{"gene":"SPPL2B","run_date":"2026-06-10T07:46:40","timeline":{"discoveries":[{"year":2006,"finding":"SPPL2b is a functional aspartyl intramembrane protease that catalyses intramembrane cleavage of tumour necrosis factor alpha (TNFα), a type II membrane-anchored protein, releasing the TNFα intracellular domain. SPPL2b localizes to the plasma membrane, distinct from SPPL2a (endosomes). The released intracellular domain triggers IL-12 expression in activated human dendritic cells, linking SPPL2b-mediated proteolysis to innate and adaptive immunity.","method":"Cellular co-expression assays, subcellular localization studies, functional readout (IL-12 production in dendritic cells)","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal functional assays in cells with defined substrates and cytokine readout, replicated concept across SPPL2a and SPPL2b, foundational mechanistic study","pmids":["16829952"],"is_preprint":false},{"year":2005,"finding":"SPPL2b is targeted through the secretory pathway to endosomal/lysosomal vesicles (not the ER), distinguishing it from SPP and SPPL3 which are ER-restricted. Mutation of the putative C-terminal active site aspartate (D/A mutation in the GXGD motif) of SPPL2b phenocopies sppl2b knockdown in zebrafish (erythrocyte accumulation in an enlarged caudal vein), establishing that its proteolytic activity is required for its in vivo function.","method":"Subcellular localization in cultured cells, antisense gripNA knockdown in zebrafish, active-site mutagenesis (D/A mutations)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — active-site mutagenesis combined with in vivo knockdown phenocopies and localization studies in a single rigorous study","pmids":["15998642"],"is_preprint":false},{"year":2007,"finding":"SPPL2b (and SPPL2a, but not SPP or SPPL3) mediates intramembrane proteolysis of Bri2 (Itm2b), a type II transmembrane protein associated with familial British and Danish dementia. This cleavage requires prior ectodomain shedding by ADAM10, producing a membrane-bound N-terminal fragment that is then cleaved by SPPL2b to generate an intracellular domain and a secreted low molecular weight C-terminal peptide.","method":"Co-expression of all SPP/SPPL family members and loss-of-function variants with Bri2 substrate; Western blot detection of cleavage products","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic comparison of all family members with loss-of-function variants, identifying SPPL2a/b as selective mediators; multiple orthogonal approaches","pmids":["17965014"],"is_preprint":false},{"year":2008,"finding":"Efficient SPPL2b-dependent intramembrane proteolysis of Bri2 requires: (1) prior ectodomain shedding by ADAM10 (ectodomain length negatively correlates with cleavage efficiency); (2) primary sequence determinants within the intracellular domain and transmembrane domain; (3) short luminal juxtamembrane sequences. Bri3 (Itm2c), a close homologue of Bri2, fails to be processed by SPPL2b even when a short membrane stub is generated, indicating that shedding alone is insufficient and substrate-intrinsic sequence determinants are required.","method":"Serial deletions, domain swapping analysis, cellular co-expression assays with SPPL2b","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — systematic domain swap and deletion analyses in cells with defined negative controls (Bri3), single lab but multiple orthogonal mutagenesis approaches","pmids":["19114711"],"is_preprint":false},{"year":2011,"finding":"The α-helical content of the Bri2 transmembrane domain (TMD) is a key determinant of SPPL2b cleavage efficiency: high α-helical content reduces intramembrane cleavage by SPPL2b. A single conserved intramembrane glycine residue significantly affects both the secondary structure of the Bri2 TMD and its intramembrane processing by SPPL2b, whereas the GXXXG dimerization motif has only minor influence.","method":"Site-directed mutagenesis of Bri2 TMD residues, circular dichroism spectroscopy, cellular SPPL2b cleavage assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis combined with circular dichroism structural readout and functional cleavage assay, two orthogonal methods in one study","pmids":["22194595"],"is_preprint":false},{"year":2022,"finding":"SPPL2b (together with SPPL2a) can process tail-anchored SNARE proteins (VAMP1, VAMP2, VAMP3, VAMP4) in addition to type II membrane proteins. Loss of SPPL2a/b activity in double-knockout mice leads to accumulation of VAMP1-4 in a cell-type- and tissue-dependent manner, identifying these SNAREs as in vivo substrates and implicating SPPL2a/b in controlling cellular levels of these membrane fusion regulators.","method":"Cellular co-expression screening of 18 SNARE proteins, SPPL2a/b inhibition in cell lines, VAMP1-4 protein level analysis in tissues and primary cells of SPPL2a/b double-deficient (dKO) mice","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo validation in dKO mice combined with cellular inhibition assays across multiple cell lines and tissues, multiple orthogonal methods","pmids":["36047592"],"is_preprint":false},{"year":2024,"finding":"An in vitro cleavage assay for SPPL2b using TNFα as substrate was developed, demonstrating that SPPL2b activity in vitro recapitulates mechanistic principles observed in cells, including the same cleavage sites and consecutive turnover of the TNFα transmembrane domain. The assay works with separately isolated protease and substrate, and was used to characterize the influence of phospholipids, detergent, and cholesterol on SPPL2b activity, and to biochemically characterize a selective small molecule inhibitor (SPL-707) of SPPL2a/b.","method":"In vitro protease cleavage assay with purified SPPL2b and TNFα substrate, lipid/detergent titration, small molecule inhibitor characterization","journal":"Chemico-biological interactions","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted in vitro biochemical assay with purified components, functional validation against cellular context, inhibitor biochemistry; single lab","pmids":["38636792"],"is_preprint":false}],"current_model":"SPPL2b is a GXGD-type aspartyl intramembrane protease localized to endosomal/lysosomal compartments and the plasma membrane that mediates regulated intramembrane proteolysis of type II-oriented transmembrane proteins (including TNFα and Bri2/Itm2b) and tail-anchored SNARE proteins (VAMP1-4); efficient cleavage of type II substrates requires prior ectodomain shedding and is governed by substrate TMD α-helical content and specific intracellular/juxtamembrane sequence determinants, while its catalytic activity—dependent on the critical GXGD active-site aspartate—is required for immune signaling (TNFα ICD-driven IL-12 production in dendritic cells) and regulation of SNARE homeostasis in vivo."},"narrative":{"mechanistic_narrative":"SPPL2b is a GXGD-type aspartyl intramembrane protease that mediates regulated intramembrane proteolysis of type II-oriented transmembrane substrates and tail-anchored SNARE proteins, linking membrane proteolysis to immune signaling and membrane protein homeostasis [PMID:16829952, PMID:36047592]. It is delivered through the secretory pathway to endosomal/lysosomal vesicles and the plasma membrane—distinct from the ER-restricted family members SPP and SPPL3—and its catalytic activity depends on the conserved C-terminal active-site aspartate within the GXGD motif, which is required for its in vivo function [PMID:15998642, PMID:16829952]. SPPL2b cleaves TNFα to release an intracellular domain that drives IL-12 expression in activated dendritic cells, and it processes Bri2/Itm2b only after prior ectodomain shedding by ADAM10, generating an intracellular domain and a secreted C-terminal peptide [PMID:16829952, PMID:17965014]. Efficient cleavage is governed by substrate-intrinsic determinants: shed ectodomain length, juxtamembrane and intracellular sequences, and the α-helical content of the transmembrane domain, where a single conserved intramembrane glycine modulates both TMD secondary structure and processing efficiency [PMID:19114711, PMID:22194595]. Beyond type II substrates, SPPL2b processes the SNARE proteins VAMP1–4, controlling their cellular levels in a tissue-dependent manner in vivo [PMID:36047592]. The protease has been reconstituted in vitro with purified components, recapitulating cellular cleavage sites and TMD turnover and enabling characterization of lipid and cholesterol dependence and a selective small-molecule inhibitor [PMID:38636792].","teleology":[{"year":2005,"claim":"Establishing where SPPL2b acts and whether its predicted catalytic residue is functionally required addressed whether it is a genuine, compartment-specific protease.","evidence":"Subcellular localization in cultured cells plus active-site (GXGD D/A) mutagenesis and antisense knockdown phenocopy in zebrafish","pmids":["15998642"],"confidence":"High","gaps":["The physiological substrate underlying the zebrafish phenotype was not defined","No direct biochemical demonstration of proteolysis at this stage"]},{"year":2006,"claim":"Identifying TNFα as a substrate and linking the released intracellular domain to cytokine output showed SPPL2b is a functional protease coupled to immune signaling.","evidence":"Cellular co-expression cleavage assays with localization studies and IL-12 readout in human dendritic cells","pmids":["16829952"],"confidence":"High","gaps":["Did not establish the requirement for prior ectodomain shedding","In vivo relevance of TNFα ICD signaling not tested"]},{"year":2007,"claim":"Systematic comparison across the SPP/SPPL family defined SPPL2a/b as selective for Bri2 and revealed a sequential shedding-then-intramembrane-cleavage mechanism.","evidence":"Co-expression