{"gene":"ADAM28","run_date":"2026-06-09T22:02:41","timeline":{"discoveries":[{"year":2000,"finding":"ADAM28 undergoes autocatalytic pro-domain removal: a glutamate-to-alanine mutation in the catalytic site abolishes pro-domain removal even though the mutant is transported to the cell surface normally, indicating maturation is at least partly autocatalytic. Mature ADAM28 is accessible to cell surface trypsinization in COS-7 cells, indicating it functions mainly on the cell surface. Processing occurs in a late Golgi compartment.","method":"Site-directed mutagenesis of catalytic glutamate, cell surface trypsinization, subcellular fractionation, expression in COS-7 cells","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — catalytic-site mutagenesis combined with cell surface accessibility assay and localization, single lab but multiple orthogonal methods","pmids":["10794709"],"is_preprint":false},{"year":2001,"finding":"Recombinant ADAM28 cleaves myelin basic protein at two sites; catalytic activity is abolished by a catalytic-site mutation and is insensitive to TIMP-1 and TIMP-2.","method":"In vitro protease assay with recombinant ADAM28, site-directed mutagenesis, TIMP inhibition assay","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis and inhibitor controls, single lab","pmids":["11389903"],"is_preprint":false},{"year":2003,"finding":"Soluble recombinant ADAM28 (MDC-L) catalyzes ectodomain shedding of CD23 (low-affinity IgE receptor); its substrate specificity on synthetic peptide libraries is similar to ADAM8 and ADAM15 but distinct from ADAM17.","method":"Synthetic peptide substrate library screening, ectodomain shedding assay with recombinant enzyme, metalloprotease inhibitor treatment","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with recombinant enzyme plus inhibitor validation, single lab with multiple substrates tested","pmids":["12777399"],"is_preprint":false},{"year":2004,"finding":"ProADAM28s (65 kDa secreted isoform) is processed and activated by active MMP-7 (matrilysin-1), generating 42- and 40-kDa active forms lacking the propeptide. Active ADAM28s cleaves IGFBP-3 in both free form and in complex with IGF-I or IGF-II; cleavage is blocked by EDTA, 1,10-phenanthroline, KB-R7785, TIMP-3, and TIMP-4.","method":"In vitro MMP-7 activation assay, immunoblotting, metalloprotease inhibitor panel, TIMP inhibition assay","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of activation and substrate cleavage with multiple inhibitor controls, single lab","pmids":["15013428"],"is_preprint":false},{"year":2005,"finding":"Mouse ADAM28 gene expression in the epididymis is regulated by testicular factors (but not androgen), whereas ADAM7 is regulated by both androgen and testicular factors. Unlike ADAM7, ADAM28 protein is not transferred to the sperm surface during epididymal transit.","method":"RT-PCR, protein analysis, castration/androgen supplementation experiments, epididymis-sperm transfer assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct in vivo manipulation (castration model) with protein-level readout, single lab","pmids":["15883027"],"is_preprint":false},{"year":2006,"finding":"ADAM28 promotes breast carcinoma cell proliferation by cleaving IGFBP-3, thereby releasing bioavailable IGF-I. ADAM28 siRNA knockdown reduces IGFBP-3 cleavage, IGF-I signaling, cell proliferation in vitro, and xenograft tumor growth in vivo.","method":"siRNA knockdown, ADAM28 inhibitor and neutralizing antibody treatment, proliferation assay, IGFBP-3 cleavage assay by immunoblot, xenograft mouse model","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal loss-of-function approaches (siRNA, inhibitor, antibody) with in vitro and in vivo validation, mechanistic substrate identified","pmids":["17047053"],"is_preprint":false},{"year":2007,"finding":"The disintegrin-like domain of secreted ADAM28 binds to PSGL-1 (P-selectin glycoprotein ligand-1) on leukocytes. This binding enhances PSGL-1/P-selectin-mediated leukocyte adhesion to endothelial cells and increases leukocyte accumulation in pulmonary microcirculation in a mouse model of endotoxin-induced inflammation.","method":"Yeast two-hybrid screen, domain-specific binding assay, PSGL-1 stable transfectants, co-immunoprecipitation, co-immunolocalization, P-selectin adhesion assay, in vivo mouse endotoxin model","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid plus reciprocal co-IP plus functional adhesion assay plus in vivo model, multiple orthogonal methods","pmids":["17597069"],"is_preprint":false},{"year":2009,"finding":"ATRA stimulation induces ADAM28 expression in human chondrocytes (HCS-2/8 cells and primary chondrocytes). Transfection of ADAM28 or its soluble alternatively spliced form augments proteoglycan release from cartilage; a mutant lacking part of the disintegrin domain does not, indicating the disintegrin domain is required for localization/substrate recognition in cartilage degradation.","method":"RT-PCR, transfection overexpression and domain-deletion mutant, proteoglycan release assay from bovine nasal cartilage","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain-deletion mutagenesis with functional readout, single lab","pmids":["19527685"],"is_preprint":false},{"year":2010,"finding":"ADAM28 cleaves CTGF at the Ala181-Tyr182 and Asp191-Pro192 bonds in the hinge region. ADAM28 selectively digests CTGF in the CTGF/VEGF165 complex, releasing biologically active VEGF165. CTGF binds to proADAM28.","method":"Yeast two-hybrid screen, protein binding assay, in vitro cleavage assay with site mapping by immunoblot, VEGF165 release assay","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with cleavage site mapping plus binding assay, single lab with multiple orthogonal methods","pmids":["20971063"],"is_preprint":false},{"year":2011,"finding":"ADAM28 binds to integrin α4β1 via its disintegrin domain. Soluble ADAM28 enhances α4β1-dependent cell adhesion to VCAM-1 and promotes lymphocyte adhesion to and transendothelial migration through endothelial monolayers. Binding is attenuated by anti-functional integrin antibodies and enhanced by Mn2+ and activating antibody 9EG7.","method":"Affymetrix microarray, RT-PCR, immunohistochemistry, cell adhesion assay with recombinant murine ADAM28, integrin-blocking antibodies, Mn2+ activation, transendothelial migration assay","journal":"Cell biology international","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional adhesion assay with recombinant protein plus integrin-blocking antibody controls and migration assay, single lab","pmids":["21332445"],"is_preprint":false},{"year":2012,"finding":"ADAM28 binds to and cleaves von Willebrand factor (VWF). High ADAM28 expression in carcinoma cells confers resistance to VWF-induced apoptosis. Knockdown of ADAM28 by shRNA increases carcinoma cell apoptosis in lung blood vessels and significantly decreases lung metastasis in xenograft models.","method":"Yeast two-hybrid screen, protein binding assay, immunoblotting of VWF cleavage, shRNA and siRNA knockdown, neutralizing antibody, mouse xenograft in vivo imaging model","journal":"Journal of the National Cancer Institute","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple loss-of-function approaches (shRNA, siRNA, antibody) with in vitro and in vivo validation, substrate binding confirmed by independent binding assay","pmids":["22636800"],"is_preprint":false},{"year":2012,"finding":"ADAM28 co-localizes and co-immunoprecipitates with TNF-α; overexpression of ADAM28 with TNF-α in HEK293 cells promotes TNF-α shedding, which is significantly reduced by ADAM28 siRNA knockdown or pharmacological inhibition. Endogenous ADAM28 in THP-1 macrophages similarly mediates TNF-α shedding.","method":"Co-immunoprecipitation, co-localization