{"gene":"TSPAN33","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2006,"finding":"TSPAN33 (Penumbra) encodes a novel tetraspanin of 283 amino acids that is targeted to the cell surface and forms disulfide-bonded homodimers. It is highly expressed in erythroid progenitors, and Penumbra knockout mice develop massive splenomegaly, macrocytic anemia, and ineffective erythropoiesis. Re-expression of Penumbra in a Penumbra-/- multipotent hematopoietic cell line (EMX) rescued erythropoietin-driven erythroid differentiation, establishing a direct positive role for TSPAN33 in erythropoiesis.","method":"Gene knockout mouse model, cell-surface targeting assay, disulfide-bond analysis, re-expression rescue experiment in Penumbra-/- cell line (EMX) with erythropoietin stimulation","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO mouse with defined cellular phenotype (anemia, ineffective erythropoiesis), rescued by re-expression in KO cell line; multiple orthogonal methods in a single focused study","pmids":["17158226"],"is_preprint":false},{"year":2013,"finding":"TSPAN33 is not expressed in resting human B cells but is strongly induced upon B cell activation. It is also upregulated in Burkitt's lymphoma-derived 2E2 cells upon activation, and is expressed in Hodgkin's lymphoma, diffuse large B cell lymphoma, and in B cells from autoimmune disease patients (rheumatoid arthritis, SLE), establishing TSPAN33 as a marker of activated and malignant B cells.","method":"Gene expression analysis in primary human B cells (resting vs. activated), flow cytometry/immunostaining in B cell lines and lymphoma samples, and in MRL/Fas(lpr/lpr) mouse spleen B cells","journal":"Clinical immunology (Orlando, Fla.)","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — expression-based characterization across multiple primary and cell-line systems, but mechanistic pathway not elucidated; single lab with multiple sample types","pmids":["24211713"],"is_preprint":false},{"year":2016,"finding":"TSPAN33 expression is induced in macrophages by TLR signaling (TLR2, TLR3, TLR4) and IFN-γ, and this induction is dependent on NOTCH signaling (diminished in Notch1/Notch2-deficient macrophages, enhanced by constitutively active NOTCH1 intracellular domain). TSPAN33 overexpression increases ADAM10 (but not ADAM17) maturation, thereby promoting NOTCH processing at the membrane and amplifying NOTCH signaling. TSPAN33 also modulates TLR-induced proinflammatory gene expression at least partly through increased NF-κB-dependent transcriptional activity, placing TSPAN33 as a TspanC8 subgroup member that acts as a positive feedback regulator of NOTCH/ADAM10 in macrophage inflammatory activation.","method":"Tspan33 overexpression and knockdown in macrophages, Notch1/Notch2-deficient macrophages, constitutively active NOTCH1 overexpression, ADAM10/ADAM17 maturation western blot, NF-κB reporter assay, TLR ligand stimulation assays","journal":"Journal of immunology (Baltimore, Md. : 1950)","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal gain/loss-of-function experiments with multiple orthogonal readouts (ADAM10 maturation, NOTCH processing, NF-κB activity, proinflammatory gene expression) in a single focused study","pmids":["27574297"],"is_preprint":false}],"current_model":"TSPAN33 (Penumbra) is a cell-surface tetraspanin that forms disulfide-bonded homodimers and plays a positive role in erythropoiesis (loss causes anemia in mice); in the immune system it is induced in activated B cells and, in macrophages, it acts downstream of TLR/NOTCH signaling to promote ADAM10 maturation, enhance NOTCH processing, and amplify NF-κB-driven proinflammatory gene expression."