{"gene":"RHBDL2","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":2004,"finding":"RHBDL2 cleaves the transmembrane domain of ephrinB3, acting as an intramembrane serine protease; the amino acid sequence at the luminal face of the transmembrane domain of a substrate determines whether it is cleaved by RHBDL2.","method":"Co-expression in mammalian cells, site-directed mutagenesis of substrate transmembrane domain","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — cleavage demonstrated in cells with mutagenesis of substrate determinants, single lab","pmids":["15047175"],"is_preprint":false},{"year":2011,"finding":"RHBDL2 cleaves EGF just outside its transmembrane domain, releasing soluble EGF and triggering EGFR activation; endogenous RHBDL2 activity was detected in tumour cell lines.","method":"Cell-based cleavage assay, EGFR activation assay, endogenous activity measurement in tumour lines","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, replicated in endogenous context, highly cited","pmids":["21494248"],"is_preprint":false},{"year":2011,"finding":"RHBDL2 cleaves thrombomodulin (TM) at its transmembrane domain, releasing soluble TM (sTM) from keratinocytes; this ectodomain shedding promotes wound healing as an autocrine/paracrine signal.","method":"shRNA knockdown, pharmacological inhibition (DCI), conditioned medium rescue, ex vivo skin culture, in vivo mouse wound model","journal":"The Journal of investigative dermatology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal in vitro and in vivo methods, functional rescue experiments","pmids":["21833011"],"is_preprint":false},{"year":2014,"finding":"RHBDL2 overexpression promotes anoikis resistance in malignant epithelial cells by cleaving EGF ligand and activating EGFR-mediated signaling, including focal adhesion kinase phosphorylation; inhibition of RHBDL2 increases apoptosis (cleaved caspase-3).","method":"Overexpression and shRNA knockdown, rhomboid protease inhibitor, EGFR inhibition, caspase-3 cleavage assay, suspension culture","journal":"TheScientificWorldJournal","confidence":"Medium","confidence_rationale":"Tier 2 — multiple methods but single lab, pathway placement via EGFR inhibition epistasis","pmids":["24977233"],"is_preprint":false},{"year":2016,"finding":"RHBDL2, but not RHBDL1 or RHBDL3, specifically sheds the ectodomain of CLEC14A at a defined cleavage site identified by site-directed mutagenesis; siRNA knockdown of endogenous RHBDL2 confirmed specificity; shed CLEC14A ectodomain inhibits sprouting angiogenesis by binding to tip cells.","method":"Co-expression cleavage assay, site-directed mutagenesis, siRNA knockdown, in vitro sprouting assays, in vivo sponge implant model","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including in vivo validation, endogenous knockdown confirmation","pmids":["26939791"],"is_preprint":false},{"year":2017,"finding":"Quantitative proteomics identified multiple novel RHBDL2 substrates in human cells including IL6R, Spint-1/HAI-1, DDR1, N-Cadherin, CLCP1/DCBLD2, KIRREL, and BCAM; these substrates are shed by endogenous RHBDL2 and a subset is resistant to metalloprotease shedding.","method":"Quantitative proteomics (SILAC-based), endogenous RHBDL2 shedding assays, metalloprotease inhibitor comparison","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1-2 — unbiased quantitative proteomics plus validation of endogenous substrates with orthogonal methods","pmids":["28779096"],"is_preprint":false},{"year":2021,"finding":"RHBDL2 cleaves the IL-11 receptor (IL-11R) at the extracellular region between Ala-370 and Ser-371 (close to the plasma membrane), generating soluble IL-11R that is biologically active for trans-signaling; critical transmembrane residues are required for cleavage; RHBDL2 can cleave IL-11R in the early secretory pathway, not only at the plasma membrane; the human mutation IL-11R-A370V prevents RHBDL2-mediated cleavage.","method":"Cleavage site mapping, site-directed mutagenesis of substrate TM helix, subcellular fractionation, trans-signaling bioassay, disease mutation analysis","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 1-2 — precise cleavage site determined, mutagenesis of both substrate and protease requirements, functional (trans-signaling) validation","pmids":["33566379"],"is_preprint":false},{"year":2021,"finding":"RHBDL2 cleaves stochastically activated Orai1/CRAC channel subunits via a 'conformational surveillance' mechanism, preventing unwanted Ca2+ signaling in unstimulated cells.","method":"Discussed as mechanistic commentary on Grieve et al. 2021 findings","journal":"Molecular cell","confidence":"Low","confidence_rationale":"Tier 