{"gene":"GZMA","run_date":"2026-06-10T01:55:21","timeline":{"discoveries":[{"year":1987,"finding":"CTLA-3 (GZMA) transcripts are expressed preferentially in cytolytic T lymphocytes but can also be detected in some non-cytotoxic lymphocytes; they are absent in cytotoxic macrophages and most natural cytotoxic cells, indicating CTLA-3/GZMA is not required for macrophage or natural cytotoxicity.","method":"Tissue expression survey by molecular hybridization across diverse cell types including CTLs, NK cells, macrophages, and mast cells","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — systematic expression survey across many cell types by a single lab, no functional reconstitution but multiple cell types tested with consistent pattern","pmids":["3495579"],"is_preprint":false},{"year":1993,"finding":"Human granzyme A (HFSP/CTLA3) is a homodimeric, trypsin-like serine protease of 60 kDa localized in granules of cytolytic T cells and NK cells; its gene maps to chromosome 5q11-q12 and spans 10 kb with a defined exon-intron structure, placing it outside known serine protease superfamily loci.","method":"Cosmid clone isolation, complete exon-intron mapping, chromosomal mapping by FISH with an 11-kb genomic probe","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct genomic cloning and chromosomal localization with functional annotation of protein biochemistry (homodimer, trypsin-like serine protease, granule localization), single lab","pmids":["8288245"],"is_preprint":false},{"year":2014,"finding":"GZMA serine protease activity promotes proinflammatory cytokine production (IFN-γ, IL-1β, IL-1α) in IL-12-stimulated splenocytes; pharmacological inhibition of serine protease activity with AEBSF diminished GZMA activity and shifted the response toward a Th2 profile (increased IL-4, STAT6A nuclear translocation, GATA3, c-Maf), demonstrating that GZMA enzymatic activity modulates Th1/Th2 balance.","method":"In vitro splenocyte assay with serine protease inhibitor AEBSF, cytokine measurement, Western blot for phospho-STAT6A nuclear translocation, in vivo estrogen treatment model","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological inhibition of enzymatic activity with defined readouts across multiple cytokine and transcription factor endpoints, single lab","pmids":["24840346"],"is_preprint":false},{"year":2020,"finding":"GZMA functions as a dissemination suppressor in Theileria annulata-transformed macrophages; CRISPR/Cas9-mediated knockdown of GZMA in attenuated macrophages restored their dissemination capacity in Rag2/γC mice, and overexpression of GZMA dampened dissemination potential in human B lymphomas.","method":"CRISPR/Cas9 knockdown in bovine macrophages, in vivo dissemination assay in Rag2/γC mice, comparative transcriptomics of 934 human cancer cell lines","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function (CRISPR) with defined in vivo phenotypic readout, single lab, complemented by human cancer cell line analysis","pmids":["32830401"],"is_preprint":false},{"year":2022,"finding":"GZMA secreted by cytotoxic cells binds to F2R (PAR1) expressed on tumor cells via the LDPRSFLL motif at the N-terminus of F2R; this interaction activates the JAK2/STAT1 signaling pathway, promotes tumor cell apoptosis, and enhances T cell-mediated tumor killing in hepatocellular carcinoma.","method":"Co-immunoprecipitation, in vivo mouse tumor model, in vitro T cell killing assays, motif mutagenesis identifying the LDPRSFLL N-terminal motif of F2R, single-cell sequencing","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP identifying GZMA-F2R binding, supported by in vivo and in vitro functional validation, motif-level mechanistic detail, single lab","pmids":["35256589"],"is_preprint":false},{"year":2023,"finding":"GZMA promotes angiogenesis in endothelial cells via interaction with F2R (PAR1); addition of GZMA recombinant protein to HUVECs co-cultured with Jurkat T cells increased F2R expression and enhanced endothelial migration and tube formation.","method":"In vitro co-culture of HUVECs and Jurkat T cells with GZMA recombinant protein, RT-PCR, angiogenesis assay, migration assay","journal":"International endodontic journal","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single recombinant protein addition experiment without genetic manipulation or mechanistic dissection of the F2R signaling cascade","pmids":["37400946"],"is_preprint":false},{"year":2023,"finding":"GZMA promotes osteoclast proliferation and inhibits osteoclast apoptosis; Cas13d-mediated inhibition of GZMA increased miR-25-3p expression, which downregulated TGF-β, leading to decreased PAR1 (PAR1/PARs pathway) expression in osteoclasts during chronic apical periodontitis.","method":"crRNA/Cas13d-mediated GZMA inhibition in osteoclasts, RT-PCR and Western blot for miR-25-3p, TGF-β, and PAR1, cell proliferation and apoptosis assays","journal":"Cellular & molecular biology letters","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, loss-of-function with defined downstream readouts but limited mechanistic depth and