},"narrative":{"mechanistic_narrative":"TSPAN33 (Penumbra) is a cell-surface tetraspanin that links membrane proteolysis to hematopoietic and inflammatory programs [PMID:17158226, PMID:27574297]. It is synthesized as a 283-residue protein that traffics to the cell surface and assembles into disulfide-bonded homodimers, and is required for normal erythropoiesis: loss in mice produces splenomegaly, macrocytic anemia, and ineffective erythropoiesis, while re-expression restores erythropoietin-driven erythroid differentiation in knockout cells [PMID:17158226]. As a TspanC8 tetraspanin, TSPAN33 promotes maturation of the metalloprotease ADAM10 (but not ADAM17), thereby enhancing NOTCH processing at the membrane [PMID:27574297]. In macrophages its own expression is induced by TLR (TLR2/3/4) and IFN-γ signaling in a NOTCH-dependent manner, and it amplifies NF-κB-dependent proinflammatory transcription, positioning it as a positive feedback regulator of the NOTCH/ADAM10 axis during inflammatory activation [PMID:27574297]. It is absent from resting B cells but strongly induced upon B cell activation and is expressed in several lymphomas and in B cells of autoimmune patients, marking activated and malignant B cells [PMID:24211713].","teleology":[{"year":2006,"claim":"Established TSPAN33 as a bona fide cell-surface tetraspanin with a non-redundant, positive role in erythroid differentiation, answering whether this novel gene had a defined physiological function.","evidence":"Knockout mouse with anemia/ineffective erythropoiesis phenotype, cell-surface targeting and disulfide-bond assays, and re-expression rescue of EPO-driven erythroid differentiation in a Penumbra-/- cell line","pmids":["17158226"],"confidence":"High","gaps":["Molecular partners mediating the erythropoietic effect not identified","No link to ADAM10/NOTCH established in this context"]},{"year":2013,"claim":"Extended TSPAN33 beyond erythropoiesis by showing it is an activation-induced marker of B cells and is expressed in lymphomas and autoimmune B cells, raising the question of its role in adaptive immunity.","evidence":"Expression profiling of resting vs. activated primary human B cells, flow cytometry/immunostaining of B cell lines, lymphoma samples, and MRL/Fas(lpr/lpr) mouse B cells","pmids":["24211713"],"confidence":"Medium","gaps":["Mechanistic role in B cell activation or transformation not defined","Correlative expression only; no functional perturbation"]},{"year":2016,"claim":"Defined the molecular mechanism by which TSPAN33 acts in macrophages, placing it in a TLR/NOTCH-induced feedback loop that promotes ADAM10 maturation and amplifies NOTCH and NF-κB signaling.","evidence":"Reciprocal overexpression/knockdown in macrophages, Notch1/Notch2-deficient macrophages, constitutively active NOTCH1, ADAM10/ADAM17 maturation westerns, NF-κB reporter and TLR ligand stimulation assays","pmids":["27574297"],"confidence":"High","gaps":["Whether the same ADAM10/NOTCH mechanism underlies the erythroid and B cell phenotypes is untested","Direct physical interaction between TSPAN33 and ADAM10 not biochemically resolved here","No structural basis for ADAM10 selectivity over ADAM17"]},{"year":null,"claim":"Whether the ADAM10/NOTCH-promoting activity defined in macrophages mechanistically explains TSPAN33's roles in erythropoiesis and in B cell activation/malignancy remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying mechanism connecting erythroid, B cell, and macrophage functions","Erythropoiesis-relevant substrates or partners unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,2]}],"complexes":[],"partners":["ADAM10"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q86UF1","full_name":"Tetraspanin-33","aliases":["Penumbra","hPen","Proerythroblast new membrane"],"length_aa":283,"mass_kda":31.5,"function":"Part of TspanC8 subgroup, composed of 6 members that interact with the transmembrane metalloprotease ADAM10. This interaction is required for ADAM10 exit from the endoplasmic reticulum and for enzymatic maturation and trafficking to the cell surface as well as substrate specificity. Different TspanC8/ADAM10 complexes have distinct substrates (PubMed:26686862, PubMed:30463011, PubMed:37516108). Plays an important role in normal erythropoiesis (By similarity). It has a role in the differentiation of erythroid progenitors (By similarity). Negatively regulates ligand-induced Notch activity probably by regulating ADAM10 activity (PubMed:26686862). Mediates docking of ADAM10 to zonula adherens by interacting with ADAM10 and, in a PDZD11-dependent manner, with the zonula adherens