{"gene":"SEPTIN8","run_date":"2026-06-10T07:46:30","timeline":{"discoveries":[{"year":2009,"finding":"Sept8 binds directly to VAMP2 (identified by yeast two-hybrid screening) and suppresses the interaction between VAMP2 and synaptophysin, thereby regulating SNARE complex availability at presynapses. Sept8 also forms a complex with syntaxin1A, and the Sept8-VAMP2 interaction is disrupted by SNAP-25.","method":"Yeast two-hybrid screening, co-immunoprecipitation, protein interaction assays in neuronal context","journal":"Journal of neurochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus co-IP with functional interaction data, single lab, multiple orthogonal methods","pmids":["19196426"],"is_preprint":false},{"year":2004,"finding":"SEPT8 interacts with SEPT4 (identified by yeast two-hybrid, confirmed by co-immunoprecipitation) in human platelets; both proteins localize surrounding α-granules and translocate to the platelet surface upon activation, suggesting a role in granular secretion.","method":"Yeast two-hybrid, co-immunoprecipitation, transmission electron microscopy, subcellular localization","journal":"Thrombosis and haemostasis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus TEM localization, single lab, two orthogonal methods","pmids":["15116257"],"is_preprint":false},{"year":2002,"finding":"SEPT8 (KIAA0202) interacts with SEPT5 (CDCrel-1); the interaction was demonstrated by yeast two-hybrid, GST pull-down assay, and immunoprecipitation in the K-562 cell line.","method":"Yeast two-hybrid, GST pull-down, co-immunoprecipitation","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — three orthogonal methods (Y2H, GST pull-down, Co-IP) in single lab","pmids":["12023038"],"is_preprint":false},{"year":2002,"finding":"SEPT8 (KIAA0202) interacts with hPFTAIRE1, a Cdc2-related kinase localized in the cytoplasm; the interaction was identified by yeast two-hybrid and confirmed by immunoprecipitation.","method":"Yeast two-hybrid, co-immunoprecipitation","journal":"Acta biochimica et biophysica Sinica","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Y2H plus single Co-IP, single lab, limited functional follow-up","pmids":["12098780"],"is_preprint":false},{"year":2012,"finding":"SEPT8 is a direct interaction partner and in vitro substrate of MAP kinase-activated protein kinase 5 (MK5); serine residues 242 and 271 on SEPT8 are phosphorylated by MK5 in vitro. MK5 and SEPT8 co-localize in the perinuclear area, cell protrusions, and with the vesicle marker synaptophysin.","method":"Yeast two-hybrid, GST pull-down, co-immunoprecipitation, FRET, in vitro kinase assay, peptide array, confocal microscopy","journal":"World journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with site mapping plus multiple orthogonal binding methods (GST pull-down, Co-IP, FRET), single lab","pmids":["22649572"],"is_preprint":false},{"year":2016,"finding":"SEPT8 reduces BACE1 protein levels and thereby decreases soluble APPβ and Aβ generation in neuronal cells via a post-translational mechanism; a disease-associated SEPT8 transcript variant increases BACE1 half-life and promotes BACE1 localization in recycling endosomes, leading to elevated Aβ.","method":"SEPT8 knockdown/overexpression in neuronal cells, BACE1 half-life measurements, subcellular fractionation/localization of BACE1 to recycling endosomes, Aβ/sAPPβ quantification","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function and gain-of-function with defined molecular readouts (BACE1 half-life, endosomal localization, Aβ levels), single lab, multiple orthogonal methods","pmids":["27084579"],"is_preprint":false},{"year":2020,"finding":"Sept8-deficient mouse platelets show impaired integrin αIIbβ3 activation, defective α-granule exocytosis, reduced aggregation (particularly to convulxin/GPVI agonist), diminished fibrinogen binding and spreading, and reduced procoagulant activity and thrombin generation; δ-granule and lysosome exocytosis were unaffected.","method":"Sept8 knockout mouse model, flow cytometry, platelet aggregation assays, fibrinogen binding under static conditions, lactadherin binding, thrombin generation assay","journal":"Thrombosis and haemostasis","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean knockout with multiple specific platelet function assays, selective phenotype (α-granule but not δ-granule), single lab but multiple orthogonal readouts","pmids":["33202444"],"is_preprint":false},{"year":2023,"finding":"Sept8-204 (a brain-specific variant) is palmitoylated at Cys469, Cys470, and Cys472 by the palmitoyltransferase ZDHHC7; this modification is reversed by depalmitoylase PPT1. Palmitoylated Sept8-204 binds F-actin and promotes filopodia outgrowth in N2a cells and neurite arborization in hippocampal neurons. A non-palmitoylatable