of all family members and loss-of-function variants with Bri2; Western blot of cleavage products","pmids":["17965014"],"confidence":"High","gaps":["Did not resolve which substrate sequence features dictate selectivity","Functional consequences of Bri2 ICD release not addressed"]},{"year":2008,"claim":"Defining the substrate determinants of Bri2 processing clarified why shedding alone is insufficient and explained substrate selectivity within close homologues.","evidence":"Serial deletions and domain swapping with Bri3 as negative control in cellular SPPL2b assays","pmids":["19114711"],"confidence":"High","gaps":["The structural basis for sequence preference was not resolved","Mechanism of TMD recognition by the protease not defined"]},{"year":2011,"claim":"Connecting TMD secondary structure to cleavage efficiency provided a biophysical principle governing intramembrane proteolysis by SPPL2b.","evidence":"Site-directed mutagenesis of Bri2 TMD with circular dichroism and cellular cleavage assays","pmids":["22194595"],"confidence":"High","gaps":["No structure of the protease–substrate complex","Whether the same α-helical principle governs other substrates untested at the time"]},{"year":2022,"claim":"Identifying VAMP1–4 as substrates expanded SPPL2b's role beyond type II proteins to control of SNARE-mediated membrane fusion regulators in vivo.","evidence":"SNARE co-expression screen, inhibition in cell lines, and VAMP level analysis in SPPL2a/b double-knockout mice","pmids":["36047592"],"confidence":"High","gaps":["Functional consequences of VAMP accumulation for membrane fusion not fully resolved","Relative contribution of SPPL2a versus SPPL2b not separated in dKO"]},{"year":2024,"claim":"Reconstituting SPPL2b activity with purified components established a defined biochemical system recapitulating cellular cleavage and enabling inhibitor and lipid-dependence studies.","evidence":"In vitro protease assay with purified SPPL2b and TNFα, lipid/cholesterol titration, and characterization of the inhibitor SPL-707","pmids":["38636792"],"confidence":"High","gaps":["No high-resolution structure derived from the reconstituted system","Lipid regulation in native membranes versus detergent not fully equated"]},{"year":null,"claim":"How SPPL2b substrate recognition is structurally encoded and how its proteolysis of distinct substrate classes is integrated into physiology remain open.","evidence":"","pmids":[],"confidence":"High","gaps":["No experimentally determined structure of SPPL2b or its protease–substrate complex","Physiological roles of SNARE versus type II substrate processing not disentangled in vivo"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,5,6]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,1,6]}],"localization":[{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[1]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[1]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,2,5]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0]}],"complexes":[],"partners":["TNF","ITM2B","ADAM10","VAMP1","VAMP2","VAMP3","VAMP4"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8TCT7","full_name":"Signal peptide peptidase-like 2B","aliases":["Intramembrane protease 4","IMP-4","Presenilin homologous protein 4","PSH4","Presenilin-like protein 1"],"length_aa":592,"mass_kda":64.6,"function":"Intramembrane-cleaving aspartic protease (I-CLiP) that cleaves type II membrane signal peptides in the hydrophobic plane of the membrane. Functions in ITM2B and TNF processing (PubMed:16829951, PubMed:16829952, PubMed:17965014, PubMed:19114711, PubMed:22194595). Catalyzes the intramembrane cleavage of the anchored fragment of shed TNF (TNF), which promotes the release of the intracellular domain (ICD) for signaling to the nucleus (PubMed:16829951, PubMed:16829952). May play a role in the regulation of innate and adaptive immunity (PubMed:16829952). Catalyzes the intramembrane cleavage of the simian foamy virus processed leader peptide gp18 of the envelope glycoprotein gp130 dependently of prior ectodomain shedding by furin or furin-like proprotein convertase (PC)-mediated cleavage proteolysis (PubMed:23132852)","subcellular_location":"Cell membrane; Golgi apparatus membrane; Lysosome membrane; Endosome membrane; Membrane","url":"https://www.uniprot.org/uniprotkb/Q8TCT7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SPPL2B","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":74,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CANX","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/SPPL2B","total_profiled":1310},"omim":[{"mim_id":"608239","title":"SIGNAL PEPTIDE PEPTIDASE-LIKE 2B; SPPL2B","url":"https://www.omim.org/entry/608239"},{"mim_id":"603904","title":"INTEGRAL MEMBRANE PROTEIN 2B; ITM2B","url":"https://www.omim.org/entry/603904"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Plasma membrane","reliability":"Approved"},{"location":"Centrosome","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SPPL2B"},"hgnc":{"alias_symbol":["IMP4","PSL1","KIAA1532"],"prev_symbol":[]},"alphafold":{"accession":"Q8TCT7","domains":[{"cath_id":"3.50.30.30","chopping":"29-165","consensus_level":"high","plddt":85.9964,"start":29,"end":165},{"cath_id":"-","chopping":"174-207_220-284_293-501","consensus_level":"medium","plddt":85.7526,"start":174,"end":501}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TCT7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TCT7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TCT7-F1-predicted_aligned_error_v6.png","plddt_mean":76.