{"gene":"RHBDL2","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2004,"finding":"RHBDL2 cleaves the intramembrane/transmembrane domain of ephrinB3, a B-type ephrin, when co-expressed in mammalian cells, reducing ephrinB3 surface levels; the amino acid sequence at the luminal face of the transmembrane domain of the substrate determines cleavage susceptibility by RHBDL2.","method":"Co-expression in mammalian cells; substrate domain mutagenesis; cell-surface detection","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct cleavage assay in mammalian cells with domain-swap mutagenesis identifying luminal determinants, single lab","pmids":["15047175"],"is_preprint":false},{"year":2011,"finding":"RHBDL2 cleaves EGF just outside its transmembrane domain, facilitating EGF secretion and triggering EGFR activation; endogenous RHBDL2 activity was detected in several tumour cell lines.","method":"Overexpression and cleavage assay in mammalian cells; EGFR activation readout; detection of endogenous activity in tumour cell lines","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct cleavage assay with signalling readout, endogenous activity confirmed, single lab","pmids":["21494248"],"is_preprint":false},{"year":2011,"finding":"RHBDL2 cleaves thrombomodulin (TM) at its transmembrane domain, releasing soluble TM (sTM) from keratinocytes; both RHBDL2 and TM are upregulated during wound healing, and RHBDL2 inhibition (by DCI or shRNA) blocks sTM shedding and impairs wound healing in cells and in vivo.","method":"shRNA knockdown; pharmacological inhibition (DCI); conditioned media assay; ex vivo skin culture; in vivo mouse wound model","journal":"The Journal of investigative dermatology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (shRNA, pharmacological inhibitor, in vivo model), functional rescue with recombinant sTM, replicated across cell and animal systems","pmids":["21833011"],"is_preprint":false},{"year":2016,"finding":"RHBDL2 (but not RHBDL1 or RHBDL3) specifically cleaves CLEC14A, shedding its ectodomain; site-directed mutagenesis identified the precise cleavage site, and siRNA knockdown of endogenous RHBDL2 validated CLEC14A shedding specificity; shed CLEC14A ectodomain inhibits sprouting angiogenesis.","method":"Co-expression cleavage assay; site-directed mutagenesis; siRNA knockdown of endogenous RHBDL2; in vitro and in vivo angiogenesis assays","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — site-directed mutagenesis identifying cleavage site, endogenous knockdown validation, functional in vitro and in vivo readouts, single lab with multiple orthogonal methods","pmids":["26939791"],"is_preprint":false},{"year":2017,"finding":"Quantitative proteomics identified a substrate repertoire of RHBDL2 in human cells including IL6R, Spint-1, DDR1, N-Cadherin, CLCP1/DCBLD2, KIRREL, and BCAM; these substrates are specifically shed by endogenous RHBDL2 and a subset is resistant to metalloprotease-mediated shedding.","method":"Quantitative proteomics (SILAC-based); endogenous RHBDL2-dependent shedding validation","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — quantitative proteomics with endogenous activity validation, multiple substrates confirmed, single lab but multiple orthogonal approaches","pmids":["28779096"],"is_preprint":false},{"year":2014,"finding":"RHBDL2 overexpression in human epithelial cells promotes cell proliferation, reduces cell adhesion, and suppresses anoikis; inhibition of RHBDL2 (by rhomboid protease inhibitor or shRNA) increases cleaved caspase-3, and EGFR inhibition similarly increases apoptosis markers, placing RHBDL2 upstream of EGFR-mediated anoikis resistance.","method":"Overexpression; shRNA knockdown; pharmacological inhibition; caspase-3 cleavage assay; FAK phosphorylation assay; suspension culture","journal":"TheScientificWorldJournal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss- and gain-of-function with multiple readouts, pathway placement via EGFR inhibitor epistasis, single lab","pmids":["24977233"],"is_preprint":false},{"year":2021,"finding":"RHBDL2 cleaves the IL-11 receptor (IL-11R) at the extracellular region between Ala-370 and Ser-371, generating soluble IL-11R capable of IL-11 trans-signaling; critical residues within the transmembrane helix are required for proteolysis; RHBDL2 can cleave IL-11R in the early secretory pathway as well as at the plasma membrane; the human mutation IL-11R-A370V prevents RHBDL2-mediated cleavage without impairing classic IL-11 signaling.","method":"Co-expression cleavage assay; cleavage site mapping; transmembrane domain mutagenesis; subcellular localization analysis; trans-signaling functional assay; disease variant analysis","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — cleavage