{"gene":"SPPL2A","run_date":"2026-04-28T20:42:08","timeline":{"discoveries":[{"year":2004,"finding":"SPPL2a is a polytopic membrane protein belonging to the GxGD-type aspartyl intramembrane protease family (SPP/SPPL family); its N-terminal extension is located in the extracellular space and is modified with N-glycans; the hydrophilic loops containing the catalytic residues face the exoplasm and the C-terminus faces the cytosol, placing it in opposite orientation to presenilins and predicting it cleaves type II-oriented substrate peptides.","method":"Comparative topology analysis using epitope-tagged constructs, glycosylation mapping, and protease protection assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal biochemical methods in single rigorous study, foundational topology paper","pmids":["15385547"],"is_preprint":false},{"year":2006,"finding":"SPPL2a is sorted to endosomes and functions as an active intramembrane protease that catalyses intramembrane cleavage of tumour necrosis factor alpha (TNFα), promoting release of the TNFα intracellular domain (ICD), which triggers IL-12 expression in activated human dendritic cells.","method":"Overexpression and catalytic-dead mutant constructs, subcellular fractionation/immunofluorescence for localization, reporter assays for ICD release and IL-12 induction in primary human dendritic cells","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1-2 — functional protease activity demonstrated with loss-of-function mutants, localization defined, physiological readout in primary cells; independently replicated","pmids":["16829952"],"is_preprint":false},{"year":2007,"finding":"SPPL2a mediates intramembrane cleavage of Fas ligand (FasL) in T-cells after prior ectodomain shedding by ADAM10; SPPL2a cleavage liberates the FasL intracellular domain (ICD), which translocates to the nucleus and inhibits gene transcription.","method":"Overexpression and dominant-negative SPPL2a in T-cell lines, immunoprecipitation, subcellular fractionation, luciferase transcription reporter assays; endogenous FasL processing validated","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 — reciprocal biochemical validation with endogenous substrate, functional nuclear ICD readout, two-protease epistasis established","pmids":["17557115"],"is_preprint":false},{"year":2007,"finding":"SPPL2a and SPPL2b, but not SPP or SPPL3, mediate intramembrane proteolysis of Bri2 (Itm2b) N-terminal fragment generated after ADAM10 ectodomain shedding, producing an intracellular domain and a secreted low-molecular-weight C-terminal peptide.","method":"Expression of all SPP/SPPL family members and catalytic-dead variants, co-transfection with Bri2 constructs, immunoprecipitation, Western blot detection of cleavage products","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — systematic comparison across all family members with loss-of-function controls, multiple cleavage products characterized","pmids":["17965014"],"is_preprint":false},{"year":2011,"finding":"Endogenous SPPL2a localises to lysosomal/late endosomal membranes. Lysosomal targeting is mediated by a canonical tyrosine-based sorting motif (YXXø at position Y498) in its cytosolic C-terminal tail, which is both necessary and sufficient to direct SPPL2a to lysosomes/late endosomes and distinguishes it from its plasma-membrane-localised homologue SPPL2b.","method":"Immunofluorescence of endogenous SPPL2a, chimeric SPPL2a/SPPL2b constructs with point mutations in sorting motif, live-cell imaging","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 2 — direct localization experiment with mutagenesis of sorting motif, functional consequence for substrate access demonstrated","pmids":["21896273"],"is_preprint":false},{"year":2012,"finding":"SPPL2a is the intramembrane protease responsible for cleavage of the invariant chain (CD74) N-terminal fragment (NTF) in lysosomes/late endosomes of B cells. Loss of SPPL2a in mice causes CD74 NTF accumulation that severely impairs endosomal membrane trafficking, reduces surface BAFF-R expression, causes MHCII accumulation, and leads to a block in B cell maturation at the T1 stage; all these defects are rescued by additional CD74 knockout.","method":"SPPL2a−/− mouse model, flow cytometry, Western blot, immunofluorescence, endocytic trafficking assays, genetic epistasis (CD74/SPPL2a double KO rescue)","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — in vivo KO with defined mechanistic phenotype and genetic rescue experiment; independently replicated by two concurrent papers","pmids":["23267015"],"is_preprint":false},{"year":2012,"finding":"SPPL2a deficiency blocks proteolytic processing of CD74 MHC II invariant chain in B cells and CD8-negative dendritic cells, causing build-up of the p8 cathepsin-S product and interfering with earlier steps in CD74 endosomal retention. Loss of SPPL2a phenocopies BAFF deficiency; B cell survival is restored by BCL2 overexpression but not by BAFF.","method":"ENU mutagenesis screen (chompB mouse), positional cloning of Sppl2a mutation, proteomic identification of CD74 as substrate, flow cytometry, Western blot","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — unbiased genetic screen identifying SPPL2a, proteomics-validated substrate, multiple phenotypic readouts; concurrent independent replication","pmids":["23267013"],"is_preprint":false},{"year":2012,"finding":"SPPL2a deficiency causes dramatic accumulation of the CD74 p8 NTF in B cells and CD8-negative dendritic cells, reduces surface IgM, IgD, and BAFF-R, and leads to a profound humoral immunodeficiency. The final step of the CD74-MHCII lysosomal processing pathway requires SPPL2a.","method":"Inactivating point mutation knock-in mouse, flow cytometry, Western blot, bone marrow transfer experiments","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — independent replication of CD74 substrate finding with distinct mouse model and orthogonal readouts","pmids":["23267016"],"is_preprint":false},{"year":2013,"finding":"SPPL2a is critical for tooth enamel formation; Sppl2a−/− mice exhibit defective enamel mineralisation and impaired maturation-stage ameloblast function with delayed and incomplete resorption of the proteinaceous enamel matrix, establishing a role for SPPL2a-mediated intramembrane proteolysis in ameloblast cellular homeostasis.","method":"Sppl2a−/− mouse model, micro-CT, histology, electron microscopy, immunohistochemistry for SPPL2a in enamel epithelium","journal":"Journal of bone and mineral research","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo KO with defined tissue phenotype, but molecular substrate in ameloblasts not yet identified","pmids":["23426979"],"is_preprint":false},{"year":2014,"finding":"SPPL2a, and to a lesser extent SPPL2b, mediate intramembrane proteolysis of the frontotemporal lobar degeneration risk factor TMEM106B, generating and then rapidly degrading a small intracellular domain from the lysosome-localised N-terminal fragment of TMEM106B. TMEM106A, a paralogue, is not a specific SPPL2a substrate.","method":"Co-expression of SPPL2a/b with TMEM106B in cell lines, catalytic-dead mutant controls, lysosomal protease inhibitors, Western blot detection of NTF and ICD","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — catalytic-dead controls and paralog specificity established, single lab","pmids":["24872421"],"is_preprint":false},{"year":2014,"finding":"SPPL2a-mediated CD74 NTF processing is conserved in human B cells; lymphoblastoid cell lines from patients with a homozygous chromosomal microdeletion disrupting the SPPL2a locus lack SPPL2a protein and accumulate CD74 NTF comparably to Sppl2a−/− mouse B cells.","method":"Patient-derived lymphoblastoid cell lines with genomic SPPL2a deletion, Western blot for SPPL2a and CD74 NTF, genomic deletion mapping","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 2 — human genetic loss-of-function validates mouse KO findings, confirms conservation of mechanism","pmids":["25035924"],"is_preprint":false},{"year":2015,"finding":"SPPL2a-mediated cleavage of the CD74 NTF is indispensable for tonic and BCR-induced PI3K/Akt signalling in transitional B cells. Accumulating CD74 NTF interacts with BCR and Syk, reducing surface IgM and dampening BCR signal transmission. SPPL2a deficiency also causes dysregulation of FOXO1, elevating transcription of pro-apoptotic genes.","method":"SPPL2a−/− mouse B cells, phospho-flow cytometry for Akt/Syk signalling, co-immunoprecipitation of CD74 NTF with BCR components, BCR trafficking assays, transcription factor analysis","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — Co-IP plus signalling epistasis with defined mechanistic pathway placement","pmids":["26157172"],"is_preprint":false},{"year":2016,"finding":"The primary SPPL2a cleavage site within CD74 is located between Y52 and F53 in the transmembrane segment. The intracellular domain of CD74 is dispensable for SPPL2a cleavage. Alanine substitution of helix-destabilising