imaging, overexpression in HEK293 cells, siRNA knockdown, pharmacological inhibition, ELISA for TNF-α","journal":"Immunology and cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus functional siRNA and inhibitor validation in two cell types, single lab","pmids":["23010875"],"is_preprint":false},{"year":2013,"finding":"ADAM28 mediates ectodomain shedding of CD200 from B-CLL cells: siRNA knockdown of ADAM28 decreases soluble CD200 release, and transfection of cloned ADAM28 into CD200+ cells enhances sCD200 release.","method":"siRNA knockdown, ADAM28 gene transfection, ELISA for soluble CD200","journal":"Leukemia research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — complementary loss- and gain-of-function with protein-level readout, single lab","pmids":["23643150"],"is_preprint":false},{"year":2013,"finding":"Src kinase induces ADAM28 gene expression in v-src-transformed epithelial cells through the MEK/ERK and PI3K/mTOR pathways. ADAM28 expression in human carcinoma cell lines correlates with phosphorylated c-Src and is suppressed by Src, MEK, and PI3K inhibitors.","method":"Oncogene transformation panel, Src/MEK/PI3K kinase inhibitors, immunoblotting for ADAM28, co-expression immunohistochemistry in human tissues","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological pathway epistasis with multiple inhibitor combinations in transformed cells plus human tissue validation, single lab","pmids":["24007880"],"is_preprint":false},{"year":2016,"finding":"ADAM28 binds C1q (identified by yeast two-hybrid); recombinant ADAM28 pre-treatment of C1q reduces C1q-induced apoptosis and autophagy in bronchial epithelial cells. ADAM28 siRNA knockdown increases C1q-induced cell death, establishing a cytoprotective function for ADAM28 in epithelial cells against C1q cytotoxicity. Binding confirmed by binding assays, immunoprecipitation, and surface plasmon resonance.","method":"Yeast two-hybrid, protein binding assay, immunoprecipitation, surface plasmon resonance, siRNA knockdown, cell death assay, autophagy (LC3-II) and apoptosis (caspase-3, p38) markers","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — SPR quantitative binding plus IP plus functional siRNA knockdown with multiple cell death readouts, single lab with multiple orthogonal methods","pmids":["26918856"],"is_preprint":false},{"year":2017,"finding":"CD19 promotes ADAM28-mediated cleavage of Notch2 in marginal zone precursors (MZP) to drive their differentiation into MZ B cells. CD19 upregulates ADAM28 expression by suppressing Foxo1. CD19-deficient MZP fail to differentiate into MZ B cells, and re-expression of CD19 rescues this.","method":"CD19 knockout mice, CD19 rescue experiments, ADAM28 expression analysis, Notch2 cleavage assay, Foxo1 expression analysis","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout epistasis with rescue, substrate cleavage assay, single lab","pmids":["28707394"],"is_preprint":false},{"year":2017,"finding":"ADAM28 siRNA knockdown in diet-induced obese mice prevents weight gain, promotes insulin sensitivity/glucose tolerance, and decreases liver TNF-α levels. ADAM28 knockout mice show reduced body weight and elevated HDL cholesterol, confirming a functional role for ADAM28 in metabolic dysfunction.","method":"siRNA knockdown in diet-induced obesity model, ADAM28 knockout mice, metabolic phenotyping (weight, glucose tolerance, insulin sensitivity, serum lipids, liver enzymes)","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two independent loss-of-function approaches (siRNA and KO mice) with metabolic phenotyping, single lab","pmids":["28430139"],"is_preprint":false},{"year":2018,"finding":"ADAM28 promotes AML leukemic cell proliferation, migration, and invasion through IGFBP-3 degradation and consequent IGF-I-induced cell proliferation. Knockout of ADAM28 in HL-60 cells reduces growth and dissemination in a xenotransplantation mouse model.","method":"ADAM28 CRISPR/Cas9 knockout, proliferation/migration/invasion assays, IGFBP-3 cleavage assay, IGF-I stimulation, xenotransplantation mouse model","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with in vitro and in vivo readouts plus substrate cleavage, single lab","pmids":["30429106"],"is_preprint":false},{"year":2018,"finding":"A neutralizing antibody (211-14) against the junctional region between the cysteine-rich domain and secreted-specific domain of ADAM28 inhibits IGFBP-3 cleavage, blocks IGF-1-stimulated proliferation, and promotes VWF-induced cell death in lung adenocarcinoma cells. In vivo, it reduces tumor growth and metastasis and prolongs survival in lung metastasis models.","method":"Human neutralizing antibody generation, IC50 determination, KD measurement by binding assay, proliferation assay, VWF-induced apoptosis assay, lung metastasis mouse model, survival analysis","journal":"Molecular cancer therapeutics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — antibody functional validation with epitope mapping, dual substrate inhibition, in vitro and in vivo, single lab","pmids":["30190423"],"is_preprint":false},{"year":2018,"finding":"Host (microenvironment)-derived ADAM28 in ADAM28 knockout mice is associated with decreased CD8+ T cell numbers in spleen and lungs and increased lung colonization by tumor cells, indicating a protective role for host ADAM28 against metastasis through T cell mobilization (not proliferation, migration, or activation of CD8+ T cells per se).","method":"ADAM28 full knockout mouse model, experimental tumor metastasis models, flow cytometry for CD8+ T cells, ex vivo CD8+ T cell characterization","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with multiple tumor models and immune phenotyping, single lab","pmids":["30647853"],"is_preprint":false},{"year":2021,"finding":"ADAM28 from both gastric cancer cells and endothelial cells (HUVECs) cleaves VWF to eliminate VWF-induced apoptosis of gastric cancer cells. ADAM28 knockdown in HUVECs increases VWF in supernatant and elevates integrin β3, p-TP53, and cleaved caspase-3 in co-cultured gastric cancer cells; these effects are reversed by VWF knockdown.","method":"ADAM28 overexpression/knockdown in gastric cancer cells and HUVECs, co-culture system, ELISA for VWF, Western blot for apoptosis markers, MTT, wound healing, flow cytometry","journal":"European journal of pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — complementary gain- and loss-of-function in two cell types with VWF rescue experiment, single lab","pmids":["33675784"],"is_preprint":false},{"year":2017,"finding":"ADAM28 localizes to HLA-G+ trophoblasts in distal anchoring columns and decidua. siRNA-mediated ADAM28 knockdown in placental explant cultures impairs trophoblast column outgrowth and increases apoptosis in matrix-invading trophoblasts.","method":"qPCR, immunofluorescence microscopy on purified placental cell populations, siRNA knockdown in Matrigel-embedded placental explant cultures, apoptosis assay","journal":"Placenta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization with functional siRNA knockdown, single lab","pmids":["28623976"],"is_preprint":false},{"year":2023,"finding":"In ADAM28 knockout mice exposed to ovalbumin, airway responsiveness, Th2 cell proportion, B cell numbers, collagen deposition, smooth muscle hyperplasia, and mucous hyperplasia are all significantly reduced compared to wild-type. Lung fibroblasts from ADAM28 KO OVA-exposed mice show decreased proliferative capacity, suggesting ADAM28 directly contributes to airway remodelling.","method":"ADAM28 knockout mice, ovalbumin sensitization/challenge model, flexiVent airway responsiveness measurement, BAL analysis, lung histology, fibroblast proliferation assay ex vivo","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO in disease model with multiple orthogonal phenotypic readouts and ex vivo fibroblast assay, single lab","pmids":["36685493"],"is_preprint":false},{"year":2018,"finding":"EBV nuclear antigen EBNA3C represses the ADAM28-ADAMDEC1 locus through interaction with KDM2B (histone lysine demethylase), which removes the activating mark H3K4me3. Concurrently, EBNA3C recruits Polycomb proteins BMI1 and SUZ12 to deposit H3K27me3 at the locus. These are two distinct repressive mechanisms.","method":"Recombinant EBV mutant viruses (RBPJ interaction motifs), ChIP for BMI1, SUZ12, H3K27me3, H3K4me3, gene expression analysis in LCLs, EBNA3C-KDM2B interaction assay","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP with recombinant virus mutants plus protein interaction data, single lab with multiple chromatin marks; this defines transcriptional regulation of ADAM28 locus","pmids":["30135119"],"is_preprint":false}],"current_model":"ADAM28 is a zinc-dependent metalloprotease (ADAM family) existing as membrane-anchored (ADAM28m) and secreted (ADAM28s) isoforms that undergoes at least partly autocatalytic pro-domain removal in the Golgi; it is activated extracellularly by MMP-7, cleaves defined substrates including IGFBP-3, VWF, CTGF, CD23, CD200, Notch2, and TNF-α, and via its disintegrin domain binds integrins α4β1/α4β7/α9β1 and PSGL-1 to regulate cell adhesion and leukocyte trafficking; in carcinoma cells ADAM28 expression is driven by Src kinase through MEK/ERK and PI3K/mTOR pathways, and its proteolytic activities promote tumor cell proliferation (by liberating IGF-I from IGFBP-3) and metastatic survival (by inactivating pro-apoptotic VWF), while host-derived ADAM28 plays a counter-protective role in anti-tumor immunity through CD8+ T cell mobilization."},"narrative":{"mechanistic_narrative":"ADAM28 is a zinc-dependent ADAM-family metalloprotease that operates principally at the cell surface and as a secreted isoform to regulate growth-factor bioavailability, cell adhesion, and apoptosis [PMID:10794709, PMID:17047053, PMID:22636800]. It matures through at least partly autocatalytic pro-domain removal in a late Golgi compartment, with a catalytic-site glutamate required for processing, and functions chiefly on the cell surface [PMID:10794709]; the secreted pro-form is further processed extracellularly by active MMP-7 [PMID:15013428]. Its catalytic activity is intrinsic and insensitive to TIMP-1/-2 but blocked by TIMP-3/-4 and broad metalloprotease inhibitors [PMID:11389903, PMID:15013428]. ADAM28 cleaves a defined set of substrates: it degrades IGFBP-3 to release bioavailable IGF-I and drive tumor cell proliferation [PMID:17047053, PMID:30429106], cleaves von Willebrand factor to protect carcinoma cells from VWF-induced apoptosis and enable metastatic survival [PMID:22636800, PMID:33675784], and sheds the ectodomains of CD23, CD200, and TNF-α [PMID:12777399, PMID:23010875, PMID:23643150] as well as cleaving CTGF to liberate active VEGF165 [PMID:20971063] and Notch2 to drive marginal-zone B cell differentiation [PMID:28707394]. Independent of catalysis, its disintegrin-like domain binds integrin α4β1 and PSGL-1 to promote leukocyte adhesion, transendothelial migration, and trafficking [PMID:17597069, PMID:21332445]. In carcinoma cells ADAM28 expression is induced by Src kinase signaling through MEK/ERK and PI3K/mTOR pathways [PMID:24007880], and a neutralizing antibody targeting ADAM28 blocks IGFBP-3 cleavage and tumor growth in vivo [PMID:30190423]. Beyond its tumor-promoting cell-intrinsic roles, ADAM28 contributes to inflammatory and remodeling phenotypes including airway remodeling, metabolic dysfunction, and trophoblast survival [PMID:28430139, PMID:28623976, PMID:36685493], while host-derived ADAM28 plays a counter-protective anti-metastatic role linked to CD8+ T cell mobilization [PMID:30647853].","teleology":[{"year":2000,"claim":"Established how ADAM28 matures and where it acts, showing that pro-domain removal is at least partly autocatalytic and that the mature enzyme functions at the cell surface.","evidence":"Catalytic-glutamate mutagenesis, cell surface trypsinization, and subcellular fractionation in COS-7 cells","pmids":["10794709"],"confidence":"High","gaps":["Does not establish whether trans-cleavage by other proteases contributes to maturation in vivo","No structural detail of the active site"]},{"year":2001,"claim":"Demonstrated intrinsic proteolytic activity of ADAM28 with defined cleavage and a TIMP-insensitivity profile distinguishing it from other metalloproteases.","evidence":"In vitro protease assay on myelin basic protein with catalytic mutant and TIMP-1/-2 controls","pmids":["11389903"],"confidence":"High","gaps":["MBP is a model substrate, not necessarily physiological","Did not test full TIMP panel"]},{"year":2003,"claim":"Identified the first ectodomain shedding substrate (CD23) and defined ADAM28 peptide specificity relative to other ADAMs.","evidence":"Synthetic peptide library screening and shedding assay with recombinant soluble enzyme","pmids":["12777399"],"confidence":"High","gaps":["Cellular context of CD23 shedding by endogenous ADAM28 not established"]},{"year":2004,"claim":"Showed that the secreted pro-isoform is activated extracellularly by MMP-7 and that active ADAM28 cleaves IGFBP-3 free and in IGF complexes, linking ADAM28 to growth-factor bioavailability.","evidence":"In vitro MMP-7 activation, immunoblotting, and a metalloprotease/TIMP inhibitor panel","pmids":["15013428"],"confidence":"High","gaps":["Whether MMP-7 is the physiological activator in tissues not shown","Membrane isoform activation route not addressed"]},{"year":2006,"claim":"Connected IGFBP-3 cleavage to a cancer phenotype, establishing ADAM28 as a driver of tumor cell proliferation via IGF-I liberation.","evidence":"siRNA, inhibitor, and neutralizing antibody loss-of-function with proliferation, cleavage assays, and breast cancer xenografts","pmids":["17047053"],"confidence":"High","gaps":["Does not address ADAM28 contributions independent of IGF-I axis"]},{"year":2007,"claim":"Revealed a non-catalytic adhesive function: the disintegrin domain binds PSGL-1 to promote leukocyte adhesion and trafficking in inflammation.","evidence":"Yeast two-hybrid, reciprocal co-IP, P-selectin adhesion assay, and mouse endotoxin model","pmids":["17597069"],"confidence":"High","gaps":["Did not resolve whether catalytic activity modulates this adhesive role"]},{"year":2010,"claim":"Expanded the substrate repertoire to CTGF, mapping cleavage sites and showing release of active VEGF165 from the CTGF/VEGF complex.","evidence":"Yeast two-hybrid, binding assay, and in vitro cleavage with site mapping","pmids":["20971063"],"confidence":"High","gaps":["In vivo angiogenic consequence not demonstrated"]},{"year":2011,"claim":"Defined a second integrin partner (α4β1) bound through the disintegrin domain, mechanistically linking ADAM28 to lymphocyte adhesion and transendothelial migration.","evidence":"Cell adhesion assays with recombinant ADAM28, integrin-blocking antibodies, and Mn2+/9EG7 activation","pmids":["21332445"],"confidence":"Medium","gaps":["Single lab; direct affinity not quantified","Physiological in vivo relevance to lymphocyte trafficking not tested"]},{"year":2012,"claim":"Established that ADAM28 cleavage of VWF confers resistance to VWF-induced apoptosis, explaining a pro-metastatic survival mechanism distinct from proliferation.","evidence":"Yeast two-hybrid, binding assay, shRNA/siRNA, neutralizing antibody, and xenograft lung metastasis imaging","pmids":["22636800"],"confidence":"High","gaps":["VWF cleavage site not mapped here","Mechanism by which VWF triggers apoptosis not fully defined"]},{"year":2012,"claim":"Added TNF-α