{"gene":"SPPL2A","run_date":"2026-06-10T07:46:40","timeline":{"discoveries":[{"year":2006,"finding":"SPPL2a localizes to endosomes and functions as an aspartyl intramembrane protease (GxGD type) that catalyzes intramembrane cleavage of TNFα, releasing the TNFα intracellular domain (ICD), which in turn triggers IL-12 expression in activated human dendritic cells.","method":"Subcellular localization by sorting assays; intramembrane proteolysis demonstrated by release of TNFα ICD; IL-12 induction measured in activated dendritic cells","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct enzymatic activity assay with localization data and functional cytokine readout, single lab but multiple orthogonal methods","pmids":["16829952"],"is_preprint":false},{"year":2007,"finding":"SPPL2a (and SPPL2b) mediate intramembrane proteolysis of the Bri2 (Itm2b) N-terminal fragment after ADAM10 ectodomain shedding, generating an intracellular domain; SPP and SPPL3 cannot process this substrate. Loss-of-function variants of SPPL2a/b abolish this cleavage.","method":"Overexpression of all SPP/SPPL family members and their loss-of-function variants; Western blot detection of cleavage products","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — systematic family-wide expression with loss-of-function mutagenesis, multiple orthogonal controls, single lab","pmids":["17965014"],"is_preprint":false},{"year":2007,"finding":"SPPL2a cleaves the membrane-anchored N-terminal fragment of FasL (generated by ADAM10 ectodomain shedding) via intramembrane proteolysis, liberating the FasL intracellular domain (ICD), which translocates to the nucleus and inhibits gene transcription.","method":"Co-expression, Western blot detection of FasL ICD fragment, nuclear translocation assay, transcription inhibition assay in T cells with endogenous FasL","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Moderate — cleavage demonstrated at endogenous and overexpressed levels, nuclear translocation and functional transcriptional readout, single lab with multiple methods","pmids":["17557115"],"is_preprint":false},{"year":2012,"finding":"SPPL2a mediates intramembrane proteolysis of the invariant chain CD74 N-terminal fragment (NTF) in endosomes/lysosomes of B cells; SPPL2a deficiency causes accumulation of CD74 NTF, severely impairing endocytic membrane trafficking, B cell receptor signaling, BAFF-R surface expression, and MHC II compartment integrity, resulting in a B cell maturation arrest at the T1 stage and disrupted humoral immunity. The B cell defect is rescued by additional CD74 ablation.","method":"SPPL2a knockout mouse (genetic epistasis/rescue by CD74 double KO), Western blot for CD74 NTF accumulation, flow cytometry, endosomal trafficking assays, BCR signaling assays","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — three independent labs (PMIDs 23267015, 23267013, 23267016) using KO mice, genetic rescue, and proteomic substrate identification, replicated across labs","pmids":["23267015","23267013","23267016"],"is_preprint":false},{"year":2012,"finding":"Proteomic analysis of SPPL2a-deficient (chompB) mice identified CD74 as a key substrate; Sppl2a deficiency blocks CD74 regulated intramembrane proteolysis in B cells and myeloid dendritic cells, causing accumulation of the p8 cathepsin S product of CD74 and interfering with endosomal retention and earlier CD74 processing steps.","method":"ENU mutagenesis screen, genetic mapping, proteomic (mass spectrometry) substrate identification, Western blot in primary B cells and DCs","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic mapping plus MS-based substrate identification plus cellular phenotype, replicated by two additional concurrent studies","pmids":["23267013"],"is_preprint":false},{"year":2012,"finding":"SPPL2a deficiency in B cells causes dramatic build-up of the CD74 p8 fragment (product of cathepsin S), low surface BAFF-R, IgM, and IgD BCR; BCL2 overexpression rescues B cell accumulation but BAFF overexpression does not, placing SPPL2a-mediated CD74 processing upstream of BCL2-dependent survival signaling.","method":"Inactivating point mutation knock-in mice, BCL2 transgenic rescue, BAFF transgenic rescue, flow cytometry, Western blot","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with BCL2 vs BAFF rescue, replicated in independent KO models","pmids":["23267016"],"is_preprint":false},{"year":2013,"finding":"SPPL2a is expressed in enamel epithelium during amelogenesis and its genetic ablation in mice causes defective maturation-stage ameloblast function, incomplete resorption of proteinaceous enamel matrix, reduced mineral content, and enamel hypoplasia, establishing SPPL2a as essential for ameloblast cellular homeostasis.","method":"Sppl2a knockout mice, histology, micro-CT mineral quantification, immunohistochemistry of ameloblast morphology","journal":"Journal of bone and mineral research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KO with specific phenotypic readout (ameloblast function, enamel mineralization), single lab","pmids":["23426979"],"is_preprint":false},{"year":2014,"finding":"SPPL2a (and to a lesser extent SPPL2b) are responsible for intramembrane cleavage of the TMEM106B N-terminal fragment generated by lysosomal protease-dependent ectodomain processing, producing a small rapidly degraded