3 — secondary commentary paper; primary experimental data is in Grieve et al. which is not directly in corpus","pmids":["34861185"],"is_preprint":false},{"year":2022,"finding":"RHBDL2 cleaves Notch1 to release the N1ICD intracellular domain and collaborates with the deubiquitinase OTUD7B to stabilize N1ICD by reducing its ubiquitination, thereby activating Notch signaling and promoting pancreatic cancer cell proliferation and migration.","method":"Co-immunoprecipitation, gain- and loss-of-function assays, RNA-seq, ubiquitination assay, Notch pathway inhibitor (IMR-1), in vivo xenograft","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP and functional assays with epistasis via inhibitor, single lab","pmids":["36351890"],"is_preprint":false},{"year":2026,"finding":"RHBDL2 functions as a non-proteolytic scaffold to stabilize the deubiquitinase USP3 through a hydrophobic interaction anchored by Val245 of RHBDL2, independently of its protease activity; stabilized USP3 deubiquitinates and stabilizes PPT1, which drives FASN-dependent de novo lipogenesis in osteosarcoma.","method":"Multi-omics analysis, structural analysis, mutagenesis (Val245), Co-IP, ubiquitination assay, in vivo tumor model, pharmacological inhibition (EGCG)","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — structural and biochemical evidence for scaffold function with mutagenesis, single lab","pmids":["42031733"],"is_preprint":false},{"year":2026,"finding":"In zebrafish, Rhbdl2 loss-of-function (CRISPR knockout) increases macrophage accumulation at wound sites and enhances regenerative growth; Rac2 protein levels are elevated in rhbdl2 mutants, and morpholino knockdown of Rac2 suppresses both elevated macrophage recruitment and enhanced regeneration, placing Rhbdl2 upstream of Rac2 in immune-regulated tissue regeneration.","method":"CRISPR-Cas9 knockout, proteomics, morpholino knockdown epistasis, live imaging of wound response","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis with proteomics, but preprint and zebrafish ortholog","pmids":["41726946"],"is_preprint":true}],"current_model":"RHBDL2 is a mammalian intramembrane serine protease that sheds the ectodomains of diverse type I transmembrane substrates (including EGF, thrombomodulin, ephrinB3, IL-6R, IL-11R, CLEC14A, DDR1, N-cadherin, and Notch1) by cleaving within or just outside their transmembrane domains, thereby activating downstream signaling pathways (EGFR, IL-11 trans-signaling, Notch); substrate recognition is governed by luminal-face transmembrane sequences, cleavage can occur in the early secretory pathway as well as at the plasma membrane, and RHBDL2 also possesses a non-proteolytic scaffold function that stabilizes the USP3 deubiquitinase through a Val245-anchored hydrophobic interface."},"narrative":{"teleology":[{"year":2004,"claim":"Establishing RHBDL2 as a functional intramembrane protease in mammalian cells resolved whether mammalian rhomboid family members possessed catalytic activity and revealed that substrate selectivity is encoded in the luminal-face transmembrane sequence of substrates.","evidence":"Co-expression cleavage assay with ephrinB3 and site-directed mutagenesis of substrate transmembrane domain in mammalian cells","pmids":["15047175"],"confidence":"Medium","gaps":["Only one substrate (ephrinB3) tested; breadth of substrate repertoire unknown","Catalytic mechanism and active-site residue requirements not characterized","No endogenous activity demonstrated"]},{"year":2011,"claim":"Identification of EGF and thrombomodulin as endogenous RHBDL2 substrates established that this protease functions as an ectodomain sheddase with physiological consequences—EGFR activation in tumour cells and wound healing in keratinocytes—broadening its role beyond intramembrane cleavage of ephrinB3.","evidence":"Cell-based cleavage assays with endogenous activity in tumour lines (EGF/EGFR); shRNA knockdown, conditioned-medium rescue, ex vivo skin culture, and in vivo mouse wound model (thrombomodulin)","pmids":["21494248","21833011"],"confidence":"High","gaps":["Full substrate repertoire still undefined","Whether RHBDL2 cleaves within or just outside the transmembrane domain was not yet precisely mapped","Regulation of RHBDL2 catalytic activity in vivo unknown"]},{"year":2014,"claim":"Demonstrating that RHBDL2-mediated EGF shedding confers anoikis resistance through EGFR and FAK signaling placed the protease in a pro-survival oncogenic circuit, explaining its potential role in tumour progression.","evidence":"Overexpression/shRNA knockdown with rhomboid inhibitor, EGFR inhibitor epistasis, caspase-3 cleavage in suspension