allow multimeric complex formation consistent with ASC-PYD fibril assemblies.\",\n      \"method\": \"Solution NMR spectroscopy, analytical ultracentrifugation, structural modeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure and binding characterization with orthogonal biophysical method (AUC); single lab\",\n      \"pmids\": [\"27432880\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The deubiquitinating enzyme USP50 binds ASC and deubiquitinates K63-linked polyubiquitin chains on ASC. USP50 knockdown in THP-1 cells and BMDMs significantly decreases procaspase-1 cleavage, IL-1β/IL-18 secretion, and ASC speck formation/oligomerization upon NLRP3 stimuli.\",\n      \"method\": \"Co-IP (USP50-ASC binding), deubiquitination assay (K63-linked Ub), siRNA knockdown of USP50, caspase-1 cleavage assay, IL-1β/IL-18 ELISA, ASC speck formation assay\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, deubiquitination assay, and loss-of-function knockdown with multiple functional readouts; single lab\",\n      \"pmids\": [\"28094437\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Tyrosine dephosphorylation of ASC is required for NLRP3 and AIM2 inflammasome activation. Site-directed mutagenesis identified tyrosine residues Y60 and Y137 of ASC as critical for inflammasome assembly and function (ASC nucleation, caspase-1 activation, IL-1β/IL-18 processing, and pyroptosis).\",\n      \"method\": \"Site-directed mutagenesis of ASC tyrosine residues (Y60, Y137), pharmacological PTPase inhibition (phenylarsine oxide), caspase-1 activation assay, IL-1β/IL-18 release assay, pyroptosis assay\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-directed mutagenesis combined with pharmacological inhibition and functional readouts; single lab\",\n      \"pmids\": [\"31333677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The E3 ubiquitin ligase Peli1 conjugates K63-linked ubiquitin chains specifically to lysine 55 of ASC, which facilitates ASC/NLRP3 interaction and ASC oligomerization, thereby promoting NLRP3 inflammasome activation. Peli1 deficiency impairs NLRP3-induced (but not AIM2, NLRP1, or NLRC4) caspase-1 activation and IL-1β maturation.\",\n      \"method\": \"Co-IP (ASC-Peli1 interaction), ubiquitination assay (K63-specific, lysine-55 mapping by mutagenesis), Peli1 knockout macrophages and mice, IL-1β secretion assay, peritonitis mouse model\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — site-specific ubiquitination mapping by mutagenesis, Peli1 KO cells and in vivo mouse model, inflammasome-selectivity analysis across multiple sensors\",\n      \"pmids\": [\"34706239\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"cAbl kinase phosphorylates ASC at tyrosine 146 (Y146), and this phosphorylation is required for ASC oligomerization and inflammasome activation. CRISPR/Cas9 deletion of cABL in THP-1 cells inhibits inflammasome function and reduces phosphorylated ASC release after LPS/ATP stimulation. Complementation of ASC-knockout cells with Y146A-mutant ASC abrogates inflammasome activation and ASC oligomerization.\",\n      \"method\": \"CRISPR/Cas9 cABL knockout, site-directed mutagenesis (Y146A), ASC phosphorylation assay, IL-18/IL-1β release assay, ASC oligomerization assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — CRISPR KO combined with Y146A mutagenesis and functional complementation; multiple orthogonal readouts in single study\",\n      \"pmids\": [\"33568399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP3 is a deubiquitinating enzyme that directly binds ASC, removes K48-linked ubiquitin chains, and stabilizes ASC protein by blocking proteasomal degradation. USP3 promotes inflammasome activation, confirmed in mouse models of alum-induced peritonitis, F. novicida infection, and flagellin-induced pneumonia.\",\n      \"method\": \"Co-IP (USP3-ASC), deubiquitination assay (K48-linked Ub), proteasome inhibitor experiments, USP3-deficient mice models (peritonitis, F. novicida, flagellin pneumonia)\",\n      \"journal\": \"Cellular & molecular immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, K48-Ub-specific deubiquitination assay, proteasomal degradation rescue, and three independent in vivo mouse models\",\n      \"pmids\": [\"36050480\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ZBTB16 (a nuclear zinc-finger/BTB domain protein) promotes SUMOylation of ASC, which controls ASC function in inflammasome assembly. Ablation of ZBTB16 in a mouse model of Muckle-Wells syndrome reduces acute inflammatory pathogenesis driven by constitutively hyperactive inflammasome.