ICD; TMEM106A, a paralog, is not a substrate of SPPL2a/b.","method":"Co-expression of GxGD protease family members, Western blot detection of cleavage products, pharmacological inhibition of lysosomal proteases, paralog comparison","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — systematic family-member comparison with protease inhibitor controls, single lab","pmids":["24872421"],"is_preprint":false},{"year":2015,"finding":"Accumulating CD74 NTF in SPPL2a-deficient B cells interacts with the BCR and Syk, impairs tonic and BCR-induced PI3K/Akt signaling, reduces surface IgM, and dysregulates FOXO1 transcription factor leading to elevated proapoptotic gene expression; SPPL2a-mediated CD74 NTF clearance is thus required to maintain appropriate BCR signaling for B cell maturation.","method":"SPPL2a KO mice, co-immunoprecipitation of CD74 NTF with BCR/Syk, phospho-flow/Western blot for PI3K/Akt pathway, FOXO1 localization, gene expression analysis","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus signaling pathway analysis plus KO rescue in SPPL2a-deficient cells, single lab with multiple orthogonal methods","pmids":["26157172"],"is_preprint":false},{"year":2016,"finding":"The primary SPPL2a cleavage site in CD74 is between Y52 and F53 within the transmembrane segment, identified by IP-MS of cleavage products. The intracellular domain of CD74 is dispensable for SPPL2a cleavage; helix-destabilizing glycines within the TM segment and specific luminal membrane-proximal residues facilitate efficient intramembrane proteolysis, while none of these determinants individually are absolutely essential.","method":"Domain-exchange experiments, IP-MS cleavage site identification, systematic alanine-scanning mutagenesis of CD74 TM and juxtamembrane regions","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct cleavage site identification by IP-MS plus systematic mutagenesis, single lab but multiple orthogonal approaches","pmids":["26987812"],"is_preprint":false},{"year":2018,"finding":"In humans, loss-of-function mutations in SPPL2A cause accumulation of CD74 NTF in HLA class II+ myeloid and lymphoid cells, selectively depleting IL-12/IL-23-producing CD1c+ conventional dendritic cells (cDC2s) and their progenitors; SPPL2a-deficient mice recapitulate cDC2 loss and show impaired IFN-γ production after BCG infection with high susceptibility to mycobacterial disease.","method":"Human patients with homozygous SPPL2A loss-of-function mutations, flow cytometry of DC subsets, in vitro mycobacterial antigen stimulation, Sppl2a KO mice BCG/M. tuberculosis infection model","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — human genetics validated in KO mouse model with mechanistic substrate (CD74 NTF accumulation) and functional immune readout, replicated across human and mouse systems","pmids":["30127434"],"is_preprint":false},{"year":2020,"finding":"SPPL2a exhibits a non-canonical ectodomain shedding activity on TNFα; conformational flexibility in the center of the TNFα TM helix (promoted by proline insertions, reduced by leucine mutations) determines the efficiency of this non-canonical shedding, as shown by biophysical characterization and molecular dynamics simulations.","method":"Proline/leucine mutagenesis of TNFα TM helix, cell-based cleavage assays, biophysical analysis (CD spectroscopy), molecular dynamics simulations","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — mutagenesis plus structural/MD analysis establishing substrate TM flexibility as determinant, single lab","pmids":["33294784"],"is_preprint":false},{"year":2020,"finding":"SPPL2a deficiency in dendritic cells alters pattern recognition receptor responses: Dectin-1 is redistributed to endosomal compartments, leading to enhanced IL-1β and reduced IL-10 and IFN-β secretion upon mycobacterial stimulation in a CD74-dependent manner.","method":"SPPL2a/CD74 double KO bone marrow-derived DCs, cytokine ELISA, selective PRR stimulation (TLR4, Dectin-1), confocal microscopy of Dectin-1 localization","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — genetic epistasis (SPPL2a vs SPPL2a/CD74 dKO) with localization and functional cytokine data, single lab","pmids":["33239420"],"is_preprint":false},{"year":2022,"finding":"SPPL2a and SPPL2b cleave tail-anchored SNARE proteins VAMP1, VAMP2, VAMP3, and VAMP4 in the endocytic/late secretory pathway; loss of SPPL2a/b in double-KO mice causes tissue- and cell-type-dependent accumulation of VAMP1-4, identifying these SNAREs as in vivo substrates and implicating SPPL2a/b in SNARE protein turnover.","method":"Cellular co-expression screen of 18 SNARE proteins, pharmacological SPPL2a/b inhibition in cell lines, Western blot in SPPL2a/b double-KO mouse tissues and primary cells","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — systematic substrate screen validated at endogenous level in multiple cell types and in vivo KO mice, single lab with multiple orthogonal methods","pmids":["36047592"],"is_preprint":false}],"current_model":"SPPL2a is a GxGD-type aspartyl intramembrane protease localized to lysosomes/late endosomes that cleaves type II transmembrane proteins (including CD74/invariant chain, TNFα, FasL, Bri2, TMEM106B, and VAMP1-4) after ectodomain shedding, releasing intracellular domains that can signal to the nucleus; its most critical in vivo role is the degradation of the CD74 N-terminal fragment, whose accumulation in SPPL2a-deficient B cells and dendritic cells disrupts BCR signaling, endosomal trafficking, cDC2 survival, and IL-12/IFN-γ-dependent antimycobacterial immunity."