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SPPL2B","jax_strain_url":"https://www.jax.org/strain/search?query=SPPL2B"},"sequence":{"accession":"Q8TCT7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8TCT7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8TCT7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TCT7"}},"corpus_meta":[{"pmid":"16829952","id":"PMC_16829952","title":"SPPL2a and SPPL2b promote intramembrane proteolysis of TNFalpha in activated dendritic cells to trigger IL-12 production.","date":"2006","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/16829952","citation_count":168,"is_preprint":false},{"pmid":"17965014","id":"PMC_17965014","title":"Regulated intramembrane proteolysis of Bri2 (Itm2b) by ADAM10 and SPPL2a/SPPL2b.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17965014","citation_count":137,"is_preprint":false},{"pmid":"15998642","id":"PMC_15998642","title":"Differential localization and identification of a critical aspartate suggest non-redundant proteolytic functions of the presenilin homologues SPPL2b and SPPL3.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15998642","citation_count":76,"is_preprint":false},{"pmid":"19114711","id":"PMC_19114711","title":"Substrate requirements for SPPL2b-dependent regulated intramembrane proteolysis.","date":"2008","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19114711","citation_count":52,"is_preprint":false},{"pmid":"22194595","id":"PMC_22194595","title":"The α-helical content of the transmembrane domain of the British dementia protein-2 (Bri2) determines its processing by signal peptide peptidase-like 2b (SPPL2b).","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22194595","citation_count":36,"is_preprint":false},{"pmid":"9364205","id":"PMC_9364205","title":"Association of a murine chromosome 9 locus (Psl1) with susceptibility to mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate.","date":"1997","source":"Molecular carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/9364205","citation_count":35,"is_preprint":false},{"pmid":"26469904","id":"PMC_26469904","title":"STK39, But Not BST1, HLA-DQB1, and SPPL2B Polymorphism, Is Associated With Han-Chinese Parkinson's Disease in Taiwan.","date":"2015","source":"Medicine","url":"https://pubmed.ncbi.nlm.nih.gov/26469904","citation_count":17,"is_preprint":false},{"pmid":"36047592","id":"PMC_36047592","title":"The intramembrane proteases SPPL2a and SPPL2b regulate the homeostasis of selected SNARE proteins.","date":"2022","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/36047592","citation_count":12,"is_preprint":false},{"pmid":"11746828","id":"PMC_11746828","title":"Confirmation of the mapping of a 12-O-tetradecanoylphorbol-13-acetate promotion susceptibility locus, Psl1, to distal mouse chromosome 9.","date":"2001","source":"Molecular carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/11746828","citation_count":8,"is_preprint":false},{"pmid":"3303077","id":"PMC_3303077","title":"Identification of the minimal replication region of the multicopy Streptomyces plasmid pSL1.","date":"1987","source":"Plasmid","url":"https://pubmed.ncbi.nlm.nih.gov/3303077","citation_count":7,"is_preprint":false},{"pmid":"3615660","id":"PMC_3615660","title":"Characterization of the incompatibility region of Streptomyces plasmid pSL1.","date":"1987","source":"Plasmid","url":"https://pubmed.ncbi.nlm.nih.gov/3615660","citation_count":5,"is_preprint":false},{"pmid":"33445784","id":"PMC_33445784","title":"The S. pombe CDK5 Orthologue Pef1 Cooperates with Three Cyclins, Clg1, Pas1 and Psl1, to Promote Pre-Meiotic DNA Replication.","date":"2021","source":"Biomolecules","url":"https://pubmed.ncbi.nlm.nih.gov/33445784","citation_count":3,"is_preprint":false},{"pmid":"38636792","id":"PMC_38636792","title":"In vitro cleavage of tumor necrosis factor α (TNFα) by Signal-Peptide-Peptidase-like 2b (SPPL2b) resembles mechanistic principles observed in the cellular context.","date":"2024","source":"Chemico-biological interactions","url":"https://pubmed.ncbi.nlm.nih.gov/38636792","citation_count":2,"is_preprint":false},{"pmid":"39800020","id":"PMC_39800020","title":"Comparative