\",\n      \"method\": \"SUMOylation assay (ASC-SUMO modification), ZBTB16 knockout in Muckle-Wells mouse model, inflammasome activation assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — SUMOylation assay and in vivo genetic model; single lab, mechanistic detail of exact SUMO site not specified in abstract\",\n      \"pmids\": [\"38123560\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Low intracellular K+ concentration causes a structural change in ASC oligomers that makes the ASC CARD domain more accessible for recruitment of the pro-caspase-1 CARD domain, thereby enhancing caspase-1 activation. This structural change in ASC is independent of NLRP3.\",\n      \"method\": \"ASC oligomerization assays under varying K+ conditions, domain accessibility experiments, caspase-1 recruitment assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic dissection of K+-dependent structural change in ASC oligomers with functional caspase-1 recruitment readout; single lab\",\n      \"pmids\": [\"37402211\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cryo-electron tomography of NLRP3-activated cells reveals that ASC puncta are composed of branched filaments with a tubular core formed by the PYD. Ribosomes and Golgi-like/endosomal vesicles permeate the ASC filament network. Mitochondria are not associated with ASC filaments but show outer-membrane discontinuities consistent with gasdermin D pores.\",\n      \"method\": \"Correlative cryo-light microscopy and cryo-electron tomography of NLRP3-activated cells\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-ET structural determination at near-native conditions in activated cells; single lab but direct structural data with organelle co-localization\",\n      \"pmids\": [\"37945612\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"In live zebrafish, CRISPR/Cas9-tagging of endogenous ASC showed strong expression in skin epithelia. Toxic stimulus triggered speck formation followed by rapid pyroptosis in keratinocytes, and macrophages engulfed and digested speck-containing pyroptotic debris. 3D ultrastructural reconstruction (CLEM) revealed compact networks of highly intercrossed filaments in in vivo specks. Full-length ASC (not PYD or CARD alone) is required to form a single compact speck and rapidly induce cell death.\",\n      \"method\": \"CRISPR/Cas9 endogenous tagging in zebrafish, live imaging, correlative light and electron microscopy (CLEM), 3D ultrastructural reconstruction\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — endogenous tagging + live in vivo imaging + CLEM ultrastructural reconstruction; orthogonal domain-deletion functional analysis\",\n      \"pmids\": [\"28701426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The ASC alternative splicing isoform ASCb (differing from ASC only in the linker length between PYD and CARD) self-associates more slowly and into less compact macrostructures than ASC, with a reduced tendency to form densely packed filaments. This kinetic/structural difference, attributable solely to linker length, explains ASCb's reduced ability to activate the inflammasome compared to ASC.\",\n      \"method\": \"Real-time NMR, dynamic light scattering (DLS), size-exclusion chromatography, transmission electron microscopy, molecular docking with procaspase-1 CARD\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal biophysical methods (NMR + DLS + SEC + TEM + docking) characterizing isoform-specific self-assembly differences\",\n      \"pmids\": [\"35007535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"A SNP in the 3' UTR of the mouse Pycard gene (DBA/2 vs. AKR alleles) regulates ASC mRNA stability without altering transcription rate, leading to higher Pycard mRNA and ASC protein levels in DBA/2 macrophages and increased inflammasome speck formation. CRISPR/Cas9 editing of the DBA/2 3'UTR SNP to the AKR allele reduced Pycard expression and inflammasome activity, confirming a post-transcriptional regulatory mechanism.