},"narrative":{"mechanistic_narrative":"SPPL2a is a GxGD-type aspartyl intramembrane protease of the endosomal/lysosomal system that cleaves the membrane-anchored N-terminal fragments of type II transmembrane proteins after their ectodomains have been shed, liberating intracellular domains and turning over residual membrane stubs [PMID:16829952, PMID:17965014]. Validated substrates include TNFα—whose released intracellular domain triggers IL-12 expression in dendritic cells [PMID:16829952]—FasL, whose liberated domain translocates to the nucleus to inhibit transcription [PMID:17557115], the Bri2/Itm2b fragment generated after ADAM10 shedding [PMID:17965014], TMEM106B [PMID:24872421], and the tail-anchored SNAREs VAMP1–4, whose accumulation in SPPL2a/b double-knockout tissues implicates the protease in SNARE turnover [PMID:36047592]. Cleavage proceeds within the transmembrane segment, with substrate processing favored by helix-destabilizing residues and conformational flexibility; in CD74 the principal scission occurs between Y52 and F53 and does not require the substrate intracellular domain [PMID:26987812, PMID:33294784]. Its most consequential physiological role is the clearance of the CD74 (invariant chain) N-terminal fragment in B cells and dendritic cells: failure to process CD74 NTF causes accumulation of the cathepsin S–derived p8 fragment, disrupts endosomal trafficking and MHC II compartment integrity, and arrests B cell maturation at the T1 stage, a defect rescued by CD74 ablation [PMID:23267015, PMID:23267013, PMID:23267016]. Accumulated CD74 NTF engages the BCR and Syk to impair PI3K/Akt signaling and dysregulate FOXO1, placing SPPL2a-dependent CD74 turnover upstream of BCL2-dependent B cell survival [PMID:23267016, PMID:26157172]. In humans, biallelic loss-of-function mutations in SPPL2A cause CD74 NTF accumulation that selectively depletes IL-12/IL-23–producing CD1c+ cDC2s and impairs IFN-γ–dependent antimycobacterial immunity [PMID:30127434]. SPPL2a is additionally required for maturation-stage ameloblast homeostasis and enamel mineralization [PMID:23426979].","teleology":[{"year":2006,"claim":"Established SPPL2a as a catalytically active endosomal intramembrane aspartyl protease with a defined substrate and a downstream signaling consequence, answering whether this GxGD family member is a functional protease.","evidence":"Subcellular sorting assays plus TNFα ICD release and IL-12 induction in activated human dendritic cells","pmids":["16829952"],"confidence":"High","gaps":["Cleavage site within TNFα not defined","Physiological requirement in vivo not yet tested"]},{"year":2007,"claim":"Defined SPPL2a substrate specificity by showing it (and SPPL2b) process the Bri2 and FasL N-terminal fragments while other family members cannot, establishing it as a dedicated intramembrane protease acting after ADAM10 shedding.","evidence":"Family-wide overexpression with loss-of-function variants, Western blot of cleavage products, and nuclear translocation/transcription assays for FasL ICD","pmids":["17965014","17557115"],"confidence":"High","gaps":["In vivo relevance of Bri2 and FasL processing not addressed","Mechanism of FasL ICD transcriptional inhibition unresolved"]},{"year":2012,"claim":"Identified CD74 as the critical in vivo substrate and explained the B cell phenotype, answering why SPPL2a loss disrupts humoral immunity.","evidence":"SPPL2a knockout and ENU-mutant mice, MS-based substrate identification, CD74 double-KO genetic rescue, and BCL2 vs BAFF epistasis","pmids":["23267015","23267013","23267016"],"confidence":"High","gaps":["Molecular mechanism by which CD74 NTF blocks trafficking not yet defined","Effect on non-B-cell lineages incompletely mapped"]},{"year":2015,"claim":"Mechanistically linked CD74 NTF accumulation to defective BCR signaling, showing the fragment engages the BCR/Syk to impair PI3K/Akt and dysregulate FOXO1.","evidence":"SPPL2a KO mice with reciprocal Co-IP of CD74 NTF with BCR/Syk and phospho-signaling/gene-expression analysis","pmids":["26157172"],"confidence":"High","gaps":["Structural basis of CD74 NTF–BCR interaction unknown","Quantitative contribution of FOXO1 dysregulation to apoptosis not isolated"]},{"year":2016,"claim":"Resolved the molecular determinants of cleavage by mapping the CD74 scission site and the TM/juxtamembrane features that promote processing, defining substrate recognition rules.","evidence":"IP-MS cleavage site identification (Y52/F53) plus domain-exchange and alanine-scanning mutagenesis","pmids":["26987812"],"confidence":"High","gaps":["No structure of SPPL2a–substrate complex","Catalytic mechanism of TM scission not directly visualized"]},{"year":2018,"claim":"Extended the mechanism to human disease, showing SPPL2A loss-of-function selectively depletes cDC2s and impairs antimycobacterial immunity via CD74 NTF accumulation.","evidence":"Human patients with homozygous LOF mutations, DC subset flow cytometry, and Sppl2a KO mouse BCG/M. tuberculosis infection models","pmids":["30127434"],"confidence":"High","gaps":["Why cDC2s are selectively vulnerable to CD74 