site precisely mapped, mutagenesis of substrate and TM domain, functional trans-signaling assay, subcellular localization determined, disease variant validation, single lab with multiple orthogonal methods","pmids":["33566379"],"is_preprint":false},{"year":2022,"finding":"RHBDL2 interacts with and cleaves Notch1, releasing the intracellular domain N1ICD; RHBDL2 also collaborates with the deubiquitinase OTUD7B to reduce ubiquitination of N1ICD, stabilizing it via the ubiquitin-proteasome pathway, thereby activating Notch signaling and promoting pancreatic cancer cell proliferation and mobility.","method":"Co-immunoprecipitation; cleavage assay; RNA-seq; ubiquitination assay; gain- and loss-of-function in vitro and in vivo; Notch inhibitor (IMR-1) rescue","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, cleavage assay, ubiquitination assay, epistasis with Notch inhibitor, in vivo confirmation, single lab","pmids":["36351890"],"is_preprint":false},{"year":2021,"finding":"RHBDL2 processes stochastically activated Orai1/CRAC channels as atypical substrates in unstimulated cells, acting as a 'conformational surveillance' mechanism that prevents unwanted Ca2+ signaling; selective processing requires CRAC channel activation state.","method":"Described via commentary on Grieve et al. (2021) in Molecular Cell; experimental basis inferred as RHBDL2 substrate cleavage assay linked to CRAC channel conformation","journal":"Molecular cell","confidence":"Low","confidence_rationale":"Tier 3 / Weak — this is a commentary/preview summarizing another study; experimental details are not directly described in this abstract","pmids":["34861185"],"is_preprint":false},{"year":2026,"finding":"RHBDL2 functions as a non-proteolytic scaffold to stabilize the deubiquitinase USP3 via a hydrophobic core interaction mediated by Val245 of RHBDL2, independently of its protease activity; stabilized USP3 then deubiquitinates PPT1, preventing its proteasomal degradation, thereby promoting FASN-dependent de novo lipogenesis in osteosarcoma; EGCG competitively disrupts the RHBDL2-USP3 interaction interface.","method":"Multi-omics; structural analysis; co-immunoprecipitation; mutagenesis (Val245); ubiquitination assay; pharmacological inhibition (EGCG); in vivo tumor model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — structural analysis with mutagenesis identifying interaction residue, ubiquitination assay, in vivo validation, single lab, preprint status unclear but published in peer-reviewed journal","pmids":["42031733"],"is_preprint":false},{"year":2026,"finding":"In zebrafish, loss of Rhbdl2 (CRISPR-Cas9 knockout) leads to enhanced regenerative growth after injury and increased macrophage accumulation at wound sites; proteomics revealed increased Rac2 protein levels in rhbdl2 mutants, and morpholino knockdown of Rac2 suppressed the elevated macrophage recruitment and enhanced regeneration phenotype, placing Rhbdl2 upstream of Rac2-dependent immune signaling.","method":"CRISPR-Cas9 knockout in zebrafish; proteomic analysis; morpholino knockdown; live imaging of macrophage recruitment; tissue regeneration assay","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with defined phenotype, proteomics, genetic epistasis via Rac2 morpholino rescue, preprint not yet peer-reviewed","pmids":["41726946"],"is_preprint":true}],"current_model":"RHBDL2 is an intramembrane serine protease that sheds the ectodomains of multiple single-pass transmembrane substrates (including EGF, ephrinB3, thrombomodulin, IL-11R, CLEC14A, IL6R, DDR1, N-Cadherin, and others) by cleaving within or just outside the transmembrane domain, with cleavage site selectivity determined by the luminal face of the substrate TM domain; it also acts as a non-proteolytic scaffold to stabilize USP3 (via Val245) independently of its catalytic activity, and it functions as a conformational surveillance protease for activated Orai1/CRAC channels, collectively placing RHBDL2 as a multifunctional regulator of EGFR signaling, Notch signaling, cytokine receptor trans-signaling, angiogenesis, wound healing, anoikis resistance, and lipid metabolic reprogramming."