\",\n      \"method\": \"QTL mapping, mRNA stability assay, ASC protein quantification, inflammasome speck formation, CRISPR/Cas9 single-base editing of 3'UTR SNP\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — CRISPR single-base editing validation of mRNA stability mechanism; multiple orthogonal methods confirming post-transcriptional regulation\",\n      \"pmids\": [\"34197316\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PYCARD/ASC, but not NLRP3 or caspase-1, is required for granuloma formation and host protection during chronic Mycobacterium tuberculosis infection in vivo, revealing an inflammasome-independent role for ASC. Pycard-/- mice showed abrupt decrease in survival with defective granuloma formation, whereas Nlrp3-/- and Casp1-/- mice showed no difference from wild-type.\",\n      \"method\": \"Pycard-/-, Nlrp3-/-, and Casp1-/- mouse aerosol infection model, survival analysis, bacterial burden measurement, histological granuloma assessment, macrophage IL-1β secretion assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with three separate knockouts in vivo, defining an inflammasome-independent function of ASC in granuloma formation\",\n      \"pmids\": [\"20808838\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ASC stabilizes HIF-1α protein in oral squamous cell carcinoma cells under normoxia by interacting with HIF-1α in both cytoplasm and nucleus. ASC interaction decreases HIF-1α hydroxylation (HIF-1α-OH) and ubiquitination without affecting VHL or PHD2 levels, thereby promoting HIF-1α-dependent transcription of genes driving cell migration and invasion.\",\n      \"method\": \"Co-IP (ASC-HIF-1α), subcellular fractionation, HIF-1α hydroxylation and ubiquitination assays, transcriptomic analysis (RNA-seq), HIF-1α synthesis inhibitor treatment, migration/invasion assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with mechanistic follow-up (hydroxylation/ubiquitination assays), subcellular localization, and functional rescue experiments; single lab\",\n      \"pmids\": [\"32883954\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Pyrin-only protein 2 (POP2) co-localizes with ASC in perinuclear specks and disrupts ASC:CLR (CIAS1/NALP3) interactions, inhibiting speck formation by NLRP3. POP2 also modulates NF-κB activity through changes in nuclear import or distribution.\",\n      \"method\": \"Co-transfection/immunofluorescence co-localization, NF-κB reporter assay, speck formation assay with NLRP3 co-expression\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-localization and reporter assays, two orthogonal readouts; single lab, no direct binding assay for POP2-ASC\",\n      \"pmids\": [\"17339483\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"NLRC4 activates an ASC- and caspase-8-dependent apoptotic pathway that is distinct from caspase-1-mediated pyroptosis. A genome-wide CRISPR/Cas9 screen and gene-targeted mouse validation confirmed that ASC and caspase-8 are required for the NLRC4-dependent alternative apoptotic program. Caspase-1 catalytic dead knock-in BMDMs confirm that caspase-1 does not compete with ASC for NLRC4 interactions.\",\n      \"method\": \"Genome-wide CRISPR/Cas9 screen, gene-targeted (caspase-8, ASC knockout) mice, caspase-1 catalytic dead knock-in mice, cell death characterization\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — unbiased genome-wide screen with multiple genetic validations in gene-targeted mice; caspase-1 knock-in provides mechanistic dissection\",\n      \"pmids\": [\"29491424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"T cell-intrinsic ASC-NLRP3-caspase-8 inflammasome in TH17 cells drives IL-1β production upon ATP stimulation of the T cell antigen receptor, promoting autocrine TH17 cell survival during experimental autoimmune encephalomyelitis (EAE).\",\n      \"method\": \"T cell-specific ASC knockout, caspase-8 activation assay in T cells, IL-1β production assay, ATP stimulation of TCR-activated TH17 and TH1 cells, EAE mouse model\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — T cell-intrinsic ASC KO with functional caspase-8 and IL-1β readouts plus in vivo EAE model; single lab\",\n      \"pmids\": [\"26998763\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Pristimerin (PM) promotes K63-linked ubiquitination of ASC at K158/161 by inhibiting the deubiquitinase USP50 (through covalent binding at Cys53), which serves as a recognition signal for SQSTM1/p62-mediated selective autophagic degradation of the AIM2-ASC complex, thereby suppressing AIM2 inflammasome activation.