mutant (Sept8-204-3CA) fails to bind F-actin and does not promote morphological changes. Genetic deletion of Sept8, Sept8-204, or Zdhhc7 causes learning/memory deficits and anxiety-like behaviors in mice.","method":"Palmitoylation site mutagenesis (3CA mutant), acylation-RAC assay, F-actin co-sedimentation, live imaging in N2a cells and hippocampal neurons, Sept8 and Zdhhc7 knockout mice, behavioral assays","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis of palmitoylation sites, in vitro F-actin binding assay, identified writer (ZDHHC7) and eraser (PPT1), in vivo knockout behavioral phenotype, multiple orthogonal methods","pmids":["38051778"],"is_preprint":false},{"year":2024,"finding":"Palmitoylation of Sept8-204 is required for Sept8-204/Sept5 co-expression to form small vesicle-like structures and co-localize with synaptophysin; expression of non-palmitoylatable Sept8-204-3CA or pharmacological inhibition with 2-BP produces large puncta that do not co-localize with synaptophysin. ZDHHC17 mediates Sept8-204 palmitoylation and PPT1 removes it, dynamically controlling vesicle-like structure size.","method":"Palmitoylation site mutagenesis (3CA mutant), pharmacological inhibition (2-BP), ZDHHC17/PPT1 knockout cells, fluorescence microscopy co-localization with synaptophysin","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis plus genetic loss-of-function of writers/erasers with morphological readout, single lab, single publication","pmids":["38308620"],"is_preprint":false},{"year":2010,"finding":"Recombinant human SEPT8 (conserved GTP-binding domain plus C-terminal domain) purified as a predominantly dimeric species in solution, with the preferred dimer interface involving the GTP-binding site as determined by homology modelling and analytical gel filtration/DLS.","method":"Recombinant protein expression, affinity chromatography, gel filtration, DLS, CD spectroscopy, homology modelling","journal":"The protein journal","confidence":"Low","confidence_rationale":"Tier 3 / Weak — biochemical characterization of purified protein, no functional mutagenesis or structural validation beyond homology model, single lab","pmids":["20544379"],"is_preprint":false},{"year":2021,"finding":"SEPTIN8 localizes with acetyl-alpha tubulin in human proximal tubule cells under normal conditions; after hypoxia-induced damage, SEPTIN8 staining becomes diffuse and appears to re-localize with actin, indicating a role in cellular organization and structural response to environmental stress.","method":"Immunofluorescence localization in human proximal tubule cells, hypoxia in vitro model, co-staining with cytoskeletal markers","journal":"Scientific reports","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single localization experiment, no functional loss-of-function linked to the localization change, single study","pmids":["33483609"],"is_preprint":false}],"current_model":"SEPTIN8 is a GTP-binding cytoskeletal protein that functions at synapses and in vesicle trafficking by directly binding VAMP2 to regulate SNARE complex availability, forming complexes with SEPT4 and SEPT5, and undergoing palmitoylation (by ZDHHC7/ZDHHC17, reversed by PPT1) at a brain-specific splice variant (Sept8-204) to bind F-actin, promote filopodia/neurite outgrowth, and enable synaptophysin co-localization; it is also phosphorylated by MK5 at Ser242/Ser271, modulates BACE1 stability and endosomal sorting to control amyloid-β generation, and is required in platelets for integrin αIIbβ3 activation and α-granule exocytosis."},"narrative":{"mechanistic_narrative":"SEPTIN8 is a GTP-binding cytoskeletal protein that operates at the interface of vesicle trafficking, regulated exocytosis, and the actin cytoskeleton, partnering with other septins and SNARE machinery to control membrane dynamics in neurons and platelets [PMID:19196426, PMID:33202444, PMID:38051778]. At presynaptic terminals it binds VAMP2 directly and suppresses the VAMP2–synaptophysin interaction, thereby gating SNARE complex availability, an interaction modulated by SNAP-25 and accompanied by complex formation with syntaxin1A [PMID:19196426]. SEPTIN8 assembles with other septins, including SEPT4 and SEPT5, the former in platelets where both surround α-granules and translocate to the surface upon activation [PMID:15116257, PMID:12023038]. In platelets, SEPTIN8 is selectively required for integrin αIIbβ3 activation and α-granule exocytosis but not δ-granule or lysosome release [PMID:33202444]. A brain-specific variant, Sept8-204, is palmitoylated at Cys469/Cys470/Cys472 by ZDHHC7 and ZDHHC17 and depalmitoylated by PPT1; this lipid modification licenses F-actin binding, filopodia and neurite outgrowth, and Sept5-dependent formation of synaptophysin-positive vesicle-like structures, while