culture","pmids":["24977233"],"confidence":"Medium","gaps":["Single-lab study; independent replication in additional cancer models needed","In vivo tumourigenesis data not provided","Whether other RHBDL2 substrates contribute to anoikis resistance unknown"]},{"year":2016,"claim":"Identification of CLEC14A as a specific RHBDL2 substrate with anti-angiogenic function upon shedding revealed a selective role among rhomboid family members (RHBDL1/3 do not cleave CLEC14A) and extended RHBDL2 biology to vascular biology.","evidence":"Co-expression cleavage, site-directed mutagenesis of cleavage site, siRNA knockdown, in vitro sprouting assay, in vivo sponge implant angiogenesis model","pmids":["26939791"],"confidence":"High","gaps":["Mechanism of RHBDL2 selectivity over RHBDL1/RHBDL3 not structurally resolved","Whether CLEC14A shedding occurs in pathological angiogenesis settings not tested"]},{"year":2017,"claim":"Unbiased quantitative proteomics greatly expanded the RHBDL2 substrate repertoire (IL-6R, DDR1, N-cadherin, KIRREL, BCAM, HAI-1, DCBLD2), demonstrating that RHBDL2 functions as a broad ectodomain sheddase complementary to—but partially non-overlapping with—metalloprotease-mediated shedding.","evidence":"SILAC-based quantitative proteomics in human cells, endogenous RHBDL2 shedding validation, metalloprotease inhibitor comparison","pmids":["28779096"],"confidence":"High","gaps":["Cleavage sites not mapped for most new substrates","Physiological significance of each shedding event not individually addressed","Redundancy with other rhomboid proteases not fully explored"]},{"year":2021,"claim":"Precise mapping of the IL-11R cleavage site (Ala370-Ser371) and demonstration that cleavage generates trans-signaling-competent soluble IL-11R—even in the early secretory pathway—revealed that RHBDL2 activity is not confined to the plasma membrane and has direct implications for cytokine signaling; the disease-associated IL-11R-A370V mutation blocks this cleavage.","evidence":"Cleavage site mapping, substrate TM-helix mutagenesis, subcellular fractionation, IL-11 trans-signaling bioassay, disease mutation analysis","pmids":["33566379"],"confidence":"High","gaps":["How RHBDL2 accesses substrates in the ER/Golgi is mechanistically unclear","In vivo consequences of impaired IL-11R shedding (A370V) not demonstrated","Whether early secretory cleavage applies to other substrates not tested"]},{"year":2022,"claim":"Discovery that RHBDL2 cleaves Notch1 to release N1ICD and cooperates with the deubiquitinase OTUD7B to stabilize N1ICD expanded RHBDL2 into the Notch signaling pathway and demonstrated functional collaboration between a rhomboid protease and a DUB.","evidence":"Co-immunoprecipitation, RNA-seq, ubiquitination assay, Notch inhibitor (IMR-1) epistasis, in vivo xenograft in pancreatic cancer","pmids":["36351890"],"confidence":"Medium","gaps":["Notch1 cleavage site not mapped","Relationship to canonical γ-secretase-mediated Notch processing unclear","Single-lab study in one cancer type"]},{"year":2026,"claim":"Identification of a protease-independent scaffold function of RHBDL2 that stabilizes USP3 via a Val245-mediated hydrophobic interface revealed a second molecular activity for the protein, linking it to deubiquitination of PPT1 and FASN-dependent lipogenesis in osteosarcoma.","evidence":"Multi-omics, structural analysis, Val245 mutagenesis, Co-IP, ubiquitination assay, in vivo tumour model, EGCG pharmacological inhibition","pmids":["42031733"],"confidence":"Medium","gaps":["Scaffold function demonstrated in a single cancer context; generality unknown","No reconstituted biochemical assay for direct RHBDL2–USP3 stabilization","Structural basis of the Val245 interface resolved only computationally"]},{"year":null,"claim":"Key unresolved questions include how RHBDL2 activity is regulated (transcriptionally, post-translationally, or by lipid environment), the structural basis for substrate selectivity among rhomboid family members, whether the scaffold function extends to additional partners, and the in vivo physiological consequences of RHBDL2 loss in mammals.","evidence":"","pmids":[],"confidence":"High","gaps":["No mammalian RHBDL2 knockout phenotype reported","No high-resolution structure of RHBDL2","Regulatory mechanisms (transcriptional or post-translational) of RHBDL2 expression and activity unknown","Relative contribution of RHBDL2 versus metalloproteases for shared substrates in vivo