{"gene":"GSDME","run_date":"2026-04-28T18:06:53","timeline":{"discoveries":[{"year":2017,"finding":"Caspase-3 cleaves GSDME (DFNA5) after Asp270 to generate a GSDME-N fragment that targets the plasma membrane and induces secondary necrosis/pyroptosis; cells lacking GSDME instead disassemble into apoptotic bodies.","method":"In vitro caspase cleavage assay, site-directed mutagenesis, GSDME knockout cells, plasma membrane targeting assay, flow cytometry","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1-2 — identified cleavage site by mutagenesis, reconstituted membrane targeting, orthogonal phenotypic readout in KO cells; highly replicated foundational paper","pmids":["28045099"],"is_preprint":false},{"year":2004,"finding":"Mutant GSDME (exon-8-skipped truncation) acts via a gain-of-function mechanism to induce necrotic cell death when transfected into mammalian cells, whereas wild-type GSDME does not cause equivalent cell death.","method":"Transfection of GFP-tagged wild-type vs. mutant DFNA5 in HEK293T and COS-1 cells; flow cytometry and fluorescence microscopy for cell death quantification","journal":"Journal of Medical Genetics","confidence":"Medium","confidence_rationale":"Tier 2 — clean gain-of-function transfection experiment with quantified cell death; single lab but orthogonal readouts","pmids":["15173223"],"is_preprint":false},{"year":2006,"finding":"GSDME is a transcriptional target of p53; p53 binds a response element in intron 1 of the DFNA5 gene and drives its expression upon genotoxic stress, and ectopic GSDME enhances etoposide-induced cell death in a p53-dependent manner.","method":"Chromatin immunoprecipitation (ChIP), reporter gene assay, p53-null vs. wild-type mouse colon, ectopic expression with etoposide treatment","journal":"Journal of Human Genetics","confidence":"High","confidence_rationale":"Tier 1-2 — ChIP identified p53 binding site, reporter assay confirmed transcriptional activity, in vivo p53+/+ vs p53-/- validation; multiple orthogonal methods","pmids":["16897187"],"is_preprint":false},{"year":2020,"finding":"GSDME-C domain is palmitoylated during chemotherapy-induced pyroptosis; palmitoylation is catalyzed by ZDHHC-2, -7, -11, and -15; 2-bromopalmitate inhibits GSDME-C palmitoylation and promotes interaction between GSDME-C and GSDME-N, blocking pyroptosis; mutation of palmitoylation sites on GSDME also diminishes pyroptosis.","method":"Palmitoylation assay, site-directed mutagenesis of palmitoylation sites, 2-BP inhibitor treatment, Co-IP between GSDME-C and GSDME-N, GSDME knockdown","journal":"Cell Death & Disease","confidence":"High","confidence_rationale":"Tier 1-2 — biochemical palmitoylation assay, mutagenesis of sites, inhibitor rescue, and Co-IP interaction data; multiple orthogonal methods in single study","pmids":["32332857"],"is_preprint":false},{"year":2020,"finding":"STAT3 directly correlates with and positively regulates GSDME expression in macrophages during atherosclerosis.","method":"ChIP/promoter analysis, STAT3 knockdown, GSDME-/-/ApoE-/- double-knockout mouse model, ox-LDL treatment of macrophages","journal":"Nature Communications","confidence":"Medium","confidence_rationale":"Tier 2 — genetic KO model combined with in vitro mechanistic data; single lab but multiple approaches","pmids":["36807553"],"is_preprint":false},{"year":2022,"finding":"OTUD4 (ovarian tumor family deubiquitinase 4) deubiquitinates and stabilizes GSDME, enhancing NPC radiosensitivity by promoting caspase-3-mediated GSDME cleavage and pyroptosis; low GSDME expression confers radioresistance.","method":"Immunoprecipitation, mass spectrometry, ubiquitination assay, OTUD4 overexpression/knockdown, in vitro and in vivo radiosensitivity assays","journal":"Journal of Experimental & Clinical Cancer Research","confidence":"High","confidence_rationale":"Tier 1-2 — reciprocal IP + MS identified the deubiquitinase, functional ubiquitination assay, in vivo validation; multiple orthogonal methods","pmids":["36411454"],"is_preprint":false},{"year":2023,"finding":"CDC20 (E3 ligase component) targets GSDME for ubiquitination-mediated proteasomal degradation in a degron-dependent manner; CDC20 knockdown increases GSDME abundance and switches cell death from apoptosis to pyroptosis.","method":"Ubiquitination assay, immunoprecipitation, cycloheximide chase, CDC20 knockdown/overexpression, syngeneic murine models","journal":"Experimental Hematology & Oncology","confidence":"High","confidence_rationale":"Tier 1-2 — biochemical ubiquitination assay, IP, protein stability assay, in vivo confirmation; multiple orthogonal methods","pmids":["37528490"],"is_preprint":false},{"year":2023,"finding":"Mannose metabolism generates the metabolite GlcNAc-6P which binds AMPK and facilitates its phosphorylation by LKB1; activated AMPK then phosphorylates GSDME at Thr6, blocking caspase-3-induced cleavage and thereby suppressing pyroptosis.","method":"AMPK knockout and GSDME knock-in (T6E and T6A) mice, metabolite binding assay, AMPK phosphorylation assay, in vitro caspase-3 cleavage assay, patient clinical data","journal":"Cell Research","confidence":"High","confidence_rationale":"Tier 1 — identified phosphorylation site by mutagenesis (T6E/T6A knock-in mice), metabolite-kinase binding assay, in vitro cleavage protection; replicated in vivo","pmids":["37460805"],"is_preprint":false},{"year":2024,"finding":"Full-length GSDME (without proteolytic cleavage) can execute pyroptosis via a cleavage-independent mechanism: intense UV-C-induced DNA damage activates PARP1 to generate PAR polymers, which are released to the cytoplasm and activate PARP5 to PARylate GSDME; PARylated GSDME undergoes conformational change relieving autoinhibition; concurrent cytochrome c-catalysed cardiolipin peroxidation generates lipid ROS sensed by PARylated GSDME, driving oxidative oligomerization and plasma membrane targeting of FL-GSDME.","method":"UV-C irradiation, PARP1/PARP5 inhibitors and knockdown, PAR polymer detection, GSDME PARylation assay, lipid ROS measurement, cardiolipin peroxidation assay, confocal membrane targeting, pyroptosis readouts","journal":"Nature Cell Biology","confidence":"High","confidence_rationale":"Tier 1 — multiple biochemical assays reconstituting a novel mechanism, PARP inhibitor rescue, PAR-GSDME interaction, lipid ROS-GSDME link, all within single rigorous study","pmids":["38997456"],"is_preprint":false},{"year":2024,"finding":"Sp1 (Specificity Protein 1) transcription factor directly binds the GSDME promoter at the -36 to -28 site and promotes GSDME gene transcription; this effect synergizes with STAT3 activity and is antagonized by DNA methylation.","method":"ChIP assay, promoter luciferase reporter assay, Sp1 knockdown/inhibition, rescue experiments with chemotherapy drugs","journal":"Cell Death & Disease","confidence":"High","confidence_rationale":"Tier 1-2 — ChIP identified binding site, reporter assay confirmed functional transcription, Sp1 KD phenocopy; multiple orthogonal methods","pmids":["38238307"],"is_preprint":false},{"year":2021,"finding":"EMT-activating transcription factors ZEB1 and ZEB2 directly bind the GSDME promoter to drive its transcriptional activation; GSDME levels positively correlate with EMT gene signatures across cancers and can be reversed when EMT is reverted.","method":"ChIP assay, ZEB1/2 knockdown, EMT induction/reversion models, bioinformatics correlation analysis","journal":"Frontiers in Cell and Developmental Biology","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP confirmed binding, functional KD with phenotypic readout; single lab","pmids":["34901025"],"is_preprint":false},{"year":2024,"finding":"circPDIA3 directly binds the GSDME-C domain and blocks its palmitoylation by ZDHHC3 and ZDHHC17, thereby enhancing the autoinhibitory effect of GSDME-C on GSDME-N and suppressing pyroptosis to promote chemoresistance in colorectal cancer.","method":"RIP, RNA pulldown, Co-IP, palmitoylation assay, ZDHHC3/17 knockdown, in vivo PDX models","journal":"Drug Resistance Updates","confidence":"High","confidence_rationale":"Tier 1-2 — biochemical RIP/pulldown confirmed RNA-protein interaction, palmitoylation assay with ZDHHC identification, Co-IP, in vivo PDX validation; multiple orthogonal