\",\n      \"method\": \"Ubiquitination assay (K63-specific, K158/161 mapping), DARTS assay (PM binding to USP50 at Cys53), co-IP (AIM2-ASC), autophagy flux assay, SQSTM1/p62 siRNA, tendinopathy mouse model\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — site-specific ubiquitination mapping, drug-target binding (DARTS), receptor-ligand delineation, and in vivo model; multiple orthogonal methods\",\n      \"pmids\": [\"37647255\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Zebrafish ASC (zASC) forms speck and filament structures when overexpressed in vitro and in vivo. Crystal structures of the N-terminal PYD and C-terminal CARD of zASC were determined. Structure-guided mutagenesis revealed functional relevance of the PYD hydrophilic surface. The fish caspase-1 homolog Caspy (not Caspy2) interacts with zASC via PYD-PYD interactions, differing from the mammalian CARD-CARD recruitment mechanism.\",\n      \"method\": \"Crystal structure determination (X-ray crystallography), structure-guided mutagenesis, co-IP (zASC-Caspy PYD-PYD interaction), in vitro and in vivo speck formation assays\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional mutagenesis validation and direct binding assays; ortholog with evolutionary insight into mammalian mechanism\",\n      \"pmids\": [\"29791979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PYCARD expression is induced in hematopoietic KG1a cells by contact-dependent stroma-derived signals (co-culture with stromal cell line HS5), and suppression of PYCARD by miRNA inhibits stroma-dependent TNF-α-induced apoptosis and caspase-3 activation.\",\n      \"method\": \"Gene expression profiling (microarray), miRNA knockdown of PYCARD, caspase-3 activation assay, TNF-α-induced apoptosis assay in co-culture\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single method (miRNA KD) with functional readout; correlative expression data with single mechanistic follow-up\",\n      \"pmids\": [\"18945969\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PYCARD/ASC is a bipartite adaptor protein (PYD + CARD) that acts as the central scaffold of inflammasome complexes: upon danger signal detection by sensors such as NLRP3, AIM2, or NLRC4, ASC-PYD polymerizes into filaments (cross-linked via CARD domains) into a micron-sized 'speck' that recruits and activates procaspase-1 via CARD-CARD interactions and procaspase-8 via PYD-DED interactions, leading to IL-1β/IL-18 maturation and pyroptotic or apoptotic cell death; ASC activity is tightly regulated by post-translational modifications including K63-linked ubiquitination (added by Peli1, removed by USP50; required for NLRP3/ASC interaction), K48-linked ubiquitination (removed by USP3 to prevent proteasomal degradation), tyrosine phosphorylation at Y60/Y137 and Y146 (by cAbl kinase; required for speck formation), and SUMOylation (promoted by ZBTB16; modulates assembly); subcellular translocation of ASC from nucleus to perinuclear area is regulated by IKKi/IKKα; extracellular ASC specks released after pyroptosis continue to process IL-1β and propagate inflammasome activation in neighboring cells in a prionoid manner; independently of inflammasome function, ASC regulates MAPK/ERK signaling via DUSP10, serves as a Bax adaptor in the p53-dependent mitochondrial apoptosis pathway, and is required for granuloma formation during chronic mycobacterial infection.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PYCARD/ASC is a bipartite (PYD + CARD) adaptor that serves as the central scaffold of inflammasome complexes, transducing danger-sensor activation into caspase activation and inflammatory cell death [#0]. Cytosolic sensors engage ASC through homotypic PYD–PYD contacts—NLRP3-PYD binds ASC-PYD with high affinity [#12] and AIM2 couples cytosolic dsDNA detection to ASC [#3]—nucleating ASC into micron-scale specks. Assembly is a two-tier process: ASC-PYD polymerizes into a rigid tubular filament core while the mobile CARD cross-links these filaments into the compact speck [#7, #8, #20], a higher-order architecture resolved at atomic and near-native resolution and recapitulated in vivo as intercrossed filament networks [#21]. Speck formation amplifies caspase-1-dependent IL-1\\u03b2 maturation but is dispensable for gasdermin-D cleavage and