not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,4,5,6,8]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[9]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1,2,4,6]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,3,6,8]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,5,9]}],"complexes":[],"partners":["EGF","THBD","EFNB3","CLEC14A","IL11RA","NOTCH1","USP3","OTUD7B"],"other_free_text":[]},"mechanistic_narrative":"RHBDL2 is a mammalian intramembrane serine protease that sheds the ectodomains of diverse type I transmembrane substrates—including EGF, thrombomodulin, ephrinB3, CLEC14A, IL-6R, IL-11R, DDR1, N-cadherin, and Notch1—by cleaving within or near their transmembrane domains, with substrate recognition governed by the amino acid sequence at the luminal face of the transmembrane helix [PMID:15047175, PMID:28779096, PMID:33566379]. Cleavage can occur both at the plasma membrane and in the early secretory pathway, and the released ectodomains activate downstream signaling cascades including EGFR-mediated survival signaling, IL-11 trans-signaling, Notch pathway activation, and anti-angiogenic signaling via shed CLEC14A [PMID:21494248, PMID:33566379, PMID:36351890, PMID:26939791]. RHBDL2-dependent EGF shedding promotes anoikis resistance in epithelial cancer cells, thrombomodulin shedding by RHBDL2 in keratinocytes promotes wound healing, and Notch1 cleavage cooperates with OTUD7B-mediated deubiquitination to drive pancreatic cancer cell proliferation [PMID:24977233, PMID:21833011, PMID:36351890]. Beyond its proteolytic function, RHBDL2 acts as a non-proteolytic scaffold that stabilizes the deubiquitinase USP3 through a Val245-anchored hydrophobic interface, thereby promoting USP3-dependent deubiquitination of PPT1 and FASN-dependent de novo lipogenesis in osteosarcoma [PMID:42031733]."},"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":99,"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":41,"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":"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},{"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}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6295,"output_tokens":2650,"usd":0.029317},"stage2":{"model":"claude-opus-4-6","input_tokens":5977,"output_tokens":2897,"usd":0.153465},"total_usd":0.182782,"stage1_batch_id":"msgbatch_01SUR8NDzVrNmyszWyJeCct7","stage2_batch_id":"msgbatch_01G7Yfutko3cDWzejrwH5AMx","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2004,\n      \"finding\": \"RHBDL2 cleaves the transmembrane domain of ephrinB3, acting as an intramembrane serine protease; the amino acid sequence at the luminal face of the transmembrane domain of a substrate determines whether it is cleaved by RHBDL2.\",\n      \"method\": \"Co-expression in mammalian cells, site-directed mutagenesis of substrate transmembrane domain\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cleavage demonstrated in cells with mutagenesis of substrate 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, releasing soluble EGF and triggering EGFR activation; endogenous RHBDL2 activity was detected in tumour cell lines.\",\n      \"method\": \"Cell-based cleavage assay, EGFR activation assay, endogenous activity measurement in tumour lines\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, replicated in endogenous context, highly cited\",\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; this ectodomain shedding promotes wound healing as an autocrine/paracrine signal.\",\n      \"method\": \"shRNA knockdown, pharmacological inhibition (DCI), conditioned medium rescue, ex vivo skin culture, in vivo mouse wound model\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal in vitro and in vivo methods, functional rescue experiments\",\n      \"pmids\": [\"21833011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RHBDL2 overexpression promotes anoikis resistance in malignant epithelial cells by cleaving EGF ligand and activating EGFR-mediated signaling, including focal adhesion kinase phosphorylation; inhibition of RHBDL2 increases apoptosis (cleaved caspase-3).\",\n      \"method\": \"Overexpression and shRNA knockdown, rhomboid protease inhibitor, EGFR inhibition, caspase-3 cleavage assay, suspension culture\",\n      \"journal\": \"TheScientificWorldJournal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple methods but single lab, pathway placement via EGFR inhibition epistasis\",\n      \"pmids\": [\"24977233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"RHBDL2, but not RHBDL1 or RHBDL3, specifically sheds the ectodomain of CLEC14A at a defined cleavage site identified by site-directed mutagenesis; siRNA knockdown of endogenous RHBDL2 confirmed specificity; shed CLEC14A ectodomain inhibits sprouting angiogenesis by binding to tip cells.