methods","pmids":["38861804"],"is_preprint":false},{"year":2023,"finding":"GSDME-N fragment overexpressed in multiple myeloma cells can penetrate mitochondrial membranes and trigger cytochrome c release, activating caspase-3/9, establishing a forward amplification loop; BAX acts upstream to promote GSDME-dependent pyroptosis via the mitochondrial pathway.","method":"GSDME-N overexpression, cytochrome c release assay, mitochondrial fractionation, BAX overexpression, Bcl-2/BAX interaction by IP, GSDME KO rescue","journal":"Acta Pharmacologica Sinica","confidence":"Medium","confidence_rationale":"Tier 2 — mitochondrial fractionation, IP of Bcl-2/BAX disruption, GSDME-N overexpression with cytochrome c readout; single lab","pmids":["36807412"],"is_preprint":false},{"year":2024,"finding":"In platelets, caspase-3 cleaves GSDME to release GSDME-N; flotillin-2 (a scaffold protein) interacts with GSDME-N and recruits it to the platelet plasma membrane, forming pores that facilitate granule release and platelet hyperactivity.","method":"GSDME-knockout mice, Co-IP identifying flotillin-2 as GSDME-N interactor, caspase-3 cleavage assay, platelet activation assays, cisplatin-treated murine model","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1-2 — GSDME KO mouse, Co-IP identifying membrane recruitment partner, in vitro cleavage assay, in vivo chemotherapy model; multiple orthogonal methods","pmids":["39378585"],"is_preprint":false},{"year":2021,"finding":"GSDME-mediated plasma membrane permeabilization during secondary necrosis is size-selective: GSDME accelerates cell lysis (SYTOX Blue influx) and mediates molecular-weight-dependent dextran influx, but phosphatidylserine exposure on the plasma membrane is independent of GSDME.","method":"GSDME KO L929sAhFas cells, dextran influx/efflux assay with different molecular weight probes, SYTOX Blue staining, annexin V staining","journal":"Cellular and Molecular Life Sciences","confidence":"Medium","confidence_rationale":"Tier 2 — GSDME KO cells with quantitative pore-size characterization using dextrans of different sizes; single lab","pmids":["34971436"],"is_preprint":false},{"year":2024,"finding":"The non-N-terminal fragment of GSDME within macrophages interacts with PDPK1, activating the PI3K-AKT pathway to facilitate M2-like macrophage polarization; inhibition of PDPK1 (by Eliprodil) blocks this GSDME-driven immunosuppressive effect.","method":"Co-IP (GSDME-C with PDPK1), flow cytometry (M2 macrophage proportion), GSDME KO in nontumor cells, single-cell sequencing, Eliprodil treatment in HCC mouse models","journal":"Cellular & Molecular Immunology","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP identifying GSDME-PDPK1 interaction, GSDME KO functional rescue, in vivo validation; single lab","pmids":["39496854"],"is_preprint":false},{"year":2026,"finding":"ALKBH4 inhibits GSDME expression at the transcriptional level by reducing H3K4me3 histone modification at the GSDME promoter region, thereby suppressing GSDME-mediated pyroptosis and decreasing sensitivity to 5-FU in gastric cancer.","method":"ChIP for H3K4me3, ALKBH4 knockdown/overexpression, GSDME promoter activity assay, 5-FU sensitivity assay","journal":"Cell Death & Disease","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP confirmed epigenetic mechanism, functional rescue experiment; single lab","pmids":["38902235"],"is_preprint":false},{"year":2020,"finding":"In caspase-1/11-deficient macrophages, NLRP3 inflammasome activation drives caspase-8 activation through ASC, which then cleaves GSDME to induce an 'incomplete pyroptosis' characterized by IL-1α but not IL-1β release (unprocessed pro-IL-1β is retained inside the pyroptotic cell in a molecular complex).","method":"Caspase-1/11 double-KO macrophages, ASC knockdown, caspase-8 inhibitor, GSDME knockdown, IL-1α/IL-1β ELISA, VX765 (caspase-1 pharmacological inhibitor)","journal":"iScience","confidence":"High","confidence_rationale":"Tier 2 — genetic KO and pharmacological inhibitor data converge, specific cytokine release phenotype linked to GSDME cleavage; multiple orthogonal tools","pmids":["32361594"],"is_preprint":false},{"year":2023,"finding":"Amphioxus GSDME (BbGSDME) is cleaved by distinct caspase homologs to yield functionally distinct N-terminal fragments (N253 and N304): N253 binds cell membrane, triggers pyroptosis, and inhibits bacterial growth; N304 negatively regulates N253-mediated cell death; evolutionarily conserved amino acids are important for both BbGSDME and human GSDME function.","method":"Caspase cleavage assay, membrane binding assay, bacterial growth inhibition assay, mutational analysis of conserved residues, amphioxus in vivo infection model","journal":"PLoS Biology","confidence":"Medium","confidence_rationale":"Tier 1-2 — in vitro caspase cleavage, membrane binding reconstitution, mutagenesis; ortholog study with direct relevance to HsGSDME mechanism","pmids":["37134086"],"is_preprint":false},{"year":2024,"finding":"In a STAT1-dependent manner, IFNγ-induced STAT1 activation drives GSDME expression; cytotoxic lymphocyte-derived granzyme B or caspase-3 then cleaves GSDME to trigger pyroptosis; GSDME deletion abolishes the antitumor efficacy of HDAC inhibitor + anti-PD1 combination, demonstrating a self-reinforcing STAT1-GSDME pyroptotic circuitry.","method":"GSDME knockout (genetic), STAT1 knockout (genetic), chromatin immunoprecipitation-seq, single-cell multiomics, HDAC inhibitor treatment, co-culture systems, orthotopic HCC mouse models","journal":"Gut","confidence":"High","confidence_rationale":"Tier 1-2 — ChIP-seq confirming chromatin accessibility, double KO experiments, in vivo orthotopic models; multiple orthogonal approaches","pmids":["39486886"],"is_preprint":false},{"year":2023,"finding":"GZMB (granzyme B) cleaves caspase-3 to activate GSDME-mediated pyroptosis in RA synovial fibroblasts; GZMB silencing reduces GSDME cleavage and pyroptosis markers (LDH, IL-1β, IL-18).","method":"GZMB siRNA knockdown in HFLS-RA and MH7A cells, Western blot for caspase-3 and GSDME cleavage, LDH assay, ELISA","journal":"Molecular Immunology","confidence":"Medium","confidence_rationale":"Tier 2 — KD with specific phenotypic readout linking GZMB-caspase-3-GSDME; single lab","pmids":["37531918"],"is_preprint":false}],"current_model":"GSDME is a gasdermin-family pore-forming protein that is canonically activated by caspase-3 cleaving after Asp270 to release a cytotoxic N-terminal domain (GSDME-N) that inserts into the plasma membrane and executes pyroptosis (secondary necrosis); this switch from apoptosis to pyroptosis depends on GSDME expression level and is regulated at the transcriptional level by p53, Sp1, ZEB1/2, and STAT1/3, and post-translationally by AMPK-mediated phosphorylation at Thr6 (inhibitory), CDC20-mediated ubiquitination (degradation), OTUD4-mediated deubiquitination (stabilization), and ZDHHC-mediated palmitoylation of GSDME-C (modulating autoinhibition); additionally, GSDME can be activated in a cleavage-independent manner through PARP1/PARP5-mediated PARylation coupled with lipid ROS-driven oxidative oligomerization, and its N-terminal fragment can penetrate mitochondrial membranes to amplify cytochrome c release in a feed-forward loop."