glycines in the CD74 transmembrane segment and distinct luminal membrane-proximal residues reduces SPPL2a cleavage efficiency, indicating that transmembrane helix flexibility and the luminal juxtamembrane domain facilitate intramembrane proteolysis.","method":"Domain-exchange constructs, alanine-scanning mutagenesis of CD74 TM and juxtamembrane region, IP-MS identification of primary cleavage site, cell-based cleavage assays","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 — systematic mutagenesis with MS-based cleavage site mapping, multiple orthogonal methods","pmids":["26987812"],"is_preprint":false},{"year":2018,"finding":"Inherited loss-of-function mutations in SPPL2A in humans cause selective depletion of IL-12/IL-23-producing CD1c+ conventional dendritic cells (cDC2s) and their progenitors due to toxic CD74 NTF accumulation, and impair IFN-γ production by mycobacterium-specific memory TH1* cells, resulting in susceptibility to mycobacterial disease (BCG disease).","method":"Whole-exome sequencing of patients, patient cell immunophenotyping (flow cytometry), in vitro stimulation assays, Sppl2a−/− mouse infection model (BCG and M. tuberculosis), genetic complementation","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 — human inborn error genetics linked to defined cellular mechanism, validated in mouse infection model with multiple orthogonal readouts","pmids":["30127434"],"is_preprint":false},{"year":2019,"finding":"SPPL2a exhibits a non-canonical ectodomain shedding activity on TNFα that is distinct from classical intramembrane cleavage; proline insertions in the TNFα transmembrane helix strongly increase this SPPL2a non-canonical shedding, while leucine substitutions decrease it. Biophysical analysis identified a flexible region in the centre of the TNFα TM domain as critical for this processing mode.","method":"TM helix mutagenesis (proline/leucine insertions), biochemical shedding assays, circular dichroism and NMR structural analysis, molecular dynamics simulations","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 1 — mutagenesis plus biophysical and structural analysis, but non-canonical activity of uncertain physiological significance; single lab","pmids":["33294784"],"is_preprint":false},{"year":2019,"finding":"SPPL2a and SPPL2b control cellular levels of VAMP1, VAMP2, VAMP3, and VAMP4 (tail-anchored SNARE proteins) by proteolytic degradation; loss of both proteases in double-KO mice causes cell-type- and tissue-dependent accumulation of VAMP1-4, establishing these SNAREs as in vivo substrates of SPPL2a/b.","method":"Cellular co-expression cleavage screen of 18 SNARE proteins, SPPL2a/b inhibitor treatment, Western blot of endogenous VAMP1-4 in primary cells and tissues from SPPL2a/b double-KO mice","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 2 — in vivo double-KO validation across multiple cell types and tissues, endogenous substrate accumulation confirmed","pmids":["36047592"],"is_preprint":false},{"year":2019,"finding":"SPPL2a and SPPL2b control LOX-1 signalling by cleaving ADAM10-generated LOX-1 N-terminal fragments (NTFs) in lysosomes and at the plasma membrane, respectively. LOX-1 NTFs self-associate via their transmembrane domain and activate MAP kinases in a ligand-independent manner, upregulating pro-atherogenic targets (ICAM-1, CTGF). SPPL2a/b-deficient mice accumulate LOX-1 NTFs and develop larger atherosclerotic plaques.","method":"Co-expression and catalytic-dead mutant assays, co-IP for LOX-1 NTF self-association, MAP kinase signalling readouts, SPPL2a/b double-KO mouse atherosclerosis model (plaque quantification)","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — substrate identification plus in vivo disease model with mechanistic pathway placement","pmids":["30819724"],"is_preprint":false}],"current_model":"SPPL2a is a GxGD-type aspartyl intramembrane protease localised to lysosomes/late endosomes via a C-terminal YXXø tyrosine sorting motif, where it sequentially processes the N-terminal fragments of type II transmembrane proteins—most critically the CD74 (MHC II invariant chain) NTF—after prior ectodomain shedding, with its primary cleavage site on CD74 mapped to Y52-F53 in the transmembrane segment; loss of SPPL2a causes toxic CD74 NTF accumulation that blocks B cell maturation at the T1 stage, impairs BCR/PI3K/Akt signalling, depletes cDC2s, and in humans causes mycobacterial susceptibility, while additional substrates include TNFα, FasL, Bri2, TMEM106B, LOX-1 NTFs, and VAMP1-4 SNAREs."},"narrative":{"teleology":[{"year":2004,"claim":"Establishing the membrane topology of SPPL2A resolved how a GxGD-type aspartyl protease could accommodate type II transmembrane substrates, because its active-site loops face the exoplasm—the opposite orientation to presenilins.","evidence":"Epitope-tagged constructs, glycosylation mapping, and protease protection assays in mammalian cells","pmids":["15385547"],"confidence":"High","gaps":["No substrate identified at this stage","Subcellular compartment of endogenous protein not determined","No structural model at atomic resolution"]},{"year":2006,"claim":"Demonstrating that SPPL2A cleaves TNFα NTF in endosomes of primary dendritic cells provided the first substrate identification and showed that the released ICD has a signalling function (IL-12 induction), establishing SPPL2A as a functionally active intramembrane protease.","evidence":"Catalytic-dead mutant overexpression, subcellular fractionation, reporter assays for ICD release and IL-12 in primary human dendritic cells","pmids":["16829952"],"confidence":"High","gaps":["Physiological importance of TNFα ICD signalling not confirmed in vivo","Other substrates not yet known"]},{"year":2007,"claim":"Identification of FasL and Bri2 as additional SPPL2A substrates demonstrated that the enzyme acts broadly on type II transmembrane protein NTFs generated by ADAM10 shedding, and that liberated ICDs can regulate nuclear transcription.","evidence":"Dominant-negative SPPL2A in T-cell lines (FasL); systematic family member comparison with catalytic-dead controls (Bri2)","pmids":["17557115","17965014"],"confidence":"High","gaps":["In vivo consequences of FasL and Bri2 ICD signalling uncharacterised","Structural basis of substrate selectivity unknown"]},{"year":2011,"claim":"Mapping the YXXφ lysosomal targeting motif at Y498 in the SPPL2A C-terminal tail explained how SPPL2A is sorted to lysosomes/late endosomes and distinguished from its plasma-membrane homologue SPPL2B, establishing compartment-specific substrate access.","evidence":"Chimeric SPPL2A/SPPL2B constructs with point mutations, immunofluorescence of endogenous protein, live-cell imaging","pmids":["21896273"],"confidence":"High","gaps":["Adaptor proteins mediating YXXφ-dependent sorting not identified","Quantitative contribution of lysosomal vs. endosomal pools unclear"]},{"year":2012,"claim":"Three independent mouse models converged on CD74 NTF as the physiologically dominant SPPL2A substrate: loss of SPPL2A caused toxic CD74 NTF accumulation that blocked B cell maturation at T1 and depleted CD8-negative DCs, with full rescue by concurrent CD74 deletion, establishing a non-redundant SPPL2A–CD74 axis in adaptive immunity.","evidence":"SPPL2A knockout, ENU mutant (chompB), and knock-in mouse models; flow cytometry, Western blot, genetic epistasis with CD74 KO, bone marrow transfer","pmids":["23267015","23267013","23267016"],"confidence":"High","gaps":["Mechanism by which accumulated CD74 NTF disrupts endosomal trafficking incompletely defined","Whether other substrates contribute to immune phenotype unclear"]},{"year":2014,"claim":"Conservation of the SPPL2A–CD74 axis was validated in humans when patient-derived cells carrying a homozygous SPPL2A deletion accumulated CD74 NTF identically to mouse knockouts, and TMEM106B was identified as a neurodegenerative-disease-relevant lysosomal substrate.","evidence":"Patient lymphoblastoid cell lines with SPPL2A genomic deletion (CD74 NTF); co-expression and catalytic-dead mutant assays (TMEM106B)","pmids":["25035924","24872421"],"confidence":"High","gaps":["Neurological phenotype in SPPL2A-deficient patients not assessed","In vivo relevance of TMEM106B cleavage to neurodegeneration untested"]},{"year":2015,"claim":"Defining how accumulated CD74 NTF physically associates with BCR and Syk to suppress tonic PI3K/Akt signalling and elevate FOXO1-dependent pro-apoptotic transcription provided a molecular explanation for the T1 B cell maturation block in SPPL2A deficiency.","evidence":"Co-immunoprecipitation of CD74 NTF with BCR components, phospho-flow cytometry for Akt/Syk, transcription factor analysis in SPPL2A−/− mouse B cells","pmids":["26157172"],"confidence":"High","gaps":["Direct structural basis of CD74 NTF–BCR interaction unknown","Whether this mechanism operates in mature B cells or germinal centres untested"]},{"year":2016,"claim":"Mass spectrometry-based mapping of the primary SPPL2A