protein PLEKHA7 (PubMed:30463011)","subcellular_location":"Cell membrane; Cell junction, adherens junction; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q86UF1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TSPAN33","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TSPAN33","total_profiled":1310},"omim":[{"mim_id":"610120","title":"TETRASPANIN 33; TSPAN33","url":"https://www.omim.org/entry/610120"},{"mim_id":"601500","title":"SMOOTHENED, FRIZZLED CLASS RECEPTOR; SMO","url":"https://www.omim.org/entry/601500"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Microtubules","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"kidney","ntpm":46.7}],"url":"https://www.proteinatlas.org/search/TSPAN33"},"hgnc":{"alias_symbol":["MGC50844","Penumbra"],"prev_symbol":[]},"alphafold":{"accession":"Q86UF1","domains":[{"cath_id":"-","chopping":"19-119_236-274","consensus_level":"high","plddt":91.2761,"start":19,"end":274}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86UF1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q86UF1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q86UF1-F1-predicted_aligned_error_v6.png","plddt_mean":89.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TSPAN33","jax_strain_url":"https://www.jax.org/strain/search?query=TSPAN33"},"sequence":{"accession":"Q86UF1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q86UF1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q86UF1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86UF1"}},"corpus_meta":[{"pmid":"7944288","id":"PMC_7944288","title":"Viability 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rats.","date":"2001","source":"Brain research","url":"https://pubmed.ncbi.nlm.nih.gov/11597598","citation_count":40,"is_preprint":false},{"pmid":"16403464","id":"PMC_16403464","title":"Focal ischemia induces expression of protease-activated receptor1 (PAR1) and PAR3 on microglia and enhances PAR4 labeling in the penumbra.","date":"2006","source":"Brain research","url":"https://pubmed.ncbi.nlm.nih.gov/16403464","citation_count":40,"is_preprint":false},{"pmid":"19333878","id":"PMC_19333878","title":"Ischemic stroke intervention requires mixed cellular protection of the penumbra.","date":"2009","source":"Current opinion in investigational drugs (London, England : 2000)","url":"https://pubmed.ncbi.nlm.nih.gov/19333878","citation_count":39,"is_preprint":false},{"pmid":"16327251","id":"PMC_16327251","title":"How healthy is the acutely reperfused ischemic penumbra?","date":"2005","source":"Cerebrovascular diseases (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/16327251","citation_count":38,"is_preprint":false},{"pmid":"11462799","id":"PMC_11462799","title":"Cell death in spinal cord injury (SCI) requires de novo protein synthesis. Calpain inhibitor E-64-d provides neuroprotection in SCI lesion and penumbra.","date":"2001","source":"Annals of the New York Academy of Sciences","url":"https://pubmed.ncbi.nlm.nih.gov/11462799","citation_count":37,"is_preprint":false},{"pmid":"23202804","id":"PMC_23202804","title":"WIN55,212-2 protects oligodendrocyte precursor cells in stroke penumbra following permanent focal cerebral ischemia in rats.","date":"2012","source":"Acta pharmacologica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/23202804","citation_count":36,"is_preprint":false},{"pmid":"31455665","id":"PMC_31455665","title":"Early molecular oxidative stress biomarkers of ischemic penumbra in acute stroke.","date":"2019","source":"Neurology","url":"https://pubmed.ncbi.nlm.nih.gov/31455665","citation_count":36,"is_preprint":false},{"pmid":"24287115","id":"PMC_24287115","title":"Lesional-targeting of neuroprotection to the inflammatory penumbra in experimental multiple sclerosis.","date":"2013","source":"Brain : a journal of neurology","url":"https://pubmed.ncbi.nlm.nih.gov/24287115","citation_count":35,"is_preprint":false},{"pmid":"12359312","id":"PMC_12359312","title":"Relationship between the activation of cyclic AMP responsive element binding protein and ischemic tolerance in the penumbra region of rat cerebral cortex.","date":"2002","source":"Neuroscience