NTF not fully explained","Therapeutic implications untested"]},{"year":2020,"claim":"Broadened understanding of substrate engagement and immune consequences, characterizing TNFα TM flexibility as a cleavage determinant and CD74-dependent rewiring of PRR responses.","evidence":"TNFα TM proline/leucine mutagenesis with CD/MD analysis; SPPL2a/CD74 double-KO BMDCs with Dectin-1 localization and cytokine profiling","pmids":["33294784","33239420"],"confidence":"Medium","gaps":["TNFα shedding determinants studied biophysically without in vivo validation","Single-lab cytokine and localization data for PRR rewiring"]},{"year":2022,"claim":"Identified VAMP1–4 SNAREs as in vivo substrates, expanding SPPL2a's role to SNARE protein turnover in the endocytic/secretory pathway.","evidence":"Co-expression screen of 18 SNAREs, pharmacological inhibition, and Western blot in SPPL2a/b double-KO mouse tissues","pmids":["36047592"],"confidence":"High","gaps":["Functional consequence of VAMP accumulation for membrane trafficking not defined","Relative SPPL2a vs SPPL2b contribution to each VAMP unresolved"]},{"year":null,"claim":"How SPPL2a substrate selection, cleavage-site choice, and tissue-specific activity are coordinated structurally and regulated remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No experimental structure of SPPL2a","Regulation of protease activity and substrate prioritization unknown","Mechanism of ameloblast requirement (#6) not connected to a defined substrate"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,1,2,9,13]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,3,9,13]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,3]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[3,4]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,9,13]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[3,5,10,12]}],"complexes":[],"partners":["CD74","TNF","FASLG","ITM2B","TMEM106B","VAMP1","VAMP2","SYK"],"other_free_text":[]}},"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":"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":"17557115","id":"PMC_17557115","title":"The Fas ligand intracellular domain is released by ADAM10 and SPPL2a cleavage in T-cells.","date":"2007","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/17557115","citation_count":114,"is_preprint":false},{"pmid":"23267015","id":"PMC_23267015","title":"The intramembrane protease SPPL2a promotes B cell development and controls endosomal traffic by cleavage of the invariant chain.","date":"2012","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/23267015","citation_count":107,"is_preprint":false},{"pmid":"30127434","id":"PMC_30127434","title":"Disruption of an antimycobacterial circuit between dendritic and helper T cells in human SPPL2a deficiency.","date":"2018","source":"Nature immunology","url":"https://pubmed.ncbi.nlm.nih.gov/30127434","citation_count":93,"is_preprint":false},{"pmid":"23267013","id":"PMC_23267013","title":"The intramembrane protease Sppl2a is required for B cell and DC development and survival via cleavage of the invariant chain.","date":"2012","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/23267013","citation_count":77,"is_preprint":false},{"pmid":"23267016","id":"PMC_23267016","title":"B cell survival, surface BCR and BAFFR expression, CD74 metabolism, and CD8- dendritic cells require the intramembrane endopeptidase SPPL2A.","date":"2012","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/23267016","citation_count":73,"is_preprint":false},{"pmid":"24872421","id":"PMC_24872421","title":"Regulated intramembrane proteolysis of the frontotemporal lobar degeneration risk factor, TMEM106B, by signal peptide peptidase-like 2a (SPPL2a).","date":"2014","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/24872421","citation_count":43,"is_preprint":false},{"pmid":"26157172","id":"PMC_26157172","title":"Processing of CD74 by the Intramembrane Protease SPPL2a Is Critical for B Cell Receptor Signaling in Transitional B Cells.","date":"2015","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/26157172","citation_count":29,"is_preprint":false},{"pmid":"1884982","id":"PMC_1884982","title":"Identification, cloning and sequencing of the replication region of Lactococcus lactis ssp. lactis biovar. diacetylactis Bu2 citrate plasmid pSL2.","date":"1991","source":"FEMS microbiology letters","url":"https://pubmed.ncbi.nlm.nih.gov/1884982","citation_count":26,"is_preprint":false},{"pmid":"26987812","id":"PMC_26987812","title":"Substrate determinants of signal peptide peptidase-like 2a (SPPL2a)-mediated intramembrane proteolysis of the invariant chain CD74.","date":"2016","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/26987812","citation_count":24,"is_preprint":false},{"pmid":"33294784","id":"PMC_33294784","title":"Non-canonical Shedding of TNFα by SPPL2a Is Determined by the Conformational Flexibility of Its Transmembrane Helix.","date":"2020","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/33294784","citation_count":22,"is_preprint":false},{"pmid":"3098895","id":"PMC_3098895","title":"Human rheumatoid factor crossidiotypes. II. Primary structure-dependent crossreactive idiotype, PSL2-CRI, present on Wa monoclonal rheumatoid factors is present on Bla and other IgM kappa monoclonal