pyroptosis, defining ASC oligomerization as a signal-amplification device [#8]; full-length ASC, not isolated domains, is required for both speck formation and rapid death [#0, #21]. ASC recruits effectors via two distinct death-fold interfaces—procaspase-1 through CARD–CARD contacts, gated by K+-dependent CARD accessibility [#0, #19], and procaspase-8 through ASC-PYD nucleation of caspase-8 tandem-DED filaments—driving an apoptotic arm including NLRC4- and TH17-associated programs [#4, #11, #27, #28]. ASC function is tuned by extensive post-translational control: K63-linked ubiquitination (added by Peli1 at K55, removed by USP50) promotes ASC/NLRP3 interaction and oligomerization [#13, #15], USP3 removes K48 chains to block proteasomal degradation [#17], tyrosine status (cAbl phosphorylation at Y146; requisite dephosphorylation at Y60/Y137) governs nucleation [#14, #16], SUMOylation via ZBTB16 modulates assembly [#18], and IKK\\u03b1/IKKi control nuclear-to-perinuclear translocation [#6]. Beyond canonical inflammasome signaling, ASC drives MAPK/ERK activation by suppressing DUSP10 [#10], acts as a Bax adaptor in p53-dependent mitochondrial apoptosis [#2], stabilizes HIF-1\\u03b1 to promote tumor cell invasion [#25], and is selectively required for granuloma formation during chronic mycobacterial infection independently of NLRP3 and caspase-1 [#24]. Released extracellular ASC specks propagate inflammation in a prionoid manner by nucleating soluble ASC in recipient macrophages [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established that ASC engages an upstream pyrin-domain sensor (MEFV/pyrin) and that this interaction modulates ASC-driven speck formation and cell fate, first placing ASC in a PYD-based signaling module.\",\n      \"evidence\": \"Yeast two-hybrid, co-transfection co-localization, and survival assay in HeLa cells\",\n      \"pmids\": [\"11498534\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural basis for the pyrin–ASC interface\", \"Endogenous physiological context not tested\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined ASC's bipartite logic—PYD as oligomerization domain, CARD as effector—showing full-length ASC oligomerizes procaspase-1 via CARD–CARD contacts to process pro-IL-1\\u03b2, answering how an adaptor converts sensing into caspase activation.\",\n      \"evidence\": \"Co-IP, FKBP-inducible oligomerization domain swap, transfection and THP-1 siRNA\",\n      \"pmids\": [\"11967258\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve filament/speck ultrastructure\", \"Sensor specificity upstream not addressed\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Revealed an inflammasome-independent role for ASC as a Bax adaptor in p53-dependent mitochondrial apoptosis, expanding ASC function beyond IL-1\\u03b2 maturation.\",\n      \"evidence\": \"Reciprocal Co-IP, cytochrome c release, Bax-null cells, siRNA in cell lines\",\n      \"pmids\": [\"14730312\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Domain requirement for Bax interaction not mapped\", \"Relationship to ASC speck formation unclear\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Showed ASC routes caspase-1 toward IL-1\\u03b2 processing and away from RIP2/NF-\\u03baB signaling, establishing ASC as a switch between inflammatory outputs.\",\n      \"evidence\": \"NF-\\u03baB reporter, Co-IP, siRNA, caspase-1-knockout macrophages\",\n      \"pmids\": [\"16585594\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of caspase-1 partitioning between partners not structurally defined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identified AIM2 as a cytosolic dsDNA sensor that recruits ASC via PYD–PYD to assemble a functional caspase-1 inflammasome, broadening the repertoire of ASC-coupled sensors.\",\n      \"evidence\": \"Co-IP domain mapping, siRNA, NF-\\u03baB reporter, caspase-1 assay, viral infection\",\n      \"pmids\": [\"19158675\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of AIM2–ASC assembly not resolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrated an inflammasome-independent ASC function in MAPK/ERK signaling through suppression of DUSP10, using genetic epistasis to separate it from NLRP3/NLRC4/caspase-1.