no direct biochemical interaction assay","pmids":["37626297"],"is_preprint":false},{"year":2024,"finding":"GZMA inhibits PDE4B activity in intestinal epithelial cells, triggering the cAMP/PKA/CREB signaling cascade; PKA interacts with CREB (interaction enhanced by GZMA), leading to CREB nuclear translocation and transactivation of GPX4, which inhibits ferroptosis and promotes CDX2-mediated cell differentiation, increasing Occludin and ZO-1 expression to improve intestinal barrier function.","method":"Western blot, qPCR, immunofluorescence, in vitro permeability assay, intestinal organoid culture, luciferase reporter assay, co-immunoprecipitation (PKA-CREB), subcellular fractionation, DSS-induced colitis mouse model","journal":"Cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, luciferase, fractionation, in vivo model) defining a mechanistic pathway, single lab","pmids":["39367435"],"is_preprint":false},{"year":2026,"finding":"GZMA (secreted by activated immune cells) activates GEF-H1 by dephosphorylating Ser886, which triggers the RhoA/ROCK pathway and subsequent phosphorylation of MLC2, LIMK, and cofilin, driving cytoskeletal remodeling and disruption of tight junction proteins (occludin, claudin-1, ZO-1, E-cadherin), thereby impairing the intestinal epithelial barrier during sepsis.","method":"In vitro immune cell–epithelial co-culture, TEER measurement, permeability assay, site-directed mutagenesis (Ser886 on GEF-H1), GEF-H1 knockout mice, CLP sepsis mouse model, high-throughput drug screening, plinabulin (GEF-H1 activator) treatment","journal":"Clinical and translational medicine","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis identifying Ser886 dephosphorylation, genetic knockout (GEF-H1 KO mice) with survival/barrier readouts, in vitro mechanistic dissection of downstream ROCK/MLC2/LIMK/cofilin phosphorylation, multiple orthogonal methods","pmids":["41943423"],"is_preprint":false},{"year":2026,"finding":"CD4+GZMA+ T cells cause endothelial cell cytotoxicity in atherosclerosis; cigarette tar promotes CIITA nuclear translocation and PRMT5-mediated H3R2 symmetric dimethylation, upregulating MHC II expression on endothelial cells, which activates CD4+Gzma+ T cells that in turn damage endothelial cells.","method":"ApoEKO mouse cigarette tar inhalation model, scRNA-seq, mass spectrometry identifying CIITA-PRMT5 interaction, CIITA knockout/knockdown, PRMT5 inhibition/knockdown, in vitro co-culture of CD4+Gzma+ T cells with endothelial cells","journal":"BMC medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout and pharmacological inhibition with defined cellular phenotype, mass spectrometry-identified interaction, but GZMA's direct enzymatic role in endothelial damage not biochemically resolved","pmids":["42237295"],"is_preprint":false}],"current_model":"GZMA (granzyme A) is a homodimeric, trypsin-like serine protease stored in cytotoxic T cell and NK cell granules that exerts multiple extracellular signaling functions beyond direct cytotoxicity: it binds F2R/PAR1 on target cells to activate JAK2/STAT1-driven apoptosis, promotes proangiogenic signaling in endothelial cells, drives intestinal barrier regulation through PDE4B inhibition and the cAMP/PKA/CREB/GPX4 axis, and disrupts the intestinal epithelial barrier during sepsis by dephosphorylating GEF-H1 Ser886 and activating the RhoA/ROCK cytoskeletal remodeling pathway."},"narrative":{"mechanistic_narrative":"GZMA is a homodimeric, trypsin-like serine protease stored in the granules of cytolytic T cells and NK cells that has emerged as a multifunctional effector acting largely through extracellular signaling on target cells [PMID:8288245, PMID:3495579]. Its enzymatic activity shapes adaptive immune output, biasing IL-12-stimulated responses toward a Th1 cytokine profile, since pharmacological inhibition of its serine protease activity shifts the response toward Th2 [PMID:24840346]. A recurrent mechanistic theme is engagement of the protease-activated receptor F2R/PAR1 on target cells: GZMA binds the LDPRSFLL N-terminal motif of F2R to activate JAK2/STAT1 signaling and drive tumor cell apoptosis while enhancing T cell-mediated killing [PMID:35256589], and the same F2R axis underlies its proangiogenic effects on endothelial cells [PMID:37400946]. In intestinal epithelium GZMA exerts opposing barrier-modulating activities through distinct routes: it inhibits PDE4B to trigger a cAMP/PKA/CREB cascade that transactivates GPX4, suppressing ferroptosis and reinforcing tight junctions via Occludin and ZO-1 [PMID:39367435], whereas in sepsis it dephosphorylates GEF-H1 at Ser886 to activate RhoA/ROCK-driven cytoskeletal remodeling that disassembles tight junction proteins and disrupts the barrier [PMID:41943423]. GZMA also functions in tissue pathology as a cytotoxic mediator, with CD4+GZMA+ T cells damaging endothelial cells in atherosclerosis [PMID:42237295] and GZMA suppressing dissemination of transformed macrophages [PMID:32830401].","teleology":[{"year":1987,"claim":"Established the cellular source of GZMA by defining where its