as a shedding substrate, implicating ADAM28 in inflammatory cytokine release by macrophages.","evidence":"Co-IP, co-localization, overexpression in HEK293, siRNA, inhibitor, and TNF-α ELISA in THP-1 cells","pmids":["23010875"],"confidence":"Medium","gaps":["Overlap/redundancy with ADAM17-mediated TNF-α shedding not delineated","Single lab"]},{"year":2013,"claim":"Identified CD200 as a shedding substrate in B-CLL cells, broadening immune-modulatory roles.","evidence":"siRNA knockdown and ADAM28 transfection with soluble CD200 ELISA","pmids":["23643150"],"confidence":"Medium","gaps":["Cleavage site not mapped","In vivo immune consequence not tested"]},{"year":2013,"claim":"Defined the upstream signaling that drives ADAM28 expression in cancer: Src acting through MEK/ERK and PI3K/mTOR.","evidence":"Oncogene transformation panel, kinase inhibitors, immunoblotting, and human tissue co-expression","pmids":["24007880"],"confidence":"Medium","gaps":["Transcription factors directly binding the ADAM28 promoter not identified","Single lab"]},{"year":2016,"claim":"Showed a cytoprotective function in epithelial cells via C1q binding, where ADAM28 reduces C1q-induced apoptosis and autophagy.","evidence":"Yeast two-hybrid, SPR, IP, siRNA, and cell death/autophagy markers in bronchial epithelial cells","pmids":["26918856"],"confidence":"High","gaps":["Whether C1q is a cleavage substrate or only a binding partner not resolved"]},{"year":2017,"claim":"Placed ADAM28 in a developmental B cell pathway, cleaving Notch2 downstream of CD19 to drive marginal-zone B cell differentiation.","evidence":"CD19 knockout/rescue mice, Notch2 cleavage assay, and Foxo1 analysis","pmids":["28707394"],"confidence":"Medium","gaps":["Direct demonstration of ADAM28 acting on Notch2 in vivo limited","Single lab"]},{"year":2017,"claim":"Demonstrated a role in trophoblast biology, where ADAM28 supports column outgrowth and protects invading trophoblasts from apoptosis.","evidence":"Immunofluorescence localization and siRNA knockdown in placental explant cultures","pmids":["28623976"],"confidence":"Medium","gaps":["Substrate responsible for trophoblast survival not identified","Single lab"]},{"year":2017,"claim":"Implicated ADAM28 in metabolic dysfunction, with loss reducing weight gain and improving glucose handling.","evidence":"siRNA in diet-induced obese mice and ADAM28 knockout mice with metabolic phenotyping","pmids":["28430139"],"confidence":"Medium","gaps":["Mechanistic substrate linking ADAM28 to metabolism not defined","Single lab"]},{"year":2018,"claim":"Extended the IGFBP-3/IGF-I proliferation mechanism to leukemia, showing ADAM28 promotes AML growth and dissemination.","evidence":"CRISPR knockout in HL-60 cells, invasion assays, IGFBP-3 cleavage, and xenotransplantation","pmids":["30429106"],"confidence":"Medium","gaps":["Single lab; relative contribution of non-IGF mechanisms not assessed"]},{"year":2018,"claim":"Provided therapeutic proof-of-concept that targeting a defined ADAM28 epitope blocks both IGFBP-3 cleavage and VWF protection, reducing tumor growth and metastasis.","evidence":"Neutralizing antibody 211-14 with IC50/KD measurement, dual substrate assays, and lung metastasis/survival models","pmids":["30190423"],"confidence":"Medium","gaps":["Single lab; human clinical validation absent"]},{"year":2018,"claim":"Distinguished a counter-protective host role, where microenvironment-derived ADAM28 limits metastasis via CD8+ T cell mobilization, contrasting with the tumor-cell-intrinsic pro-metastatic function.","evidence":"ADAM28 knockout mice in experimental metastasis models with CD8+ T cell flow cytometry","pmids":["30647853"],"confidence":"Medium","gaps":["Molecular substrate mediating T cell mobilization not identified","Single lab"]},{"year":2018,"claim":"Defined transcriptional repression of the ADAM28 locus by EBV EBNA3C through two epigenetic mechanisms, providing regulatory context for ADAM28 silencing.","evidence":"Recombinant EBV mutants, ChIP for H3K4me3/H3K27me3/BMI1/SUZ12, and EBNA3C-KDM2B interaction in LCLs","pmids":["30135119"],"confidence":"Medium","gaps":["Endogenous (non-viral) regulators of this chromatin state not addressed"]},{"year":2021,"claim":"Generalized the VWF-cleavage survival mechanism to gastric cancer with stromal endothelial ADAM28 contributing, linking VWF removal to integrin β3/p53/caspase-3 signaling.","evidence":"Overexpression/knockdown in gastric cancer cells and HUVECs in co-culture with VWF rescue","pmids":["33675784"],"confidence":"Medium","gaps":["Single lab; direct cleavage of VWF in this system inferred from supernatant levels"]},{"year":2023,"claim":"Implicated ADAM28 in airway remodeling, with knockout reducing Th2 responses, fibrosis, and fibroblast proliferation in an asthma model.","evidence":"ADAM28 knockout mice in ovalbumin model with flexiVent, BAL, histology, and ex vivo fibroblast assays","pmids":["36685493"],"confidence":"Medium","gaps":["Substrate driving remodeling not identified","Single lab"]},{"year":null,"claim":"The molecular substrates and effectors connecting ADAM28 to its metabolic, trophoblast, airway-remodeling, and host anti-metastatic immune phenotypes remain undefined, as does a structural basis for distinguishing its catalytic from disintegrin-dependent functions.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of the catalytic or disintegrin domain in the corpus","Substrates mediating the in vivo metabolic and immune phenotypes unidentified","Relative contributions of membrane vs secreted isoforms in vivo unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[1,3,5,10]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[2,8,11,12,15]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[6,9]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5,10,18]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[0]},{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[3,6]}],"pathway":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[2,8,11,12,15]}],"complexes":[],"partners":["PSGL-1","ITGA4","VWF","IGFBP3","CTGF","C1QA","NOTCH2","MMP7"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UKQ2","full_name":"Disintegrin and metalloproteinase domain-containing protein 28","aliases":["Epididymal metalloproteinase-like, disintegrin-like, and cysteine-rich protein II","eMDC II","Metalloproteinase-like, disintegrin-like, and cysteine-rich protein L","MDC-L"],"length_aa":775,"mass_kda":87.1,"function":"May play a role in the adhesive and proteolytic events that occur during lymphocyte emigration or may function in ectodomain shedding of lymphocyte surface target proteins, such as FASL and CD40L. May be involved in sperm maturation","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/Q9UKQ2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ADAM28","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":[{"gene":"CANX","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/ADAM28","total_profiled":1310},"omim":[{"mim_id":"607310","title":"A DISINTEGRIN AND METALLOPROTEINASE DOMAIN 7; ADAM7","url":"https://www.omim.org/entry/607310"},{"mim_id":"606393","title":"A DISINTEGRIN AND METALLOPROTEINASE DOMAIN-LIKE PROTEIN DECYSIN 1; ADAMDEC1","url":"https://www.omim.org/entry/606393"},{"mim_id":"606188","title":"A DISINTEGRIN AND METALLOPROTEINASE DOMAIN 28; ADAM28","url":"https://www.omim.org/entry/606188"},{"mim_id":"144755","title":"HYPEROSTOSIS CRANIALIS INTERNA; HCIN","url":"https://www.omim.org/entry/144755"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Mitochondria","reliability":"Additional"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"epididymis","ntpm":336.5},{"tissue":"stomach 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Mature ADAM28 is accessible to cell surface trypsinization in COS-7 cells, indicating it functions mainly on the cell surface. Processing occurs in a late Golgi compartment.