cleavage site to Y52–F53 in the CD74 transmembrane helix, combined with systematic alanine scanning, revealed that helix flexibility (glycine residues) and the luminal juxtamembrane domain are key determinants of intramembrane proteolysis.","evidence":"IP-MS cleavage site identification, domain-exchange and alanine-scanning mutagenesis, cell-based cleavage assays","pmids":["26987812"],"confidence":"High","gaps":["Atomic-resolution structure of SPPL2A–substrate complex not available","Rules for predicting novel substrates not generalised"]},{"year":2018,"claim":"Human inborn errors of SPPL2A were shown to selectively deplete cDC2s and impair IFN-γ production by mycobacterium-specific TH1* cells, establishing SPPL2A deficiency as a Mendelian cause of susceptibility to mycobacterial disease (BCG disease).","evidence":"Whole-exome sequencing, patient immunophenotyping, in vitro stimulation, Sppl2a−/− mouse BCG/M. tuberculosis infection model, genetic complementation","pmids":["30127434"],"confidence":"High","gaps":["Full clinical spectrum of human SPPL2A deficiency beyond mycobacterial susceptibility not defined","Whether cDC2 loss is entirely CD74 NTF-dependent not formally demonstrated in patients"]},{"year":2019,"claim":"Expansion of the SPPL2A substrate repertoire to include VAMP1–4 SNAREs (degradative clearance) and LOX-1 NTFs (suppression of ligand-independent MAP kinase signalling and atherosclerosis) demonstrated that SPPL2A functions beyond immune regulation in vesicular trafficking homeostasis and cardiovascular pathology.","evidence":"Systematic SNARE cleavage screen and SPPL2A/B double-KO mouse tissues (VAMPs); co-IP, signalling assays, and atherosclerosis plaque quantification in double-KO mice (LOX-1)","pmids":["36047592","30819724"],"confidence":"High","gaps":["Relative contribution of SPPL2A vs. SPPL2B to individual SNARE and LOX-1 processing in vivo unclear","Tissue-specific substrate hierarchy not mapped"]},{"year":2019,"claim":"A non-canonical ectodomain shedding activity of SPPL2A on TNFα, governed by transmembrane helix flexibility, raised the possibility that SPPL2A can operate in processing modes beyond classical intramembrane proteolysis.","evidence":"TM helix proline/leucine mutagenesis, CD and NMR structural analysis, molecular dynamics simulations","pmids":["33294784"],"confidence":"Medium","gaps":["Physiological relevance of non-canonical shedding not demonstrated in vivo","Whether non-canonical activity applies to other substrates unknown","Single-lab observation"]},{"year":null,"claim":"Major open questions include the atomic structure of SPPL2A, the complete substrate repertoire across tissues, generalizable rules predicting substrate recognition, the full clinical phenotype of human SPPL2A deficiency beyond mycobacterial susceptibility, and whether accumulated CD74 NTF is the sole driver of cDC2 depletion in patients.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of SPPL2A or SPPL2A–substrate complex","Tissue-specific substrate hierarchy and redundancy with SPPL2B not systematically defined","Therapeutic implications of modulating SPPL2A activity unexplored"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,3,5,6,7,9,12,14,15,16]}],"localization":[{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[4,5,6,7,9]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[1,4,5]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[5,6,7,10,11,13]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,2,3,5,12,15]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,11,16]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[15]}],"complexes":[],"partners":["CD74","TNF","FASLG","ITM2B","TMEM106B","OLR1","VAMP1","VAMP2"],"other_free_text":[]},"mechanistic_narrative":"SPPL2A is a GxGD-type aspartyl intramembrane protease that resides on lysosomal/late endosomal membranes—directed there by a C-terminal YXXφ tyrosine sorting motif—where it sequentially cleaves the N-terminal fragments (NTFs) of type II transmembrane proteins following their ectodomain shedding [PMID:15385547, PMID:21896273]. Its most critical physiological substrate is the CD74 (MHC class II invariant chain) NTF: SPPL2A cleaves CD74 between Y52 and F53 in the transmembrane segment, and loss of this activity causes toxic CD74 NTF accumulation that blocks B cell maturation at the T1 stage, impairs BCR/PI3K/Akt signalling, depletes cDC2 dendritic cells, and in humans causes Mendelian susceptibility to mycobacterial disease [PMID:23267015, PMID:26987812, PMID:30127434]. Additional validated substrates include TNFα, FasL, Bri2, TMEM106B, LOX-1 NTFs, and VAMP1–4 SNAREs, through which SPPL2A regulates inflammatory cytokine signalling, apoptotic ligand turnover, and vesicular trafficking [PMID:16829952, PMID:17557115, PMID:17965014, PMID:36047592, PMID:30819724]. SPPL2A-mediated clearance of LOX-1 NTFs suppresses ligand-independent MAP kinase activation and limits atherosclerotic plaque formation in vivo [PMID:30819724]."},"prefetch_data":{"uniprot":{"accession":"Q8TCT8","full_name":"Signal peptide peptidase-like 2A","aliases":["Intramembrane protease 3","IMP-3","Presenilin-like protein 2"],"length_aa":520,"mass_kda":58.1,"function":"Intramembrane-cleaving aspartic protease (I-CLiP) that cleaves type II membrane signal peptides in the hydrophobic plane of the membrane. Functions in FASLG, ITM2B and TNF processing (PubMed:16829951, PubMed:16829952, PubMed:17557115, PubMed:17965014). 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:16829952). Also responsible for the intramembrane cleavage of Fas antigen ligand FASLG, which promotes the release of the intracellular FasL domain (FasL ICD) (PubMed:17557115). Essential for degradation of the invariant chain CD74 that plays a central role in the function of antigen-presenting cells in the immune system (By similarity). Plays a role in the regulation of innate and adaptive immunity (PubMed:16829952). Catalyzes the intramembrane cleavage of the simian foamy virus envelope glycoprotein gp130 independently of prior ectodomain shedding by furin or furin-like proprotein convertase (PC)-mediated cleavage proteolysis (PubMed:23132852)","subcellular_location":"Late endosome membrane; Lysosome membrane; Membrane","url":"https://www.uniprot.org/uniprotkb/Q8TCT8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SPPL2A","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":true,"resolved_as":"IMP3","ensg_id":"ENSG00000177971","cell_line_id":"CID001067","localizations":[{"compartment":"nucleolus_gc","grade":3}],"interactors":[{"gene":"MPHOSPH10","stoichiometry":4.0},{"gene":"IMP4","stoichiometry":0.2},{"gene":"UTP3","stoichiometry":0.2},{"gene":"DIEXF","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001067","total_profiled":1310},"omim":[{"mim_id":"619549","title":"IMMUNODEFICIENCY 86; IMD86","url":"https://www.omim.org/entry/619549"},{"mim_id":"613744","title":"SPASTIC PARAPLEGIA 51, AUTOSOMAL RECESSIVE; SPG51","url":"https://www.omim.org/entry/613744"},{"mim_id":"613413","title":"TRANSMEMBRANE PROTEIN 106B; TMEM106B","url":"https://www.omim.org/entry/613413"},{"mim_id":"608238","title":"SIGNAL PEPTIDE PEPTIDASE-LIKE 2A; SPPL2A","url":"https://www.omim.org/entry/608238"},{"mim_id":"607244","title":"ADAPTOR-RELATED PROTEIN COMPLEX 4, EPSILON-1 SUBUNIT; AP4E1","url":"https://www.omim.org/entry/607244"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Vesicles","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SPPL2A"},"hgnc":{"alias_symbol":["IMP3","PSL2"],"prev_symbol":[]},"alphafold":{"accession":"Q8TCT8","domains":[{"cath_id":"3.50.30.30","chopping":"27-162","consensus_level":"high","plddt":84.8936,"start":27,"end":162},{"cath_id":"-","chopping":"171-494","consensus_level":"high","plddt":84.1935,"start":171,"end":494}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TCT8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TCT8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TCT8-F1-predicted_aligned_error_v6.png","plddt_mean":79.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SPPL2A","jax_strain_url":"https://www.jax.org/strain/search?query=SPPL2A"},"sequence":{"accession":"Q8TCT8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8TCT8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8TCT8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TCT8"}},"corpus_meta":[{"pmid":"15753088","id":"PMC_15753088","title":"The RNA-binding protein IMP-3 is a translational activator of insulin-like growth factor II leader-3 mRNA during proliferation of human K562 leukemia cells.