\",\n      \"evidence\": \"shRNA, microarray network analysis, MAPK phosphorylation, multiple knockout macrophages\",\n      \"pmids\": [\"21487011\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct ASC–DUSP10 interaction not demonstrated\", \"Domain requirement unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Discovered that released extracellular ASC specks propagate inflammation in a prionoid fashion and act as autoantigens, redefining the speck as an intercellular danger signal.\",\n      \"evidence\": \"Confocal imaging, cell-free IL-1\\u03b2 maturation, phagocytosis/lysosomal damage assays, patient autoantibody detection\",\n      \"pmids\": [\"24952505\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Determinants of speck stability in the extracellular milieu unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined spatial regulation of ASC by IKK\\u03b1/IKKi-controlled nuclear-to-perinuclear translocation with PP2A relief, explaining how resting macrophages restrain inflammasome assembly.\",\n      \"evidence\": \"Co-IP, subcellular fractionation, kinase-dead mutants, PP2A recruitment, macrophage assays\",\n      \"pmids\": [\"25266676\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phosphosites on ASC mediating retention not mapped\", \"Single lab\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Solved the ASC filament architecture—rigid PYD core with mobile CARD—and validated PYD–PYD interfaces functionally, providing the structural basis for speck assembly.\",\n      \"evidence\": \"Cryo-EM, solid-state NMR, structure-based mutagenesis in macrophages (mouse)\",\n      \"pmids\": [\"26464513\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"CARD cross-linking geometry not resolved at the same resolution\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed ASC-PYD nucleates procaspase-8 tandem-DED filaments via a heterotypic death-fold interaction overlapping the self-association surface, mechanistically coupling ASC to the apoptotic caspase-8 arm.\",\n      \"evidence\": \"In vitro filament nucleation, in vivo co-localization, ASC-PYD mutagenesis, Co-IP\",\n      \"pmids\": [\"26468282\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Competition between caspase-8 and self-association in cells not quantified\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Dissected speck assembly into PYD filament formation followed by CARD cross-linking and showed speck formation amplifies IL-1\\u03b2 processing but is dispensable for gasdermin-D cleavage/pyroptosis, defining ASC oligomerization as a signal amplifier.\",\n      \"evidence\": \"Systematic PYD/CARD mutagenesis in primary macrophages, IL-1\\u03b2, gasdermin-D, pyroptosis readouts\",\n      \"pmids\": [\"27329339\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why pyroptosis bypasses speck requirement not fully explained\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Quantified NLRP3-PYD/ASC-PYD binding showing higher affinity for the sensor than for self-association, clarifying how NLRP3 seeds ASC fibril nucleation.\",\n      \"evidence\": \"Solution NMR, analytical ultracentrifugation, structural modeling\",\n      \"pmids\": [\"27432880\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In-cell relevance of measured affinities not tested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified a T cell-intrinsic ASC-NLRP3-caspase-8 inflammasome that sustains TH17 survival via autocrine IL-1\\u03b2, extending ASC function to adaptive immunity in autoimmunity.\",\n      \"evidence\": \"T cell-specific ASC KO, caspase-8/IL-1\\u03b2 assays, TCR/ATP stimulation, EAE model\",\n      \"pmids\": [\"26998763\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Caspase-8 vs caspase-1 dependence in T cells not fully separated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Imaged endogenous ASC speck assembly, pyroptosis, and clearance in live zebrafish and resolved in vivo speck ultrastructure as intercrossed filament networks, validating filament-based assembly under native conditions.\",\n      \"evidence\": \"CRISPR endogenous tagging, live imaging, CLEM, 3D reconstruction (zebrafish)\",\n      \"pmids\": [\"28701426\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative kinetics of human speck assembly not directly transferable\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified USP50 as a K63-deubiquitinase of ASC required for NLRP3-driven caspase-1 cleavage and speck formation, establishing ubiquitin editing of ASC as a checkpoint.