},"narrative":{"teleology":[{"year":2004,"claim":"The first indication that GSDME harbors intrinsic cytotoxic capacity came from showing that a disease-linked truncation mutant (exon-8 skip) induces necrotic cell death in a gain-of-function manner, whereas the full-length protein is autoinhibited.","evidence":"Transfection of GFP-tagged wild-type versus exon-8-skipped GSDME in HEK293T/COS-1 cells with flow cytometry and microscopy readouts","pmids":["15173223"],"confidence":"Medium","gaps":["Mechanism of autoinhibition by C-terminal domain not yet defined","No identification of the cleavage site or activating protease"]},{"year":2006,"claim":"Establishing that p53 directly transactivates GSDME via an intronic response element linked GSDME expression to the DNA damage response and explained why genotoxic stress potentiates GSDME-dependent cell death.","evidence":"ChIP for p53 binding to intron 1, promoter-reporter assay, p53+/+ versus p53−/− mouse colon, etoposide treatment","pmids":["16897187"],"confidence":"High","gaps":["Whether other transcription factors cooperate with p53 at the GSDME locus was unknown","The downstream effector mechanism (pore formation) had not been identified"]},{"year":2017,"claim":"The central activating mechanism was resolved: caspase-3 cleaves GSDME after Asp270 to liberate a pore-forming N-terminal fragment that targets the plasma membrane and switches apoptosis to pyroptosis — establishing GSDME as a gasdermin-family executioner.","evidence":"In vitro caspase cleavage, D270A mutagenesis, GSDME-KO cells showing apoptotic body formation instead of pyroptosis, plasma membrane targeting assay","pmids":["28045099"],"confidence":"High","gaps":["Whether caspases other than caspase-3 can activate GSDME","Pore structure and stoichiometry undefined","Role of the C-terminal domain in autoinhibition not mechanistically dissected"]},{"year":2020,"claim":"Post-translational palmitoylation of the GSDME C-terminal domain by ZDHHC-family enzymes was shown to relieve autoinhibition by weakening the GSDME-C/GSDME-N intramolecular interaction, thereby licensing pyroptosis upon cleavage.","evidence":"Palmitoylation assay, 2-bromopalmitate inhibitor treatment, Co-IP of GSDME-C with GSDME-N, mutagenesis of palmitoylation sites","pmids":["32332857"],"confidence":"High","gaps":["Identity of which specific palmitoylation sites are critical was incompletely resolved","Whether palmitoylation affects membrane targeting of GSDME-N directly"]},{"year":2020,"claim":"In inflammasome contexts lacking caspase-1/11, an alternative route to GSDME activation was identified: ASC-dependent caspase-8 cleaves GSDME to execute 'incomplete pyroptosis' with IL-1α but not IL-1β release, broadening the upstream activators of GSDME.","evidence":"Caspase-1/11 double-KO macrophages, ASC knockdown, caspase-8 inhibitor, GSDME knockdown, cytokine ELISA","pmids":["32361594"],"confidence":"High","gaps":["Whether granzyme B can also directly cleave GSDME independent of caspase-3 in vivo","Structural basis for differential cytokine trapping"]},{"year":2021,"claim":"Characterization of GSDME pore selectivity showed that membrane permeabilization is size-dependent, accelerating lysis and molecular influx in a graded manner, while phosphatidylserine exposure is GSDME-independent — separating pore formation from apoptotic signaling.","evidence":"GSDME-KO L929 cells, dextran influx assays with graded molecular weight probes, SYTOX Blue, annexin V","pmids":["34971436"],"confidence":"Medium","gaps":["Pore diameter and stoichiometry not structurally resolved","Single cell line used"]},{"year":2021,"claim":"EMT transcription factors ZEB1/ZEB2 were identified as direct transcriptional activators of GSDME, linking epithelial-mesenchymal transition status to pyroptotic competence across cancer types.","evidence":"ChIP for ZEB1/ZEB2 at GSDME promoter, ZEB1/2 knockdown, EMT induction/reversion models","pmids":["34901025"],"confidence":"Medium","gaps":["Single-lab finding without independent replication","Whether ZEB-driven GSDME upregulation occurs in normal developmental EMT"]},{"year":2022,"claim":"GSDME protein stability was shown to be controlled by OTUD4-mediated deubiquitination, which stabilizes GSDME and enhances caspase-3-dependent pyroptosis — revealing ubiquitin-dependent turnover as a regulatory axis.","evidence":"Reciprocal IP and mass spectrometry identifying OTUD4, ubiquitination assays, OTUD4 overexpression/knockdown, in vivo radiosensitivity in NPC models","pmids":["36411454"],"confidence":"High","gaps":["Identity of the E3 ligase opposing OTUD4 was not defined in this study","Specific ubiquitin chain type on GSDME not characterized"]},{"year":2023,"claim":"CDC20 was identified as the E3 ligase component targeting GSDME for ubiquitin-dependent proteasomal degradation; CDC20 depletion shifts the apoptosis-pyroptosis balance by stabilizing GSDME protein.","evidence":"Ubiquitination assay, cycloheximide chase, CDC20 knockdown/overexpression, syngeneic murine tumor models","pmids":["37528490"],"confidence":"High","gaps":["Whether CDC20 and OTUD4 compete at the same ubiquitin sites on GSDME","Degron motif on GSDME not fully mapped"]},{"year":2023,"claim":"A mitochondrial feed-forward loop was established: GSDME-N penetrates mitochondrial membranes to release cytochrome c, which activates caspase-9/3 to generate more GSDME-N, amplifying pyroptosis; BAX acts upstream to initiate this cascade.","evidence":"GSDME-N overexpression, mitochondrial fractionation, cytochrome c release assay, BAX overexpression, Bcl-2/BAX IP in multiple myeloma cells","pmids":["36807412"],"confidence":"Medium","gaps":["Single-lab finding in one cancer type","Structural basis for GSDME-N insertion into mitochondrial versus plasma membranes unknown"]},{"year":2023,"claim":"Evolutionary conservation of the gasdermin pore mechanism was demonstrated through amphioxus GSDME, showing that distinct caspase cleavage sites generate functionally opposing N-terminal fragments — one pyroptotic and bactericidal, the other inhibitory — and that key residues are functionally conserved in human GSDME.","evidence":"Amphioxus caspase cleavage assay, membrane binding, bacterial killing assay, mutagenesis of conserved residues, in vivo infection model","pmids":["37134086"],"confidence":"Medium","gaps":["Whether human GSDME produces an analogous inhibitory fragment","In vivo relevance of dual-fragment mechanism in mammals"]},{"year":2023,"claim":"AMPK-mediated phosphorylation at Thr6 was identified as a metabolically regulated brake on GSDME: mannose-derived GlcNAc-6P activates AMPK via LKB1, and pThr6-GSDME resists caspase-3 cleavage, directly linking cellular metabolism to pyroptotic susceptibility.","evidence":"AMPK-KO and GSDME T6E/T6A knock-in mice, metabolite binding assay, in vitro caspase-3 cleavage, clinical correlation","pmids":["37460805"],"confidence":"High","gaps":["Whether other kinases also phosphorylate GSDME-N","Structural basis for how pThr6 blocks caspase-3 access to Asp270"]},{"year":2024,"claim":"A cleavage-independent activation route was discovered: UV-C-induced DNA damage activates PARP1, generating free PAR polymers that activate cytoplasmic PARP5 to PARylate full-length GSDME; concurrent lipid ROS from cardiolipin peroxidation drive oxidative oligomerization of PARylated GSDME, enabling pore formation without proteolytic processing.","evidence":"PARP1/5 inhibitors and knockdown, PAR polymer detection, GSDME PARylation assay, lipid ROS measurement, cardiolipin peroxidation assay, confocal membrane targeting","pmids":["38997456"],"confidence":"High","gaps":["Whether PARylation-dependent activation occurs under physiological (non-UV-C) stresses","Structural details of PARylated GSDME oligomer","Whether other gasdermins share this non-canonical activation route"]},{"year":2024,"claim":"Sp1 was identified as a direct transcriptional activator binding the GSDME proximal promoter (−36 to −28), synergizing with STAT3 and antagonized by promoter DNA methylation — integrating transcriptional and epigenetic control of GSDME expression.","evidence":"ChIP for Sp1 at GSDME promoter, luciferase reporter, Sp1 knockdown/inhibition, chemotherapy rescue experiments","pmids":["38238307"],"confidence":"High","gaps":["Relative contribution of Sp1 versus p53 and ZEB1/2 in different cell types unresolved","Mechanism of DNA methylation–Sp1 interplay not fully dissected"]},{"year":2024,"claim":"In a non-canonical cell biology context, GSDME-N in platelets is recruited to the plasma membrane by flotillin-2, forming pores that drive granule release and platelet hyperactivation during chemotherapy — extending GSDME function beyond classical pyroptosis to hemostasis.","evidence":"GSDME-KO mice, Co-IP identifying flotillin-2, caspase-3 cleavage assay, platelet activation assays, cisplatin murine model","pmids":["39378585"],"confidence":"High","gaps":["Whether flotillin-2 is required for GSDME-N membrane targeting in nucleated cells","In vivo thrombotic consequences beyond the cisplatin model"]},{"year":2024,"claim":"STAT1 was shown to drive GSDME transcription downstream of IFNγ, linking adaptive immune signaling (cytotoxic lymphocyte granzyme B/caspase-3) to tumor cell pyroptosis and establishing