genomic analysis of Fusarium oxysporum f. sp. lycopersici reveals telomeric duplications of a lineage-specific region carrying SIX8 and PSL1 and genome-wide expansion of Foxy transposable elements.","date":"2025","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/39800020","citation_count":2,"is_preprint":false},{"pmid":"24700353","id":"PMC_24700353","title":"Fine mapping reveals that promotion susceptibility locus 1 (Psl1) is a compound locus with multiple genes that modify susceptibility to skin tumor development.","date":"2014","source":"G3 (Bethesda, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/24700353","citation_count":1,"is_preprint":false},{"pmid":"41239050","id":"PMC_41239050","title":"Selenite versus selenium nanoparticles in foliar application: enhancing alfalfa (Medicago sativa L.) and Bacillus subtilis PSL-1 mediated remediation of pyrene-contaminated soil.","date":"2025","source":"Environmental geochemistry and health","url":"https://pubmed.ncbi.nlm.nih.gov/41239050","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9512,"output_tokens":2213,"usd":0.030866,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9219,"output_tokens":2608,"usd":0.055648,"stage2_stop_reason":"end_turn"},"total_usd":0.086514,"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\": 2006,\n      \"finding\": \"SPPL2b is a functional aspartyl intramembrane protease that catalyses intramembrane cleavage of tumour necrosis factor alpha (TNFα), a type II membrane-anchored protein, releasing the TNFα intracellular domain. SPPL2b localizes to the plasma membrane, distinct from SPPL2a (endosomes). The released intracellular domain triggers IL-12 expression in activated human dendritic cells, linking SPPL2b-mediated proteolysis to innate and adaptive immunity.\",\n      \"method\": \"Cellular co-expression assays, subcellular localization studies, functional readout (IL-12 production in dendritic cells)\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal functional assays in cells with defined substrates and cytokine readout, replicated concept across SPPL2a and SPPL2b, foundational mechanistic study\",\n      \"pmids\": [\"16829952\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"SPPL2b is targeted through the secretory pathway to endosomal/lysosomal vesicles (not the ER), distinguishing it from SPP and SPPL3 which are ER-restricted. Mutation of the putative C-terminal active site aspartate (D/A mutation in the GXGD motif) of SPPL2b phenocopies sppl2b knockdown in zebrafish (erythrocyte accumulation in an enlarged caudal vein), establishing that its proteolytic activity is required for its in vivo function.\",\n      \"method\": \"Subcellular localization in cultured cells, antisense gripNA knockdown in zebrafish, active-site mutagenesis (D/A mutations)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — active-site mutagenesis combined with in vivo knockdown phenocopies and localization studies in a single rigorous study\",\n      \"pmids\": [\"15998642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SPPL2b (and SPPL2a, but not SPP or SPPL3) mediates intramembrane proteolysis of Bri2 (Itm2b), a type II transmembrane protein associated with familial British and Danish dementia. This cleavage requires prior ectodomain shedding by ADAM10, producing a membrane-bound N-terminal fragment that is then cleaved by SPPL2b to generate an intracellular domain and a secreted low molecular weight C-terminal peptide.\",\n      \"method\": \"Co-expression of all SPP/SPPL family members and loss-of-function variants with Bri2 substrate; Western blot detection of cleavage products\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic comparison of all family members with loss-of-function variants, identifying SPPL2a/b as selective mediators; multiple orthogonal approaches\",\n      \"pmids\": [\"17965014\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Efficient SPPL2b-dependent intramembrane proteolysis of Bri2 requires: (1) prior ectodomain shedding by ADAM10 (ectodomain length negatively correlates with cleavage efficiency); (2) primary sequence determinants within the intracellular domain and transmembrane domain; (3) short luminal juxtamembrane sequences. Bri3 (Itm2c), a close homologue of Bri2, fails to be processed by SPPL2b even when a short membrane stub is generated, indicating that shedding alone is insufficient and substrate-intrinsic sequence determinants are required.\",\n      \"method\": \"Serial deletions, domain swapping analysis, cellular co-expression assays with SPPL2b\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic domain swap and deletion analyses in cells with defined negative controls (Bri3), single lab but multiple orthogonal mutagenesis approaches\",\n      \"pmids\": [\"19114711\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The α-helical content of the Bri2 transmembrane domain (TMD) is a key determinant of SPPL2b cleavage efficiency: high α-helical content reduces intramembrane cleavage by SPPL2b. A single conserved intramembrane glycine residue significantly affects both the secondary structure of the Bri2 TMD and its intramembrane processing by SPPL2b, whereas the GXXXG dimerization motif has only minor influence.