\",\n      \"evidence\": \"Co-IP, K63-DUB assay, USP50 siRNA, caspase-1/IL-1\\u03b2/speck readouts in THP-1 and BMDM\",\n      \"pmids\": [\"28094437\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitination site on ASC not mapped in this study\", \"Apparent context-dependent role versus later degradation findings\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Used an unbiased CRISPR screen and gene-targeted mice to show NLRC4 drives an ASC- and caspase-8-dependent apoptotic program distinct from caspase-1 pyroptosis, formalizing ASC's apoptotic arm.\",\n      \"evidence\": \"Genome-wide CRISPR screen, ASC/caspase-8 KO mice, caspase-1 catalytic-dead knock-in\",\n      \"pmids\": [\"29491424\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular trigger choosing apoptosis vs pyroptosis not defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Determined crystal structures of zebrafish ASC PYD and CARD and revealed a PYD–PYD caspase recruitment mode in fish, providing evolutionary contrast to mammalian CARD–CARD recruitment.\",\n      \"evidence\": \"X-ray crystallography, structure-guided mutagenesis, Co-IP of zASC–Caspy (zebrafish)\",\n      \"pmids\": [\"29791979\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mammalian PYD surface usage not directly compared functionally here\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealed an inflammasome-independent ASC function stabilizing HIF-1\\u03b1 by reducing its hydroxylation and ubiquitination, linking ASC to tumor cell migration and invasion.\",\n      \"evidence\": \"Co-IP, fractionation, HIF-1\\u03b1 hydroxylation/ubiquitination assays, RNA-seq, invasion assays in OSCC\",\n      \"pmids\": [\"32883954\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct enzymatic mechanism by which ASC blocks hydroxylation unclear\", \"Single lab/tumor context\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrated via genetic epistasis that ASC, but not NLRP3 or caspase-1, is required for granuloma formation and host protection in chronic M. tuberculosis infection, cementing an inflammasome-independent in vivo role.\",\n      \"evidence\": \"Aerosol infection of Pycard-/-, Nlrp3-/-, Casp1-/- mice; survival, burden, histology\",\n      \"pmids\": [\"20808838\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular effector pathway downstream of ASC in granuloma formation unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed cAbl phosphorylates ASC at Y146 to enable oligomerization and inflammasome activation, defining a positive tyrosine-phosphorylation input to assembly.\",\n      \"evidence\": \"CRISPR cABL KO, Y146A complementation, phospho/oligomerization/IL-1\\u03b2 assays in THP-1\",\n      \"pmids\": [\"33568399\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconciliation with required dephosphorylation at other tyrosines not addressed here\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified Peli1-mediated K63 ubiquitination of ASC at K55 as a selective promoter of NLRP3 (but not AIM2/NLRP1/NLRC4) inflammasome activation, mapping a sensor-specific ubiquitin switch.\",\n      \"evidence\": \"Co-IP, K63/K55-mapping mutagenesis, Peli1 KO cells and mice, peritonitis model\",\n      \"pmids\": [\"34706239\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why the modification is NLRP3-selective not mechanistically explained\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed a 3'UTR SNP controls Pycard mRNA stability and ASC protein dosage to set inflammasome activity, establishing post-transcriptional regulation of ASC abundance.\",\n      \"evidence\": \"QTL mapping, mRNA stability, CRISPR single-base 3'UTR editing in mouse macrophages\",\n      \"pmids\": [\"34197316\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"trans-acting factor binding the SNP region not identified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified USP3 as a K48-deubiquitinase that stabilizes ASC against proteasomal degradation, defining abundance control as a determinant of inflammasome output in vivo.\",\n      \"evidence\": \"Co-IP, K48-DUB assay, proteasome inhibition, USP3-deficient mouse models\",\n      \"pmids\": [\"36050480\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E3 ligase opposing USP3 on K48 chains not identified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed the ASCb splice isoform, differing only in PYD–CARD linker length, self-assembles more slowly into less compact structures, explaining isoform-specific reduction in inflammasome activity.