},"narrative":{"mechanistic_narrative":"RHBDL2 is an intramembrane serine protease that sheds the ectodomains of diverse single-pass transmembrane substrates, positioning it as a broad regulator of receptor signaling, tissue repair, and cell survival [PMID:21833011, PMID:28779096]. It cleaves substrates within or just outside the transmembrane domain, with cleavage selectivity dictated by the luminal face of the substrate TM domain, as established for ephrinB3 and for the IL-11 receptor, where the precise scissile bond (Ala-370/Ser-371) was mapped and a human A370V variant abolishes cleavage without affecting classic signaling [PMID:15047175, PMID:33566379]. Its validated substrate repertoire includes EGF—whose cleavage drives EGF secretion and EGFR activation [PMID:21494248]—thrombomodulin, whose shedding from keratinocytes is required for wound healing [PMID:21833011], CLEC14A, whose shed ectodomain restrains sprouting angiogenesis [PMID:26939791], and an endogenously confirmed set comprising IL6R, Spint-1, DDR1, N-Cadherin, CLCP1/DCBLD2, KIRREL, and BCAM [PMID:28779096]. Through EGFR-coupled signaling RHBDL2 promotes proliferation, reduces adhesion, and confers anoikis resistance [PMID:24977233], and it activates Notch signaling by cleaving Notch1 to release N1ICD while collaborating with the deubiquitinase OTUD7B to stabilize N1ICD against proteasomal degradation [PMID:36351890]. Beyond proteolysis, RHBDL2 acts as a non-proteolytic scaffold: a Val245-mediated hydrophobic interaction stabilizes the deubiquitinase USP3 independently of catalytic activity, driving a USP3–PPT1–FASN axis of de novo lipogenesis [PMID:42031733]. In zebrafish, loss of Rhbdl2 enhances regenerative growth and macrophage recruitment through elevated Rac2, placing it upstream of Rac2-dependent immune signaling during injury [PMID:41726946].","teleology":[{"year":2004,"claim":"Established RHBDL2 as a functional intramembrane protease and defined the substrate determinant of its activity—the luminal face of the substrate TM domain controls cleavage susceptibility.","evidence":"Co-expression cleavage of ephrinB3 in mammalian cells with substrate domain-swap mutagenesis","pmids":["15047175"],"confidence":"Medium","gaps":["No physiological readout for ephrinB3 cleavage shown","Endogenous RHBDL2 activity not yet demonstrated","Single lab"]},{"year":2011,"claim":"Connected RHBDL2 cleavage to defined signaling and physiological outputs—EGF shedding to drive EGFR activation, and thrombomodulin shedding required for wound healing.","evidence":"Cleavage assays with EGFR activation readout in tumour cell lines; shRNA/DCI inhibition with ex vivo skin and in vivo mouse wound models, rescued by recombinant sTM","pmids":["21494248","21833011"],"confidence":"High","gaps":["Structural basis of substrate selection not resolved","Relative contribution of multiple substrates to wound phenotype unclear"]},{"year":2014,"claim":"Placed RHBDL2 upstream of EGFR-mediated anoikis resistance, linking its protease activity to cell survival and adhesion programs.","evidence":"Gain/loss-of-function with caspase-3 and FAK readouts and EGFR-inhibitor epistasis in suspension culture","pmids":["24977233"],"confidence":"Medium","gaps":["Direct substrate driving anoikis resistance not identified","Single lab"]},{"year":2016,"claim":"Demonstrated rhomboid-family selectivity (RHBDL2 but not RHBDL1/3) and mapped a precise CLEC14A cleavage site, linking shedding to suppression of angiogenesis.","evidence":"Site-directed mutagenesis, siRNA knockdown of endogenous RHBDL2, in vitro and in vivo angiogenesis assays","pmids":["26939791"],"confidence":"High","gaps":["In vivo contribution of endogenous RHBDL2 to vascular biology not established"]},{"year":2017,"claim":"Defined the endogenous substrate repertoire systematically, distinguishing RHBDL2 shedding from metalloprotease-mediated shedding.","evidence":"SILAC quantitative proteomics with endogenous RHBDL2-dependent shedding validation","pmids":["28779096"],"confidence":"High","gaps":["Physiological consequence of most identified substrates not characterized","Cleavage sites for most substrates not mapped"]},{"year":2021,"claim":"Extended RHBDL2 into cytokine trans-signaling and to atypical conformational substrate sensing—generating soluble IL-11R for trans-signaling, and surveying activated Orai1/CRAC channels.","evidence":"IL-11R cleavage site mapping, TM mutagenesis, subcellular localization, trans-signaling assay and disease variant analysis; CRAC channel surveillance described via Molecular Cell commentary","pmids":["33566379","34861185"],"confidence":"Medium","gaps":["CRAC channel surveillance is a commentary without primary experimental detail here","In vivo relevance of IL-11R shedding not established"]},{"year":2022,"claim":"Showed RHBDL2 activates Notch signaling not only by cleaving Notch1 but by co-stabilizing the released N1ICD via OTUD7B-dependent deubiquitination, revealing a combined proteolytic and stabilization mechanism in cancer.","evidence":"Co-IP, cleavage and ubiquitination assays, RNA-seq, in vitro/in vivo gain/loss-of-function with Notch-inhibitor rescue in pancreatic cancer","pmids":["36351890"],"confidence":"Medium","gaps":["Direct vs indirect role in N1ICD ubiquitin regulation not