non-palmitoylatable mutants fail in these functions, and in vivo loss of Sept8 or Zdhhc7 produces learning, memory, and anxiety phenotypes [PMID:38051778, PMID:38308620]. SEPTIN8 is a direct substrate of MK5, which phosphorylates it at Ser242 and Ser271 [PMID:22649572], and it limits amyloid-β generation by reducing BACE1 levels post-translationally, with a disease-associated transcript variant extending BACE1 half-life and shifting it to recycling endosomes to elevate Aβ [PMID:27084579].","teleology":[{"year":2002,"claim":"Establishing that SEPT8 physically associates with other septins defined it as a member of an oligomeric septin network rather than an isolated protein.","evidence":"Yeast two-hybrid, GST pull-down, and co-IP showing SEPT8–SEPT5 interaction in K-562 cells, plus an interaction with the kinase hPFTAIRE1","pmids":["12023038","12098780"],"confidence":"Medium","gaps":["Stoichiometry and architecture of the SEPT8-containing complex not resolved","Functional consequence of the hPFTAIRE1 interaction not established"]},{"year":2004,"claim":"Linking SEPT8 to SEPT4 around platelet α-granules connected the protein to regulated secretion in a non-neuronal cell type.","evidence":"Yeast two-hybrid, reciprocal co-IP, and TEM localization in human platelets","pmids":["15116257"],"confidence":"Medium","gaps":["Causal role in secretion not tested at this stage","Mechanism of granule association unknown"]},{"year":2009,"claim":"Identifying VAMP2 as a direct binding partner positioned SEPT8 as a regulator of SNARE complex assembly at presynapses.","evidence":"Yeast two-hybrid screen plus co-IP and interaction assays showing SEPT8 suppresses VAMP2–synaptophysin binding and forms a complex with syntaxin1A","pmids":["19196426"],"confidence":"Medium","gaps":["Effect on actual neurotransmitter release not measured","Whether SNAP-25 competition is physiologically regulated unknown"]},{"year":2010,"claim":"Biochemical characterization tested the oligomeric state of the SEPT8 GTP-binding module in isolation.","evidence":"Recombinant expression with gel filtration, DLS, CD, and homology modelling indicating a dimer via the GTP-binding interface","pmids":["20544379"],"confidence":"Low","gaps":["No experimental structure beyond a homology model","No mutagenesis validating the proposed dimer interface","Relevance to native heteromeric filaments unclear"]},{"year":2012,"claim":"Defining SEPT8 as an MK5 substrate introduced a post-translational regulatory input on the protein.","evidence":"Y2H, GST pull-down, co-IP, FRET, and in vitro kinase assay with peptide-array site mapping to Ser242/Ser271","pmids":["22649572"],"confidence":"High","gaps":["Functional consequence of Ser242/Ser271 phosphorylation in cells not determined","Phosphorylation not demonstrated in vivo"]},{"year":2016,"claim":"Connecting SEPT8 to BACE1 turnover and endosomal sorting linked it to amyloid-β generation.","evidence":"Knockdown/overexpression in neuronal cells with BACE1 half-life, recycling-endosome localization, and Aβ/sAPPβ readouts","pmids":["27084579"],"confidence":"Medium","gaps":["Direct molecular interaction with BACE1 not shown","In vivo relevance to Alzheimer pathology untested"]},{"year":2020,"claim":"A knockout mouse established a selective, causal requirement for Sept8 in platelet integrin activation and α-granule exocytosis.","evidence":"Sept8 knockout platelets analyzed by flow cytometry, aggregation, fibrinogen/lactadherin binding, and thrombin generation assays","pmids":["33202444"],"confidence":"High","gaps":["Molecular mechanism coupling Sept8 to αIIbβ3 inside-out signaling unresolved","Why δ-granule and lysosome exocytosis are spared unexplained"]},{"year":2023,"claim":"Identifying palmitoylation of the brain-specific Sept8-204 variant as a switch for F-actin binding tied a defined lipid modification to neuronal morphogenesis and behavior.","evidence":"Palmitoylation-site mutagenesis (3CA), acyl-RAC, F-actin co-sedimentation, live imaging, and Sept8/Zdhhc7 knockout behavioral assays","pmids":["38051778"],"confidence":"High","gaps":["How palmitoylation structurally enables F-actin binding unknown","Behavioral deficits not mechanistically traced to a single circuit"]},{"year":2024,"claim":"Extending the palmitoylation switch to vesicle-like structure formation showed it dynamically controls Sept8-204/Sept5 assembly and synaptophysin co-localization.","evidence":"3CA mutant, 2-BP inhibition, ZDHHC17/PPT1 knockout cells with synaptophysin co-localization microscopy","pmids":["38308620"],"confidence":"Medium","gaps":["Identity and cargo of the vesicle-like structures undefined","Functional output for synaptic transmission not measured"]},{"year":null,"claim":"How SEPT8's septin-filament assembly, palmitoylation-driven actin coupling, SNARE