\",\n      \"evidence\": \"Real-time NMR, DLS, SEC, TEM, docking with procaspase-1 CARD\",\n      \"pmids\": [\"35007535\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological abundance and regulation of ASCb in tissues not quantified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Resolved ASC puncta in NLRP3-activated cells as branched PYD-cored filaments permeated by ribosomes and vesicles, providing near-native cellular context for speck architecture.\",\n      \"evidence\": \"Correlative cryo-light microscopy and cryo-electron tomography\",\n      \"pmids\": [\"37945612\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of organelle entrapment in the filament network unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed low intracellular K+ structurally exposes the ASC CARD to enhance procaspase-1 recruitment independently of NLRP3, linking the canonical K+-efflux trigger directly to ASC conformation.\",\n      \"evidence\": \"ASC oligomerization under varying K+, CARD accessibility and caspase-1 recruitment assays\",\n      \"pmids\": [\"37402211\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural model of the K+-sensitive conformational change not solved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed ZBTB16-promoted SUMOylation of ASC controls inflammasome assembly, and its ablation mitigates Muckle-Wells pathology, adding SUMO as a regulatory modification.\",\n      \"evidence\": \"SUMOylation assay, ZBTB16 KO in Muckle-Wells mouse model\",\n      \"pmids\": [\"38123560\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Exact SUMO site on ASC not defined\", \"Direct vs indirect ZBTB16 effect unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated that K63 ubiquitination of ASC at K158/161 (driven pharmacologically via USP50 inhibition) targets the AIM2-ASC complex for SQSTM1/p62-mediated selective autophagy, defining an autophagic route for inflammasome termination.\",\n      \"evidence\": \"Site-specific ubiquitination, DARTS drug-target binding, Co-IP, autophagy flux, p62 siRNA, tendinopathy model\",\n      \"pmids\": [\"37647255\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological (non-pharmacological) trigger of this pathway not defined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Linked stroma-induced PYCARD expression to TNF-\\u03b1-dependent apoptosis in hematopoietic cells, an early correlative hint of microenvironment-regulated ASC function.\",\n      \"evidence\": \"Expression profiling, miRNA knockdown, caspase-3/apoptosis assays in co-culture\",\n      \"pmids\": [\"18945969\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single knockdown method with correlative expression data; mechanism not established\", \"No direct ASC interaction partner identified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the many competing post-translational inputs (K63/K48 ubiquitination, tyrosine phosphorylation/dephosphorylation, SUMOylation) are integrated in time and space to license a single speck per cell, and how inflammasome-independent ASC functions are mechanistically partitioned from the canonical pathway, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model coupling modification state to assembly kinetics\", \"Structural basis of CARD cross-linking in the speck incomplete\", \"Mechanistic effectors of inflammasome-independent roles (granuloma, HIF-1\\u03b1, MAPK) undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 2, 4, 11]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [7, 8, 20, 21]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [9, 10, 25]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 6, 21]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6, 25]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 3, 8, 15]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [2, 4, 27]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [19, 10]}\n    ],\n    \"complexes\": [\"NLRP3 inflammasome\", \"AIM2 inflammasome\", \"NLRC4 inflammasome\", \"ASC speck/filament\"],\n    \"partners\": [\"NLRP3\", \"AIM2\", \"CASP1\", \"CASP8\", \"BAX\", \"MEFV\", \"Peli1\", \"USP3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}