a self-reinforcing STAT1–GSDME circuit required for immunotherapy efficacy.","evidence":"GSDME-KO and STAT1-KO, ChIP-seq, single-cell multiomics, HDAC inhibitor + anti-PD1 in orthotopic HCC models","pmids":["39486886"],"confidence":"High","gaps":["Whether STAT1 and STAT3 compete or cooperate at the GSDME locus under different cytokine contexts","Mechanism by which pyroptosis feeds back to amplify STAT1 signaling"]},{"year":2024,"claim":"circPDIA3 was shown to directly bind the GSDME-C domain and block its palmitoylation by ZDHHC3/17, strengthening autoinhibition and suppressing pyroptosis — revealing circular RNA as a post-translational modulator of gasdermin activation.","evidence":"RIP, RNA pulldown, Co-IP, palmitoylation assay, ZDHHC3/17 knockdown, PDX models in colorectal cancer","pmids":["38861804"],"confidence":"High","gaps":["Whether other circRNAs or RNAs regulate other gasdermin family members","Stoichiometry of circPDIA3–GSDME-C interaction"]},{"year":2024,"claim":"The GSDME C-terminal fragment was found to have a signaling function beyond autoinhibition: interaction with PDPK1 activates PI3K–AKT to drive M2-like macrophage polarization, suggesting GSDME cleavage products have immunomodulatory roles in the tumor microenvironment.","evidence":"Co-IP of GSDME-C with PDPK1, GSDME KO, single-cell sequencing, Eliprodil inhibitor treatment in HCC mouse models","pmids":["39496854"],"confidence":"Medium","gaps":["Single-lab finding; PDPK1 interaction awaits independent confirmation","Whether GSDME-C reaches macrophages in trans or acts cell-autonomously"]},{"year":null,"claim":"Key unresolved questions include the cryo-EM structure of the GSDME pore, the mechanism by which PARylation relieves autoinhibition at the structural level, whether the cleavage-independent activation pathway operates under physiological (non-UV-C) stimuli, and how the opposing ubiquitin ligase (CDC20) and deubiquitinase (OTUD4) activities are coordinated in different tissues.","evidence":"","pmids":[],"confidence":"Low","gaps":["No cryo-EM or high-resolution structure of GSDME pore available","PARylation sites on GSDME not mapped","Physiological relevance of cleavage-independent pathway beyond UV-C not tested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,8,14]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,14]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,13,14]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[12]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[0,7,8,17]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[17,19,20]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[7,15]},{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[13]}],"complexes":[],"partners":["CASP3","OTUD4","CDC20","FLOT2","PDPK1","PARP1","AMPK"],"other_free_text":[]},"mechanistic_narrative":"GSDME is a gasdermin-family pore-forming protein that serves as a molecular switch converting apoptosis into pyroptosis, with broad roles in innate immunity, chemosensitivity, and inflammatory cell death. Caspase-3 cleaves GSDME after Asp270 to release a cytotoxic N-terminal fragment (GSDME-N) that oligomerizes and forms size-selective pores in the plasma membrane, executing secondary necrosis/pyroptosis; in cells lacking GSDME, apoptosis instead proceeds to orderly disassembly into apoptotic bodies [PMID:28045099, PMID:34971436]. GSDME-N also penetrates mitochondrial membranes to trigger cytochrome c release and amplify caspase-3 activation in a feed-forward loop [PMID:36807412], and full-length GSDME can execute pyroptosis independently of cleavage through PARP1/PARP5-mediated PARylation coupled with lipid ROS-driven oxidative oligomerization [PMID:38997456]. GSDME activity is regulated at multiple levels: transcriptionally by p53, Sp1, ZEB1/2, STAT1, and STAT3 [PMID:16897187, PMID:38238307, PMID:34901025, PMID:39486886]; post-translationally by inhibitory AMPK phosphorylation at Thr6 [PMID:37460805], CDC20-mediated ubiquitin-dependent degradation opposed by OTUD4 deubiquitination [PMID:37528490, PMID:36411454], and ZDHHC-catalyzed palmitoylation of the autoinhibitory C-terminal domain that relieves intramolecular suppression [PMID:32332857, PMID:38861804]."},"prefetch_data":{"uniprot":{"accession":"O60443","full_name":"Gasdermin-E","aliases":["Inversely correlated with estrogen receptor expression 1","ICERE-1","Non-syndromic hearing impairment protein 5"],"length_aa":496,"mass_kda":54.6,"function":"Precursor of a pore-forming protein that converts non-inflammatory apoptosis to pyroptosis (PubMed:27281216, PubMed:28459430, PubMed:33852854, PubMed:35594856, PubMed:36607699). This form constitutes the precursor of the pore-forming protein: upon cleavage, the released N-terminal moiety (Gasdermin-E, N-terminal) binds to membranes and forms pores, triggering pyroptosis (PubMed:28459430) Pore-forming protein produced by cleavage by CASP3 or granzyme B (GZMB), which converts non-inflammatory apoptosis to pyroptosis or promotes granzyme-mediated pyroptosis, respectively (PubMed:27281216, PubMed:28459430, PubMed:32188940, PubMed:33852854, PubMed:35594856). After cleavage, moves to the plasma membrane, homooligomerizes within the membrane and forms pores of 10-15 nanometers (nm) of inner diameter, allowing the release of mature interleukins (IL1B and IL16) and triggering pyroptosis (PubMed:28459430, PubMed:32188940, PubMed:33852854, PubMed:35594856). Binds to inner leaflet lipids, bisphosphorylated phosphatidylinositols, such as phosphatidylinositol (4,5)-bisphosphate (PubMed:28459430). Cleavage by CASP3 switches CASP3-mediated apoptosis induced by TNF or danger signals, such as chemotherapy drugs, to pyroptosis (PubMed:27281216, PubMed:28459430, PubMed:32188940). Mediates secondary necrosis downstream of the mitochondrial apoptotic pathway and CASP3 activation as well as in response to viral agents (PubMed:28045099). Exhibits bactericidal activity (PubMed:27281216). Cleavage by GZMB promotes tumor suppressor activity by triggering robust anti-tumor immunity (PubMed:21522185, PubMed:32188940). Suppresses tumors by mediating granzyme-mediated pyroptosis in target cells of natural killer (NK) cells: cleavage by granzyme B (GZMB), delivered to target cells from NK-cells, triggers pyroptosis of tumor cells and tumor suppression (PubMed:31953257, PubMed:32188940). May play a role in the p53/TP53-regulated cellular response to DNA damage (PubMed:16897187) (Microbial infection) Pore-forming protein, which promotes maternal placental pyroptosis in response to Zika virus infection, contributing to adverse fetal outcomes","subcellular_location":"Cytoplasm, cytosol","url":"https://www.uniprot.org/uniprotkb/O60443/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/GSDME","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/GSDME","total_profiled":1310},"omim":[{"mim_id":"608798","title":"GASDERMIN E; GSDME","url":"https://www.omim.org/entry/608798"},{"mim_id":"600994","title":"DEAFNESS, AUTOSOMAL DOMINANT 5; DFNA5","url":"https://www.omim.org/entry/600994"},{"mim_id":"600636","title":"CASPASE 3, APOPTOSIS-RELATED CYSTEINE PROTEASE; 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disease","url":"https://pubmed.ncbi.nlm.nih.gov/38902235","citation_count":17,"is_preprint":false},{"pmid":"36895972","id":"PMC_36895972","title":"Endogenous HMGB1 regulates GSDME-mediated pyroptosis via ROS/ERK1/2/caspase-3/GSDME signaling in neuroblastoma.","date":"2023","source":"American journal of cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/36895972","citation_count":17,"is_preprint":false},{"pmid":"36807412","id":"PMC_36807412","title":"Proteasomal inhibitors induce myeloma cell pyroptosis via the BAX/GSDME pathway.","date":"2023","source":"Acta pharmacologica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/36807412","citation_count":17,"is_preprint":false},{"pmid":"39870303","id":"PMC_39870303","title":"Hypericin photoactivation induces triple-negative breast cancer cells pyroptosis by targeting the ROS/CALR/Caspase-3/GSDME pathway.","date":"2025","source":"Journal of advanced research","url":"https://pubmed.ncbi.nlm.nih.gov/39870303","citation_count":16,"is_preprint":false},{"pmid":"39915005","id":"PMC_39915005","title":"Clofarabine induces tumor