fully separated","Single lab"]},{"year":2026,"claim":"Revealed a protease-independent scaffolding function—Val245-mediated stabilization of USP3—coupling RHBDL2 to a USP3–PPT1–FASN lipogenic axis.","evidence":"Structural analysis, Val245 mutagenesis, Co-IP, ubiquitination assay, EGCG disruption and in vivo osteosarcoma model","pmids":["42031733"],"confidence":"Medium","gaps":["Structural model of the RHBDL2–USP3 interface not fully resolved","Generality of scaffold function beyond osteosarcoma unknown"]},{"year":2026,"claim":"Placed Rhbdl2 as a brake on regenerative growth and macrophage recruitment via Rac2 in an in vivo vertebrate injury model.","evidence":"Zebrafish CRISPR-Cas9 knockout, proteomics, Rac2 morpholino epistasis and live imaging (preprint)","pmids":["41726946"],"confidence":"Medium","gaps":["Preprint, not yet peer-reviewed","Substrate linking Rhbdl2 to Rac2 levels unidentified","Mammalian relevance untested"]},{"year":null,"claim":"How RHBDL2 substrate selection, subcellular site of cleavage, and the switch between proteolytic and scaffolding modes are regulated in vivo remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of RHBDL2 with a substrate or with USP3","Determinants governing proteolytic vs non-proteolytic activity unknown","In vivo physiological functions in mammals largely uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,3,4,6,7]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,2,3,6]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[9]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,1,6]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[2,3,4,6]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,5,7]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[9]}],"complexes":[],"partners":["USP3","OTUD7B","NOTCH1","CLEC14A","IL11RA","IL6R","DDR1","CDH2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9NX52","full_name":"Rhomboid-related protein 2","aliases":["Rhomboid-like protein 2"],"length_aa":303,"mass_kda":34.0,"function":"Involved in regulated intramembrane proteolysis and the subsequent release of functional polypeptides from their membrane anchors. Known substrate: EFNB3","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q9NX52/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RHBDL2","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RHBDL2","total_profiled":1310},"omim":[{"mim_id":"614404","title":"RHOMBOID 5 HOMOLOG 2; RHBDF2","url":"https://www.omim.org/entry/614404"},{"mim_id":"614403","title":"RHOMBOID 5 HOMOLOG 1; RHBDF1","url":"https://www.omim.org/entry/614403"},{"mim_id":"608962","title":"RHOMBOID-LIKE 2; RHBDL2","url":"https://www.omim.org/entry/608962"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"esophagus","ntpm":20.9}],"url":"https://www.proteinatlas.org/search/RHBDL2"},"hgnc":{"alias_symbol":["FLJ20435"],"prev_symbol":[]},"alphafold":{"accession":"Q9NX52","domains":[{"cath_id":"1.20.1540.10","chopping":"72-298","consensus_level":"high","plddt":90.0336,"start":72,"end":298}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NX52","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NX52-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NX52-F1-predicted_aligned_error_v6.png","plddt_mean":85.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RHBDL2","jax_strain_url":"https://www.jax.org/strain/search?query=RHBDL2"},"sequence":{"accession":"Q9NX52","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NX52.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NX52/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NX52"}},"corpus_meta":[{"pmid":"21494248","id":"PMC_21494248","title":"Mammalian EGF receptor activation by the rhomboid protease RHBDL2.","date":"2011","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/21494248","citation_count":100,"is_preprint":false},{"pmid":"15047175","id":"PMC_15047175","title":"Intramembrane cleavage of ephrinB3 by the human rhomboid family protease, RHBDL2.","date":"2004","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/15047175","citation_count":71,"is_preprint":false},{"pmid":"21833011","id":"PMC_21833011","title":"Functions of rhomboid family protease RHBDL2 and thrombomodulin in wound healing.","date":"2011","source":"The Journal of investigative dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/21833011","citation_count":59,"is_preprint":false},{"pmid":"28779096","id":"PMC_28779096","title":"Quantitative proteomics screen identifies a substrate repertoire of rhomboid protease RHBDL2 in human cells and implicates it in epithelial homeostasis.","date":"2017","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/28779096","citation_count":42,"is_preprint":false},{"pmid":"33566379","id":"PMC_33566379","title":"Interleukin-11 (IL-11) receptor cleavage by the rhomboid protease RHBDL2 induces IL-11 trans-signaling.","date":"2021","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/33566379","citation_count":33,"is_preprint":false},{"pmid":"26939791","id":"PMC_26939791","title":"Sprouting angiogenesis is regulated by shedding of the C-type lectin family 14, member A (CLEC14A) ectodomain, catalyzed by rhomboid-like 2 protein (RHBDL2).","date":"2016","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/26939791","citation_count":32,"is_preprint":false},{"pmid":"36351890","id":"PMC_36351890","title":"RHBDL2 promotes the proliferation, migration, and invasion of pancreatic cancer by stabilizing the N1ICD via the OTUD7B and activating the Notch signaling pathway.","date":"2022","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/36351890","citation_count":24,"is_preprint":false},{"pmid":"24977233","id":"PMC_24977233","title":"RHBDL2 is a critical membrane protease for anoikis resistance in human malignant epithelial cells.","date":"2014","source":"TheScientificWorldJournal","url":"https://pubmed.ncbi.nlm.nih.gov/24977233","citation_count":19,"is_preprint":false},{"pmid":"41726946","id":"PMC_41726946","title":"Rhomboid protease Rhbdl2 regulates macrophage recruitment and wound regeneration in zebrafish.","date":"2026","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/41726946","citation_count":0,"is_preprint":false},{"pmid":"42031733","id":"PMC_42031733","title":"RHBDL2 drives lipid metabolic reprogramming in osteosarcoma via USP3-mediated deubiquitination of PPT1.","date":"2026","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/42031733","citation_count":0,"is_preprint":false},{"pmid":"34861185","id":"PMC_34861185","title":"Signaling is silver, silence is golden: RHBDL2 intramembrane proteolysis prevents stochastic Ca2+ signaling in unstimulated cells.","date":"2021","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/34861185","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7058,"output_tokens":3070,"usd":0.033612,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10398,"output_tokens":3514,"usd":0.06992,"stage2_stop_reason":"end_turn"},"total_usd":0.103532,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2004,\n      \"finding\": \"RHBDL2 cleaves the intramembrane/transmembrane domain of ephrinB3, a B-type ephrin, when co-expressed in mammalian cells, reducing ephrinB3 surface levels; the amino acid sequence at the luminal face of the transmembrane domain of the substrate determines cleavage susceptibility by RHBDL2.\",\n      \"method\": \"Co-expression in mammalian cells; substrate domain mutagenesis; cell-surface detection\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct cleavage assay in mammalian cells with domain-swap mutagenesis identifying luminal determinants, single lab\",\n      \"pmids\": [\"15047175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"RHBDL2 cleaves EGF just outside its transmembrane domain, facilitating EGF secretion and triggering EGFR activation; endogenous RHBDL2 activity was detected in several tumour cell lines.\",\n      \"method\": \"Overexpression and cleavage assay in mammalian cells; EGFR activation readout; detection of endogenous activity in tumour cell lines\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct cleavage assay with signalling readout, endogenous activity confirmed, single lab\",\n      \"pmids\": [\"21494248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"RHBDL2 cleaves thrombomodulin (TM) at its transmembrane domain, releasing soluble TM (sTM) from keratinocytes; both RHBDL2 and TM are upregulated during wound healing, and RHBDL2 inhibition (by DCI or shRNA) blocks sTM shedding and impairs wound healing in cells and in vivo.\",\n      \"method\": \"shRNA knockdown; pharmacological inhibition (DCI); conditioned media assay; ex vivo skin culture; in vivo mouse wound model\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (shRNA, pharmacological inhibitor, in vivo model), functional rescue with recombinant sTM, replicated across cell and animal systems\",\n      \"pmids\": [\"21833011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"RHBDL2 (but not RHBDL1 or RHBDL3) specifically cleaves CLEC14A, shedding its ectodomain; site-directed mutagenesis identified the precise cleavage site, and siRNA knockdown of endogenous RHBDL2 validated CLEC14A shedding specificity; shed CLEC14A ectodomain inhibits sprouting angiogenesis.