transcripts are and are not expressed, distinguishing CTL/NK-associated cytotoxicity from macrophage and natural cytotoxicity.","evidence":"Molecular hybridization expression survey across CTLs, NK cells, macrophages, and mast cells","pmids":["3495579"],"confidence":"Medium","gaps":["Expression survey does not establish protein function","Does not address the basis of expression in some non-cytotoxic lymphocytes"]},{"year":1993,"claim":"Defined GZMA's molecular identity as a homodimeric trypsin-like serine protease in cytotoxic granules and mapped its gene, providing the biochemical foundation for later functional studies.","evidence":"Cosmid cloning, exon-intron mapping, and FISH chromosomal localization to 5q11-q12","pmids":["8288245"],"confidence":"Medium","gaps":["No substrate identified at this stage","Granule localization shown but secretion and target engagement not addressed"]},{"year":2014,"claim":"Showed that GZMA enzymatic activity is not merely cytotoxic but modulates the Th1/Th2 cytokine balance, expanding its role into immune regulation.","evidence":"Splenocyte assays with serine protease inhibitor AEBSF, cytokine and phospho-STAT6A readouts, in vivo estrogen model","pmids":["24840346"],"confidence":"Medium","gaps":["Direct protease substrates driving the cytokine shift not identified","AEBSF is a broad serine protease inhibitor, not GZMA-specific"]},{"year":2020,"claim":"Demonstrated a tumor/pathogen-context function in which GZMA suppresses dissemination of transformed macrophages, implicating it in malignancy beyond direct killing.","evidence":"CRISPR/Cas9 knockdown in bovine macrophages, in vivo dissemination in Rag2/γC mice, cancer cell line transcriptomics","pmids":["32830401"],"confidence":"Medium","gaps":["Molecular mechanism of dissemination suppression unresolved","Whether protease activity is required not tested"]},{"year":2022,"claim":"Identified F2R/PAR1 as a direct GZMA receptor and mapped the binding motif, defining a JAK2/STAT1 apoptotic signaling axis on target cells.","evidence":"Co-IP, F2R LDPRSFLL motif mutagenesis, in vivo tumor model, and T cell killing assays in hepatocellular carcinoma","pmids":["35256589"],"confidence":"Medium","gaps":["Single Co-IP without independent structural confirmation of the interface","Whether cleavage of F2R or non-catalytic binding drives signaling not fully separated"]},{"year":2023,"claim":"Extended the GZMA-F2R axis to endothelial biology, linking it to proangiogenic responses.","evidence":"HUVEC/Jurkat co-culture with recombinant GZMA, RT-PCR, migration and tube formation assays","pmids":["37400946"],"confidence":"Low","gaps":["Single recombinant-protein addition experiment without genetic manipulation","Downstream F2R signaling cascade in endothelium not dissected"]},{"year":2023,"claim":"Connected GZMA to bone remodeling via osteoclast survival, placing it within a miR-25-3p/TGF-β/PAR1 regulatory circuit.","evidence":"Cas13d-mediated GZMA inhibition in osteoclasts with RT-PCR/Western readouts and proliferation/apoptosis assays","pmids":["37626297"],"confidence":"Low","gaps":["No direct biochemical interaction assay","Causal ordering of the miR-25-3p/TGF-β/PAR1 axis relative to GZMA unclear"]},{"year":2024,"claim":"Defined a barrier-protective intestinal pathway in which GZMA inhibits PDE4B to drive cAMP/PKA/CREB-dependent GPX4 transactivation, suppressing ferroptosis and strengthening tight junctions.","evidence":"Co-IP (PKA-CREB), luciferase reporter, subcellular fractionation, intestinal organoids, and DSS colitis mouse model","pmids":["39367435"],"confidence":"Medium","gaps":["Whether GZMA directly cleaves or binds PDE4B not biochemically resolved","Receptor or entry route into epithelial cells not defined"]},{"year":2026,"claim":"Established a barrier-disruptive GZMA mechanism in sepsis via Ser886 dephosphorylation of GEF-H1 and RhoA/ROCK-driven cytoskeletal remodeling, contrasting with its protective intestinal role.","evidence":"Site-directed mutagenesis of GEF-H1 Ser886, GEF-H1 KO mice, CLP sepsis model, TEER/permeability assays, plinabulin treatment","pmids":["41943423"],"confidence":"High","gaps":["How a protease produces a net dephosphorylation of GEF-H1 not mechanistically reconciled","Reconciliation with the opposing PDE4B/GPX4 barrier-protective pathway unresolved"]},{"year":2026,"claim":"Implicated CD4+GZMA+ T cells as effectors of endothelial cytotoxicity in atherosclerosis, downstream of cigarette-tar-induced MHC II upregulation.","evidence":"ApoEKO tar inhalation model, scRNA-seq, mass spectrometry of CIITA-PRMT5, CIITA/PRMT5 perturbation, and CD4+Gzma+ T cell/endothelial co-culture","pmids":["42237295"],"confidence":"Medium","gaps":["GZMA's direct enzymatic role in endothelial damage not biochemically resolved","Whether F2R or another receptor mediates the cytotoxicity not tested"]},{"year":null,"claim":"It remains unresolved how GZMA reconciles its opposing barrier-protective (PDE4B/GPX4) and barrier-disruptive (GEF-H1/RhoA) intestinal activities, and which catalytic substrates underlie each non-canonical signaling