cell apoptosis, GSDME-related pyroptosis, and CD8+ T-cell antitumor activity via the non-canonical P53/STING pathway.","date":"2025","source":"Journal for immunotherapy of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/39915005","citation_count":16,"is_preprint":false},{"pmid":"34901025","id":"PMC_34901025","title":"Epithelial-Mesenchymal Transition Induces GSDME Transcriptional Activation for Inflammatory Pyroptosis.","date":"2021","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/34901025","citation_count":16,"is_preprint":false},{"pmid":"38843953","id":"PMC_38843953","title":"Pharmacological inhibition of cGAS ameliorates postoperative cognitive dysfunction by suppressing caspase-3/GSDME-dependent pyroptosis.","date":"2024","source":"Neurochemistry 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immunology","url":"https://pubmed.ncbi.nlm.nih.gov/37531918","citation_count":15,"is_preprint":false},{"pmid":"37134086","id":"PMC_37134086","title":"Characterization of GSDME in amphioxus provides insights into the functional evolution of GSDM-mediated pyroptosis.","date":"2023","source":"PLoS biology","url":"https://pubmed.ncbi.nlm.nih.gov/37134086","citation_count":13,"is_preprint":false},{"pmid":"35038006","id":"PMC_35038006","title":"AudioGene: refining the natural history of KCNQ4, GSDME, WFS1, and COCH-associated hearing loss.","date":"2022","source":"Human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/35038006","citation_count":13,"is_preprint":false},{"pmid":"39378585","id":"PMC_39378585","title":"GSDME-mediated pyroptosis contributes to chemotherapy-induced platelet hyperactivity and thrombotic 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sciences","url":"https://pubmed.ncbi.nlm.nih.gov/32486382","citation_count":12,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":54529,"output_tokens":5404,"usd":0.122324},"stage2":{"model":"claude-opus-4-6","input_tokens":8940,"output_tokens":4697,"usd":0.243188},"total_usd":0.365512,"stage1_batch_id":"msgbatch_01DzrsgVgYngY1hvM3DU1yF2","stage2_batch_id":"msgbatch_01KA9b7iRzKFdhoBUT7vCvSe","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2017,\n      \"finding\": \"Caspase-3 cleaves GSDME (DFNA5) after Asp270 to generate a GSDME-N fragment that targets the plasma membrane and induces secondary necrosis/pyroptosis; cells lacking GSDME instead disassemble into apoptotic bodies.\",\n      \"method\": \"In vitro caspase cleavage assay, site-directed mutagenesis, GSDME knockout cells, plasma membrane targeting assay, flow cytometry\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — identified cleavage site by mutagenesis, reconstituted membrane targeting, orthogonal phenotypic readout in KO cells; highly replicated foundational paper\",\n      \"pmids\": [\"28045099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Mutant GSDME (exon-8-skipped truncation) acts via a gain-of-function mechanism to induce necrotic cell death when transfected into mammalian cells, whereas wild-type GSDME does not cause equivalent cell death.\",\n      \"method\": \"Transfection of GFP-tagged wild-type vs. mutant DFNA5 in HEK293T and COS-1 cells; flow cytometry and fluorescence microscopy for cell death quantification\",\n      \"journal\": \"Journal of Medical Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean gain-of-function transfection experiment with quantified cell death; single lab but orthogonal readouts\",\n      \"pmids\": [\"15173223\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"GSDME is a transcriptional target of p53; p53 binds a response element in intron 1 of the DFNA5 gene and drives its expression upon genotoxic stress, and ectopic GSDME enhances etoposide-induced cell death in a p53-dependent manner.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP), reporter gene assay, p53-null vs. wild-type mouse colon, ectopic expression with etoposide treatment\",\n      \"journal\": \"Journal of Human Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — ChIP identified p53 binding site, reporter assay confirmed transcriptional activity, in vivo p53+/+ vs p53-/- validation; multiple orthogonal methods\",\n      \"pmids\": [\"16897187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GSDME-C domain is palmitoylated during chemotherapy-induced pyroptosis; palmitoylation is catalyzed by ZDHHC-2, -7, -11, and -15; 2-bromopalmitate inhibits GSDME-C palmitoylation and promotes interaction between GSDME-C and GSDME-N, blocking pyroptosis; mutation of palmitoylation sites on GSDME also diminishes pyroptosis.\",\n      \"method\": \"Palmitoylation assay, site-directed mutagenesis of palmitoylation sites, 2-BP inhibitor treatment, Co-IP between GSDME-C and GSDME-N, GSDME knockdown\",\n      \"journal\": \"Cell Death & Disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — biochemical palmitoylation assay, mutagenesis of sites, inhibitor rescue, and Co-IP interaction data; multiple orthogonal methods in single study\",\n      \"pmids\": [\"32332857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"STAT3 directly correlates with and positively regulates GSDME expression in macrophages during atherosclerosis.\",\n      \"method\": \"ChIP/promoter analysis, STAT3 knockdown, GSDME-/-/ApoE-/- double-knockout mouse model, ox-LDL treatment of macrophages\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO model combined with in vitro mechanistic data; single lab but multiple approaches\",\n      \"pmids\": [\"36807553\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"OTUD4 (ovarian tumor family deubiquitinase 4) deubiquitinates and stabilizes GSDME, enhancing NPC radiosensitivity by promoting caspase-3-mediated GSDME cleavage and pyroptosis; low GSDME expression confers radioresistance.\",\n      \"method\": \"Immunoprecipitation, mass spectrometry, ubiquitination assay, OTUD4 overexpression/knockdown, in vitro and in vivo radiosensitivity assays\",\n      \"journal\": \"Journal of Experimental & Clinical Cancer Research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — reciprocal IP + MS identified the deubiquitinase, functional ubiquitination assay, in vivo validation; multiple orthogonal methods\",\n      \"pmids\": [\"36411454\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CDC20 (E3 ligase component) targets GSDME for ubiquitination-mediated proteasomal degradation in a degron-dependent manner; CDC20 knockdown increases GSDME abundance and switches cell death from apoptosis to pyroptosis.\",\n      \"method\": \"Ubiquitination assay, immunoprecipitation, cycloheximide chase, CDC20 knockdown/overexpression, syngeneic murine models\",\n      \"journal\": \"Experimental Hematology & Oncology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — biochemical ubiquitination assay, IP, protein stability assay, in vivo confirmation; multiple orthogonal methods\",\n      \"pmids\": [\"37528490\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Mannose metabolism generates the metabolite GlcNAc-6P which binds AMPK and facilitates its phosphorylation by LKB1; activated AMPK then phosphorylates GSDME at Thr6, blocking caspase-3-induced cleavage and thereby suppressing pyroptosis.\",\n      \"method\": \"AMPK knockout and GSDME knock-in (T6E and T6A) mice, metabolite binding assay, AMPK phosphorylation assay, in vitro caspase-3 cleavage assay, patient clinical data\",\n      \"journal\": \"Cell Research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — identified phosphorylation site by mutagenesis (T6E/T6A knock-in mice), metabolite-kinase binding assay, in vitro cleavage protection; replicated in vivo\",\n      \"pmids\": [\"37460805\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Full-length GSDME (without proteolytic cleavage) can execute pyroptosis via a cleavage-independent mechanism: intense UV-C-induced DNA damage activates PARP1 to generate PAR polymers, which are released to the cytoplasm and activate PARP5 to PARylate GSDME; PARylated GSDME undergoes conformational change relieving autoinhibition; concurrent cytochrome c-catalysed cardiolipin peroxidation generates lipid ROS sensed by PARylated GSDME, driving oxidative oligomerization and plasma membrane targeting of FL-GSDME.