letters","url":"https://pubmed.ncbi.nlm.nih.gov/12359312","citation_count":35,"is_preprint":false},{"pmid":"15856211","id":"PMC_15856211","title":"Co-induction of alphaB-crystallin and MAPKAPK-2 in astrocytes in the penumbra after transient focal cerebral ischemia.","date":"2005","source":"Experimental brain research","url":"https://pubmed.ncbi.nlm.nih.gov/15856211","citation_count":34,"is_preprint":false},{"pmid":"18075295","id":"PMC_18075295","title":"Upregulation of Beclin 1 in the ischemic penumbra.","date":"2007","source":"Autophagy","url":"https://pubmed.ncbi.nlm.nih.gov/18075295","citation_count":31,"is_preprint":false},{"pmid":"16871404","id":"PMC_16871404","title":"Neuroprotective cannabinoid receptor antagonist SR141716A prevents downregulation of excitotoxic NMDA receptors in the ischemic penumbra.","date":"2006","source":"Acta neuropathologica","url":"https://pubmed.ncbi.nlm.nih.gov/16871404","citation_count":31,"is_preprint":false},{"pmid":"36219377","id":"PMC_36219377","title":"Reinventing the Penumbra - the Emerging Clockwork of a Multi-modal Mechanistic Paradigm.","date":"2022","source":"Translational stroke research","url":"https://pubmed.ncbi.nlm.nih.gov/36219377","citation_count":30,"is_preprint":false},{"pmid":"34970953","id":"PMC_34970953","title":"Penumbra in Acute Ischemic Stroke.","date":"2021","source":"Current neurovascular research","url":"https://pubmed.ncbi.nlm.nih.gov/34970953","citation_count":30,"is_preprint":false},{"pmid":"31945213","id":"PMC_31945213","title":"Rapid degeneration of neurons in the penumbra region following a small, focal ischemic stroke.","date":"2020","source":"The European journal of neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/31945213","citation_count":30,"is_preprint":false},{"pmid":"30854452","id":"PMC_30854452","title":"[18F] FDG Positron Emission Tomography (PET) Tumor and Penumbra Imaging Features Predict Recurrence in Non-Small Cell Lung Cancer.","date":"2019","source":"Tomography (Ann Arbor, Mich.)","url":"https://pubmed.ncbi.nlm.nih.gov/30854452","citation_count":29,"is_preprint":false},{"pmid":"12823888","id":"PMC_12823888","title":"Differential cerebral protein synthesis and heat shock protein 70 expression in the core and penumbra of rat brain after transient focal ischemia.","date":"2003","source":"Neurosurgery","url":"https://pubmed.ncbi.nlm.nih.gov/12823888","citation_count":27,"is_preprint":false},{"pmid":"17158226","id":"PMC_17158226","title":"Penumbra encodes a novel tetraspanin that is highly expressed in erythroid progenitors and promotes effective erythropoiesis.","date":"2006","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/17158226","citation_count":27,"is_preprint":false},{"pmid":"26722460","id":"PMC_26722460","title":"Down-regulated Na(+)/K(+)-ATPase activity in ischemic penumbra after focal cerebral ischemia/reperfusion in rats.","date":"2015","source":"International journal of clinical and experimental pathology","url":"https://pubmed.ncbi.nlm.nih.gov/26722460","citation_count":27,"is_preprint":false},{"pmid":"19156869","id":"PMC_19156869","title":"p75 NTR expression is induced in isolated neurons of the penumbra after ischemia by cortical devascularization.","date":"2009","source":"Journal of neuroscience research","url":"https://pubmed.ncbi.nlm.nih.gov/19156869","citation_count":27,"is_preprint":false},{"pmid":"12911466","id":"PMC_12911466","title":"Bedside biochemical monitoring of the penumbra zone surrounding an evacuated acute subdural haematoma.","date":"2003","source":"Acta neurologica Scandinavica","url":"https://pubmed.ncbi.nlm.nih.gov/12911466","citation_count":27,"is_preprint":false},{"pmid":"26531025","id":"PMC_26531025","title":"Expression of neuronal and signaling proteins in penumbra around a photothrombotic infarction core in rat cerebral cortex.","date":"2015","source":"Biochemistry. Biokhimiia","url":"https://pubmed.ncbi.nlm.nih.gov/26531025","citation_count":26,"is_preprint":false},{"pmid":"27093556","id":"PMC_27093556","title":"Precise Characterization of the Penumbra Revealed by MRI: A Modified Photothrombotic Stroke Model