outcome.","evidence":"No single study integrates the divergent GZMA target-cell pathways or defines the proteolytic substrate set","pmids":[],"confidence":"Low","gaps":["No unified model of context-dependent GZMA signaling","Direct enzymatic substrates for most signaling outputs unidentified","Mechanism of GZMA entry/access to intracellular targets like PDE4B and GEF-H1 unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,2,8]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[1,2]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[4,5]}],"localization":[{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[1]},{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[4,8]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,2,9]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4,7,8]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[4,7]}],"complexes":[],"partners":["F2R","PDE4B","GEF-H1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P12544","full_name":"Granzyme A","aliases":["CTL tryptase","Cytotoxic T-lymphocyte proteinase 1","Fragmentin-1","Granzyme-1","Hanukkah factor","H factor","HF"],"length_aa":262,"mass_kda":29.0,"function":"Abundant protease in the cytosolic granules of cytotoxic T-cells and NK-cells which activates caspase-independent pyroptosis when delivered into the target cell through the immunological synapse (PubMed:12819770, PubMed:32299851, PubMed:3257574, PubMed:3262682, PubMed:3263427). It cleaves after Lys or Arg (PubMed:12819770, PubMed:32299851). Once delivered into the target cell, acts by catalyzing cleavage of gasdermin-B (GSDMB), releasing the pore-forming moiety of GSDMB, thereby triggering pyroptosis and target cell death (PubMed:32299851, PubMed:34022140, PubMed:36157507, PubMed:36899106). Cleaves APEX1 after 'Lys-31' and destroys its oxidative repair activity (PubMed:12524539). Cleaves the nucleosome assembly protein SET after 'Lys-189', which disrupts its nucleosome assembly activity and allows the SET complex to translocate into the nucleus to nick and degrade the DNA (PubMed:11555662, PubMed:12628186, PubMed:16818237)","subcellular_location":"Secreted; Cytoplasmic granule","url":"https://www.uniprot.org/uniprotkb/P12544/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/GZMA","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/GZMA","total_profiled":1310},"omim":[{"mim_id":"611221","title":"GASDERMIN B; GSDMB","url":"https://www.omim.org/entry/611221"},{"mim_id":"606609","title":"3-PRIME @REPAIR EXONUCLEASE 1; TREX1","url":"https://www.omim.org/entry/606609"},{"mim_id":"605772","title":"ESTROGEN RECEPTOR-BINDING SITE-ASSOCIATED ANTIGEN, 9; EBAG9","url":"https://www.omim.org/entry/605772"},{"mim_id":"603846","title":"NADH-UBIQUINONE OXIDOREDUCTASE Fe-S PROTEIN 3; NDUFS3","url":"https://www.omim.org/entry/603846"},{"mim_id":"602365","title":"CATHEPSIN C; CTSC","url":"https://www.omim.org/entry/602365"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"lymphoid tissue","ntpm":63.9}],"url":"https://www.proteinatlas.org/search/GZMA"},"hgnc":{"alias_symbol":[],"prev_symbol":["HFSP","CTLA3"]},"alphafold":{"accession":"P12544","domains":[{"cath_id":"2.40.10.10","chopping":"44-133_250-262","consensus_level":"medium","plddt":97.2533,"start":44,"end":262},{"cath_id":"2.40.10.10","chopping":"135-248","consensus_level":"medium","plddt":96.3054,"start":135,"end":248}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P12544","model_url":"https://alphafold.ebi.ac.uk/files/AF-P12544-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P12544-F1-predicted_aligned_error_v6.png","plddt_mean":91.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=GZMA","jax_strain_url":"https://www.jax.org/strain/search?query=GZMA"},"sequence":{"accession":"P12544","fasta_url":"https://rest.uniprot.org/uniprotkb/P12544.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P12544/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P12544"}},"corpus_meta":[{"pmid":"3495579","id":"PMC_3495579","title":"CTLA-1 and CTLA-3 serine esterase transcripts are detected mostly in cytotoxic T cells, but not only and not always.","date":"1987","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/3495579","citation_count":64,"is_preprint":false},{"pmid":"35256589","id":"PMC_35256589","title":"Heterogeneity induced GZMA-F2R communication inefficient impairs antitumor immunotherapy of PD-1 mAb through JAK2/STAT1 signal suppression in hepatocellular carcinoma.","date":"2022","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/35256589","citation_count":34,"is_preprint":false},{"pmid":"29950013","id":"PMC_29950013","title":"HFSP: high speed homology-driven function annotation of proteins.","date":"2018","source":"Bioinformatics (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/29950013","citation_count":32,"is_preprint":false},{"pmid":"39367435","id":"PMC_39367435","title":"GZMA suppressed GPX4-mediated ferroptosis to improve intestinal 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HFSP Workshop on Genetic Control of Vertebrate Development cosponsored by the Human Frontier