\",\n      \"method\": \"UV-C irradiation, PARP1/PARP5 inhibitors and knockdown, PAR polymer detection, GSDME PARylation assay, lipid ROS measurement, cardiolipin peroxidation assay, confocal membrane targeting, pyroptosis readouts\",\n      \"journal\": \"Nature Cell Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple biochemical assays reconstituting a novel mechanism, PARP inhibitor rescue, PAR-GSDME interaction, lipid ROS-GSDME link, all within single rigorous study\",\n      \"pmids\": [\"38997456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Sp1 (Specificity Protein 1) transcription factor directly binds the GSDME promoter at the -36 to -28 site and promotes GSDME gene transcription; this effect synergizes with STAT3 activity and is antagonized by DNA methylation.\",\n      \"method\": \"ChIP assay, promoter luciferase reporter assay, Sp1 knockdown/inhibition, rescue experiments with chemotherapy drugs\",\n      \"journal\": \"Cell Death & Disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — ChIP identified binding site, reporter assay confirmed functional transcription, Sp1 KD phenocopy; multiple orthogonal methods\",\n      \"pmids\": [\"38238307\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"EMT-activating transcription factors ZEB1 and ZEB2 directly bind the GSDME promoter to drive its transcriptional activation; GSDME levels positively correlate with EMT gene signatures across cancers and can be reversed when EMT is reverted.\",\n      \"method\": \"ChIP assay, ZEB1/2 knockdown, EMT induction/reversion models, bioinformatics correlation analysis\",\n      \"journal\": \"Frontiers in Cell and Developmental Biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP confirmed binding, functional KD with phenotypic readout; single lab\",\n      \"pmids\": [\"34901025\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"circPDIA3 directly binds the GSDME-C domain and blocks its palmitoylation by ZDHHC3 and ZDHHC17, thereby enhancing the autoinhibitory effect of GSDME-C on GSDME-N and suppressing pyroptosis to promote chemoresistance in colorectal cancer.\",\n      \"method\": \"RIP, RNA pulldown, Co-IP, palmitoylation assay, ZDHHC3/17 knockdown, in vivo PDX models\",\n      \"journal\": \"Drug Resistance Updates\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — biochemical RIP/pulldown confirmed RNA-protein interaction, palmitoylation assay with ZDHHC identification, Co-IP, in vivo PDX validation; multiple orthogonal methods\",\n      \"pmids\": [\"38861804\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"GSDME-N fragment overexpressed in multiple myeloma cells can penetrate mitochondrial membranes and trigger cytochrome c release, activating caspase-3/9, establishing a forward amplification loop; BAX acts upstream to promote GSDME-dependent pyroptosis via the mitochondrial pathway.\",\n      \"method\": \"GSDME-N overexpression, cytochrome c release assay, mitochondrial fractionation, BAX overexpression, Bcl-2/BAX interaction by IP, GSDME KO rescue\",\n      \"journal\": \"Acta Pharmacologica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mitochondrial fractionation, IP of Bcl-2/BAX disruption, GSDME-N overexpression with cytochrome c readout; single lab\",\n      \"pmids\": [\"36807412\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In platelets, caspase-3 cleaves GSDME to release GSDME-N; flotillin-2 (a scaffold protein) interacts with GSDME-N and recruits it to the platelet plasma membrane, forming pores that facilitate granule release and platelet hyperactivity.\",\n      \"method\": \"GSDME-knockout mice, Co-IP identifying flotillin-2 as GSDME-N interactor, caspase-3 cleavage assay, platelet activation assays, cisplatin-treated murine model\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — GSDME KO mouse, Co-IP identifying membrane recruitment partner, in vitro cleavage assay, in vivo chemotherapy model; multiple orthogonal methods\",\n      \"pmids\": [\"39378585\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"GSDME-mediated plasma membrane permeabilization during secondary necrosis is size-selective: GSDME accelerates cell lysis (SYTOX Blue influx) and mediates molecular-weight-dependent dextran influx, but phosphatidylserine exposure on the plasma membrane is independent of GSDME.\",\n      \"method\": \"GSDME KO L929sAhFas cells, dextran influx/efflux assay with different molecular weight probes, SYTOX Blue staining, annexin V staining\",\n      \"journal\": \"Cellular and Molecular Life Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — GSDME KO cells with quantitative pore-size characterization using dextrans of different sizes; single lab\",\n      \"pmids\": [\"34971436\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The non-N-terminal fragment of GSDME within macrophages interacts with PDPK1, activating the PI3K-AKT pathway to facilitate M2-like macrophage polarization; inhibition of PDPK1 (by Eliprodil) blocks this GSDME-driven immunosuppressive effect.\",\n      \"method\": \"Co-IP (GSDME-C with PDPK1), flow cytometry (M2 macrophage proportion), GSDME KO in nontumor cells, single-cell sequencing, Eliprodil treatment in HCC mouse models\",\n      \"journal\": \"Cellular & Molecular Immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP identifying GSDME-PDPK1 interaction, GSDME KO functional rescue, in vivo validation; single lab\",\n      \"pmids\": [\"39496854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"ALKBH4 inhibits GSDME expression at the transcriptional level by reducing H3K4me3 histone modification at the GSDME promoter region, thereby suppressing GSDME-mediated pyroptosis and decreasing sensitivity to 5-FU in gastric cancer.\",\n      \"method\": \"ChIP for H3K4me3, ALKBH4 knockdown/overexpression, GSDME promoter activity assay, 5-FU sensitivity assay\",\n      \"journal\": \"Cell Death & Disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP confirmed epigenetic mechanism, functional rescue experiment; single lab\",\n      \"pmids\": [\"38902235\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In caspase-1/11-deficient macrophages, NLRP3 inflammasome activation drives caspase-8 activation through ASC, which then cleaves GSDME to induce an 'incomplete pyroptosis' characterized by IL-1α but not IL-1β release (unprocessed pro-IL-1β is retained inside the pyroptotic cell in a molecular complex).\",\n      \"method\": \"Caspase-1/11 double-KO macrophages, ASC knockdown, caspase-8 inhibitor, GSDME knockdown, IL-1α/IL-1β ELISA, VX765 (caspase-1 pharmacological inhibitor)\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO and pharmacological inhibitor data converge, specific cytokine release phenotype linked to GSDME cleavage; multiple orthogonal tools\",\n      \"pmids\": [\"32361594\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Amphioxus GSDME (BbGSDME) is cleaved by distinct caspase homologs to yield functionally distinct N-terminal fragments (N253 and N304): N253 binds cell membrane, triggers pyroptosis, and inhibits bacterial growth; N304 negatively regulates N253-mediated cell death; evolutionarily conserved amino acids are important for both BbGSDME and human GSDME function.\",\n      \"method\": \"Caspase cleavage assay, membrane binding assay, bacterial growth inhibition assay, mutational analysis of conserved residues, amphioxus in vivo infection model\",\n      \"journal\": \"PLoS Biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro caspase cleavage, membrane binding reconstitution, mutagenesis; ortholog study with direct relevance to HsGSDME mechanism\",\n      \"pmids\": [\"37134086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In a STAT1-dependent manner, IFNγ-induced STAT1 activation drives GSDME expression; cytotoxic lymphocyte-derived granzyme B or caspase-3 then cleaves GSDME to trigger pyroptosis; GSDME deletion abolishes the antitumor efficacy of HDAC inhibitor + anti-PD1 combination, demonstrating a self-reinforcing STAT1-GSDME pyroptotic circuitry.