regulation, and BACE1 sorting are integrated into a single mechanistic logic across neurons and platelets remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of native SEPT8-containing heterofilaments","Unknown whether MK5 phosphorylation, palmitoylation, and partner binding are coordinately regulated","Cell-type-specific determinants of SEPT8 function not defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[7]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[7,9]}],"localization":[{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[8]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[7,10]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[5]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1,6]}],"pathway":[{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[6]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[0,7]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,8]}],"complexes":[],"partners":["VAMP2","STX1A","SEPT4","SEPT5","MK5","ZDHHC7","ZDHHC17","PPT1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q92599","full_name":"Septin-8","aliases":[],"length_aa":483,"mass_kda":55.8,"function":"Filament-forming cytoskeletal GTPase (By similarity). May play a role in platelet secretion (PubMed:15116257). Seems to participate in the process of SNARE complex formation in synaptic vesicles (By similarity) Stabilizes BACE1 protein levels and promotes the sorting and accumulation of BACE1 to the recycling or endosomal compartments, modulating the beta-amyloidogenic processing of APP","subcellular_location":"Cytoplasm; Cytoplasm, cytoskeleton; Synapse; Cell projection, axon; Cytoplasmic vesicle, secretory vesicle, synaptic vesicle membrane; Presynapse","url":"https://www.uniprot.org/uniprotkb/Q92599/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SEPTIN8","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1090,"dependency_fraction":0.0009174311926605505},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000164402","cell_line_id":"CID000732","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"cytoskeleton","grade":3},{"compartment":"membrane","grade":2}],"interactors":[{"gene":"SEPT11","stoichiometry":10.0},{"gene":"SEPT2","stoichiometry":10.0},{"gene":"SEPT6","stoichiometry":10.0},{"gene":"SEPT7","stoichiometry":10.0},{"gene":"SEPT9","stoichiometry":10.0},{"gene":"SEPT10","stoichiometry":10.0},{"gene":"SEPT5","stoichiometry":4.0},{"gene":"SEPT3","stoichiometry":4.0},{"gene":"COPB2","stoichiometry":0.2},{"gene":"COPE","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000732","total_profiled":1310},"omim":[],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"},{"location":"Actin filaments","reliability":"Additional"},{"location":"Primary cilium","reliability":"Additional"},{"location":"Mid piece","reliability":"Additional"},{"location":"Principal piece","reliability":"Additional"},{"location":"Annulus","reliability":"Additional"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"brain","ntpm":749.6}],"url":"https://www.proteinatlas.org/search/SEPTIN8"},"hgnc":{"alias_symbol":["KIAA0202","SEP2","Septin-8"],"prev_symbol":["SEPT8"]},"alphafold":{"accession":"Q92599","domains":[{"cath_id":"3.40.50.300","chopping":"36-307","consensus_level":"high","plddt":88.9557,"start":36,"end":307},{"cath_id":"1.20.5","chopping":"332-405","consensus_level":"medium","plddt":85.8553,"start":332,"end":405}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q92599","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q92599-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q92599-F1-predicted_aligned_error_v6.png","plddt_mean":76.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SEPTIN8","jax_strain_url":"https://www.jax.org/strain/search?query=SEPTIN8"},"sequence":{"accession":"Q92599","fasta_url":"https://rest.uniprot.org/uniprotkb/Q92599.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q92599/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q92599"}},"corpus_meta":[{"pmid":"19196426","id":"PMC_19196426","title":"Sept8 controls the binding of vesicle-associated membrane protein 2 to synaptophysin.","date":"2009","source":"Journal of neurochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19196426","citation_count":51,"is_preprint":false},{"pmid":"15116257","id":"PMC_15116257","title":"The novel human platelet septin SEPT8 is an interaction partner of SEPT4.","date":"2004","source":"Thrombosis and haemostasis","url":"https://pubmed.ncbi.nlm.nih.gov/15116257","citation_count":28,"is_preprint":false},{"pmid":"12023038","id":"PMC_12023038","title":"Human septin-septin interaction: CDCrel-1 partners with KIAA0202.","date":"2002","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/12023038","citation_count":27,"is_preprint":false},{"pmid":"12909369","id":"PMC_12909369","title":"Isolation