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15753088","citation_count":192,"is_preprint":false,"source_track":"pubmed_title"},{"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":165,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"20562850","id":"PMC_20562850","title":"Poorly differentiated carcinoma of the thyroid: validation of the Turin proposal and analysis of IMP3 expression.","date":"2010","source":"Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc","url":"https://pubmed.ncbi.nlm.nih.gov/20562850","citation_count":140,"is_preprint":false,"source_track":"pubmed_title"},{"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":135,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"27510448","id":"PMC_27510448","title":"CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs.","date":"2016","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/27510448","citation_count":129,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"18223334","id":"PMC_18223334","title":"The oncofetal protein IMP3: a novel biomarker for endometrial serous carcinoma.","date":"2008","source":"The American journal of surgical 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in vitro and cell-based protease assays with loss-of-function variants; cytokine (IL-12) production readout in dendritic cells\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct enzymatic activity demonstrated with loss-of-function variants, subcellular localization established, downstream signaling consequence (IL-12) measured; original discovery paper with multiple orthogonal methods\",\n      \"pmids\": [\"16829952\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SPPL2a cleaves the membrane-anchored N-terminal fragment of Bri2 (Itm2b) within its transmembrane domain to produce an intracellular domain (ICD) and a secreted low-molecular-weight C-terminal peptide; this cleavage is selective for SPPL2a and SPPL2b but not SPP or SPPL3, and requires prior ectodomain shedding by ADAM10.\",\n      \"method\": \"Overexpression of all SPP/SPPL family members and their catalytic loss-of-function variants in cell lines; immunoblotting for cleavage products\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — substrate identification with systematic family-member comparison and catalytic mutant controls, replicated with multiple constructs\",\n      \"pmids\": [\"17965014\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SPPL2a cleaves the membrane-anchored 17-kDa N-terminal remnant of FasL (generated by ADAM10 ectodomain shedding) to release the FasL intracellular domain (ICD), which translocates to the nucleus and inhibits gene transcription.\",\n      \"method\": \"Overexpression and knockdown of SPPL2a in T-cells; immunoblotting for FasL fragments; nuclear fractionation; transcriptional reporter assays\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — substrate cleavage demonstrated with endogenous FasL in T-cells, nuclear translocation of ICD shown by fractionation, functional transcriptional consequence measured\",\n      \"pmids\": [\"17557115\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SPPL2a mediates intramembrane proteolysis of the CD74 (invariant chain) N-terminal fragment (NTF) in late endosomes/lysosomes of B lymphocytes; accumulation of the CD74 NTF in SPPL2a-deficient mice severely impairs endocytic membrane trafficking, reduces BAFF-R surface expression, and causes a developmental block at the transitional T1 B cell stage, leading to loss of mature B cell subsets and defective humoral immunity.\",\n      \"method\": \"SPPL2a knockout mouse model (genetic loss-of-function); CD74 NTF accumulation shown by immunoblot and immunofluorescence; B cell subset analysis by flow cytometry; rescue by additional CD74 ablation confirming epistasis\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined cellular phenotype, genetic epistasis (CD74 double KO rescue), substrate accumulation directly demonstrated; independently replicated in two concurrent papers\",\n      \"pmids\": [\"23267015\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SPPL2a is required for proteolytic processing of CD74 in B cells and CD8-negative dendritic cells; its inactivation causes dramatic accumulation of the p8 CD74 fragment (product of cathepsin S), blocks earlier steps in CD74 endosomal processing, and results in loss of mature B cells and CD8− DCs, mirroring BAFF deficiency.\",\n      \"method\": \"ENU-induced mutant mouse (chompB) mapped to Sppl2a; proteomic (MS) identification of CD74 as substrate; flow cytometry for immune cell populations; BCL2 overexpression rescue experiment\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — substrate identified by proteomics, genetic model with defined phenotype, independent replication of CD74/B cell finding\",\n      \"pmids\": [\"23267013\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SPPL2a deficiency blocks CD74 processing in B cells and CD8− DCs, causing accumulation of the p8 CD74 NTF; B cell survival can be rescued by BCL2 overexpression but not by BAFF, and cells display reduced surface BAFF-R and IgM/IgD BCR.\",\n      \"method\": \"Inactivating mutation knockin mouse; flow cytometry; BCL2 and BAFF rescue experiments; CD74 processing immunoblot\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — third independent laboratory confirming same substrate and B cell phenotype with additional mechanistic detail (BCL2/BAFF rescue)\",\n      \"pmids\": [\"23267016\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"SPPL2a is expressed in enamel epithelium during amelogenesis, and its knockout in mice causes defective enamel maturation with reduced mineral content and delayed resorption of the proteinaceous enamel matrix, indicating that SPPL2a-mediated intramembrane proteolysis is essential for ameloblast homeostasis.\",\n      \"method\": \"Sppl2a knockout mouse; histology; microCT mineral content analysis; immunostaining of ameloblasts\",\n      \"journal\": \"Journal of bone and mineral research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined tissue phenotype, but substrate in ameloblasts not directly identified in this study\",\n      \"pmids\": [\"23426979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SPPL2a (and to a lesser extent SPPL2b) mediates intramembrane proteolysis of TMEM106B, a frontotemporal lobar degeneration risk factor; processing is lysosome-dependent and generates a rapidly degraded intracellular domain from the N-terminal membrane-bound fragment.\",\n      \"method\": \"Overexpression of SPPL2a/b and GxGD catalytic mutants; inhibitor treatment; lysosomal inhibitors; immunoblotting for TMEM106B fragments; selectivity confirmed by comparison with paralog TMEM106A\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — substrate cleavage shown with catalytic mutant controls and inhibitors in cell-based assay; single laboratory\",\n      \"pmids\": [\"24872421\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SPPL2a-mediated cleavage of the CD74 NTF is required for tonic and BCR-induced PI3K/Akt signaling in transitional B cells; the accumulating CD74 NTF interacts with BCR and Syk, reducing surface IgM and impairing signal transduction, leading to dysregulation of FOXO1 and elevated transcription of proapoptotic genes.\",\n      \"method\": \"Sppl2a−/− B cells; phospho-flow cytometry for PI3K/Akt/Syk pathway; BCR internalization assay; co-immunoprecipitation of CD74 NTF with BCR and Syk; FOXO1 target gene expression\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP identifies molecular interaction (CD74 NTF–BCR/Syk), signaling pathway placed by loss-of-function with multiple pathway readouts, mechanistic epistasis established\",\n      \"pmids\": [\"26157172\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The primary SPPL2a cleavage site on CD74 NTF is between Y52 and F53 within the transmembrane segment; domain-exchange experiments show the CD74 intracellular domain is dispensable, while helix-destabilizing glycines in the TM segment and residues in the luminal juxtamembrane region facilitate intramembrane cleavage.\",\n      \"method\": \"IP-MS identification of cleavage product; domain-exchange constructs; alanine-scanning mutagenesis of CD74 TM and juxtamembrane regions; cell-based cleavage assays with immunoblotting\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — MS-based cleavage site mapping combined with systematic mutagenesis; defines substrate determinants at residue level\",\n      \"pmids\": [\"26987812\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Human loss-of-function mutations in SPPL2A cause accumulation of CD74 NTF in HLA class II+ myeloid and lymphoid cells, which selectively depletes IL-12/IL-23-producing CD1c+ conventional dendritic cells (cDC2s) and their progenitors, and impairs IFN-γ production by mycobacterium-specific memory TH1* cells, underlying susceptibility to mycobacterial disease.\",\n      \"method\": \"Human genetic study (homozygous LOF mutations); CD74 NTF accumulation confirmed in patient cells; cDC2 depletion by flow cytometry; Sppl2a−/− mouse BCG/M. tuberculosis infection model; in vitro antigen stimulation of patient T cells\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — human genetics validated in mouse model; CD74 NTF accumulation and cDC2 depletion mechanistically linked; multiple orthogonal methods across human and mouse\",\n      \"pmids\": [\"30127434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SPPL2a exhibits non-canonical ectodomain shedding activity on TNFα in addition to intramembrane cleavage; this shedding is enhanced by proline insertions that increase conformational flexibility of the TNFα TM helix and reduced by leucine substitutions, indicating that TM helix flexibility is a key substrate determinant for SPPL2a non-canonical shedding.