Science Program, European Science Foundation, and European Molecular Biology Organization, Les Diablerets, Switzerland, May 26-30, 1991.","date":"1992","source":"The New biologist","url":"https://pubmed.ncbi.nlm.nih.gov/1346970","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.10.14.25337919","title":"Interpretable machine learning applied to high-dimensional salivary proteomics accurately classifies pediatric inflammatory bowel diseases","date":"2025-10-17","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.14.25337919","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.07.31.25332518","title":"Genomic and molecular associations with preoperative immune checkpoint inhibition in patients with stage III clear cell renal cell carcinoma","date":"2025-08-02","source":"bioRxiv","url":"https://doi.org/10.1101/2025.07.31.25332518","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.08.06.668764","title":"Angiogenic CD8 T cells from PWH induce Granzymes-dependent PAR1 activation promoting endothelial inflammation","date":"2025-08-08","source":"bioRxiv","url":"https://doi.org/10.1101/2025.08.06.668764","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.02.03.636324","title":"Single-cell RNA-seq reveals altered plasma cell subsets and decreased cytotoxicity of NK cells in patients with Kawasaki disease","date":"2025-02-08","source":"bioRxiv","url":"https://doi.org/10.1101/2025.02.03.636324","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.12.17.628857","title":"TCR clonality and TCR clonal expansion in the  <i>in situ</i>  microenvironment of non-small cell lung cancer","date":"2025-01-04","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.17.628857","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.06.22.24306750","title":"Peripheral blood<i>GATA2</i>expression impacts<i>RNF213</i>mutation penetrance and clinical severity in moyamoya disease","date":"2024-06-28","source":"bioRxiv","url":"https://doi.org/10.1101/2024.06.22.24306750","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.09.03.610972","title":"IL-6 and IL-27 negatively regulate CRTAM-expressing CD4  <sup>+</sup>  T-cells associated with lymphoid-driven synovitis","date":"2024-09-06","source":"bioRxiv","url":"https://doi.org/10.1101/2024.09.03.610972","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.06.19.24309192","title":"A surgical window of opportunity trial evaluating the effect of the PCSK9 inhibitor evolocumab on tumoral MHC-I expression and CD8<sup>+</sup> infiltration in glioma","date":"2024-06-20","source":"bioRxiv","url":"https://doi.org/10.1101/2024.06.19.24309192","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.08.01.25332809","title":"Advancing Non-Invasive Respiratory Diagnostics: Multiplex Nasal Biomarker Profiling for Stratification of Airway Inflammatory Diseases","date":"2025-08-05","source":"bioRxiv","url":"https://doi.org/10.1101/2025.08.01.25332809","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":17015,"output_tokens":2931,"usd":0.047505,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10231,"output_tokens":3609,"usd":0.07069,"stage2_stop_reason":"end_turn"},"total_usd":0.118195,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1987,\n      \"finding\": \"CTLA-3 (GZMA) transcripts are expressed preferentially in cytolytic T lymphocytes but can also be detected in some non-cytotoxic lymphocytes; they are absent in cytotoxic macrophages and most natural cytotoxic cells, indicating CTLA-3/GZMA is not required for macrophage or natural cytotoxicity.\",\n      \"method\": \"Tissue expression survey by molecular hybridization across diverse cell types including CTLs, NK cells, macrophages, and mast cells\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — systematic expression survey across many cell types by a single lab, no functional reconstitution but multiple cell types tested with consistent pattern\",\n      \"pmids\": [\"3495579\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Human granzyme A (HFSP/CTLA3) is a homodimeric, trypsin-like serine protease of 60 kDa localized in granules of cytolytic T cells and NK cells; its gene maps to chromosome 5q11-q12 and spans 10 kb with a defined exon-intron structure, placing it outside known serine protease superfamily loci.\",\n      \"method\": \"Cosmid clone isolation, complete exon-intron mapping, chromosomal mapping by FISH with an 11-kb genomic probe\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct genomic cloning and chromosomal localization with functional annotation of protein biochemistry (homodimer, trypsin-like serine protease, granule localization), single lab\",\n      \"pmids\": [\"8288245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"GZMA serine protease activity promotes proinflammatory cytokine production (IFN-γ, IL-1β, IL-1α) in IL-12-stimulated splenocytes; pharmacological inhibition of serine protease activity with AEBSF diminished GZMA activity and shifted the response toward a Th2 profile (increased IL-4, STAT6A nuclear translocation, GATA3, c-Maf), demonstrating that GZMA enzymatic activity modulates Th1/Th2 balance.