of new splice isoforms, characterization and expression analysis of the human septin SEPT8 (KIAA0202).","date":"2003","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/12909369","citation_count":24,"is_preprint":false},{"pmid":"27084579","id":"PMC_27084579","title":"SEPT8 modulates β-amyloidogenic processing of APP by affecting the sorting and accumulation of BACE1.","date":"2016","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/27084579","citation_count":19,"is_preprint":false},{"pmid":"22649572","id":"PMC_22649572","title":"Septin 8 is an interaction partner and in vitro substrate of MK5.","date":"2012","source":"World journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22649572","citation_count":18,"is_preprint":false},{"pmid":"33483609","id":"PMC_33483609","title":"Sept8/SEPTIN8 involvement in cellular structure and kidney damage is identified by genetic mapping and a novel human tubule hypoxic model.","date":"2021","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/33483609","citation_count":13,"is_preprint":false},{"pmid":"15923366","id":"PMC_15923366","title":"Immunoreactivity of the septins SEPT4, SEPT5, and SEPT8 in the human eye.","date":"2005","source":"The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society","url":"https://pubmed.ncbi.nlm.nih.gov/15923366","citation_count":9,"is_preprint":false},{"pmid":"12098780","id":"PMC_12098780","title":"KIAA0202, a human septin family member, interacting with hPFTAIRE1.","date":"2002","source":"Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/12098780","citation_count":9,"is_preprint":false},{"pmid":"38051778","id":"PMC_38051778","title":"Palmitoylated Sept8-204 modulates learning and anxiety by regulating filopodia arborization and actin dynamics.","date":"2023","source":"Science signaling","url":"https://pubmed.ncbi.nlm.nih.gov/38051778","citation_count":7,"is_preprint":false},{"pmid":"33202444","id":"PMC_33202444","title":"Impaired Platelet Function in Sept8-Deficient Mice In Vitro.","date":"2020","source":"Thrombosis and haemostasis","url":"https://pubmed.ncbi.nlm.nih.gov/33202444","citation_count":7,"is_preprint":false},{"pmid":"17709200","id":"PMC_17709200","title":"Localization of septin 8 in murine retina, and spatiotemporal expression of septin 8 in a murine model of photoreceptor cell degeneration.","date":"2007","source":"Neuroscience letters","url":"https://pubmed.ncbi.nlm.nih.gov/17709200","citation_count":5,"is_preprint":false},{"pmid":"20544379","id":"PMC_20544379","title":"Cloning, overexpression, purification and preliminary characterization of human septin 8.","date":"2010","source":"The protein journal","url":"https://pubmed.ncbi.nlm.nih.gov/20544379","citation_count":2,"is_preprint":false},{"pmid":"38308620","id":"PMC_38308620","title":"Palmitoylation is required for Sept8-204 and Sept5 to form vesicle-like structure and colocalize with synaptophysin.","date":"2024","source":"Journal of cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/38308620","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8244,"output_tokens":2902,"usd":0.034131,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10203,"output_tokens":3299,"usd":0.066745,"stage2_stop_reason":"end_turn"},"total_usd":0.100876,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"Sept8 binds directly to VAMP2 (identified by yeast two-hybrid screening) and suppresses the interaction between VAMP2 and synaptophysin, thereby regulating SNARE complex availability at presynapses. Sept8 also forms a complex with syntaxin1A, and the Sept8-VAMP2 interaction is disrupted by SNAP-25.\",\n      \"method\": \"Yeast two-hybrid screening, co-immunoprecipitation, protein interaction assays in neuronal context\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus co-IP with functional interaction data, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"19196426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"SEPT8 interacts with SEPT4 (identified by yeast two-hybrid, confirmed by co-immunoprecipitation) in human platelets; both proteins localize surrounding α-granules and translocate to the platelet surface upon activation, suggesting a role in granular secretion.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, transmission electron microscopy, subcellular localization\",\n      \"journal\": \"Thrombosis and haemostasis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus TEM localization, single lab, two orthogonal methods\",\n      \"pmids\": [\"15116257\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"SEPT8 (KIAA0202) interacts with SEPT5 (CDCrel-1); the interaction was demonstrated by yeast two-hybrid, GST pull-down assay, and immunoprecipitation in the K-562 cell line.