\",\n      \"method\": \"Proline/leucine mutagenesis of TNFα TM domain; cell-based cleavage assays; biophysical analysis (CD spectroscopy, MD simulations) of TM helix flexibility; immunoblotting for shedding products\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis combined with structural/biophysical analysis; single laboratory but multiple orthogonal methods\",\n      \"pmids\": [\"33294784\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SPPL2a (and SPPL2b) cleave the tail-anchored SNARE proteins VAMP1, VAMP2, VAMP3, and VAMP4 in the endocytic/late secretory pathway; loss of SPPL2a/b in double-knockout mice leads to accumulation of these VAMPs in a cell-type- and tissue-dependent manner, identifying them as bona fide in vivo substrates.\",\n      \"method\": \"Screen of 18 SNARE proteins by co-expression cleavage assay; endogenous VAMP1-4 levels assessed upon pharmacological SPPL2a/b inhibition; SPPL2a/b double-KO mouse tissue analysis by immunoblot\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — substrate screen validated at endogenous level and in vivo in dKO mice; multiple substrates confirmed with multiple methods\",\n      \"pmids\": [\"36047592\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SPPL2a is a lysosomal/late-endosomal GxGD-type aspartyl intramembrane protease that sequentially cleaves type II transmembrane proteins after ectodomain shedding, with validated substrates including CD74 (invariant chain), TNFα, FasL, Bri2, TMEM106B, and VAMP1-4; its proteolysis of the CD74 N-terminal fragment is the critical step controlling B cell maturation, BCR/PI3K-Akt signaling, cDC2 survival, and antimycobacterial immunity, and TM-domain flexibility of substrates is a key determinant of cleavage efficiency.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2004,\n      \"finding\": \"SPPL2a is a polytopic membrane protein belonging to the GxGD-type aspartyl intramembrane protease family (SPP/SPPL family); its N-terminal extension is located in the extracellular space and is modified with N-glycans; the hydrophilic loops containing the catalytic residues face the exoplasm and the C-terminus faces the cytosol, placing it in opposite orientation to presenilins and predicting it cleaves type II-oriented substrate peptides.\",\n      \"method\": \"Comparative topology analysis using epitope-tagged constructs, glycosylation mapping, and protease protection assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal biochemical methods in single rigorous study, foundational topology paper\",\n      \"pmids\": [\"15385547\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"SPPL2a is sorted to endosomes and functions as an active intramembrane protease that catalyses intramembrane cleavage of tumour necrosis factor alpha (TNFα), promoting release of the TNFα intracellular domain (ICD), which triggers IL-12 expression in activated human dendritic cells.\",\n      \"method\": \"Overexpression and catalytic-dead mutant constructs, subcellular fractionation/immunofluorescence for localization, reporter assays for ICD release and IL-12 induction in primary human dendritic cells\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — functional protease activity demonstrated with loss-of-function mutants, localization defined, physiological readout in primary cells; independently replicated\",\n      \"pmids\": [\"16829952\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SPPL2a mediates intramembrane cleavage of Fas ligand (FasL) in T-cells after prior ectodomain shedding by ADAM10; SPPL2a cleavage liberates the FasL intracellular domain (ICD), which translocates to the nucleus and inhibits gene transcription.\",\n      \"method\": \"Overexpression and dominant-negative SPPL2a in T-cell lines, immunoprecipitation, subcellular fractionation, luciferase transcription reporter assays; endogenous FasL processing validated\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal biochemical validation with endogenous substrate, functional nuclear ICD readout, two-protease epistasis established\",\n      \"pmids\": [\"17557115\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SPPL2a and SPPL2b, but not SPP or SPPL3, mediate intramembrane proteolysis of Bri2 (Itm2b) N-terminal fragment generated after ADAM10 ectodomain shedding, producing an intracellular domain and a secreted low-molecular-weight C-terminal peptide.\",\n      \"method\": \"Expression of all SPP/SPPL family members and catalytic-dead variants, co-transfection with Bri2 constructs, immunoprecipitation, Western blot detection of cleavage products\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — systematic comparison across all family members with loss-of-function controls, multiple cleavage products characterized\",\n      \"pmids\": [\"17965014\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Endogenous SPPL2a localises to lysosomal/late endosomal membranes. Lysosomal targeting is mediated by a canonical tyrosine-based sorting motif (YXXø at position Y498) in its cytosolic C-terminal tail, which is both necessary and sufficient to direct SPPL2a to lysosomes/late endosomes and distinguishes it from its plasma-membrane-localised homologue SPPL2b.\",\n      \"method\": \"Immunofluorescence of endogenous SPPL2a, chimeric SPPL2a/SPPL2b constructs with point mutations in sorting motif, live-cell imaging\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct localization experiment with mutagenesis of sorting motif, functional consequence for substrate access demonstrated\",\n      \"pmids\": [\"21896273\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SPPL2a is the intramembrane protease responsible for cleavage of the invariant chain (CD74) N-terminal fragment (NTF) in lysosomes/late endosomes of B cells. Loss of SPPL2a in mice causes CD74 NTF accumulation that severely impairs endosomal membrane trafficking, reduces surface BAFF-R expression, causes MHCII accumulation, and leads to a block in B cell maturation at the T1 stage; all these defects are rescued by additional CD74 knockout.\",\n      \"method\": \"SPPL2a−/− mouse model, flow cytometry, Western blot, immunofluorescence, endocytic trafficking assays, genetic epistasis (CD74/SPPL2a double KO rescue)\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo KO with defined mechanistic phenotype and genetic rescue experiment; independently replicated by two concurrent papers\",\n      \"pmids\": [\"23267015\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SPPL2a deficiency blocks proteolytic processing of CD74 MHC II invariant chain in B cells and CD8-negative dendritic cells, causing build-up of the p8 cathepsin-S product and interfering with earlier steps in CD74 endosomal retention. Loss of SPPL2a phenocopies BAFF deficiency; B cell survival is restored by BCL2 overexpression but not by BAFF.\",\n      \"method\": \"ENU mutagenesis screen (chompB mouse), positional cloning of Sppl2a mutation, proteomic identification of CD74 as substrate, flow cytometry, Western blot\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — unbiased genetic screen identifying SPPL2a, proteomics-validated substrate, multiple phenotypic readouts; concurrent independent replication\",\n      \"pmids\": [\"23267013\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SPPL2a deficiency causes dramatic accumulation of the CD74 p8 NTF in B cells and CD8-negative dendritic cells, reduces surface IgM, IgD, and BAFF-R, and leads to a profound humoral immunodeficiency. The final step of the CD74-MHCII lysosomal processing pathway requires SPPL2a.\",\n      \"method\": \"Inactivating point mutation knock-in mouse, flow cytometry, Western blot, bone marrow transfer experiments\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — independent replication of CD74 substrate finding with distinct mouse model and orthogonal readouts\",\n      \"pmids\": [\"23267016\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"SPPL2a is critical for tooth enamel formation; Sppl2a−/− mice exhibit defective enamel mineralisation and impaired maturation-stage ameloblast function with delayed and incomplete resorption of the proteinaceous enamel matrix, establishing a role for SPPL2a-mediated intramembrane proteolysis in ameloblast cellular homeostasis.