Study.","date":"2016","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/27093556","citation_count":26,"is_preprint":false},{"pmid":"23142152","id":"PMC_23142152","title":"Development of a pattern in biochemical parameters in the core and penumbra during infarct evolution after transient MCAO in rats.","date":"2012","source":"Neurochemistry international","url":"https://pubmed.ncbi.nlm.nih.gov/23142152","citation_count":26,"is_preprint":false},{"pmid":"24211713","id":"PMC_24211713","title":"TSPAN33 is a novel marker of activated and malignant B cells.","date":"2013","source":"Clinical immunology (Orlando, Fla.)","url":"https://pubmed.ncbi.nlm.nih.gov/24211713","citation_count":25,"is_preprint":false},{"pmid":"17662498","id":"PMC_17662498","title":"Altered expression of sodium transporters in ischemic penumbra after focal cerebral ischemia in rats.","date":"2007","source":"Neuroscience research","url":"https://pubmed.ncbi.nlm.nih.gov/17662498","citation_count":25,"is_preprint":false},{"pmid":"27651773","id":"PMC_27651773","title":"Endoplasmic reticulum stress-induced apoptosis in the penumbra aggravates secondary damage in rats with traumatic brain injury.","date":"2016","source":"Neural regeneration research","url":"https://pubmed.ncbi.nlm.nih.gov/27651773","citation_count":24,"is_preprint":false},{"pmid":"21616101","id":"PMC_21616101","title":"Role of activating transcription factor 3 in ischemic penumbra region following transient middle cerebral artery occlusion and reperfusion injury.","date":"2011","source":"Neuroscience research","url":"https://pubmed.ncbi.nlm.nih.gov/21616101","citation_count":24,"is_preprint":false},{"pmid":"31888302","id":"PMC_31888302","title":"Sequential Transcriptome Changes in the Penumbra after Ischemic Stroke.","date":"2019","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/31888302","citation_count":23,"is_preprint":false},{"pmid":"31493239","id":"PMC_31493239","title":"Expression of Histone Deacetylases HDAC1 and HDAC2 and Their Role in Apoptosis in the Penumbra Induced by Photothrombotic Stroke.","date":"2019","source":"Molecular neurobiology","url":"https://pubmed.ncbi.nlm.nih.gov/31493239","citation_count":22,"is_preprint":false},{"pmid":"18157632","id":"PMC_18157632","title":"Cross-talk between calpain and caspase-3 in penumbra and core during focal cerebral ischemia-reperfusion.","date":"2007","source":"Cellular and molecular neurobiology","url":"https://pubmed.ncbi.nlm.nih.gov/18157632","citation_count":22,"is_preprint":false},{"pmid":"22805876","id":"PMC_22805876","title":"Painted turtle cortex is resistant to an in vitro mimic of the ischemic mammalian penumbra.","date":"2012","source":"Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/22805876","citation_count":22,"is_preprint":false},{"pmid":"27574297","id":"PMC_27574297","title":"The Tetraspanin TSPAN33 Controls TLR-Triggered Macrophage Activation through Modulation of NOTCH Signaling.","date":"2016","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/27574297","citation_count":21,"is_preprint":false},{"pmid":"24824264","id":"PMC_24824264","title":"Research progress in traumatic brain penumbra.","date":"2014","source":"Chinese medical journal","url":"https://pubmed.ncbi.nlm.nih.gov/24824264","citation_count":21,"is_preprint":false},{"pmid":"20926974","id":"PMC_20926974","title":"Expression of connexin29 and 32 in the penumbra region after traumatic brain injury of mice.","date":"2010","source":"Neuroreport","url":"https://pubmed.ncbi.nlm.nih.gov/20926974","citation_count":21,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":52945,"output_tokens":1499,"usd":0.09066,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":7766,"output_tokens":1741,"usd":0.041177,"stage2_stop_reason":"end_turn"},"total_usd":0.131837,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"Looking through the 100 abstracts, I need to identify papers about the canonical TSPAN33 protein (also known as Penumbra/MGC50844). Most of these papers are about the ischemic penumbra (brain tissue around stroke lesions) — a completely different meaning of \"penumbra.\" I need to find only papers about the TSPAN33 tetraspanin protein.