\",\n      \"method\": \"In vitro splenocyte assay with serine protease inhibitor AEBSF, cytokine measurement, Western blot for phospho-STAT6A nuclear translocation, in vivo estrogen treatment model\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological inhibition of enzymatic activity with defined readouts across multiple cytokine and transcription factor endpoints, single lab\",\n      \"pmids\": [\"24840346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GZMA functions as a dissemination suppressor in Theileria annulata-transformed macrophages; CRISPR/Cas9-mediated knockdown of GZMA in attenuated macrophages restored their dissemination capacity in Rag2/γC mice, and overexpression of GZMA dampened dissemination potential in human B lymphomas.\",\n      \"method\": \"CRISPR/Cas9 knockdown in bovine macrophages, in vivo dissemination assay in Rag2/γC mice, comparative transcriptomics of 934 human cancer cell lines\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function (CRISPR) with defined in vivo phenotypic readout, single lab, complemented by human cancer cell line analysis\",\n      \"pmids\": [\"32830401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"GZMA secreted by cytotoxic cells binds to F2R (PAR1) expressed on tumor cells via the LDPRSFLL motif at the N-terminus of F2R; this interaction activates the JAK2/STAT1 signaling pathway, promotes tumor cell apoptosis, and enhances T cell-mediated tumor killing in hepatocellular carcinoma.\",\n      \"method\": \"Co-immunoprecipitation, in vivo mouse tumor model, in vitro T cell killing assays, motif mutagenesis identifying the LDPRSFLL N-terminal motif of F2R, single-cell sequencing\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP identifying GZMA-F2R binding, supported by in vivo and in vitro functional validation, motif-level mechanistic detail, single lab\",\n      \"pmids\": [\"35256589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"GZMA promotes angiogenesis in endothelial cells via interaction with F2R (PAR1); addition of GZMA recombinant protein to HUVECs co-cultured with Jurkat T cells increased F2R expression and enhanced endothelial migration and tube formation.\",\n      \"method\": \"In vitro co-culture of HUVECs and Jurkat T cells with GZMA recombinant protein, RT-PCR, angiogenesis assay, migration assay\",\n      \"journal\": \"International endodontic journal\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single recombinant protein addition experiment without genetic manipulation or mechanistic dissection of the F2R signaling cascade\",\n      \"pmids\": [\"37400946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"GZMA promotes osteoclast proliferation and inhibits osteoclast apoptosis; Cas13d-mediated inhibition of GZMA increased miR-25-3p expression, which downregulated TGF-β, leading to decreased PAR1 (PAR1/PARs pathway) expression in osteoclasts during chronic apical periodontitis.\",\n      \"method\": \"crRNA/Cas13d-mediated GZMA inhibition in osteoclasts, RT-PCR and Western blot for miR-25-3p, TGF-β, and PAR1, cell proliferation and apoptosis assays\",\n      \"journal\": \"Cellular & molecular biology letters\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, loss-of-function with defined downstream readouts but limited mechanistic depth and no direct biochemical interaction assay\",\n      \"pmids\": [\"37626297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"GZMA inhibits PDE4B activity in intestinal epithelial cells, triggering the cAMP/PKA/CREB signaling cascade; PKA interacts with CREB (interaction enhanced by GZMA), leading to CREB nuclear translocation and transactivation of GPX4, which inhibits ferroptosis and promotes CDX2-mediated cell differentiation, increasing Occludin and ZO-1 expression to improve intestinal barrier function.\",\n      \"method\": \"Western blot, qPCR, immunofluorescence, in vitro permeability assay, intestinal organoid culture, luciferase reporter assay, co-immunoprecipitation (PKA-CREB), subcellular fractionation, DSS-induced colitis mouse model\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, luciferase, fractionation, in vivo model) defining a mechanistic pathway, single lab\",\n      \"pmids\": [\"39367435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"GZMA (secreted by activated immune cells) activates GEF-H1 by dephosphorylating Ser886, which triggers the RhoA/ROCK pathway and subsequent phosphorylation of MLC2, LIMK, and cofilin, driving cytoskeletal remodeling and disruption of tight junction proteins (occludin, claudin-1, ZO-1, E-cadherin), thereby impairing the intestinal epithelial barrier during sepsis.