\",\n      \"method\": \"Site-directed mutagenesis of Bri2 TMD residues, circular dichroism spectroscopy, cellular SPPL2b cleavage assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis combined with circular dichroism structural readout and functional cleavage assay, two orthogonal methods in one study\",\n      \"pmids\": [\"22194595\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SPPL2b (together with SPPL2a) can process tail-anchored SNARE proteins (VAMP1, VAMP2, VAMP3, VAMP4) in addition to type II membrane proteins. Loss of SPPL2a/b activity in double-knockout mice leads to accumulation of VAMP1-4 in a cell-type- and tissue-dependent manner, identifying these SNAREs as in vivo substrates and implicating SPPL2a/b in controlling cellular levels of these membrane fusion regulators.\",\n      \"method\": \"Cellular co-expression screening of 18 SNARE proteins, SPPL2a/b inhibition in cell lines, VAMP1-4 protein level analysis in tissues and primary cells of SPPL2a/b double-deficient (dKO) mice\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo validation in dKO mice combined with cellular inhibition assays across multiple cell lines and tissues, multiple orthogonal methods\",\n      \"pmids\": [\"36047592\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"An in vitro cleavage assay for SPPL2b using TNFα as substrate was developed, demonstrating that SPPL2b activity in vitro recapitulates mechanistic principles observed in cells, including the same cleavage sites and consecutive turnover of the TNFα transmembrane domain. The assay works with separately isolated protease and substrate, and was used to characterize the influence of phospholipids, detergent, and cholesterol on SPPL2b activity, and to biochemically characterize a selective small molecule inhibitor (SPL-707) of SPPL2a/b.\",\n      \"method\": \"In vitro protease cleavage assay with purified SPPL2b and TNFα substrate, lipid/detergent titration, small molecule inhibitor characterization\",\n      \"journal\": \"Chemico-biological interactions\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted in vitro biochemical assay with purified components, functional validation against cellular context, inhibitor biochemistry; single lab\",\n      \"pmids\": [\"38636792\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SPPL2b is a GXGD-type aspartyl intramembrane protease localized to endosomal/lysosomal compartments and the plasma membrane that mediates regulated intramembrane proteolysis of type II-oriented transmembrane proteins (including TNFα and Bri2/Itm2b) and tail-anchored SNARE proteins (VAMP1-4); efficient cleavage of type II substrates requires prior ectodomain shedding and is governed by substrate TMD α-helical content and specific intracellular/juxtamembrane sequence determinants, while its catalytic activity—dependent on the critical GXGD active-site aspartate—is required for immune signaling (TNFα ICD-driven IL-12 production in dendritic cells) and regulation of SNARE homeostasis in vivo.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SPPL2b is a GXGD-type aspartyl intramembrane protease that mediates regulated intramembrane proteolysis of type II-oriented transmembrane substrates and tail-anchored SNARE proteins, linking membrane proteolysis to immune signaling and membrane protein homeostasis [#0, #5]. It is delivered through the secretory pathway to endosomal/lysosomal vesicles and the plasma membrane—distinct from the ER-restricted family members SPP and SPPL3—and its catalytic activity depends on the conserved C-terminal active-site aspartate within the GXGD motif, which is required for its in vivo function [#1, #0]. SPPL2b cleaves TNFα to release an intracellular domain that drives IL-12 expression in activated dendritic cells, and it processes Bri2/Itm2b only after prior ectodomain shedding by ADAM10, generating an intracellular domain and a secreted C-terminal peptide [#0, #2]. Efficient cleavage is governed by substrate-intrinsic determinants: shed ectodomain length, juxtamembrane and intracellular sequences, and the α-helical content of the transmembrane domain, where a single conserved intramembrane glycine modulates both TMD secondary structure and processing efficiency [#3, #4]. Beyond type II substrates, SPPL2b processes the SNARE proteins VAMP1–4, controlling their cellular levels in a tissue-dependent manner in vivo [#5]. The protease has been reconstituted in vitro with purified components, recapitulating cellular cleavage sites and TMD turnover and enabling characterization of lipid and cholesterol dependence and a selective small-molecule inhibitor [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Establishing where SPPL2b acts and whether its predicted catalytic residue is functionally required addressed whether it is a genuine, compartment-specific protease.