\",\n      \"method\": \"Co-expression cleavage assay, site-directed mutagenesis, siRNA knockdown, in vitro sprouting assays, in vivo sponge implant model\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including in vivo validation, endogenous knockdown confirmation\",\n      \"pmids\": [\"26939791\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Quantitative proteomics identified multiple novel RHBDL2 substrates in human cells including IL6R, Spint-1/HAI-1, DDR1, N-Cadherin, CLCP1/DCBLD2, KIRREL, and BCAM; these substrates are shed by endogenous RHBDL2 and a subset is resistant to metalloprotease shedding.\",\n      \"method\": \"Quantitative proteomics (SILAC-based), endogenous RHBDL2 shedding assays, metalloprotease inhibitor comparison\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — unbiased quantitative proteomics plus validation of endogenous substrates with orthogonal methods\",\n      \"pmids\": [\"28779096\"],\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 (close to the plasma membrane), generating soluble IL-11R that is biologically active for trans-signaling; critical transmembrane residues are required for cleavage; RHBDL2 can cleave IL-11R in the early secretory pathway, not only at the plasma membrane; the human mutation IL-11R-A370V prevents RHBDL2-mediated cleavage.\",\n      \"method\": \"Cleavage site mapping, site-directed mutagenesis of substrate TM helix, subcellular fractionation, trans-signaling bioassay, disease mutation analysis\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — precise cleavage site determined, mutagenesis of both substrate and protease requirements, functional (trans-signaling) validation\",\n      \"pmids\": [\"33566379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RHBDL2 cleaves stochastically activated Orai1/CRAC channel subunits via a 'conformational surveillance' mechanism, preventing unwanted Ca2+ signaling in unstimulated cells.\",\n      \"method\": \"Discussed as mechanistic commentary on Grieve et al. 2021 findings\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — secondary commentary paper; primary experimental data is in Grieve et al. which is not directly in corpus\",\n      \"pmids\": [\"34861185\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RHBDL2 cleaves Notch1 to release the N1ICD intracellular domain and collaborates with the deubiquitinase OTUD7B to stabilize N1ICD by reducing its ubiquitination, thereby activating Notch signaling and promoting pancreatic cancer cell proliferation and migration.\",\n      \"method\": \"Co-immunoprecipitation, gain- and loss-of-function assays, RNA-seq, ubiquitination assay, Notch pathway inhibitor (IMR-1), in vivo xenograft\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP and functional assays with epistasis via inhibitor, single lab\",\n      \"pmids\": [\"36351890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RHBDL2 functions as a non-proteolytic scaffold to stabilize the deubiquitinase USP3 through a hydrophobic interaction anchored by Val245 of RHBDL2, independently of its protease activity; stabilized USP3 deubiquitinates and stabilizes PPT1, which drives FASN-dependent de novo lipogenesis in osteosarcoma.\",\n      \"method\": \"Multi-omics analysis, structural analysis, mutagenesis (Val245), Co-IP, ubiquitination assay, in vivo tumor model, pharmacological inhibition (EGCG)\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — structural and biochemical evidence for scaffold function with mutagenesis, single lab\",\n      \"pmids\": [\"42031733\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"In zebrafish, Rhbdl2 loss-of-function (CRISPR knockout) increases macrophage accumulation at wound sites and enhances regenerative growth; Rac2 protein levels are elevated in rhbdl2 mutants, and morpholino knockdown of Rac2 suppresses both elevated macrophage recruitment and enhanced regeneration, placing Rhbdl2 upstream of Rac2 in immune-regulated tissue regeneration.\",\n      \"method\": \"CRISPR-Cas9 knockout, proteomics, morpholino knockdown epistasis, live imaging of wound response\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with proteomics, but preprint and zebrafish ortholog\",\n      \"pmids\": [\"41726946\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"RHBDL2 is a mammalian intramembrane serine protease that sheds the ectodomains of diverse type I transmembrane substrates (including EGF, thrombomodulin, ephrinB3, IL-6R, IL-11R, CLEC14A, DDR1, N-cadherin, and Notch1) by cleaving within or just outside their transmembrane domains, thereby activating downstream signaling pathways (EGFR, IL-11 trans-signaling, Notch); substrate recognition is governed by luminal-face transmembrane sequences, cleavage can occur in the early secretory pathway as well as at the plasma membrane, and RHBDL2 also possesses a non-proteolytic scaffold function that stabilizes the USP3 deubiquitinase through a Val245-anchored hydrophobic interface.