\",\n      \"method\": \"In vitro immune cell–epithelial co-culture, TEER measurement, permeability assay, site-directed mutagenesis (Ser886 on GEF-H1), GEF-H1 knockout mice, CLP sepsis mouse model, high-throughput drug screening, plinabulin (GEF-H1 activator) treatment\",\n      \"journal\": \"Clinical and translational medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis identifying Ser886 dephosphorylation, genetic knockout (GEF-H1 KO mice) with survival/barrier readouts, in vitro mechanistic dissection of downstream ROCK/MLC2/LIMK/cofilin phosphorylation, multiple orthogonal methods\",\n      \"pmids\": [\"41943423\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"CD4+GZMA+ T cells cause endothelial cell cytotoxicity in atherosclerosis; cigarette tar promotes CIITA nuclear translocation and PRMT5-mediated H3R2 symmetric dimethylation, upregulating MHC II expression on endothelial cells, which activates CD4+Gzma+ T cells that in turn damage endothelial cells.\",\n      \"method\": \"ApoEKO mouse cigarette tar inhalation model, scRNA-seq, mass spectrometry identifying CIITA-PRMT5 interaction, CIITA knockout/knockdown, PRMT5 inhibition/knockdown, in vitro co-culture of CD4+Gzma+ T cells with endothelial cells\",\n      \"journal\": \"BMC medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout and pharmacological inhibition with defined cellular phenotype, mass spectrometry-identified interaction, but GZMA's direct enzymatic role in endothelial damage not biochemically resolved\",\n      \"pmids\": [\"42237295\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"GZMA (granzyme A) is a homodimeric, trypsin-like serine protease stored in cytotoxic T cell and NK cell granules that exerts multiple extracellular signaling functions beyond direct cytotoxicity: it binds F2R/PAR1 on target cells to activate JAK2/STAT1-driven apoptosis, promotes proangiogenic signaling in endothelial cells, drives intestinal barrier regulation through PDE4B inhibition and the cAMP/PKA/CREB/GPX4 axis, and disrupts the intestinal epithelial barrier during sepsis by dephosphorylating GEF-H1 Ser886 and activating the RhoA/ROCK cytoskeletal remodeling pathway.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GZMA is a homodimeric, trypsin-like serine protease stored in the granules of cytolytic T cells and NK cells that has emerged as a multifunctional effector acting largely through extracellular signaling on target cells [#1, #0]. Its enzymatic activity shapes adaptive immune output, biasing IL-12-stimulated responses toward a Th1 cytokine profile, since pharmacological inhibition of its serine protease activity shifts the response toward Th2 [#2]. A recurrent mechanistic theme is engagement of the protease-activated receptor F2R/PAR1 on target cells: GZMA binds the LDPRSFLL N-terminal motif of F2R to activate JAK2/STAT1 signaling and drive tumor cell apoptosis while enhancing T cell-mediated killing [#4], and the same F2R axis underlies its proangiogenic effects on endothelial cells [#5]. In intestinal epithelium GZMA exerts opposing barrier-modulating activities through distinct routes: it inhibits PDE4B to trigger a cAMP/PKA/CREB cascade that transactivates GPX4, suppressing ferroptosis and reinforcing tight junctions via Occludin and ZO-1 [#7], whereas in sepsis it dephosphorylates GEF-H1 at Ser886 to activate RhoA/ROCK-driven cytoskeletal remodeling that disassembles tight junction proteins and disrupts the barrier [#8]. GZMA also functions in tissue pathology as a cytotoxic mediator, with CD4+GZMA+ T cells damaging endothelial cells in atherosclerosis [#9] and GZMA suppressing dissemination of transformed macrophages [#3].\",\n  \"teleology\": [\n    {\n      \"year\": 1987,\n      \"claim\": \"Established the cellular source of GZMA by defining where its transcripts are and are not expressed, distinguishing CTL/NK-associated cytotoxicity from macrophage and natural cytotoxicity.\",\n      \"evidence\": \"Molecular hybridization expression survey across CTLs, NK cells, macrophages, and mast cells\",\n      \"pmids\": [\"3495579\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Expression survey does not establish protein function\", \"Does not address the basis of expression in some non-cytotoxic lymphocytes\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Defined GZMA's molecular identity as a homodimeric trypsin-like serine protease in cytotoxic granules and mapped its gene, providing the biochemical foundation for later functional studies.\",\n      \"evidence\": \"Cosmid cloning, exon-intron mapping, and FISH chromosomal localization to 5q11-q12\",\n      \"pmids\": [\"8288245\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No substrate identified at this stage\", \"Granule localization shown but secretion and target engagement not addressed\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed that GZMA enzymatic activity is not merely cytotoxic but modulates the Th1/Th2 cytokine balance, expanding its role into immune regulation.\",\n      \"evidence\": \"Splenocyte assays with serine protease inhibitor AEBSF, cytokine and phospho-STAT6A readouts, in vivo estrogen model\",\n      \"pmids\": [\"24840346\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct protease substrates driving the cytokine shift not identified\", \"AEBSF is a broad serine protease inhibitor, not GZMA-specific\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrated a tumor/pathogen-context function in which GZMA suppresses dissemination of transformed macrophages, implicating it in malignancy beyond direct killing.