autoantibodies.","date":"1987","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/3098895","citation_count":21,"is_preprint":false},{"pmid":"23426979","id":"PMC_23426979","title":"The intramembrane protease SPPL2A is critical for tooth enamel formation.","date":"2013","source":"Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research","url":"https://pubmed.ncbi.nlm.nih.gov/23426979","citation_count":16,"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":"33239420","id":"PMC_33239420","title":"Deficiency of the Intramembrane Protease SPPL2a Alters Antimycobacterial Cytokine Responses of Dendritic Cells.","date":"2020","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/33239420","citation_count":9,"is_preprint":false},{"pmid":"32198923","id":"PMC_32198923","title":"Impaired proteolysis by SPPL2a causes CD74 fragment accumulation that can be recognized by anti-CD74 autoantibodies in human ankylosing spondylitis.","date":"2020","source":"European journal of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/32198923","citation_count":9,"is_preprint":false},{"pmid":"28731783","id":"PMC_28731783","title":"Identification of SPPL2a Inhibitors by Multiparametric Analysis of a High-Content Ultra-High-Throughput Screen.","date":"2017","source":"SLAS discovery : advancing life sciences R & D","url":"https://pubmed.ncbi.nlm.nih.gov/28731783","citation_count":9,"is_preprint":false},{"pmid":"37896114","id":"PMC_37896114","title":"Alleviation of Cadmium Toxicity in Thai Rice Cultivar (PSL2) Using Biofertilizer Containing Indigenous Cadmium-Resistant Microbial Consortia.","date":"2023","source":"Plants (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/37896114","citation_count":3,"is_preprint":false},{"pmid":"36310421","id":"PMC_36310421","title":"On the track of intramembrane clippers: the SPPL2a/b proteases caught in the act in animal models.","date":"2022","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/36310421","citation_count":2,"is_preprint":false},{"pmid":"30127437","id":"PMC_30127437","title":"Old vaccines, new diseases: when BCG meets SPPL2a.","date":"2018","source":"Nature immunology","url":"https://pubmed.ncbi.nlm.nih.gov/30127437","citation_count":1,"is_preprint":false},{"pmid":"39586751","id":"PMC_39586751","title":"Mendelian Susceptibility to Mycobacterial Disease with Signal Peptide Peptidase-like 2A (SPPL2A) Deficiency: A Case Report.","date":"2024","source":"Iranian journal of allergy, asthma, and immunology","url":"https://pubmed.ncbi.nlm.nih.gov/39586751","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11820,"output_tokens":3740,"usd":0.04578,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11452,"output_tokens":3202,"usd":0.068655,"stage2_stop_reason":"end_turn"},"total_usd":0.114435,"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\": \"SPPL2a localizes to endosomes and functions as an aspartyl intramembrane protease (GxGD type) that catalyzes intramembrane cleavage of TNFα, releasing the TNFα intracellular domain (ICD), which in turn triggers IL-12 expression in activated human dendritic cells.\",\n      \"method\": \"Subcellular localization by sorting assays; intramembrane proteolysis demonstrated by release of TNFα ICD; IL-12 induction measured in activated dendritic cells\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct enzymatic activity assay with localization data and functional cytokine readout, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"16829952\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SPPL2a (and SPPL2b) mediate intramembrane proteolysis of the Bri2 (Itm2b) N-terminal fragment after ADAM10 ectodomain shedding, generating an intracellular domain; SPP and SPPL3 cannot process this substrate. Loss-of-function variants of SPPL2a/b abolish this cleavage.\",\n      \"method\": \"Overexpression of all SPP/SPPL family members and their loss-of-function variants; Western blot detection of cleavage products\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic family-wide expression with loss-of-function mutagenesis, multiple orthogonal controls, single lab\",\n      \"pmids\": [\"17965014\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SPPL2a cleaves the membrane-anchored N-terminal fragment of FasL (generated by ADAM10 ectodomain shedding) via intramembrane proteolysis, liberating the FasL intracellular domain (ICD), which translocates to the nucleus and inhibits gene transcription.\",\n      \"method\": \"Co-expression, Western blot detection of FasL ICD fragment, nuclear translocation assay, transcription inhibition assay in T cells with endogenous FasL\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cleavage demonstrated at endogenous and overexpressed levels, nuclear translocation and functional transcriptional readout, single lab with multiple methods\",\n      \"pmids\": [\"17557115\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SPPL2a mediates intramembrane proteolysis of the invariant chain CD74 N-terminal fragment (NTF) in endosomes/lysosomes of B cells; SPPL2a deficiency causes accumulation of CD74 NTF, severely impairing endocytic membrane trafficking, B cell receptor signaling, BAFF-R surface expression, and MHC II compartment integrity, resulting in a B cell maturation arrest at the T1 stage and disrupted humoral immunity. The B cell defect is rescued by additional CD74 ablation.