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RHBDL2 is a mammalian intramembrane serine protease that sheds the ectodomains of diverse type I transmembrane substrates—including EGF, thrombomodulin, ephrinB3, CLEC14A, IL-6R, IL-11R, DDR1, N-cadherin, and Notch1—by cleaving within or near their transmembrane domains, with substrate recognition governed by the amino acid sequence at the luminal face of the transmembrane helix [PMID:15047175, PMID:28779096, PMID:33566379]. Cleavage can occur both at the plasma membrane and in the early secretory pathway, and the released ectodomains activate downstream signaling cascades including EGFR-mediated survival signaling, IL-11 trans-signaling, Notch pathway activation, and anti-angiogenic signaling via shed CLEC14A [PMID:21494248, PMID:33566379, PMID:36351890, PMID:26939791]. RHBDL2-dependent EGF shedding promotes anoikis resistance in epithelial cancer cells, thrombomodulin shedding by RHBDL2 in keratinocytes promotes wound healing, and Notch1 cleavage cooperates with OTUD7B-mediated deubiquitination to drive pancreatic cancer cell proliferation [PMID:24977233, PMID:21833011, PMID:36351890]. Beyond its proteolytic function, RHBDL2 acts as a non-proteolytic scaffold that stabilizes the deubiquitinase USP3 through a Val245-anchored hydrophobic interface, thereby promoting USP3-dependent deubiquitination of PPT1 and FASN-dependent de novo lipogenesis in osteosarcoma [PMID:42031733].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Establishing RHBDL2 as a functional intramembrane protease in mammalian cells resolved whether mammalian rhomboid family members possessed catalytic activity and revealed that substrate selectivity is encoded in the luminal-face transmembrane sequence of substrates.\",\n      \"evidence\": \"Co-expression cleavage assay with ephrinB3 and site-directed mutagenesis of substrate transmembrane domain in mammalian cells\",\n      \"pmids\": [\"15047175\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Only one substrate (ephrinB3) tested; breadth of substrate repertoire unknown\",\n        \"Catalytic mechanism and active-site residue requirements not characterized\",\n        \"No endogenous activity demonstrated\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identification of EGF and thrombomodulin as endogenous RHBDL2 substrates established that this protease functions as an ectodomain sheddase with physiological consequences—EGFR activation in tumour cells and wound healing in keratinocytes—broadening its role beyond intramembrane cleavage of ephrinB3.\",\n      \"evidence\": \"Cell-based cleavage assays with endogenous activity in tumour lines (EGF/EGFR); shRNA knockdown, conditioned-medium rescue, ex vivo skin culture, and in vivo mouse wound model (thrombomodulin)\",\n      \"pmids\": [\"21494248\", \"21833011\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Full substrate repertoire still undefined\",\n        \"Whether RHBDL2 cleaves within or just outside the transmembrane domain was not yet precisely mapped\",\n        \"Regulation of RHBDL2 catalytic activity in vivo unknown\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrating that RHBDL2-mediated EGF shedding confers anoikis resistance through EGFR and FAK signaling placed the protease in a pro-survival oncogenic circuit, explaining its potential role in tumour progression.\",\n      \"evidence\": \"Overexpression/shRNA knockdown with rhomboid inhibitor, EGFR inhibitor epistasis, caspase-3 cleavage in suspension culture\",\n      \"pmids\": [\"24977233\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-lab study; independent replication in additional cancer models needed\",\n        \"In vivo tumourigenesis data not provided\",\n        \"Whether other RHBDL2 substrates contribute to anoikis resistance unknown\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identification of CLEC14A as a specific RHBDL2 substrate with anti-angiogenic function upon shedding revealed a selective role among rhomboid family members (RHBDL1/3 do not cleave CLEC14A) and extended RHBDL2 biology to vascular biology.