\",\n      \"method\": \"Site-directed mutagenesis of catalytic glutamate, cell surface trypsinization, subcellular fractionation, expression in COS-7 cells\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — catalytic-site mutagenesis combined with cell surface accessibility assay and localization, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"10794709\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Recombinant ADAM28 cleaves myelin basic protein at two sites; catalytic activity is abolished by a catalytic-site mutation and is insensitive to TIMP-1 and TIMP-2.\",\n      \"method\": \"In vitro protease assay with recombinant ADAM28, site-directed mutagenesis, TIMP inhibition assay\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis and inhibitor controls, single lab\",\n      \"pmids\": [\"11389903\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Soluble recombinant ADAM28 (MDC-L) catalyzes ectodomain shedding of CD23 (low-affinity IgE receptor); its substrate specificity on synthetic peptide libraries is similar to ADAM8 and ADAM15 but distinct from ADAM17.\",\n      \"method\": \"Synthetic peptide substrate library screening, ectodomain shedding assay with recombinant enzyme, metalloprotease inhibitor treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with recombinant enzyme plus inhibitor validation, single lab with multiple substrates tested\",\n      \"pmids\": [\"12777399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ProADAM28s (65 kDa secreted isoform) is processed and activated by active MMP-7 (matrilysin-1), generating 42- and 40-kDa active forms lacking the propeptide. Active ADAM28s cleaves IGFBP-3 in both free form and in complex with IGF-I or IGF-II; cleavage is blocked by EDTA, 1,10-phenanthroline, KB-R7785, TIMP-3, and TIMP-4.\",\n      \"method\": \"In vitro MMP-7 activation assay, immunoblotting, metalloprotease inhibitor panel, TIMP inhibition assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of activation and substrate cleavage with multiple inhibitor controls, single lab\",\n      \"pmids\": [\"15013428\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Mouse ADAM28 gene expression in the epididymis is regulated by testicular factors (but not androgen), whereas ADAM7 is regulated by both androgen and testicular factors. Unlike ADAM7, ADAM28 protein is not transferred to the sperm surface during epididymal transit.\",\n      \"method\": \"RT-PCR, protein analysis, castration/androgen supplementation experiments, epididymis-sperm transfer assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct in vivo manipulation (castration model) with protein-level readout, single lab\",\n      \"pmids\": [\"15883027\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ADAM28 promotes breast carcinoma cell proliferation by cleaving IGFBP-3, thereby releasing bioavailable IGF-I. ADAM28 siRNA knockdown reduces IGFBP-3 cleavage, IGF-I signaling, cell proliferation in vitro, and xenograft tumor growth in vivo.\",\n      \"method\": \"siRNA knockdown, ADAM28 inhibitor and neutralizing antibody treatment, proliferation assay, IGFBP-3 cleavage assay by immunoblot, xenograft mouse model\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal loss-of-function approaches (siRNA, inhibitor, antibody) with in vitro and in vivo validation, mechanistic substrate identified\",\n      \"pmids\": [\"17047053\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The disintegrin-like domain of secreted ADAM28 binds to PSGL-1 (P-selectin glycoprotein ligand-1) on leukocytes. This binding enhances PSGL-1/P-selectin-mediated leukocyte adhesion to endothelial cells and increases leukocyte accumulation in pulmonary microcirculation in a mouse model of endotoxin-induced inflammation.\",\n      \"method\": \"Yeast two-hybrid screen, domain-specific binding assay, PSGL-1 stable transfectants, co-immunoprecipitation, co-immunolocalization, P-selectin adhesion assay, in vivo mouse endotoxin model\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast two-hybrid plus reciprocal co-IP plus functional adhesion assay plus in vivo model, multiple orthogonal methods\",\n      \"pmids\": [\"17597069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ATRA stimulation induces ADAM28 expression in human chondrocytes (HCS-2/8 cells and primary chondrocytes). Transfection of ADAM28 or its soluble alternatively spliced form augments proteoglycan release from cartilage; a mutant lacking part of the disintegrin domain does not, indicating the disintegrin domain is required for localization/substrate recognition in cartilage degradation.\",\n      \"method\": \"RT-PCR, transfection overexpression and domain-deletion mutant, proteoglycan release assay from bovine nasal cartilage\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-deletion mutagenesis with functional readout, single lab\",\n      \"pmids\": [\"19527685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ADAM28 cleaves CTGF at the Ala181-Tyr182 and Asp191-Pro192 bonds in the hinge region. ADAM28 selectively digests CTGF in the CTGF/VEGF165 complex, releasing biologically active VEGF165. CTGF binds to proADAM28.\",\n      \"method\": \"Yeast two-hybrid screen, protein binding assay, in vitro cleavage assay with site mapping by immunoblot, VEGF165 release assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with cleavage site mapping plus binding assay, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"20971063\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"ADAM28 binds to integrin α4β1 via its disintegrin domain. Soluble ADAM28 enhances α4β1-dependent cell adhesion to VCAM-1 and promotes lymphocyte adhesion to and transendothelial migration through endothelial monolayers. Binding is attenuated by anti-functional integrin antibodies and enhanced by Mn2+ and activating antibody 9EG7.\",\n      \"method\": \"Affymetrix microarray, RT-PCR, immunohistochemistry, cell adhesion assay with recombinant murine ADAM28, integrin-blocking antibodies, Mn2+ activation, transendothelial migration assay\",\n      \"journal\": \"Cell biology international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional adhesion assay with recombinant protein plus integrin-blocking antibody controls and migration assay, single lab\",\n      \"pmids\": [\"21332445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ADAM28 binds to and cleaves von Willebrand factor (VWF). High ADAM28 expression in carcinoma cells confers resistance to VWF-induced apoptosis. Knockdown of ADAM28 by shRNA increases carcinoma cell apoptosis in lung blood vessels and significantly decreases lung metastasis in xenograft models.\",\n      \"method\": \"Yeast two-hybrid screen, protein binding assay, immunoblotting of VWF cleavage, shRNA and siRNA knockdown, neutralizing antibody, mouse xenograft in vivo imaging model\",\n      \"journal\": \"Journal of the National Cancer Institute\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple loss-of-function approaches (shRNA, siRNA, antibody) with in vitro and in vivo validation, substrate binding confirmed by independent binding assay\",\n      \"pmids\": [\"22636800\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ADAM28 co-localizes and co-immunoprecipitates with TNF-α; overexpression of ADAM28 with TNF-α in HEK293 cells promotes TNF-α shedding, which is significantly reduced by ADAM28 siRNA knockdown or pharmacological inhibition. Endogenous ADAM28 in THP-1 macrophages similarly mediates TNF-α shedding.