\",\n      \"method\": \"Sppl2a−/− mouse model, micro-CT, histology, electron microscopy, immunohistochemistry for SPPL2a in enamel epithelium\",\n      \"journal\": \"Journal of bone and mineral research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo KO with defined tissue phenotype, but molecular substrate in ameloblasts not yet identified\",\n      \"pmids\": [\"23426979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SPPL2a, and to a lesser extent SPPL2b, mediate intramembrane proteolysis of the frontotemporal lobar degeneration risk factor TMEM106B, generating and then rapidly degrading a small intracellular domain from the lysosome-localised N-terminal fragment of TMEM106B. TMEM106A, a paralogue, is not a specific SPPL2a substrate.\",\n      \"method\": \"Co-expression of SPPL2a/b with TMEM106B in cell lines, catalytic-dead mutant controls, lysosomal protease inhibitors, Western blot detection of NTF and ICD\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — catalytic-dead controls and paralog specificity established, single lab\",\n      \"pmids\": [\"24872421\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SPPL2a-mediated CD74 NTF processing is conserved in human B cells; lymphoblastoid cell lines from patients with a homozygous chromosomal microdeletion disrupting the SPPL2a locus lack SPPL2a protein and accumulate CD74 NTF comparably to Sppl2a−/− mouse B cells.\",\n      \"method\": \"Patient-derived lymphoblastoid cell lines with genomic SPPL2a deletion, Western blot for SPPL2a and CD74 NTF, genomic deletion mapping\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — human genetic loss-of-function validates mouse KO findings, confirms conservation of mechanism\",\n      \"pmids\": [\"25035924\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SPPL2a-mediated cleavage of the CD74 NTF is indispensable for tonic and BCR-induced PI3K/Akt signalling in transitional B cells. Accumulating CD74 NTF interacts with BCR and Syk, reducing surface IgM and dampening BCR signal transmission. SPPL2a deficiency also causes dysregulation of FOXO1, elevating transcription of pro-apoptotic genes.\",\n      \"method\": \"SPPL2a−/− mouse B cells, phospho-flow cytometry for Akt/Syk signalling, co-immunoprecipitation of CD74 NTF with BCR components, BCR trafficking assays, transcription factor analysis\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus signalling epistasis with defined mechanistic pathway placement\",\n      \"pmids\": [\"26157172\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The primary SPPL2a cleavage site within CD74 is located between Y52 and F53 in the transmembrane segment. The intracellular domain of CD74 is dispensable for SPPL2a cleavage. Alanine substitution of helix-destabilising glycines in the CD74 transmembrane segment and distinct luminal membrane-proximal residues reduces SPPL2a cleavage efficiency, indicating that transmembrane helix flexibility and the luminal juxtamembrane domain facilitate intramembrane proteolysis.\",\n      \"method\": \"Domain-exchange constructs, alanine-scanning mutagenesis of CD74 TM and juxtamembrane region, IP-MS identification of primary cleavage site, cell-based cleavage assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — systematic mutagenesis with MS-based cleavage site mapping, multiple orthogonal methods\",\n      \"pmids\": [\"26987812\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Inherited loss-of-function mutations in SPPL2A in humans cause selective depletion of IL-12/IL-23-producing CD1c+ conventional dendritic cells (cDC2s) and their progenitors due to toxic CD74 NTF accumulation, and impair IFN-γ production by mycobacterium-specific memory TH1* cells, resulting in susceptibility to mycobacterial disease (BCG disease).\",\n      \"method\": \"Whole-exome sequencing of patients, patient cell immunophenotyping (flow cytometry), in vitro stimulation assays, Sppl2a−/− mouse infection model (BCG and M. tuberculosis), genetic complementation\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — human inborn error genetics linked to defined cellular mechanism, validated in mouse infection model with multiple orthogonal readouts\",\n      \"pmids\": [\"30127434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SPPL2a exhibits a non-canonical ectodomain shedding activity on TNFα that is distinct from classical intramembrane cleavage; proline insertions in the TNFα transmembrane helix strongly increase this SPPL2a non-canonical shedding, while leucine substitutions decrease it. Biophysical analysis identified a flexible region in the centre of the TNFα TM domain as critical for this processing mode.\",\n      \"method\": \"TM helix mutagenesis (proline/leucine insertions), biochemical shedding assays, circular dichroism and NMR structural analysis, molecular dynamics simulations\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis plus biophysical and structural analysis, but non-canonical activity of uncertain physiological significance; single lab\",\n      \"pmids\": [\"33294784\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SPPL2a and SPPL2b control cellular levels of VAMP1, VAMP2, VAMP3, and VAMP4 (tail-anchored SNARE proteins) by proteolytic degradation; loss of both proteases in double-KO mice causes cell-type- and tissue-dependent accumulation of VAMP1-4, establishing these SNAREs as in vivo substrates of SPPL2a/b.\",\n      \"method\": \"Cellular co-expression cleavage screen of 18 SNARE proteins, SPPL2a/b inhibitor treatment, Western blot of endogenous VAMP1-4 in primary cells and tissues from SPPL2a/b double-KO mice\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo double-KO validation across multiple cell types and tissues, endogenous substrate accumulation confirmed\",\n      \"pmids\": [\"36047592\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SPPL2a and SPPL2b control LOX-1 signalling by cleaving ADAM10-generated LOX-1 N-terminal fragments (NTFs) in lysosomes and at the plasma membrane, respectively. LOX-1 NTFs self-associate via their transmembrane domain and activate MAP kinases in a ligand-independent manner, upregulating pro-atherogenic targets (ICAM-1, CTGF). SPPL2a/b-deficient mice accumulate LOX-1 NTFs and develop larger atherosclerotic plaques.\",\n      \"method\": \"Co-expression and catalytic-dead mutant assays, co-IP for LOX-1 NTF self-association, MAP kinase signalling readouts, SPPL2a/b double-KO mouse atherosclerosis model (plaque quantification)\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — substrate identification plus in vivo disease model with mechanistic pathway placement\",\n      \"pmids\": [\"30819724\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SPPL2a is a GxGD-type aspartyl intramembrane protease localised to lysosomes/late endosomes via a C-terminal YXXø tyrosine sorting motif, where it sequentially processes the N-terminal fragments of type II transmembrane proteins—most critically the CD74 (MHC II invariant chain) NTF—after prior ectodomain shedding, with its primary cleavage site on CD74 mapped to Y52-F53 in the transmembrane segment; loss of SPPL2a causes toxic CD74 NTF accumulation that blocks B cell maturation at the T1 stage, impairs BCR/PI3K/Akt signalling, depletes cDC2s, and in humans causes mycobacterial susceptibility, while additional substrates include TNFα, FasL, Bri2, TMEM106B, LOX-1 NTFs, and VAMP1-4 SNAREs.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"SPPL2A is a GxGD-type intramembrane aspartyl protease that resides in late endosomes and lysosomes, where it cleaves the transmembrane domains of type II membrane proteins and tail-anchored SNAREs following ectodomain shedding [PMID:16829952, PMID:36047592]. Its validated substrates include the CD74 N-terminal fragment, TNFα, FasL, Bri2, TMEM106B, and VAMP1–4, with transmembrane helix flexibility serving as a key determinant of cleavage efficiency [PMID:17965014, PMID:17557115, PMID:24872421, PMID:33294784, PMID:26987812]. The physiologically most consequential substrate is CD74: failure to clear the CD74 NTF disrupts BCR/PI3K-Akt signaling, blocks B cell maturation at the transitional T1 stage, depletes IL-12/IL-23-producing cDC2 dendritic cells, and impairs antimycobacterial immunity [PMID:23267015, PMID:26157172, PMID:30127434]. Biallelic loss-of-function mutations in human SPPL2A cause Mendelian susceptibility to mycobacterial disease through CD74 NTF accumulation and selective cDC2 depletion [PMID:30127434].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Establishing SPPL2a as a functional intramembrane protease and identifying TNFα as its first substrate resolved whether SPPL2a had enzymatic activity and placed it in endosomal signaling via ICD-triggered IL-12 expression in dendritic cells.\",\n      \"evidence\": \"Subcellular fractionation, catalytic-dead mutants, and cytokine readouts in human dendritic cells\",\n      \"pmids\": [\"16829952\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic mechanism at atomic level not resolved\", \"Whether TNFα cleavage occurs in vivo under physiological conditions not tested\", \"Range of additional substrates unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identification of Bri2 and FasL as substrates, each requiring prior ADAM10 shedding, established that SPPL2a acts on diverse type II transmembrane proteins in a two-step proteolytic cascade and that released ICDs can have distinct nuclear signaling functions.