\",\n      \"method\": \"SPPL2a knockout mouse (genetic epistasis/rescue by CD74 double KO), Western blot for CD74 NTF accumulation, flow cytometry, endosomal trafficking assays, BCR signaling assays\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — three independent labs (PMIDs 23267015, 23267013, 23267016) using KO mice, genetic rescue, and proteomic substrate identification, replicated across labs\",\n      \"pmids\": [\"23267015\", \"23267013\", \"23267016\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Proteomic analysis of SPPL2a-deficient (chompB) mice identified CD74 as a key substrate; Sppl2a deficiency blocks CD74 regulated intramembrane proteolysis in B cells and myeloid dendritic cells, causing accumulation of the p8 cathepsin S product of CD74 and interfering with endosomal retention and earlier CD74 processing steps.\",\n      \"method\": \"ENU mutagenesis screen, genetic mapping, proteomic (mass spectrometry) substrate identification, Western blot in primary B cells and DCs\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic mapping plus MS-based substrate identification plus cellular phenotype, replicated by two additional concurrent studies\",\n      \"pmids\": [\"23267013\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SPPL2a deficiency in B cells causes dramatic build-up of the CD74 p8 fragment (product of cathepsin S), low surface BAFF-R, IgM, and IgD BCR; BCL2 overexpression rescues B cell accumulation but BAFF overexpression does not, placing SPPL2a-mediated CD74 processing upstream of BCL2-dependent survival signaling.\",\n      \"method\": \"Inactivating point mutation knock-in mice, BCL2 transgenic rescue, BAFF transgenic rescue, flow cytometry, Western blot\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with BCL2 vs BAFF rescue, replicated in independent KO models\",\n      \"pmids\": [\"23267016\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"SPPL2a is expressed in enamel epithelium during amelogenesis and its genetic ablation in mice causes defective maturation-stage ameloblast function, incomplete resorption of proteinaceous enamel matrix, reduced mineral content, and enamel hypoplasia, establishing SPPL2a as essential for ameloblast cellular homeostasis.\",\n      \"method\": \"Sppl2a knockout mice, histology, micro-CT mineral quantification, immunohistochemistry of ameloblast morphology\",\n      \"journal\": \"Journal of bone and mineral research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KO with specific phenotypic readout (ameloblast function, enamel mineralization), single lab\",\n      \"pmids\": [\"23426979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SPPL2a (and to a lesser extent SPPL2b) are responsible for intramembrane cleavage of the TMEM106B N-terminal fragment generated by lysosomal protease-dependent ectodomain processing, producing a small rapidly degraded ICD; TMEM106A, a paralog, is not a substrate of SPPL2a/b.\",\n      \"method\": \"Co-expression of GxGD protease family members, Western blot detection of cleavage products, pharmacological inhibition of lysosomal proteases, paralog comparison\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — systematic family-member comparison with protease inhibitor controls, single lab\",\n      \"pmids\": [\"24872421\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Accumulating CD74 NTF in SPPL2a-deficient B cells interacts with the BCR and Syk, impairs tonic and BCR-induced PI3K/Akt signaling, reduces surface IgM, and dysregulates FOXO1 transcription factor leading to elevated proapoptotic gene expression; SPPL2a-mediated CD74 NTF clearance is thus required to maintain appropriate BCR signaling for B cell maturation.\",\n      \"method\": \"SPPL2a KO mice, co-immunoprecipitation of CD74 NTF with BCR/Syk, phospho-flow/Western blot for PI3K/Akt pathway, FOXO1 localization, gene expression analysis\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus signaling pathway analysis plus KO rescue in SPPL2a-deficient cells, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"26157172\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The primary SPPL2a cleavage site in CD74 is between Y52 and F53 within the transmembrane segment, identified by IP-MS of cleavage products. The intracellular domain of CD74 is dispensable for SPPL2a cleavage; helix-destabilizing glycines within the TM segment and specific luminal membrane-proximal residues facilitate efficient intramembrane proteolysis, while none of these determinants individually are absolutely essential.\",\n      \"method\": \"Domain-exchange experiments, IP-MS cleavage site identification, systematic alanine-scanning mutagenesis of CD74 TM and juxtamembrane regions\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct cleavage site identification by IP-MS plus systematic mutagenesis, single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"26987812\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In humans, loss-of-function mutations in SPPL2A cause accumulation of CD74 NTF in HLA class II+ myeloid and lymphoid cells, selectively depleting IL-12/IL-23-producing CD1c+ conventional dendritic cells (cDC2s) and their progenitors; SPPL2a-deficient mice recapitulate cDC2 loss and show impaired IFN-γ production after BCG infection with high susceptibility to mycobacterial disease.