\",\n      \"method\": \"Co-expression cleavage assay; site-directed mutagenesis; siRNA knockdown of endogenous RHBDL2; in vitro and in vivo angiogenesis assays\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — site-directed mutagenesis identifying cleavage site, endogenous knockdown validation, functional in vitro and in vivo readouts, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"26939791\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Quantitative proteomics identified a substrate repertoire of RHBDL2 in human cells including IL6R, Spint-1, DDR1, N-Cadherin, CLCP1/DCBLD2, KIRREL, and BCAM; these substrates are specifically shed by endogenous RHBDL2 and a subset is resistant to metalloprotease-mediated shedding.\",\n      \"method\": \"Quantitative proteomics (SILAC-based); endogenous RHBDL2-dependent shedding validation\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — quantitative proteomics with endogenous activity validation, multiple substrates confirmed, single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"28779096\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RHBDL2 overexpression in human epithelial cells promotes cell proliferation, reduces cell adhesion, and suppresses anoikis; inhibition of RHBDL2 (by rhomboid protease inhibitor or shRNA) increases cleaved caspase-3, and EGFR inhibition similarly increases apoptosis markers, placing RHBDL2 upstream of EGFR-mediated anoikis resistance.\",\n      \"method\": \"Overexpression; shRNA knockdown; pharmacological inhibition; caspase-3 cleavage assay; FAK phosphorylation assay; suspension culture\",\n      \"journal\": \"TheScientificWorldJournal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss- and gain-of-function with multiple readouts, pathway placement via EGFR inhibitor epistasis, single lab\",\n      \"pmids\": [\"24977233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RHBDL2 cleaves the IL-11 receptor (IL-11R) at the extracellular region between Ala-370 and Ser-371, generating soluble IL-11R capable of IL-11 trans-signaling; critical residues within the transmembrane helix are required for proteolysis; RHBDL2 can cleave IL-11R in the early secretory pathway as well as at the plasma membrane; the human mutation IL-11R-A370V prevents RHBDL2-mediated cleavage without impairing classic IL-11 signaling.\",\n      \"method\": \"Co-expression cleavage assay; cleavage site mapping; transmembrane domain mutagenesis; subcellular localization analysis; trans-signaling functional assay; disease variant analysis\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — cleavage site precisely mapped, mutagenesis of substrate and TM domain, functional trans-signaling assay, subcellular localization determined, disease variant validation, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"33566379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RHBDL2 interacts with and cleaves Notch1, releasing the intracellular domain N1ICD; RHBDL2 also collaborates with the deubiquitinase OTUD7B to reduce ubiquitination of N1ICD, stabilizing it via the ubiquitin-proteasome pathway, thereby activating Notch signaling and promoting pancreatic cancer cell proliferation and mobility.\",\n      \"method\": \"Co-immunoprecipitation; cleavage assay; RNA-seq; ubiquitination assay; gain- and loss-of-function in vitro and in vivo; Notch inhibitor (IMR-1) rescue\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, cleavage assay, ubiquitination assay, epistasis with Notch inhibitor, in vivo confirmation, single lab\",\n      \"pmids\": [\"36351890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RHBDL2 processes stochastically activated Orai1/CRAC channels as atypical substrates in unstimulated cells, acting as a 'conformational surveillance' mechanism that prevents unwanted Ca2+ signaling; selective processing requires CRAC channel activation state.\",\n      \"method\": \"Described via commentary on Grieve et al. (2021) in Molecular Cell; experimental basis inferred as RHBDL2 substrate cleavage assay linked to CRAC channel conformation\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — this is a commentary/preview summarizing another study; experimental details are not directly described in this abstract\",\n      \"pmids\": [\"34861185\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RHBDL2 functions as a non-proteolytic scaffold to stabilize the deubiquitinase USP3 via a hydrophobic core interaction mediated by Val245 of RHBDL2, independently of its protease activity; stabilized USP3 then deubiquitinates PPT1, preventing its proteasomal degradation, thereby promoting FASN-dependent de novo lipogenesis in osteosarcoma; EGCG competitively disrupts the RHBDL2-USP3 interaction interface.