\",\n      \"evidence\": \"CRISPR/Cas9 knockdown in bovine macrophages, in vivo dissemination in Rag2/\\u03b3C mice, cancer cell line transcriptomics\",\n      \"pmids\": [\"32830401\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism of dissemination suppression unresolved\", \"Whether protease activity is required not tested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified F2R/PAR1 as a direct GZMA receptor and mapped the binding motif, defining a JAK2/STAT1 apoptotic signaling axis on target cells.\",\n      \"evidence\": \"Co-IP, F2R LDPRSFLL motif mutagenesis, in vivo tumor model, and T cell killing assays in hepatocellular carcinoma\",\n      \"pmids\": [\"35256589\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Co-IP without independent structural confirmation of the interface\", \"Whether cleavage of F2R or non-catalytic binding drives signaling not fully separated\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Extended the GZMA-F2R axis to endothelial biology, linking it to proangiogenic responses.\",\n      \"evidence\": \"HUVEC/Jurkat co-culture with recombinant GZMA, RT-PCR, migration and tube formation assays\",\n      \"pmids\": [\"37400946\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single recombinant-protein addition experiment without genetic manipulation\", \"Downstream F2R signaling cascade in endothelium not dissected\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected GZMA to bone remodeling via osteoclast survival, placing it within a miR-25-3p/TGF-\\u03b2/PAR1 regulatory circuit.\",\n      \"evidence\": \"Cas13d-mediated GZMA inhibition in osteoclasts with RT-PCR/Western readouts and proliferation/apoptosis assays\",\n      \"pmids\": [\"37626297\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct biochemical interaction assay\", \"Causal ordering of the miR-25-3p/TGF-\\u03b2/PAR1 axis relative to GZMA unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined a barrier-protective intestinal pathway in which GZMA inhibits PDE4B to drive cAMP/PKA/CREB-dependent GPX4 transactivation, suppressing ferroptosis and strengthening tight junctions.\",\n      \"evidence\": \"Co-IP (PKA-CREB), luciferase reporter, subcellular fractionation, intestinal organoids, and DSS colitis mouse model\",\n      \"pmids\": [\"39367435\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether GZMA directly cleaves or binds PDE4B not biochemically resolved\", \"Receptor or entry route into epithelial cells not defined\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Established a barrier-disruptive GZMA mechanism in sepsis via Ser886 dephosphorylation of GEF-H1 and RhoA/ROCK-driven cytoskeletal remodeling, contrasting with its protective intestinal role.\",\n      \"evidence\": \"Site-directed mutagenesis of GEF-H1 Ser886, GEF-H1 KO mice, CLP sepsis model, TEER/permeability assays, plinabulin treatment\",\n      \"pmids\": [\"41943423\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How a protease produces a net dephosphorylation of GEF-H1 not mechanistically reconciled\", \"Reconciliation with the opposing PDE4B/GPX4 barrier-protective pathway unresolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Implicated CD4+GZMA+ T cells as effectors of endothelial cytotoxicity in atherosclerosis, downstream of cigarette-tar-induced MHC II upregulation.\",\n      \"evidence\": \"ApoEKO tar inhalation model, scRNA-seq, mass spectrometry of CIITA-PRMT5, CIITA/PRMT5 perturbation, and CD4+Gzma+ T cell/endothelial co-culture\",\n      \"pmids\": [\"42237295\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"GZMA's direct enzymatic role in endothelial damage not biochemically resolved\", \"Whether F2R or another receptor mediates the cytotoxicity not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how GZMA reconciles its opposing barrier-protective (PDE4B/GPX4) and barrier-disruptive (GEF-H1/RhoA) intestinal activities, and which catalytic substrates underlie each non-canonical signaling outcome.\",\n      \"evidence\": \"No single study integrates the divergent GZMA target-cell pathways or defines the proteolytic substrate set\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unified model of context-dependent GZMA signaling\", \"Direct enzymatic substrates for most signaling outputs unidentified\", \"Mechanism of GZMA entry/access to intracellular targets like PDE4B and GEF-H1 unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 2, 8]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [4, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [4, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 2, 9]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4, 7, 8]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [4, 7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"F2R\",\n      \"PDE4B\",\n      \"GEF-H1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":5,"faith_total":5,"faith_pct":100.0}}