\",\n      \"method\": \"Human patients with homozygous SPPL2A loss-of-function mutations, flow cytometry of DC subsets, in vitro mycobacterial antigen stimulation, Sppl2a KO mice BCG/M. tuberculosis infection model\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — human genetics validated in KO mouse model with mechanistic substrate (CD74 NTF accumulation) and functional immune readout, replicated across human and mouse systems\",\n      \"pmids\": [\"30127434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SPPL2a exhibits a non-canonical ectodomain shedding activity on TNFα; conformational flexibility in the center of the TNFα TM helix (promoted by proline insertions, reduced by leucine mutations) determines the efficiency of this non-canonical shedding, as shown by biophysical characterization and molecular dynamics simulations.\",\n      \"method\": \"Proline/leucine mutagenesis of TNFα TM helix, cell-based cleavage assays, biophysical analysis (CD spectroscopy), molecular dynamics simulations\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — mutagenesis plus structural/MD analysis establishing substrate TM flexibility as determinant, single lab\",\n      \"pmids\": [\"33294784\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SPPL2a deficiency in dendritic cells alters pattern recognition receptor responses: Dectin-1 is redistributed to endosomal compartments, leading to enhanced IL-1β and reduced IL-10 and IFN-β secretion upon mycobacterial stimulation in a CD74-dependent manner.\",\n      \"method\": \"SPPL2a/CD74 double KO bone marrow-derived DCs, cytokine ELISA, selective PRR stimulation (TLR4, Dectin-1), confocal microscopy of Dectin-1 localization\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — genetic epistasis (SPPL2a vs SPPL2a/CD74 dKO) with localization and functional cytokine data, single lab\",\n      \"pmids\": [\"33239420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SPPL2a and SPPL2b cleave tail-anchored SNARE proteins VAMP1, VAMP2, VAMP3, and VAMP4 in the endocytic/late secretory pathway; loss of SPPL2a/b in double-KO mice causes tissue- and cell-type-dependent accumulation of VAMP1-4, identifying these SNAREs as in vivo substrates and implicating SPPL2a/b in SNARE protein turnover.\",\n      \"method\": \"Cellular co-expression screen of 18 SNARE proteins, pharmacological SPPL2a/b inhibition in cell lines, Western blot in SPPL2a/b double-KO mouse tissues and primary cells\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic substrate screen validated at endogenous level in multiple cell types and in vivo KO mice, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"36047592\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SPPL2a is a GxGD-type aspartyl intramembrane protease localized to lysosomes/late endosomes that cleaves type II transmembrane proteins (including CD74/invariant chain, TNFα, FasL, Bri2, TMEM106B, and VAMP1-4) after ectodomain shedding, releasing intracellular domains that can signal to the nucleus; its most critical in vivo role is the degradation of the CD74 N-terminal fragment, whose accumulation in SPPL2a-deficient B cells and dendritic cells disrupts BCR signaling, endosomal trafficking, cDC2 survival, and IL-12/IFN-γ-dependent antimycobacterial immunity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SPPL2a is a GxGD-type aspartyl intramembrane protease of the endosomal/lysosomal system that cleaves the membrane-anchored N-terminal fragments of type II transmembrane proteins after their ectodomains have been shed, liberating intracellular domains and turning over residual membrane stubs [#0, #1]. Validated substrates include TNFα—whose released intracellular domain triggers IL-12 expression in dendritic cells [#0]—FasL, whose liberated domain translocates to the nucleus to inhibit transcription [#2], the Bri2/Itm2b fragment generated after ADAM10 shedding [#1], TMEM106B [#7], and the tail-anchored SNAREs VAMP1–4, whose accumulation in SPPL2a/b double-knockout tissues implicates the protease in SNARE turnover [#13]. Cleavage proceeds within the transmembrane segment, with substrate processing favored by helix-destabilizing residues and conformational flexibility; in CD74 the principal scission occurs between Y52 and F53 and does not require the substrate intracellular domain [#9, #11]. Its most consequential physiological role is the clearance of the CD74 (invariant chain) N-terminal fragment in B cells and dendritic cells: failure to process CD74 NTF causes accumulation of the cathepsin S–derived p8 fragment, disrupts endosomal trafficking and MHC II compartment integrity, and arrests B cell maturation at the T1 stage, a defect rescued by CD74 ablation [#3, #4]. Accumulated CD74 NTF engages the BCR and Syk to impair PI3K/Akt signaling and dysregulate FOXO1, placing SPPL2a-dependent CD74 turnover upstream of BCL2-dependent B cell survival [#5, #8]. In humans, biallelic loss-of-function mutations in SPPL2A cause CD74 NTF accumulation that selectively depletes IL-12/IL-23–producing CD1c+ cDC2s and impairs IFN-γ–dependent antimycobacterial immunity [#10]. SPPL2a is additionally required for maturation-stage ameloblast homeostasis and enamel mineralization [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established SPPL2a as a catalytically active endosomal intramembrane aspartyl protease with a defined substrate and a downstream signaling consequence, answering whether this GxGD family member is a functional protease.