\",\n      \"evidence\": \"Co-expression cleavage, site-directed mutagenesis of cleavage site, siRNA knockdown, in vitro sprouting assay, in vivo sponge implant angiogenesis model\",\n      \"pmids\": [\"26939791\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Mechanism of RHBDL2 selectivity over RHBDL1/RHBDL3 not structurally resolved\",\n        \"Whether CLEC14A shedding occurs in pathological angiogenesis settings not tested\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Unbiased quantitative proteomics greatly expanded the RHBDL2 substrate repertoire (IL-6R, DDR1, N-cadherin, KIRREL, BCAM, HAI-1, DCBLD2), demonstrating that RHBDL2 functions as a broad ectodomain sheddase complementary to—but partially non-overlapping with—metalloprotease-mediated shedding.\",\n      \"evidence\": \"SILAC-based quantitative proteomics in human cells, endogenous RHBDL2 shedding validation, metalloprotease inhibitor comparison\",\n      \"pmids\": [\"28779096\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Cleavage sites not mapped for most new substrates\",\n        \"Physiological significance of each shedding event not individually addressed\",\n        \"Redundancy with other rhomboid proteases not fully explored\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Precise mapping of the IL-11R cleavage site (Ala370-Ser371) and demonstration that cleavage generates trans-signaling-competent soluble IL-11R—even in the early secretory pathway—revealed that RHBDL2 activity is not confined to the plasma membrane and has direct implications for cytokine signaling; the disease-associated IL-11R-A370V mutation blocks this cleavage.\",\n      \"evidence\": \"Cleavage site mapping, substrate TM-helix mutagenesis, subcellular fractionation, IL-11 trans-signaling bioassay, disease mutation analysis\",\n      \"pmids\": [\"33566379\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How RHBDL2 accesses substrates in the ER/Golgi is mechanistically unclear\",\n        \"In vivo consequences of impaired IL-11R shedding (A370V) not demonstrated\",\n        \"Whether early secretory cleavage applies to other substrates not tested\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Discovery that RHBDL2 cleaves Notch1 to release N1ICD and cooperates with the deubiquitinase OTUD7B to stabilize N1ICD expanded RHBDL2 into the Notch signaling pathway and demonstrated functional collaboration between a rhomboid protease and a DUB.\",\n      \"evidence\": \"Co-immunoprecipitation, RNA-seq, ubiquitination assay, Notch inhibitor (IMR-1) epistasis, in vivo xenograft in pancreatic cancer\",\n      \"pmids\": [\"36351890\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Notch1 cleavage site not mapped\",\n        \"Relationship to canonical γ-secretase-mediated Notch processing unclear\",\n        \"Single-lab study in one cancer type\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identification of a protease-independent scaffold function of RHBDL2 that stabilizes USP3 via a Val245-mediated hydrophobic interface revealed a second molecular activity for the protein, linking it to deubiquitination of PPT1 and FASN-dependent lipogenesis in osteosarcoma.\",\n      \"evidence\": \"Multi-omics, structural analysis, Val245 mutagenesis, Co-IP, ubiquitination assay, in vivo tumour model, EGCG pharmacological inhibition\",\n      \"pmids\": [\"42031733\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Scaffold function demonstrated in a single cancer context; generality unknown\",\n        \"No reconstituted biochemical assay for direct RHBDL2–USP3 stabilization\",\n        \"Structural basis of the Val245 interface resolved only computationally\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include how RHBDL2 activity is regulated (transcriptionally, post-translationally, or by lipid environment), the structural basis for substrate selectivity among rhomboid family members, whether the scaffold function extends to additional partners, and the in vivo physiological consequences of RHBDL2 loss in mammals.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No mammalian RHBDL2 knockout phenotype reported\",\n        \"No high-resolution structure of RHBDL2\",\n        \"Regulatory mechanisms (transcriptional or post-translational) of RHBDL2 expression and activity unknown\",\n        \"Relative contribution of RHBDL2 versus metalloproteases for shared substrates in vivo not established\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 4, 5, 6, 8]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 2, 4, 6]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 3, 6, 8]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 5, 9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"EGF\",\n      \"THBD\",\n      \"EFNB3\",\n      \"CLEC14A\",\n      \"IL11RA\",\n      \"NOTCH1\",\n      \"USP3\",\n      \"OTUD7B\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}