\",\n      \"method\": \"Co-immunoprecipitation, co-localization imaging, overexpression in HEK293 cells, siRNA knockdown, pharmacological inhibition, ELISA for TNF-α\",\n      \"journal\": \"Immunology and cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus functional siRNA and inhibitor validation in two cell types, single lab\",\n      \"pmids\": [\"23010875\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ADAM28 mediates ectodomain shedding of CD200 from B-CLL cells: siRNA knockdown of ADAM28 decreases soluble CD200 release, and transfection of cloned ADAM28 into CD200+ cells enhances sCD200 release.\",\n      \"method\": \"siRNA knockdown, ADAM28 gene transfection, ELISA for soluble CD200\",\n      \"journal\": \"Leukemia research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — complementary loss- and gain-of-function with protein-level readout, single lab\",\n      \"pmids\": [\"23643150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Src kinase induces ADAM28 gene expression in v-src-transformed epithelial cells through the MEK/ERK and PI3K/mTOR pathways. ADAM28 expression in human carcinoma cell lines correlates with phosphorylated c-Src and is suppressed by Src, MEK, and PI3K inhibitors.\",\n      \"method\": \"Oncogene transformation panel, Src/MEK/PI3K kinase inhibitors, immunoblotting for ADAM28, co-expression immunohistochemistry in human tissues\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological pathway epistasis with multiple inhibitor combinations in transformed cells plus human tissue validation, single lab\",\n      \"pmids\": [\"24007880\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ADAM28 binds C1q (identified by yeast two-hybrid); recombinant ADAM28 pre-treatment of C1q reduces C1q-induced apoptosis and autophagy in bronchial epithelial cells. ADAM28 siRNA knockdown increases C1q-induced cell death, establishing a cytoprotective function for ADAM28 in epithelial cells against C1q cytotoxicity. Binding confirmed by binding assays, immunoprecipitation, and surface plasmon resonance.\",\n      \"method\": \"Yeast two-hybrid, protein binding assay, immunoprecipitation, surface plasmon resonance, siRNA knockdown, cell death assay, autophagy (LC3-II) and apoptosis (caspase-3, p38) markers\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — SPR quantitative binding plus IP plus functional siRNA knockdown with multiple cell death readouts, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"26918856\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CD19 promotes ADAM28-mediated cleavage of Notch2 in marginal zone precursors (MZP) to drive their differentiation into MZ B cells. CD19 upregulates ADAM28 expression by suppressing Foxo1. CD19-deficient MZP fail to differentiate into MZ B cells, and re-expression of CD19 rescues this.\",\n      \"method\": \"CD19 knockout mice, CD19 rescue experiments, ADAM28 expression analysis, Notch2 cleavage assay, Foxo1 expression analysis\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout epistasis with rescue, substrate cleavage assay, single lab\",\n      \"pmids\": [\"28707394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ADAM28 siRNA knockdown in diet-induced obese mice prevents weight gain, promotes insulin sensitivity/glucose tolerance, and decreases liver TNF-α levels. ADAM28 knockout mice show reduced body weight and elevated HDL cholesterol, confirming a functional role for ADAM28 in metabolic dysfunction.\",\n      \"method\": \"siRNA knockdown in diet-induced obesity model, ADAM28 knockout mice, metabolic phenotyping (weight, glucose tolerance, insulin sensitivity, serum lipids, liver enzymes)\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two independent loss-of-function approaches (siRNA and KO mice) with metabolic phenotyping, single lab\",\n      \"pmids\": [\"28430139\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ADAM28 promotes AML leukemic cell proliferation, migration, and invasion through IGFBP-3 degradation and consequent IGF-I-induced cell proliferation. Knockout of ADAM28 in HL-60 cells reduces growth and dissemination in a xenotransplantation mouse model.\",\n      \"method\": \"ADAM28 CRISPR/Cas9 knockout, proliferation/migration/invasion assays, IGFBP-3 cleavage assay, IGF-I stimulation, xenotransplantation mouse model\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with in vitro and in vivo readouts plus substrate cleavage, single lab\",\n      \"pmids\": [\"30429106\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"A neutralizing antibody (211-14) against the junctional region between the cysteine-rich domain and secreted-specific domain of ADAM28 inhibits IGFBP-3 cleavage, blocks IGF-1-stimulated proliferation, and promotes VWF-induced cell death in lung adenocarcinoma cells. In vivo, it reduces tumor growth and metastasis and prolongs survival in lung metastasis models.\",\n      \"method\": \"Human neutralizing antibody generation, IC50 determination, KD measurement by binding assay, proliferation assay, VWF-induced apoptosis assay, lung metastasis mouse model, survival analysis\",\n      \"journal\": \"Molecular cancer therapeutics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — antibody functional validation with epitope mapping, dual substrate inhibition, in vitro and in vivo, single lab\",\n      \"pmids\": [\"30190423\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Host (microenvironment)-derived ADAM28 in ADAM28 knockout mice is associated with decreased CD8+ T cell numbers in spleen and lungs and increased lung colonization by tumor cells, indicating a protective role for host ADAM28 against metastasis through T cell mobilization (not proliferation, migration, or activation of CD8+ T cells per se).\",\n      \"method\": \"ADAM28 full knockout mouse model, experimental tumor metastasis models, flow cytometry for CD8+ T cells, ex vivo CD8+ T cell characterization\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with multiple tumor models and immune phenotyping, single lab\",\n      \"pmids\": [\"30647853\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ADAM28 from both gastric cancer cells and endothelial cells (HUVECs) cleaves VWF to eliminate VWF-induced apoptosis of gastric cancer cells. ADAM28 knockdown in HUVECs increases VWF in supernatant and elevates integrin β3, p-TP53, and cleaved caspase-3 in co-cultured gastric cancer cells; these effects are reversed by VWF knockdown.\",\n      \"method\": \"ADAM28 overexpression/knockdown in gastric cancer cells and HUVECs, co-culture system, ELISA for VWF, Western blot for apoptosis markers, MTT, wound healing, flow cytometry\",\n      \"journal\": \"European journal of pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — complementary gain- and loss-of-function in two cell types with VWF rescue experiment, single lab\",\n      \"pmids\": [\"33675784\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ADAM28 localizes to HLA-G+ trophoblasts in distal anchoring columns and decidua. siRNA-mediated ADAM28 knockdown in placental explant cultures impairs trophoblast column outgrowth and increases apoptosis in matrix-invading trophoblasts.\",\n      \"method\": \"qPCR, immunofluorescence microscopy on purified placental cell populations, siRNA knockdown in Matrigel-embedded placental explant cultures, apoptosis assay\",\n      \"journal\": \"Placenta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization with functional siRNA knockdown, single lab\",\n      \"pmids\": [\"28623976\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In ADAM28 knockout mice exposed to ovalbumin, airway responsiveness, Th2 cell proportion, B cell numbers, collagen deposition, smooth muscle hyperplasia, and mucous hyperplasia are all significantly reduced compared to wild-type. Lung fibroblasts from ADAM28 KO OVA-exposed mice show decreased proliferative capacity, suggesting ADAM28 directly contributes to airway remodelling.