\",\n      \"method\": \"GSDME knockout (genetic), STAT1 knockout (genetic), chromatin immunoprecipitation-seq, single-cell multiomics, HDAC inhibitor treatment, co-culture systems, orthotopic HCC mouse models\",\n      \"journal\": \"Gut\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — ChIP-seq confirming chromatin accessibility, double KO experiments, in vivo orthotopic models; multiple orthogonal approaches\",\n      \"pmids\": [\"39486886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"GZMB (granzyme B) cleaves caspase-3 to activate GSDME-mediated pyroptosis in RA synovial fibroblasts; GZMB silencing reduces GSDME cleavage and pyroptosis markers (LDH, IL-1β, IL-18).\",\n      \"method\": \"GZMB siRNA knockdown in HFLS-RA and MH7A cells, Western blot for caspase-3 and GSDME cleavage, LDH assay, ELISA\",\n      \"journal\": \"Molecular Immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KD with specific phenotypic readout linking GZMB-caspase-3-GSDME; single lab\",\n      \"pmids\": [\"37531918\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"GSDME is a gasdermin-family pore-forming protein that is canonically activated by caspase-3 cleaving after Asp270 to release a cytotoxic N-terminal domain (GSDME-N) that inserts into the plasma membrane and executes pyroptosis (secondary necrosis); this switch from apoptosis to pyroptosis depends on GSDME expression level and is regulated at the transcriptional level by p53, Sp1, ZEB1/2, and STAT1/3, and post-translationally by AMPK-mediated phosphorylation at Thr6 (inhibitory), CDC20-mediated ubiquitination (degradation), OTUD4-mediated deubiquitination (stabilization), and ZDHHC-mediated palmitoylation of GSDME-C (modulating autoinhibition); additionally, GSDME can be activated in a cleavage-independent manner through PARP1/PARP5-mediated PARylation coupled with lipid ROS-driven oxidative oligomerization, and its N-terminal fragment can penetrate mitochondrial membranes to amplify cytochrome c release in a feed-forward loop.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"GSDME is a gasdermin-family pore-forming protein that serves as a molecular switch converting apoptosis into pyroptosis, with broad roles in innate immunity, chemosensitivity, and inflammatory cell death. Caspase-3 cleaves GSDME after Asp270 to release a cytotoxic N-terminal fragment (GSDME-N) that oligomerizes and forms size-selective pores in the plasma membrane, executing secondary necrosis/pyroptosis; in cells lacking GSDME, apoptosis instead proceeds to orderly disassembly into apoptotic bodies [PMID:28045099, PMID:34971436]. GSDME-N also penetrates mitochondrial membranes to trigger cytochrome c release and amplify caspase-3 activation in a feed-forward loop [PMID:36807412], and full-length GSDME can execute pyroptosis independently of cleavage through PARP1/PARP5-mediated PARylation coupled with lipid ROS-driven oxidative oligomerization [PMID:38997456]. GSDME activity is regulated at multiple levels: transcriptionally by p53, Sp1, ZEB1/2, STAT1, and STAT3 [PMID:16897187, PMID:38238307, PMID:34901025, PMID:39486886]; post-translationally by inhibitory AMPK phosphorylation at Thr6 [PMID:37460805], CDC20-mediated ubiquitin-dependent degradation opposed by OTUD4 deubiquitination [PMID:37528490, PMID:36411454], and ZDHHC-catalyzed palmitoylation of the autoinhibitory C-terminal domain that relieves intramolecular suppression [PMID:32332857, PMID:38861804].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"The first indication that GSDME harbors intrinsic cytotoxic capacity came from showing that a disease-linked truncation mutant (exon-8 skip) induces necrotic cell death in a gain-of-function manner, whereas the full-length protein is autoinhibited.\",\n      \"evidence\": \"Transfection of GFP-tagged wild-type versus exon-8-skipped GSDME in HEK293T/COS-1 cells with flow cytometry and microscopy readouts\",\n      \"pmids\": [\"15173223\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of autoinhibition by C-terminal domain not yet defined\", \"No identification of the cleavage site or activating protease\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Establishing that p53 directly transactivates GSDME via an intronic response element linked GSDME expression to the DNA damage response and explained why genotoxic stress potentiates GSDME-dependent cell death.\",\n      \"evidence\": \"ChIP for p53 binding to intron 1, promoter-reporter assay, p53+/+ versus p53−/− mouse colon, etoposide treatment\",\n      \"pmids\": [\"16897187\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other transcription factors cooperate with p53 at the GSDME locus was unknown\", \"The downstream effector mechanism (pore formation) had not been identified\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"The central activating mechanism was resolved: caspase-3 cleaves GSDME after Asp270 to liberate a pore-forming N-terminal fragment that targets the plasma membrane and switches apoptosis to pyroptosis — establishing GSDME as a gasdermin-family executioner.\",\n      \"evidence\": \"In vitro caspase cleavage, D270A mutagenesis, GSDME-KO cells showing apoptotic body formation instead of pyroptosis, plasma membrane targeting assay\",\n      \"pmids\": [\"28045099\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether caspases other than caspase-3 can activate GSDME\", \"Pore structure and stoichiometry undefined\", \"Role of the C-terminal domain in autoinhibition not mechanistically dissected\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Post-translational palmitoylation of the GSDME C-terminal domain by ZDHHC-family enzymes was shown to relieve autoinhibition by weakening the GSDME-C/GSDME-N intramolecular interaction, thereby licensing pyroptosis upon cleavage.\",\n      \"evidence\": \"Palmitoylation assay, 2-bromopalmitate inhibitor treatment, Co-IP of GSDME-C with GSDME-N, mutagenesis of palmitoylation sites\",\n      \"pmids\": [\"32332857\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of which specific palmitoylation sites are critical was incompletely resolved\", \"Whether palmitoylation affects membrane targeting of GSDME-N directly\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"In inflammasome contexts lacking caspase-1/11, an alternative route to GSDME activation was identified: ASC-dependent caspase-8 cleaves GSDME to execute 'incomplete pyroptosis' with IL-1α but not IL-1β release, broadening the upstream activators of GSDME.\",\n      \"evidence\": \"Caspase-1/11 double-KO macrophages, ASC knockdown, caspase-8 inhibitor, GSDME knockdown, cytokine ELISA\",\n      \"pmids\": [\"32361594\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether granzyme B can also directly cleave GSDME independent of caspase-3 in vivo\", \"Structural basis for differential cytokine trapping\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Characterization of GSDME pore selectivity showed that membrane permeabilization is size-dependent, accelerating lysis and molecular influx in a graded manner, while phosphatidylserine exposure is GSDME-independent — separating pore formation from apoptotic signaling.\",\n      \"evidence\": \"GSDME-KO L929 cells, dextran influx assays with graded molecular weight probes, SYTOX Blue, annexin V\",\n      \"pmids\": [\"34971436\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Pore diameter and stoichiometry not structurally resolved\", \"Single cell line used\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"EMT transcription factors ZEB1/ZEB2 were identified as direct transcriptional activators of GSDME, linking epithelial-mesenchymal transition status to pyroptotic competence across cancer types.\",\n      \"evidence\": \"ChIP for ZEB1/ZEB2 at GSDME promoter, ZEB1/2 knockdown, EMT induction/reversion models\",\n      \"pmids\": [\"34901025\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab finding without independent replication\", \"Whether ZEB-driven GSDME upregulation occurs in normal developmental EMT\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"GSDME protein stability was shown to be controlled by OTUD4-mediated deubiquitination, which stabilizes GSDME and enhances caspase-3-dependent pyroptosis — revealing ubiquitin-dependent turnover as a regulatory axis.