\",\n      \"evidence\": \"Subcellular localization in cultured cells plus active-site (GXGD D/A) mutagenesis and antisense knockdown phenocopy in zebrafish\",\n      \"pmids\": [\"15998642\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The physiological substrate underlying the zebrafish phenotype was not defined\", \"No direct biochemical demonstration of proteolysis at this stage\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identifying TNFα as a substrate and linking the released intracellular domain to cytokine output showed SPPL2b is a functional protease coupled to immune signaling.\",\n      \"evidence\": \"Cellular co-expression cleavage assays with localization studies and IL-12 readout in human dendritic cells\",\n      \"pmids\": [\"16829952\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish the requirement for prior ectodomain shedding\", \"In vivo relevance of TNFα ICD signaling not tested\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Systematic comparison across the SPP/SPPL family defined SPPL2a/b as selective for Bri2 and revealed a sequential shedding-then-intramembrane-cleavage mechanism.\",\n      \"evidence\": \"Co-expression of all family members and loss-of-function variants with Bri2; Western blot of cleavage products\",\n      \"pmids\": [\"17965014\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve which substrate sequence features dictate selectivity\", \"Functional consequences of Bri2 ICD release not addressed\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defining the substrate determinants of Bri2 processing clarified why shedding alone is insufficient and explained substrate selectivity within close homologues.\",\n      \"evidence\": \"Serial deletions and domain swapping with Bri3 as negative control in cellular SPPL2b assays\",\n      \"pmids\": [\"19114711\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The structural basis for sequence preference was not resolved\", \"Mechanism of TMD recognition by the protease not defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Connecting TMD secondary structure to cleavage efficiency provided a biophysical principle governing intramembrane proteolysis by SPPL2b.\",\n      \"evidence\": \"Site-directed mutagenesis of Bri2 TMD with circular dichroism and cellular cleavage assays\",\n      \"pmids\": [\"22194595\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of the protease–substrate complex\", \"Whether the same α-helical principle governs other substrates untested at the time\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identifying VAMP1–4 as substrates expanded SPPL2b's role beyond type II proteins to control of SNARE-mediated membrane fusion regulators in vivo.\",\n      \"evidence\": \"SNARE co-expression screen, inhibition in cell lines, and VAMP level analysis in SPPL2a/b double-knockout mice\",\n      \"pmids\": [\"36047592\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequences of VAMP accumulation for membrane fusion not fully resolved\", \"Relative contribution of SPPL2a versus SPPL2b not separated in dKO\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Reconstituting SPPL2b activity with purified components established a defined biochemical system recapitulating cellular cleavage and enabling inhibitor and lipid-dependence studies.\",\n      \"evidence\": \"In vitro protease assay with purified SPPL2b and TNFα, lipid/cholesterol titration, and characterization of the inhibitor SPL-707\",\n      \"pmids\": [\"38636792\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No high-resolution structure derived from the reconstituted system\", \"Lipid regulation in native membranes versus detergent not fully equated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How SPPL2b substrate recognition is structurally encoded and how its proteolysis of distinct substrate classes is integrated into physiology remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No experimentally determined structure of SPPL2b or its protease–substrate complex\", \"Physiological roles of SNARE versus type II substrate processing not disentangled in vivo\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 5, 6]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 1, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2, 5]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"TNF\", \"ITM2B\", \"ADAM10\", \"VAMP1\", \"VAMP2\", \"VAMP3\", \"VAMP4\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}