\",\n      \"method\": \"Multi-omics; structural analysis; co-immunoprecipitation; mutagenesis (Val245); ubiquitination assay; pharmacological inhibition (EGCG); in vivo tumor model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — structural analysis with mutagenesis identifying interaction residue, ubiquitination assay, in vivo validation, single lab, preprint status unclear but published in peer-reviewed journal\",\n      \"pmids\": [\"42031733\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"In zebrafish, loss of Rhbdl2 (CRISPR-Cas9 knockout) leads to enhanced regenerative growth after injury and increased macrophage accumulation at wound sites; proteomics revealed increased Rac2 protein levels in rhbdl2 mutants, and morpholino knockdown of Rac2 suppressed the elevated macrophage recruitment and enhanced regeneration phenotype, placing Rhbdl2 upstream of Rac2-dependent immune signaling.\",\n      \"method\": \"CRISPR-Cas9 knockout in zebrafish; proteomic analysis; morpholino knockdown; live imaging of macrophage recruitment; tissue regeneration assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with defined phenotype, proteomics, genetic epistasis via Rac2 morpholino rescue, preprint not yet peer-reviewed\",\n      \"pmids\": [\"41726946\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"RHBDL2 is an intramembrane serine protease that sheds the ectodomains of multiple single-pass transmembrane substrates (including EGF, ephrinB3, thrombomodulin, IL-11R, CLEC14A, IL6R, DDR1, N-Cadherin, and others) by cleaving within or just outside the transmembrane domain, with cleavage site selectivity determined by the luminal face of the substrate TM domain; it also acts as a non-proteolytic scaffold to stabilize USP3 (via Val245) independently of its catalytic activity, and it functions as a conformational surveillance protease for activated Orai1/CRAC channels, collectively placing RHBDL2 as a multifunctional regulator of EGFR signaling, Notch signaling, cytokine receptor trans-signaling, angiogenesis, wound healing, anoikis resistance, and lipid metabolic reprogramming.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RHBDL2 is an intramembrane serine protease that sheds the ectodomains of diverse single-pass transmembrane substrates, positioning it as a broad regulator of receptor signaling, tissue repair, and cell survival [#2, #4]. It cleaves substrates within or just outside the transmembrane domain, with cleavage selectivity dictated by the luminal face of the substrate TM domain, as established for ephrinB3 and for the IL-11 receptor, where the precise scissile bond (Ala-370/Ser-371) was mapped and a human A370V variant abolishes cleavage without affecting classic signaling [#0, #6]. Its validated substrate repertoire includes EGF—whose cleavage drives EGF secretion and EGFR activation [#1]—thrombomodulin, whose shedding from keratinocytes is required for wound healing [#2], CLEC14A, whose shed ectodomain restrains sprouting angiogenesis [#3], and an endogenously confirmed set comprising IL6R, Spint-1, DDR1, N-Cadherin, CLCP1/DCBLD2, KIRREL, and BCAM [#4]. Through EGFR-coupled signaling RHBDL2 promotes proliferation, reduces adhesion, and confers anoikis resistance [#5], and it activates Notch signaling by cleaving Notch1 to release N1ICD while collaborating with the deubiquitinase OTUD7B to stabilize N1ICD against proteasomal degradation [#7]. Beyond proteolysis, RHBDL2 acts as a non-proteolytic scaffold: a Val245-mediated hydrophobic interaction stabilizes the deubiquitinase USP3 independently of catalytic activity, driving a USP3–PPT1–FASN axis of de novo lipogenesis [#9]. In zebrafish, loss of Rhbdl2 enhances regenerative growth and macrophage recruitment through elevated Rac2, placing it upstream of Rac2-dependent immune signaling during injury [#10].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Established RHBDL2 as a functional intramembrane protease and defined the substrate determinant of its activity—the luminal face of the substrate TM domain controls cleavage susceptibility.\",\n      \"evidence\": \"Co-expression cleavage of ephrinB3 in mammalian cells with substrate domain-swap mutagenesis\",\n      \"pmids\": [\"15047175\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No physiological readout for ephrinB3 cleavage shown\", \"Endogenous RHBDL2 activity not yet demonstrated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Connected RHBDL2 cleavage to defined signaling and physiological outputs—EGF shedding to drive EGFR activation, and thrombomodulin shedding required for wound healing.\",\n      \"evidence\": \"Cleavage assays with EGFR activation readout in tumour cell lines; shRNA/DCI inhibition with ex vivo skin and in vivo mouse wound models, rescued by recombinant sTM\",\n      \"pmids\": [\"21494248\", \"21833011\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Structural basis of substrate selection not resolved\", \"Relative contribution of multiple substrates to wound phenotype unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Placed RHBDL2 upstream of EGFR-mediated anoikis resistance, linking its protease activity to cell survival and adhesion programs.