\",\n      \"method\": \"Yeast two-hybrid, GST pull-down, co-immunoprecipitation\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — three orthogonal methods (Y2H, GST pull-down, Co-IP) in single lab\",\n      \"pmids\": [\"12023038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"SEPT8 (KIAA0202) interacts with hPFTAIRE1, a Cdc2-related kinase localized in the cytoplasm; the interaction was identified by yeast two-hybrid and confirmed by immunoprecipitation.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation\",\n      \"journal\": \"Acta biochimica et biophysica Sinica\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Y2H plus single Co-IP, single lab, limited functional follow-up\",\n      \"pmids\": [\"12098780\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SEPT8 is a direct interaction partner and in vitro substrate of MAP kinase-activated protein kinase 5 (MK5); serine residues 242 and 271 on SEPT8 are phosphorylated by MK5 in vitro. MK5 and SEPT8 co-localize in the perinuclear area, cell protrusions, and with the vesicle marker synaptophysin.\",\n      \"method\": \"Yeast two-hybrid, GST pull-down, co-immunoprecipitation, FRET, in vitro kinase assay, peptide array, confocal microscopy\",\n      \"journal\": \"World journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with site mapping plus multiple orthogonal binding methods (GST pull-down, Co-IP, FRET), single lab\",\n      \"pmids\": [\"22649572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SEPT8 reduces BACE1 protein levels and thereby decreases soluble APPβ and Aβ generation in neuronal cells via a post-translational mechanism; a disease-associated SEPT8 transcript variant increases BACE1 half-life and promotes BACE1 localization in recycling endosomes, leading to elevated Aβ.\",\n      \"method\": \"SEPT8 knockdown/overexpression in neuronal cells, BACE1 half-life measurements, subcellular fractionation/localization of BACE1 to recycling endosomes, Aβ/sAPPβ quantification\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function and gain-of-function with defined molecular readouts (BACE1 half-life, endosomal localization, Aβ levels), single lab, multiple orthogonal methods\",\n      \"pmids\": [\"27084579\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Sept8-deficient mouse platelets show impaired integrin αIIbβ3 activation, defective α-granule exocytosis, reduced aggregation (particularly to convulxin/GPVI agonist), diminished fibrinogen binding and spreading, and reduced procoagulant activity and thrombin generation; δ-granule and lysosome exocytosis were unaffected.\",\n      \"method\": \"Sept8 knockout mouse model, flow cytometry, platelet aggregation assays, fibrinogen binding under static conditions, lactadherin binding, thrombin generation assay\",\n      \"journal\": \"Thrombosis and haemostasis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean knockout with multiple specific platelet function assays, selective phenotype (α-granule but not δ-granule), single lab but multiple orthogonal readouts\",\n      \"pmids\": [\"33202444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Sept8-204 (a brain-specific variant) is palmitoylated at Cys469, Cys470, and Cys472 by the palmitoyltransferase ZDHHC7; this modification is reversed by depalmitoylase PPT1. Palmitoylated Sept8-204 binds F-actin and promotes filopodia outgrowth in N2a cells and neurite arborization in hippocampal neurons. A non-palmitoylatable mutant (Sept8-204-3CA) fails to bind F-actin and does not promote morphological changes. Genetic deletion of Sept8, Sept8-204, or Zdhhc7 causes learning/memory deficits and anxiety-like behaviors in mice.\",\n      \"method\": \"Palmitoylation site mutagenesis (3CA mutant), acylation-RAC assay, F-actin co-sedimentation, live imaging in N2a cells and hippocampal neurons, Sept8 and Zdhhc7 knockout mice, behavioral assays\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis of palmitoylation sites, in vitro F-actin binding assay, identified writer (ZDHHC7) and eraser (PPT1), in vivo knockout behavioral phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"38051778\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Palmitoylation of Sept8-204 is required for Sept8-204/Sept5 co-expression to form small vesicle-like structures and co-localize with synaptophysin; expression of non-palmitoylatable Sept8-204-3CA or pharmacological inhibition with 2-BP produces large puncta that do not co-localize with synaptophysin. ZDHHC17 mediates Sept8-204 palmitoylation and PPT1 removes it, dynamically controlling vesicle-like structure size.