\",\n      \"evidence\": \"Systematic SPP/SPPL family comparison with catalytic mutants for Bri2; SPPL2a knockdown in T cells with nuclear fractionation and transcriptional reporters for FasL\",\n      \"pmids\": [\"17965014\", \"17557115\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No in vivo confirmation of Bri2/FasL processing at endogenous levels in knockout models\", \"Structural basis for paralog selectivity (SPPL2a vs. SPPL2b) not determined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Three independent genetic studies in mice converged on CD74 as the physiologically critical SPPL2a substrate, demonstrating that CD74 NTF accumulation blocks B cell maturation at the T1 stage and depletes CD8⁻ dendritic cells, with rescue by CD74 ablation proving epistasis.\",\n      \"evidence\": \"Sppl2a knockout, ENU-mutant (chompB), and knockin mouse models; flow cytometry; CD74 double-KO rescue; proteomics-based substrate identification; BCL2 overexpression rescue\",\n      \"pmids\": [\"23267015\", \"23267013\", \"23267016\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CD74 NTF accumulation mechanistically disrupts membrane trafficking not fully resolved\", \"Whether SPPL2a has non-redundant roles independent of CD74 in vivo unclear\", \"Molecular mechanism of cDC loss not delineated\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstration that SPPL2a knockout mice have defective enamel maturation extended the protease's physiological relevance beyond the immune system, though the relevant ameloblast substrate was not identified.\",\n      \"evidence\": \"Sppl2a knockout mouse; histology and microCT mineral analysis of teeth\",\n      \"pmids\": [\"23426979\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Substrate in ameloblasts not identified\", \"Whether the enamel phenotype is CD74-dependent not tested\", \"Single study without independent replication\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identification of TMEM106B as an SPPL2a substrate linked the protease to a frontotemporal lobar degeneration risk factor, broadening the neurobiological significance of its substrate repertoire.\",\n      \"evidence\": \"Overexpression of SPPL2a/b and catalytic mutants with lysosomal inhibitors; immunoblotting for TMEM106B fragments\",\n      \"pmids\": [\"24872421\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vivo validation in knockout model\", \"Functional consequence of TMEM106B cleavage on neurodegeneration-related pathways not established\", \"Single laboratory study\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Mechanistic dissection of the B cell block revealed that CD74 NTF physically interacts with BCR and Syk to impair tonic PI3K/Akt signaling, deregulate FOXO1, and upregulate proapoptotic genes, explaining why transitional B cells die rather than mature.\",\n      \"evidence\": \"Sppl2a⁻/⁻ B cells; co-immunoprecipitation of CD74 NTF with BCR/Syk; phospho-flow cytometry for PI3K/Akt pathway; FOXO1 target gene profiling\",\n      \"pmids\": [\"26157172\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CD74 NTF–BCR interaction is direct or mediated by other partners not resolved\", \"Signaling pathway in cDC2 cells not separately characterized\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Mapping the primary cleavage site to Y52–F53 and showing that helix-destabilizing glycines and luminal juxtamembrane residues facilitate cleavage defined the substrate-side determinants of SPPL2a intramembrane proteolysis at residue resolution.\",\n      \"evidence\": \"IP-MS of cleavage products; domain-exchange constructs and alanine-scanning mutagenesis of CD74 TM and juxtamembrane regions\",\n      \"pmids\": [\"26987812\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No crystal/cryo-EM structure of SPPL2a–substrate complex\", \"Enzyme-side active-site residues contacting the substrate not mapped\", \"Whether the same determinants apply to all substrates not tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Human biallelic SPPL2A loss-of-function mutations were shown to cause Mendelian susceptibility to mycobacterial disease by depleting cDC2 cells and impairing IFN-γ production from mycobacterium-specific TH1* cells, translating the mouse B cell/DC phenotype to human immunodeficiency.\",\n      \"evidence\": \"Human genetic study with homozygous LOF mutations; patient cell flow cytometry and CD74 NTF accumulation; Sppl2a⁻/⁻ mouse BCG/M. tuberculosis infection\",\n      \"pmids\": [\"30127434\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SPPL2A deficiency affects susceptibility to pathogens beyond mycobacteria not explored\", \"Extent of B cell deficiency in human patients less characterized than in mice\", \"Therapeutic rescue strategies not addressed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Discovery of non-canonical ectodomain shedding activity by SPPL2a, modulated by TM helix flexibility, expanded the catalytic repertoire beyond intramembrane cleavage and established TM conformational dynamics as a biophysical determinant of substrate processing.\",\n      \"evidence\": \"Proline/leucine TM mutagenesis of TNFα; CD spectroscopy and MD simulations; cell-based cleavage assays\",\n      \"pmids\": [\"33294784\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether non-canonical shedding occurs for substrates other than TNFα unknown\", \"Physiological relevance of shedding versus intramembrane cleavage in vivo unclear\", \"No structural data for SPPL2a itself\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"A systematic screen identified VAMP1–4 as in vivo SPPL2a/b substrates, revealing that the protease also processes tail-anchored SNAREs in the endocytic/late secretory pathway and thereby contributes to vesicular trafficking homeostasis.\",\n      \"evidence\": \"Screen of 18 SNARE proteins by co-expression cleavage; endogenous VAMP accumulation upon inhibitor treatment and in SPPL2a/b double-KO mouse tissues\",\n      \"pmids\": [\"36047592\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of SPPL2a versus SPPL2b to VAMP processing in each tissue not delineated\", \"Functional consequence of VAMP accumulation on vesicular trafficking not characterized\", \"Whether VAMP cleavage is constitutive or regulated unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"No high-resolution structure of SPPL2a exists, and the enzyme-side determinants of substrate recognition, the regulation of its catalytic activity, and the full in vivo substrate repertoire beyond the immune system remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No cryo-EM or crystal structure of SPPL2a alone or in complex with substrate\", \"Regulation of SPPL2a activity (post-translational modifications, cofactors) unknown\", \"Substrate repertoire in non-immune tissues (brain, enamel) not systematically explored\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 7, 9, 12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [3, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [3, 4, 5, 8, 10]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 2, 9, 12]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"CD74\",\n      \"TNF\",\n      \"FASLG\",\n      \"ITM2B\",\n      \"TMEM106B\",\n      \"VAMP1\",\n      \"VAMP2\",\n      \"VAMP3\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"SPPL2A is a GxGD-type aspartyl intramembrane protease that resides on lysosomal/late endosomal membranes—directed there by a C-terminal YXXφ tyrosine sorting motif—where it sequentially cleaves the N-terminal fragments (NTFs) of type II transmembrane proteins following their ectodomain shedding [PMID:15385547, PMID:21896273]. Its most critical physiological substrate is the CD74 (MHC class II invariant chain) NTF: SPPL2A cleaves CD74 between Y52 and F53 in the transmembrane segment, and loss of this activity causes toxic CD74 NTF accumulation that blocks B cell maturation at the T1 stage, impairs BCR/PI3K/Akt signalling, depletes cDC2 dendritic cells, and in humans causes Mendelian susceptibility to mycobacterial disease [PMID:23267015, PMID:26987812, PMID:30127434]. Additional validated substrates include TNFα, FasL, Bri2, TMEM106B, LOX-1 NTFs, and VAMP1–4 SNAREs, through which SPPL2A regulates inflammatory cytokine signalling, apoptotic ligand turnover, and vesicular trafficking [PMID:16829952, PMID:17557115, PMID:17965014, PMID:36047592, PMID:30819724]. SPPL2A-mediated clearance of LOX-1 NTFs suppresses ligand-independent MAP kinase activation and limits atherosclerotic plaque formation in vivo [PMID:30819724].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Establishing the membrane topology of SPPL2A resolved how a GxGD-type aspartyl protease could accommodate type II transmembrane substrates, because its active-site loops face the exoplasm—the opposite orientation to presenilins.