\",\n      \"evidence\": \"Reciprocal IP and mass spectrometry identifying OTUD4, ubiquitination assays, OTUD4 overexpression/knockdown, in vivo radiosensitivity in NPC models\",\n      \"pmids\": [\"36411454\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the E3 ligase opposing OTUD4 was not defined in this study\", \"Specific ubiquitin chain type on GSDME not characterized\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"CDC20 was identified as the E3 ligase component targeting GSDME for ubiquitin-dependent proteasomal degradation; CDC20 depletion shifts the apoptosis-pyroptosis balance by stabilizing GSDME protein.\",\n      \"evidence\": \"Ubiquitination assay, cycloheximide chase, CDC20 knockdown/overexpression, syngeneic murine tumor models\",\n      \"pmids\": [\"37528490\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CDC20 and OTUD4 compete at the same ubiquitin sites on GSDME\", \"Degron motif on GSDME not fully mapped\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"A mitochondrial feed-forward loop was established: GSDME-N penetrates mitochondrial membranes to release cytochrome c, which activates caspase-9/3 to generate more GSDME-N, amplifying pyroptosis; BAX acts upstream to initiate this cascade.\",\n      \"evidence\": \"GSDME-N overexpression, mitochondrial fractionation, cytochrome c release assay, BAX overexpression, Bcl-2/BAX IP in multiple myeloma cells\",\n      \"pmids\": [\"36807412\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab finding in one cancer type\", \"Structural basis for GSDME-N insertion into mitochondrial versus plasma membranes unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Evolutionary conservation of the gasdermin pore mechanism was demonstrated through amphioxus GSDME, showing that distinct caspase cleavage sites generate functionally opposing N-terminal fragments — one pyroptotic and bactericidal, the other inhibitory — and that key residues are functionally conserved in human GSDME.\",\n      \"evidence\": \"Amphioxus caspase cleavage assay, membrane binding, bacterial killing assay, mutagenesis of conserved residues, in vivo infection model\",\n      \"pmids\": [\"37134086\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether human GSDME produces an analogous inhibitory fragment\", \"In vivo relevance of dual-fragment mechanism in mammals\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"AMPK-mediated phosphorylation at Thr6 was identified as a metabolically regulated brake on GSDME: mannose-derived GlcNAc-6P activates AMPK via LKB1, and pThr6-GSDME resists caspase-3 cleavage, directly linking cellular metabolism to pyroptotic susceptibility.\",\n      \"evidence\": \"AMPK-KO and GSDME T6E/T6A knock-in mice, metabolite binding assay, in vitro caspase-3 cleavage, clinical correlation\",\n      \"pmids\": [\"37460805\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other kinases also phosphorylate GSDME-N\", \"Structural basis for how pThr6 blocks caspase-3 access to Asp270\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"A cleavage-independent activation route was discovered: UV-C-induced DNA damage activates PARP1, generating free PAR polymers that activate cytoplasmic PARP5 to PARylate full-length GSDME; concurrent lipid ROS from cardiolipin peroxidation drive oxidative oligomerization of PARylated GSDME, enabling pore formation without proteolytic processing.\",\n      \"evidence\": \"PARP1/5 inhibitors and knockdown, PAR polymer detection, GSDME PARylation assay, lipid ROS measurement, cardiolipin peroxidation assay, confocal membrane targeting\",\n      \"pmids\": [\"38997456\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PARylation-dependent activation occurs under physiological (non-UV-C) stresses\", \"Structural details of PARylated GSDME oligomer\", \"Whether other gasdermins share this non-canonical activation route\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Sp1 was identified as a direct transcriptional activator binding the GSDME proximal promoter (−36 to −28), synergizing with STAT3 and antagonized by promoter DNA methylation — integrating transcriptional and epigenetic control of GSDME expression.\",\n      \"evidence\": \"ChIP for Sp1 at GSDME promoter, luciferase reporter, Sp1 knockdown/inhibition, chemotherapy rescue experiments\",\n      \"pmids\": [\"38238307\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of Sp1 versus p53 and ZEB1/2 in different cell types unresolved\", \"Mechanism of DNA methylation–Sp1 interplay not fully dissected\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"In a non-canonical cell biology context, GSDME-N in platelets is recruited to the plasma membrane by flotillin-2, forming pores that drive granule release and platelet hyperactivation during chemotherapy — extending GSDME function beyond classical pyroptosis to hemostasis.\",\n      \"evidence\": \"GSDME-KO mice, Co-IP identifying flotillin-2, caspase-3 cleavage assay, platelet activation assays, cisplatin murine model\",\n      \"pmids\": [\"39378585\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether flotillin-2 is required for GSDME-N membrane targeting in nucleated cells\", \"In vivo thrombotic consequences beyond the cisplatin model\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"STAT1 was shown to drive GSDME transcription downstream of IFNγ, linking adaptive immune signaling (cytotoxic lymphocyte granzyme B/caspase-3) to tumor cell pyroptosis and establishing a self-reinforcing STAT1–GSDME circuit required for immunotherapy efficacy.\",\n      \"evidence\": \"GSDME-KO and STAT1-KO, ChIP-seq, single-cell multiomics, HDAC inhibitor + anti-PD1 in orthotopic HCC models\",\n      \"pmids\": [\"39486886\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether STAT1 and STAT3 compete or cooperate at the GSDME locus under different cytokine contexts\", \"Mechanism by which pyroptosis feeds back to amplify STAT1 signaling\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"circPDIA3 was shown to directly bind the GSDME-C domain and block its palmitoylation by ZDHHC3/17, strengthening autoinhibition and suppressing pyroptosis — revealing circular RNA as a post-translational modulator of gasdermin activation.\",\n      \"evidence\": \"RIP, RNA pulldown, Co-IP, palmitoylation assay, ZDHHC3/17 knockdown, PDX models in colorectal cancer\",\n      \"pmids\": [\"38861804\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other circRNAs or RNAs regulate other gasdermin family members\", \"Stoichiometry of circPDIA3–GSDME-C interaction\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"The GSDME C-terminal fragment was found to have a signaling function beyond autoinhibition: interaction with PDPK1 activates PI3K–AKT to drive M2-like macrophage polarization, suggesting GSDME cleavage products have immunomodulatory roles in the tumor microenvironment.\",\n      \"evidence\": \"Co-IP of GSDME-C with PDPK1, GSDME KO, single-cell sequencing, Eliprodil inhibitor treatment in HCC mouse models\",\n      \"pmids\": [\"39496854\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab finding; PDPK1 interaction awaits independent confirmation\", \"Whether GSDME-C reaches macrophages in trans or acts cell-autonomously\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the cryo-EM structure of the GSDME pore, the mechanism by which PARylation relieves autoinhibition at the structural level, whether the cleavage-independent activation pathway operates under physiological (non-UV-C) stimuli, and how the opposing ubiquitin ligase (CDC20) and deubiquitinase (OTUD4) activities are coordinated in different tissues.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No cryo-EM or high-resolution structure of GSDME pore available\", \"PARylation sites on GSDME not mapped\", \"Physiological relevance of cleavage-independent pathway beyond UV-C not tested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 8, 14]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 13, 14]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [0, 7, 8, 17]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [17, 19, 20]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [7, 15]},\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [13]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"CASP3\",\n      \"OTUD4\",\n      \"CDC20\",\n      \"FLOT2\",\n      \"PDPK1\",\n      \"PARP1\",\n      \"AMPK\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}