\",\n      \"method\": \"Palmitoylation site mutagenesis (3CA mutant), pharmacological inhibition (2-BP), ZDHHC17/PPT1 knockout cells, fluorescence microscopy co-localization with synaptophysin\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis plus genetic loss-of-function of writers/erasers with morphological readout, single lab, single publication\",\n      \"pmids\": [\"38308620\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Recombinant human SEPT8 (conserved GTP-binding domain plus C-terminal domain) purified as a predominantly dimeric species in solution, with the preferred dimer interface involving the GTP-binding site as determined by homology modelling and analytical gel filtration/DLS.\",\n      \"method\": \"Recombinant protein expression, affinity chromatography, gel filtration, DLS, CD spectroscopy, homology modelling\",\n      \"journal\": \"The protein journal\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — biochemical characterization of purified protein, no functional mutagenesis or structural validation beyond homology model, single lab\",\n      \"pmids\": [\"20544379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SEPTIN8 localizes with acetyl-alpha tubulin in human proximal tubule cells under normal conditions; after hypoxia-induced damage, SEPTIN8 staining becomes diffuse and appears to re-localize with actin, indicating a role in cellular organization and structural response to environmental stress.\",\n      \"method\": \"Immunofluorescence localization in human proximal tubule cells, hypoxia in vitro model, co-staining with cytoskeletal markers\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single localization experiment, no functional loss-of-function linked to the localization change, single study\",\n      \"pmids\": [\"33483609\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SEPTIN8 is a GTP-binding cytoskeletal protein that functions at synapses and in vesicle trafficking by directly binding VAMP2 to regulate SNARE complex availability, forming complexes with SEPT4 and SEPT5, and undergoing palmitoylation (by ZDHHC7/ZDHHC17, reversed by PPT1) at a brain-specific splice variant (Sept8-204) to bind F-actin, promote filopodia/neurite outgrowth, and enable synaptophysin co-localization; it is also phosphorylated by MK5 at Ser242/Ser271, modulates BACE1 stability and endosomal sorting to control amyloid-β generation, and is required in platelets for integrin αIIbβ3 activation and α-granule exocytosis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SEPTIN8 is a GTP-binding cytoskeletal protein that operates at the interface of vesicle trafficking, regulated exocytosis, and the actin cytoskeleton, partnering with other septins and SNARE machinery to control membrane dynamics in neurons and platelets [#0, #6, #7]. At presynaptic terminals it binds VAMP2 directly and suppresses the VAMP2–synaptophysin interaction, thereby gating SNARE complex availability, an interaction modulated by SNAP-25 and accompanied by complex formation with syntaxin1A [#0]. SEPTIN8 assembles with other septins, including SEPT4 and SEPT5, the former in platelets where both surround α-granules and translocate to the surface upon activation [#1, #2]. In platelets, SEPTIN8 is selectively required for integrin αIIbβ3 activation and α-granule exocytosis but not δ-granule or lysosome release [#6]. A brain-specific variant, Sept8-204, is palmitoylated at Cys469/Cys470/Cys472 by ZDHHC7 and ZDHHC17 and depalmitoylated by PPT1; this lipid modification licenses F-actin binding, filopodia and neurite outgrowth, and Sept5-dependent formation of synaptophysin-positive vesicle-like structures, while non-palmitoylatable mutants fail in these functions, and in vivo loss of Sept8 or Zdhhc7 produces learning, memory, and anxiety phenotypes [#7, #8]. SEPTIN8 is a direct substrate of MK5, which phosphorylates it at Ser242 and Ser271 [#4], and it limits amyloid-β generation by reducing BACE1 levels post-translationally, with a disease-associated transcript variant extending BACE1 half-life and shifting it to recycling endosomes to elevate Aβ [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Establishing that SEPT8 physically associates with other septins defined it as a member of an oligomeric septin network rather than an isolated protein.\",\n      \"evidence\": \"Yeast two-hybrid, GST pull-down, and co-IP showing SEPT8–SEPT5 interaction in K-562 cells, plus an interaction with the kinase hPFTAIRE1\",\n      \"pmids\": [\"12023038\", \"12098780\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Stoichiometry and architecture of the SEPT8-containing complex not resolved\", \"Functional consequence of the hPFTAIRE1 interaction not established\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Linking SEPT8 to SEPT4 around platelet α-granules connected the protein to regulated secretion in a non-neuronal cell type.