\n\nRelevant papers:\n- **PMID 17158226** (paper [83]): \"Penumbra encodes a novel tetraspanin that is highly expressed in erythroid progenitors and promotes effective erythropoiesis\" — This is clearly about the TSPAN33/Penumbra protein.\n- **PMID 24211713** (paper [90]): \"TSPAN33 is a novel marker of activated and malignant B cells\" — About TSPAN33 in B cells.\n- **PMID 27574297** (paper [98]): \"The Tetraspanin TSPAN33 Controls TLR-Triggered Macrophage Activation through Modulation of NOTCH Signaling\" — About TSPAN33 in macrophages.\n\nAll other papers are about ischemic/traumatic penumbra in neuroscience, spinal cord injury, etc. — not about the TSPAN33 protein.\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2006,\n      \"finding\": \"TSPAN33 (Penumbra) encodes a novel tetraspanin of 283 amino acids that is targeted to the cell surface and forms disulfide-bonded homodimers. It is highly expressed in erythroid progenitors, and Penumbra knockout mice develop massive splenomegaly, macrocytic anemia, and ineffective erythropoiesis. Re-expression of Penumbra in a Penumbra-/- multipotent hematopoietic cell line (EMX) rescued erythropoietin-driven erythroid differentiation, establishing a direct positive role for TSPAN33 in erythropoiesis.\",\n      \"method\": \"Gene knockout mouse model, cell-surface targeting assay, disulfide-bond analysis, re-expression rescue experiment in Penumbra-/- cell line (EMX) with erythropoietin stimulation\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO mouse with defined cellular phenotype (anemia, ineffective erythropoiesis), rescued by re-expression in KO cell line; multiple orthogonal methods in a single focused study\",\n      \"pmids\": [\"17158226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TSPAN33 is not expressed in resting human B cells but is strongly induced upon B cell activation. It is also upregulated in Burkitt's lymphoma-derived 2E2 cells upon activation, and is expressed in Hodgkin's lymphoma, diffuse large B cell lymphoma, and in B cells from autoimmune disease patients (rheumatoid arthritis, SLE), establishing TSPAN33 as a marker of activated and malignant B cells.\",\n      \"method\": \"Gene expression analysis in primary human B cells (resting vs. activated), flow cytometry/immunostaining in B cell lines and lymphoma samples, and in MRL/Fas(lpr/lpr) mouse spleen B cells\",\n      \"journal\": \"Clinical immunology (Orlando, Fla.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — expression-based characterization across multiple primary and cell-line systems, but mechanistic pathway not elucidated; single lab with multiple sample types\",\n      \"pmids\": [\"24211713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TSPAN33 expression is induced in macrophages by TLR signaling (TLR2, TLR3, TLR4) and IFN-γ, and this induction is dependent on NOTCH signaling (diminished in Notch1/Notch2-deficient macrophages, enhanced by constitutively active NOTCH1 intracellular domain). TSPAN33 overexpression increases ADAM10 (but not ADAM17) maturation, thereby promoting NOTCH processing at the membrane and amplifying NOTCH signaling. TSPAN33 also modulates TLR-induced proinflammatory gene expression at least partly through increased NF-κB-dependent transcriptional activity, placing TSPAN33 as a TspanC8 subgroup member that acts as a positive feedback regulator of NOTCH/ADAM10 in macrophage inflammatory activation.\",\n      \"method\": \"Tspan33 overexpression and knockdown in macrophages, Notch1/Notch2-deficient macrophages, constitutively active NOTCH1 overexpression, ADAM10/ADAM17 maturation western blot, NF-κB reporter assay, TLR ligand stimulation assays\",\n      \"journal\": \"Journal of immunology (Baltimore, Md. : 1950)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal gain/loss-of-function experiments with multiple orthogonal readouts (ADAM10 maturation, NOTCH processing, NF-κB activity, proinflammatory gene expression) in a single focused study\",\n      \"pmids\": [\"27574297\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TSPAN33 (Penumbra) is a cell-surface tetraspanin that forms disulfide-bonded homodimers and plays a positive role in erythropoiesis (loss causes anemia in mice); in the immune system it is induced in activated B cells and, in macrophages, it acts downstream of TLR/NOTCH signaling to promote ADAM10 maturation, enhance NOTCH processing, and amplify NF-κB-driven proinflammatory gene expression.