\",\n      \"method\": \"ADAM28 knockout mice, ovalbumin sensitization/challenge model, flexiVent airway responsiveness measurement, BAL analysis, lung histology, fibroblast proliferation assay ex vivo\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO in disease model with multiple orthogonal phenotypic readouts and ex vivo fibroblast assay, single lab\",\n      \"pmids\": [\"36685493\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"EBV nuclear antigen EBNA3C represses the ADAM28-ADAMDEC1 locus through interaction with KDM2B (histone lysine demethylase), which removes the activating mark H3K4me3. Concurrently, EBNA3C recruits Polycomb proteins BMI1 and SUZ12 to deposit H3K27me3 at the locus. These are two distinct repressive mechanisms.\",\n      \"method\": \"Recombinant EBV mutant viruses (RBPJ interaction motifs), ChIP for BMI1, SUZ12, H3K27me3, H3K4me3, gene expression analysis in LCLs, EBNA3C-KDM2B interaction assay\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP with recombinant virus mutants plus protein interaction data, single lab with multiple chromatin marks; this defines transcriptional regulation of ADAM28 locus\",\n      \"pmids\": [\"30135119\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ADAM28 is a zinc-dependent metalloprotease (ADAM family) existing as membrane-anchored (ADAM28m) and secreted (ADAM28s) isoforms that undergoes at least partly autocatalytic pro-domain removal in the Golgi; it is activated extracellularly by MMP-7, cleaves defined substrates including IGFBP-3, VWF, CTGF, CD23, CD200, Notch2, and TNF-α, and via its disintegrin domain binds integrins α4β1/α4β7/α9β1 and PSGL-1 to regulate cell adhesion and leukocyte trafficking; in carcinoma cells ADAM28 expression is driven by Src kinase through MEK/ERK and PI3K/mTOR pathways, and its proteolytic activities promote tumor cell proliferation (by liberating IGF-I from IGFBP-3) and metastatic survival (by inactivating pro-apoptotic VWF), while host-derived ADAM28 plays a counter-protective role in anti-tumor immunity through CD8+ T cell mobilization.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ADAM28 is a zinc-dependent ADAM-family metalloprotease that operates principally at the cell surface and as a secreted isoform to regulate growth-factor bioavailability, cell adhesion, and apoptosis [#0, #5, #10]. It matures through at least partly autocatalytic pro-domain removal in a late Golgi compartment, with a catalytic-site glutamate required for processing, and functions chiefly on the cell surface [#0]; the secreted pro-form is further processed extracellularly by active MMP-7 [#3]. Its catalytic activity is intrinsic and insensitive to TIMP-1/-2 but blocked by TIMP-3/-4 and broad metalloprotease inhibitors [#1, #3]. ADAM28 cleaves a defined set of substrates: it degrades IGFBP-3 to release bioavailable IGF-I and drive tumor cell proliferation [#5, #17], cleaves von Willebrand factor to protect carcinoma cells from VWF-induced apoptosis and enable metastatic survival [#10, #20], and sheds the ectodomains of CD23, CD200, and TNF-\\u03b1 [#2, #11, #12] as well as cleaving CTGF to liberate active VEGF165 [#8] and Notch2 to drive marginal-zone B cell differentiation [#15]. Independent of catalysis, its disintegrin-like domain binds integrin \\u03b14\\u03b21 and PSGL-1 to promote leukocyte adhesion, transendothelial migration, and trafficking [#6, #9]. In carcinoma cells ADAM28 expression is induced by Src kinase signaling through MEK/ERK and PI3K/mTOR pathways [#13], and a neutralizing antibody targeting ADAM28 blocks IGFBP-3 cleavage and tumor growth in vivo [#18]. Beyond its tumor-promoting cell-intrinsic roles, ADAM28 contributes to inflammatory and remodeling phenotypes including airway remodeling, metabolic dysfunction, and trophoblast survival [#16, #21, #22], while host-derived ADAM28 plays a counter-protective anti-metastatic role linked to CD8+ T cell mobilization [#19].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established how ADAM28 matures and where it acts, showing that pro-domain removal is at least partly autocatalytic and that the mature enzyme functions at the cell surface.\",\n      \"evidence\": \"Catalytic-glutamate mutagenesis, cell surface trypsinization, and subcellular fractionation in COS-7 cells\",\n      \"pmids\": [\"10794709\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not establish whether trans-cleavage by other proteases contributes to maturation in vivo\", \"No structural detail of the active site\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrated intrinsic proteolytic activity of ADAM28 with defined cleavage and a TIMP-insensitivity profile distinguishing it from other metalloproteases.\",\n      \"evidence\": \"In vitro protease assay on myelin basic protein with catalytic mutant and TIMP-1/-2 controls\",\n      \"pmids\": [\"11389903\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"MBP is a model substrate, not necessarily physiological\", \"Did not test full TIMP panel\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identified the first ectodomain shedding substrate (CD23) and defined ADAM28 peptide specificity relative to other ADAMs.\",\n      \"evidence\": \"Synthetic peptide library screening and shedding assay with recombinant soluble enzyme\",\n      \"pmids\": [\"12777399\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular context of CD23 shedding by endogenous ADAM28 not established\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Showed that the secreted pro-isoform is activated extracellularly by MMP-7 and that active ADAM28 cleaves IGFBP-3 free and in IGF complexes, linking ADAM28 to growth-factor bioavailability.\",\n      \"evidence\": \"In vitro MMP-7 activation, immunoblotting, and a metalloprotease/TIMP inhibitor panel\",\n      \"pmids\": [\"15013428\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MMP-7 is the physiological activator in tissues not shown\", \"Membrane isoform activation route not addressed\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Connected IGFBP-3 cleavage to a cancer phenotype, establishing ADAM28 as a driver of tumor cell proliferation via IGF-I liberation.\",\n      \"evidence\": \"siRNA, inhibitor, and neutralizing antibody loss-of-function with proliferation, cleavage assays, and breast cancer xenografts\",\n      \"pmids\": [\"17047053\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not address ADAM28 contributions independent of IGF-I axis\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Revealed a non-catalytic adhesive function: the disintegrin domain binds PSGL-1 to promote leukocyte adhesion and trafficking in inflammation.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal co-IP, P-selectin adhesion assay, and mouse endotoxin model\",\n      \"pmids\": [\"17597069\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve whether catalytic activity modulates this adhesive role\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Expanded the substrate repertoire to CTGF, mapping cleavage sites and showing release of active VEGF165 from the CTGF/VEGF complex.\",\n      \"evidence\": \"Yeast two-hybrid, binding assay, and in vitro cleavage with site mapping\",\n      \"pmids\": [\"20971063\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo angiogenic consequence not demonstrated\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined a second integrin partner (\\u03b14\\u03b21) bound through the disintegrin domain, mechanistically linking ADAM28 to lymphocyte adhesion and transendothelial migration.