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal co-IP, and TEM localization in human platelets\",\n      \"pmids\": [\"15116257\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal role in secretion not tested at this stage\", \"Mechanism of granule association unknown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identifying VAMP2 as a direct binding partner positioned SEPT8 as a regulator of SNARE complex assembly at presynapses.\",\n      \"evidence\": \"Yeast two-hybrid screen plus co-IP and interaction assays showing SEPT8 suppresses VAMP2–synaptophysin binding and forms a complex with syntaxin1A\",\n      \"pmids\": [\"19196426\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Effect on actual neurotransmitter release not measured\", \"Whether SNAP-25 competition is physiologically regulated unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Biochemical characterization tested the oligomeric state of the SEPT8 GTP-binding module in isolation.\",\n      \"evidence\": \"Recombinant expression with gel filtration, DLS, CD, and homology modelling indicating a dimer via the GTP-binding interface\",\n      \"pmids\": [\"20544379\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No experimental structure beyond a homology model\", \"No mutagenesis validating the proposed dimer interface\", \"Relevance to native heteromeric filaments unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defining SEPT8 as an MK5 substrate introduced a post-translational regulatory input on the protein.\",\n      \"evidence\": \"Y2H, GST pull-down, co-IP, FRET, and in vitro kinase assay with peptide-array site mapping to Ser242/Ser271\",\n      \"pmids\": [\"22649572\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of Ser242/Ser271 phosphorylation in cells not determined\", \"Phosphorylation not demonstrated in vivo\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Connecting SEPT8 to BACE1 turnover and endosomal sorting linked it to amyloid-β generation.\",\n      \"evidence\": \"Knockdown/overexpression in neuronal cells with BACE1 half-life, recycling-endosome localization, and Aβ/sAPPβ readouts\",\n      \"pmids\": [\"27084579\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular interaction with BACE1 not shown\", \"In vivo relevance to Alzheimer pathology untested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"A knockout mouse established a selective, causal requirement for Sept8 in platelet integrin activation and α-granule exocytosis.\",\n      \"evidence\": \"Sept8 knockout platelets analyzed by flow cytometry, aggregation, fibrinogen/lactadherin binding, and thrombin generation assays\",\n      \"pmids\": [\"33202444\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism coupling Sept8 to αIIbβ3 inside-out signaling unresolved\", \"Why δ-granule and lysosome exocytosis are spared unexplained\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identifying palmitoylation of the brain-specific Sept8-204 variant as a switch for F-actin binding tied a defined lipid modification to neuronal morphogenesis and behavior.\",\n      \"evidence\": \"Palmitoylation-site mutagenesis (3CA), acyl-RAC, F-actin co-sedimentation, live imaging, and Sept8/Zdhhc7 knockout behavioral assays\",\n      \"pmids\": [\"38051778\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How palmitoylation structurally enables F-actin binding unknown\", \"Behavioral deficits not mechanistically traced to a single circuit\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extending the palmitoylation switch to vesicle-like structure formation showed it dynamically controls Sept8-204/Sept5 assembly and synaptophysin co-localization.\",\n      \"evidence\": \"3CA mutant, 2-BP inhibition, ZDHHC17/PPT1 knockout cells with synaptophysin co-localization microscopy\",\n      \"pmids\": [\"38308620\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity and cargo of the vesicle-like structures undefined\", \"Functional output for synaptic transmission not measured\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How SEPT8's septin-filament assembly, palmitoylation-driven actin coupling, SNARE regulation, and BACE1 sorting are integrated into a single mechanistic logic across neurons and platelets remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of native SEPT8-containing heterofilaments\", \"Unknown whether MK5 phosphorylation, palmitoylation, and partner binding are coordinately regulated\", \"Cell-type-specific determinants of SEPT8 function not defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [7, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [7, 10]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [0, 7]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"VAMP2\", \"STX1A\", \"SEPT4\", \"SEPT5\", \"MK5\", \"ZDHHC7\", \"ZDHHC17\", \"PPT1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}