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TSPAN33 (Penumbra) is a cell-surface tetraspanin that links membrane proteolysis to hematopoietic and inflammatory programs [#0, #2]. It is synthesized as a 283-residue protein that traffics to the cell surface and assembles into disulfide-bonded homodimers, and is required for normal erythropoiesis: loss in mice produces splenomegaly, macrocytic anemia, and ineffective erythropoiesis, while re-expression restores erythropoietin-driven erythroid differentiation in knockout cells [#0]. As a TspanC8 tetraspanin, TSPAN33 promotes maturation of the metalloprotease ADAM10 (but not ADAM17), thereby enhancing NOTCH processing at the membrane [#2]. In macrophages its own expression is induced by TLR (TLR2/3/4) and IFN-\\u03b3 signaling in a NOTCH-dependent manner, and it amplifies NF-\\u03baB-dependent proinflammatory transcription, positioning it as a positive feedback regulator of the NOTCH/ADAM10 axis during inflammatory activation [#2]. It is absent from resting B cells but strongly induced upon B cell activation and is expressed in several lymphomas and in B cells of autoimmune patients, marking activated and malignant B cells [#1].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established TSPAN33 as a bona fide cell-surface tetraspanin with a non-redundant, positive role in erythroid differentiation, answering whether this novel gene had a defined physiological function.\",\n      \"evidence\": \"Knockout mouse with anemia/ineffective erythropoiesis phenotype, cell-surface targeting and disulfide-bond assays, and re-expression rescue of EPO-driven erythroid differentiation in a Penumbra-/- cell line\",\n      \"pmids\": [\"17158226\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular partners mediating the erythropoietic effect not identified\", \"No link to ADAM10/NOTCH established in this context\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Extended TSPAN33 beyond erythropoiesis by showing it is an activation-induced marker of B cells and is expressed in lymphomas and autoimmune B cells, raising the question of its role in adaptive immunity.\",\n      \"evidence\": \"Expression profiling of resting vs. activated primary human B cells, flow cytometry/immunostaining of B cell lines, lymphoma samples, and MRL/Fas(lpr/lpr) mouse B cells\",\n      \"pmids\": [\"24211713\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic role in B cell activation or transformation not defined\", \"Correlative expression only; no functional perturbation\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined the molecular mechanism by which TSPAN33 acts in macrophages, placing it in a TLR/NOTCH-induced feedback loop that promotes ADAM10 maturation and amplifies NOTCH and NF-\\u03baB signaling.\",\n      \"evidence\": \"Reciprocal overexpression/knockdown in macrophages, Notch1/Notch2-deficient macrophages, constitutively active NOTCH1, ADAM10/ADAM17 maturation westerns, NF-\\u03baB reporter and TLR ligand stimulation assays\",\n      \"pmids\": [\"27574297\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the same ADAM10/NOTCH mechanism underlies the erythroid and B cell phenotypes is untested\", \"Direct physical interaction between TSPAN33 and ADAM10 not biochemically resolved here\", \"No structural basis for ADAM10 selectivity over ADAM17\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether the ADAM10/NOTCH-promoting activity defined in macrophages mechanistically explains TSPAN33's roles in erythropoiesis and in B cell activation/malignancy remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying mechanism connecting erythroid, B cell, and macrophage functions\", \"Erythropoiesis-relevant substrates or partners unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ADAM10\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}