\",\n      \"evidence\": \"Subcellular sorting assays plus TNFα ICD release and IL-12 induction in activated human dendritic cells\",\n      \"pmids\": [\"16829952\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cleavage site within TNFα not defined\", \"Physiological requirement in vivo not yet tested\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined SPPL2a substrate specificity by showing it (and SPPL2b) process the Bri2 and FasL N-terminal fragments while other family members cannot, establishing it as a dedicated intramembrane protease acting after ADAM10 shedding.\",\n      \"evidence\": \"Family-wide overexpression with loss-of-function variants, Western blot of cleavage products, and nuclear translocation/transcription assays for FasL ICD\",\n      \"pmids\": [\"17965014\", \"17557115\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of Bri2 and FasL processing not addressed\", \"Mechanism of FasL ICD transcriptional inhibition unresolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified CD74 as the critical in vivo substrate and explained the B cell phenotype, answering why SPPL2a loss disrupts humoral immunity.\",\n      \"evidence\": \"SPPL2a knockout and ENU-mutant mice, MS-based substrate identification, CD74 double-KO genetic rescue, and BCL2 vs BAFF epistasis\",\n      \"pmids\": [\"23267015\", \"23267013\", \"23267016\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism by which CD74 NTF blocks trafficking not yet defined\", \"Effect on non-B-cell lineages incompletely mapped\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Mechanistically linked CD74 NTF accumulation to defective BCR signaling, showing the fragment engages the BCR/Syk to impair PI3K/Akt and dysregulate FOXO1.\",\n      \"evidence\": \"SPPL2a KO mice with reciprocal Co-IP of CD74 NTF with BCR/Syk and phospho-signaling/gene-expression analysis\",\n      \"pmids\": [\"26157172\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of CD74 NTF–BCR interaction unknown\", \"Quantitative contribution of FOXO1 dysregulation to apoptosis not isolated\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Resolved the molecular determinants of cleavage by mapping the CD74 scission site and the TM/juxtamembrane features that promote processing, defining substrate recognition rules.\",\n      \"evidence\": \"IP-MS cleavage site identification (Y52/F53) plus domain-exchange and alanine-scanning mutagenesis\",\n      \"pmids\": [\"26987812\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of SPPL2a–substrate complex\", \"Catalytic mechanism of TM scission not directly visualized\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Extended the mechanism to human disease, showing SPPL2A loss-of-function selectively depletes cDC2s and impairs antimycobacterial immunity via CD74 NTF accumulation.\",\n      \"evidence\": \"Human patients with homozygous LOF mutations, DC subset flow cytometry, and Sppl2a KO mouse BCG/M. tuberculosis infection models\",\n      \"pmids\": [\"30127434\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why cDC2s are selectively vulnerable to CD74 NTF not fully explained\", \"Therapeutic implications untested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Broadened understanding of substrate engagement and immune consequences, characterizing TNFα TM flexibility as a cleavage determinant and CD74-dependent rewiring of PRR responses.\",\n      \"evidence\": \"TNFα TM proline/leucine mutagenesis with CD/MD analysis; SPPL2a/CD74 double-KO BMDCs with Dectin-1 localization and cytokine profiling\",\n      \"pmids\": [\"33294784\", \"33239420\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"TNFα shedding determinants studied biophysically without in vivo validation\", \"Single-lab cytokine and localization data for PRR rewiring\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified VAMP1–4 SNAREs as in vivo substrates, expanding SPPL2a's role to SNARE protein turnover in the endocytic/secretory pathway.\",\n      \"evidence\": \"Co-expression screen of 18 SNAREs, pharmacological inhibition, and Western blot in SPPL2a/b double-KO mouse tissues\",\n      \"pmids\": [\"36047592\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of VAMP accumulation for membrane trafficking not defined\", \"Relative SPPL2a vs SPPL2b contribution to each VAMP unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How SPPL2a substrate selection, cleavage-site choice, and tissue-specific activity are coordinated structurally and regulated remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No experimental structure of SPPL2a\", \"Regulation of protease activity and substrate prioritization unknown\", \"Mechanism of ameloblast requirement (#6) not connected to a defined substrate\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 1, 2, 9, 13]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 3, 9, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 9, 13]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [3, 5, 10, 12]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CD74\", \"TNF\", \"FASLG\", \"ITM2B\", \"TMEM106B\", \"VAMP1\", \"VAMP2\", \"SYK\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}