\",\n      \"evidence\": \"Epitope-tagged constructs, glycosylation mapping, and protease protection assays in mammalian cells\",\n      \"pmids\": [\"15385547\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No substrate identified at this stage\", \"Subcellular compartment of endogenous protein not determined\", \"No structural model at atomic resolution\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Demonstrating that SPPL2A cleaves TNFα NTF in endosomes of primary dendritic cells provided the first substrate identification and showed that the released ICD has a signalling function (IL-12 induction), establishing SPPL2A as a functionally active intramembrane protease.\",\n      \"evidence\": \"Catalytic-dead mutant overexpression, subcellular fractionation, reporter assays for ICD release and IL-12 in primary human dendritic cells\",\n      \"pmids\": [\"16829952\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological importance of TNFα ICD signalling not confirmed in vivo\", \"Other substrates not yet known\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identification of FasL and Bri2 as additional SPPL2A substrates demonstrated that the enzyme acts broadly on type II transmembrane protein NTFs generated by ADAM10 shedding, and that liberated ICDs can regulate nuclear transcription.\",\n      \"evidence\": \"Dominant-negative SPPL2A in T-cell lines (FasL); systematic family member comparison with catalytic-dead controls (Bri2)\",\n      \"pmids\": [\"17557115\", \"17965014\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo consequences of FasL and Bri2 ICD signalling uncharacterised\", \"Structural basis of substrate selectivity unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Mapping the YXXφ lysosomal targeting motif at Y498 in the SPPL2A C-terminal tail explained how SPPL2A is sorted to lysosomes/late endosomes and distinguished from its plasma-membrane homologue SPPL2B, establishing compartment-specific substrate access.\",\n      \"evidence\": \"Chimeric SPPL2A/SPPL2B constructs with point mutations, immunofluorescence of endogenous protein, live-cell imaging\",\n      \"pmids\": [\"21896273\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Adaptor proteins mediating YXXφ-dependent sorting not identified\", \"Quantitative contribution of lysosomal vs. endosomal pools unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Three independent mouse models converged on CD74 NTF as the physiologically dominant SPPL2A substrate: loss of SPPL2A caused toxic CD74 NTF accumulation that blocked B cell maturation at T1 and depleted CD8-negative DCs, with full rescue by concurrent CD74 deletion, establishing a non-redundant SPPL2A–CD74 axis in adaptive immunity.\",\n      \"evidence\": \"SPPL2A knockout, ENU mutant (chompB), and knock-in mouse models; flow cytometry, Western blot, genetic epistasis with CD74 KO, bone marrow transfer\",\n      \"pmids\": [\"23267015\", \"23267013\", \"23267016\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which accumulated CD74 NTF disrupts endosomal trafficking incompletely defined\", \"Whether other substrates contribute to immune phenotype unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Conservation of the SPPL2A–CD74 axis was validated in humans when patient-derived cells carrying a homozygous SPPL2A deletion accumulated CD74 NTF identically to mouse knockouts, and TMEM106B was identified as a neurodegenerative-disease-relevant lysosomal substrate.\",\n      \"evidence\": \"Patient lymphoblastoid cell lines with SPPL2A genomic deletion (CD74 NTF); co-expression and catalytic-dead mutant assays (TMEM106B)\",\n      \"pmids\": [\"25035924\", \"24872421\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Neurological phenotype in SPPL2A-deficient patients not assessed\", \"In vivo relevance of TMEM106B cleavage to neurodegeneration untested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defining how accumulated CD74 NTF physically associates with BCR and Syk to suppress tonic PI3K/Akt signalling and elevate FOXO1-dependent pro-apoptotic transcription provided a molecular explanation for the T1 B cell maturation block in SPPL2A deficiency.\",\n      \"evidence\": \"Co-immunoprecipitation of CD74 NTF with BCR components, phospho-flow cytometry for Akt/Syk, transcription factor analysis in SPPL2A−/− mouse B cells\",\n      \"pmids\": [\"26157172\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct structural basis of CD74 NTF–BCR interaction unknown\", \"Whether this mechanism operates in mature B cells or germinal centres untested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Mass spectrometry-based mapping of the primary SPPL2A cleavage site to Y52–F53 in the CD74 transmembrane helix, combined with systematic alanine scanning, revealed that helix flexibility (glycine residues) and the luminal juxtamembrane domain are key determinants of intramembrane proteolysis.\",\n      \"evidence\": \"IP-MS cleavage site identification, domain-exchange and alanine-scanning mutagenesis, cell-based cleavage assays\",\n      \"pmids\": [\"26987812\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution structure of SPPL2A–substrate complex not available\", \"Rules for predicting novel substrates not generalised\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Human inborn errors of SPPL2A were shown to selectively deplete cDC2s and impair IFN-γ production by mycobacterium-specific TH1* cells, establishing SPPL2A deficiency as a Mendelian cause of susceptibility to mycobacterial disease (BCG disease).\",\n      \"evidence\": \"Whole-exome sequencing, patient immunophenotyping, in vitro stimulation, Sppl2a−/− mouse BCG/M. tuberculosis infection model, genetic complementation\",\n      \"pmids\": [\"30127434\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full clinical spectrum of human SPPL2A deficiency beyond mycobacterial susceptibility not defined\", \"Whether cDC2 loss is entirely CD74 NTF-dependent not formally demonstrated in patients\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Expansion of the SPPL2A substrate repertoire to include VAMP1–4 SNAREs (degradative clearance) and LOX-1 NTFs (suppression of ligand-independent MAP kinase signalling and atherosclerosis) demonstrated that SPPL2A functions beyond immune regulation in vesicular trafficking homeostasis and cardiovascular pathology.\",\n      \"evidence\": \"Systematic SNARE cleavage screen and SPPL2A/B double-KO mouse tissues (VAMPs); co-IP, signalling assays, and atherosclerosis plaque quantification in double-KO mice (LOX-1)\",\n      \"pmids\": [\"36047592\", \"30819724\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of SPPL2A vs. SPPL2B to individual SNARE and LOX-1 processing in vivo unclear\", \"Tissue-specific substrate hierarchy not mapped\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"A non-canonical ectodomain shedding activity of SPPL2A on TNFα, governed by transmembrane helix flexibility, raised the possibility that SPPL2A can operate in processing modes beyond classical intramembrane proteolysis.\",\n      \"evidence\": \"TM helix proline/leucine mutagenesis, CD and NMR structural analysis, molecular dynamics simulations\",\n      \"pmids\": [\"33294784\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological relevance of non-canonical shedding not demonstrated in vivo\", \"Whether non-canonical activity applies to other substrates unknown\", \"Single-lab observation\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Major open questions include the atomic structure of SPPL2A, the complete substrate repertoire across tissues, generalizable rules predicting substrate recognition, the full clinical phenotype of human SPPL2A deficiency beyond mycobacterial susceptibility, and whether accumulated CD74 NTF is the sole driver of cDC2 depletion in patients.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No high-resolution structure of SPPL2A or SPPL2A–substrate complex\", \"Tissue-specific substrate hierarchy and redundancy with SPPL2B not systematically defined\", \"Therapeutic implications of modulating SPPL2A activity unexplored\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 3, 5, 6, 7, 9, 12, 14, 15, 16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [4, 5, 6, 7, 9]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [1, 4, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [5, 6, 7, 10, 11, 13]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 2, 3, 5, 12, 15]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 11, 16]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [15]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"CD74\",\n      \"TNF\",\n      \"FASLG\",\n      \"ITM2B\",\n      \"TMEM106B\",\n      \"OLR1\",\n      \"VAMP1\",\n      \"VAMP2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}