\",\n      \"evidence\": \"Gain/loss-of-function with caspase-3 and FAK readouts and EGFR-inhibitor epistasis in suspension culture\",\n      \"pmids\": [\"24977233\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct substrate driving anoikis resistance not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrated rhomboid-family selectivity (RHBDL2 but not RHBDL1/3) and mapped a precise CLEC14A cleavage site, linking shedding to suppression of angiogenesis.\",\n      \"evidence\": \"Site-directed mutagenesis, siRNA knockdown of endogenous RHBDL2, in vitro and in vivo angiogenesis assays\",\n      \"pmids\": [\"26939791\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"In vivo contribution of endogenous RHBDL2 to vascular biology not established\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defined the endogenous substrate repertoire systematically, distinguishing RHBDL2 shedding from metalloprotease-mediated shedding.\",\n      \"evidence\": \"SILAC quantitative proteomics with endogenous RHBDL2-dependent shedding validation\",\n      \"pmids\": [\"28779096\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Physiological consequence of most identified substrates not characterized\", \"Cleavage sites for most substrates not mapped\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Extended RHBDL2 into cytokine trans-signaling and to atypical conformational substrate sensing—generating soluble IL-11R for trans-signaling, and surveying activated Orai1/CRAC channels.\",\n      \"evidence\": \"IL-11R cleavage site mapping, TM mutagenesis, subcellular localization, trans-signaling assay and disease variant analysis; CRAC channel surveillance described via Molecular Cell commentary\",\n      \"pmids\": [\"33566379\", \"34861185\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"CRAC channel surveillance is a commentary without primary experimental detail here\", \"In vivo relevance of IL-11R shedding not established\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed RHBDL2 activates Notch signaling not only by cleaving Notch1 but by co-stabilizing the released N1ICD via OTUD7B-dependent deubiquitination, revealing a combined proteolytic and stabilization mechanism in cancer.\",\n      \"evidence\": \"Co-IP, cleavage and ubiquitination assays, RNA-seq, in vitro/in vivo gain/loss-of-function with Notch-inhibitor rescue in pancreatic cancer\",\n      \"pmids\": [\"36351890\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct vs indirect role in N1ICD ubiquitin regulation not fully separated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Revealed a protease-independent scaffolding function—Val245-mediated stabilization of USP3—coupling RHBDL2 to a USP3–PPT1–FASN lipogenic axis.\",\n      \"evidence\": \"Structural analysis, Val245 mutagenesis, Co-IP, ubiquitination assay, EGCG disruption and in vivo osteosarcoma model\",\n      \"pmids\": [\"42031733\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Structural model of the RHBDL2–USP3 interface not fully resolved\", \"Generality of scaffold function beyond osteosarcoma unknown\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Placed Rhbdl2 as a brake on regenerative growth and macrophage recruitment via Rac2 in an in vivo vertebrate injury model.\",\n      \"evidence\": \"Zebrafish CRISPR-Cas9 knockout, proteomics, Rac2 morpholino epistasis and live imaging (preprint)\",\n      \"pmids\": [\"41726946\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"Substrate linking Rhbdl2 to Rac2 levels unidentified\", \"Mammalian relevance untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RHBDL2 substrate selection, subcellular site of cleavage, and the switch between proteolytic and scaffolding modes are regulated in vivo remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No structure of RHBDL2 with a substrate or with USP3\", \"Determinants governing proteolytic vs non-proteolytic activity unknown\", \"In vivo physiological functions in mammals largely uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 6, 7]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 2, 3, 6]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1, 6]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [2, 3, 4, 6]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 5, 7]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"USP3\", \"OTUD7B\", \"Notch1\", \"CLEC14A\", \"IL11RA\", \"IL6R\", \"DDR1\", \"CDH2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}