\",\n      \"evidence\": \"Cell adhesion assays with recombinant ADAM28, integrin-blocking antibodies, and Mn2+/9EG7 activation\",\n      \"pmids\": [\"21332445\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; direct affinity not quantified\", \"Physiological in vivo relevance to lymphocyte trafficking not tested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Established that ADAM28 cleavage of VWF confers resistance to VWF-induced apoptosis, explaining a pro-metastatic survival mechanism distinct from proliferation.\",\n      \"evidence\": \"Yeast two-hybrid, binding assay, shRNA/siRNA, neutralizing antibody, and xenograft lung metastasis imaging\",\n      \"pmids\": [\"22636800\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"VWF cleavage site not mapped here\", \"Mechanism by which VWF triggers apoptosis not fully defined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Added TNF-\\u03b1 as a shedding substrate, implicating ADAM28 in inflammatory cytokine release by macrophages.\",\n      \"evidence\": \"Co-IP, co-localization, overexpression in HEK293, siRNA, inhibitor, and TNF-\\u03b1 ELISA in THP-1 cells\",\n      \"pmids\": [\"23010875\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Overlap/redundancy with ADAM17-mediated TNF-\\u03b1 shedding not delineated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identified CD200 as a shedding substrate in B-CLL cells, broadening immune-modulatory roles.\",\n      \"evidence\": \"siRNA knockdown and ADAM28 transfection with soluble CD200 ELISA\",\n      \"pmids\": [\"23643150\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cleavage site not mapped\", \"In vivo immune consequence not tested\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined the upstream signaling that drives ADAM28 expression in cancer: Src acting through MEK/ERK and PI3K/mTOR.\",\n      \"evidence\": \"Oncogene transformation panel, kinase inhibitors, immunoblotting, and human tissue co-expression\",\n      \"pmids\": [\"24007880\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Transcription factors directly binding the ADAM28 promoter not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed a cytoprotective function in epithelial cells via C1q binding, where ADAM28 reduces C1q-induced apoptosis and autophagy.\",\n      \"evidence\": \"Yeast two-hybrid, SPR, IP, siRNA, and cell death/autophagy markers in bronchial epithelial cells\",\n      \"pmids\": [\"26918856\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether C1q is a cleavage substrate or only a binding partner not resolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Placed ADAM28 in a developmental B cell pathway, cleaving Notch2 downstream of CD19 to drive marginal-zone B cell differentiation.\",\n      \"evidence\": \"CD19 knockout/rescue mice, Notch2 cleavage assay, and Foxo1 analysis\",\n      \"pmids\": [\"28707394\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct demonstration of ADAM28 acting on Notch2 in vivo limited\", \"Single lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrated a role in trophoblast biology, where ADAM28 supports column outgrowth and protects invading trophoblasts from apoptosis.\",\n      \"evidence\": \"Immunofluorescence localization and siRNA knockdown in placental explant cultures\",\n      \"pmids\": [\"28623976\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Substrate responsible for trophoblast survival not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Implicated ADAM28 in metabolic dysfunction, with loss reducing weight gain and improving glucose handling.\",\n      \"evidence\": \"siRNA in diet-induced obese mice and ADAM28 knockout mice with metabolic phenotyping\",\n      \"pmids\": [\"28430139\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic substrate linking ADAM28 to metabolism not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Extended the IGFBP-3/IGF-I proliferation mechanism to leukemia, showing ADAM28 promotes AML growth and dissemination.\",\n      \"evidence\": \"CRISPR knockout in HL-60 cells, invasion assays, IGFBP-3 cleavage, and xenotransplantation\",\n      \"pmids\": [\"30429106\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; relative contribution of non-IGF mechanisms not assessed\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Provided therapeutic proof-of-concept that targeting a defined ADAM28 epitope blocks both IGFBP-3 cleavage and VWF protection, reducing tumor growth and metastasis.\",\n      \"evidence\": \"Neutralizing antibody 211-14 with IC50/KD measurement, dual substrate assays, and lung metastasis/survival models\",\n      \"pmids\": [\"30190423\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; human clinical validation absent\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Distinguished a counter-protective host role, where microenvironment-derived ADAM28 limits metastasis via CD8+ T cell mobilization, contrasting with the tumor-cell-intrinsic pro-metastatic function.\",\n      \"evidence\": \"ADAM28 knockout mice in experimental metastasis models with CD8+ T cell flow cytometry\",\n      \"pmids\": [\"30647853\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular substrate mediating T cell mobilization not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined transcriptional repression of the ADAM28 locus by EBV EBNA3C through two epigenetic mechanisms, providing regulatory context for ADAM28 silencing.\",\n      \"evidence\": \"Recombinant EBV mutants, ChIP for H3K4me3/H3K27me3/BMI1/SUZ12, and EBNA3C-KDM2B interaction in LCLs\",\n      \"pmids\": [\"30135119\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous (non-viral) regulators of this chromatin state not addressed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Generalized the VWF-cleavage survival mechanism to gastric cancer with stromal endothelial ADAM28 contributing, linking VWF removal to integrin \\u03b23/p53/caspase-3 signaling.\",\n      \"evidence\": \"Overexpression/knockdown in gastric cancer cells and HUVECs in co-culture with VWF rescue\",\n      \"pmids\": [\"33675784\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; direct cleavage of VWF in this system inferred from supernatant levels\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Implicated ADAM28 in airway remodeling, with knockout reducing Th2 responses, fibrosis, and fibroblast proliferation in an asthma model.\",\n      \"evidence\": \"ADAM28 knockout mice in ovalbumin model with flexiVent, BAL, histology, and ex vivo fibroblast assays\",\n      \"pmids\": [\"36685493\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Substrate driving remodeling not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular substrates and effectors connecting ADAM28 to its metabolic, trophoblast, airway-remodeling, and host anti-metastatic immune phenotypes remain undefined, as does a structural basis for distinguishing its catalytic from disintegrin-dependent functions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the catalytic or disintegrin domain in the corpus\", \"Substrates mediating the in vivo metabolic and immune phenotypes unidentified\", \"Relative contributions of membrane vs secreted isoforms in vivo unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [1, 3, 5, 10]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [2, 8, 11, 12, 15]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [6, 9]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 10, 18]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [3, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [2, 8, 11, 12, 15]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"PSGL-1\",\n      \"ITGA4\",\n      \"VWF\",\n      \"IGFBP3\",\n      \"CTGF\",\n      \"C1QA\",\n      \"NOTCH2\",\n      \"MMP7\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}