{"gene":"SPINT2","run_date":"2026-06-10T07:46:40","timeline":{"discoveries":[{"year":2005,"finding":"HAI-2 (SPINT2) potently inhibits hepsin-mediated activation of pro-HGF into biologically active HGF, with IC50 of 1.3 nM; inhibition is due to Kunitz domain-1, as shown by HAI-1B Kunitz domain mutants (R260A, K401A) losing inhibitory activity.","method":"In vitro enzymatic assay with soluble recombinant hepsin and soluble HAI-2; Kunitz domain mutagenesis","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay with defined IC50, active-site mutagenesis identifying KD-1 as the inhibitory domain; single lab but multiple orthogonal methods","pmids":["15792801"],"is_preprint":false},{"year":2005,"finding":"Restoration of HAI-2/SPINT2 expression in a RCC cell line reduced in vitro colony formation, whereas a missense mutant (P111S) had no significant effect, placing HAI-2 as a functional tumor suppressor in RCC. Increased cell motility from HAI-2 inactivation was abrogated by ERK/MAPK and PLC-γ inhibitors.","method":"Re-expression of wild-type vs. P111S mutant SPINT2 in RCC cell line; pharmacological inhibitor treatment; colony formation assay; cell motility assay","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function rescue experiment with pathway inhibitors; single lab with multiple functional assays","pmids":["15930277"],"is_preprint":false},{"year":2009,"finding":"Genetic inactivation of Spint2 in mice causes defects in neural tube closure and placental labyrinth development with loss of epithelial polarity, leading to embryonic death. Simultaneous inactivation of matriptase (St14) fully restores placental development and embryonic survival, establishing matriptase inhibition as an essential HAI-2 function. Neural tube defects persisted at lower frequency even without matriptase, indicating HAI-2 also inhibits additional serine proteases required for neural development.","method":"Genetic knockout (Spint2-/- mice); genetic epistasis (Spint2/St14 double knockout); phenotypic analysis of placentation and neural tube","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — genetic epistasis in vivo with double-knockout rescue; replicated across multiple genetic combinations","pmids":["19592578"],"is_preprint":false},{"year":2009,"finding":"Loss-of-function mutations in SPINT2 cause syndromic congenital sodium diarrhea. SPINT2 mutations (splice, missense, nonsense) were associated with loss of protein synthesis or failure to inhibit the serine protease trypsin in vitro, demonstrating that HAI-2 protease inhibitory activity is required for normal intestinal sodium homeostasis.","method":"Genetic linkage/SNP scan identifying SPINT2 mutations in families with syndromic CSD; in vitro trypsin inhibition assay with patient-derived mutant proteins","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — human genetics combined with functional in vitro assay confirming loss of inhibitory activity; multiple mutations in multiple families","pmids":["19185281"],"is_preprint":false},{"year":2009,"finding":"HAI-2/PB Kunitz domain 1 (KD-1) is critical for anti-invasive and anti-tumorigenic function in hepatocellular carcinoma cells: KD-1 inactivating mutant abolished anti-invasion activity in vitro and tumor suppression in vivo, while KD-2 inactivating mutant did not.","method":"Ectopic expression of wild-type and KD-1 vs. KD-2 mutant HAI-2 in HCC cell lines; Matrigel invasion assay; in vivo tumorigenicity assay","journal":"International journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain-specific mutagenesis with functional readout in vitro and in vivo; single lab","pmids":["19107935"],"is_preprint":false},{"year":2012,"finding":"Genetic epistasis establishes that prostasin (PRSS8) acts downstream of matriptase and upstream of HAI-2 in a proteolytic cascade: hypomorphic Prss8 mutations rescue early embryonic lethality, mid-gestation placental failure, and neural tube defects in HAI-2-deficient mice. Biochemical analysis revealed prostasin is required for conversion of matriptase zymogen to active matriptase (paradoxically upstream), while prostasin zymogen activation is matriptase-independent. Inactivation of c-Met, PAR-2, or ENaC alpha did not rescue lethality.","method":"In vivo genetic epistasis (compound knockout mice: HAI-2/Prss8, HAI-2/c-Met, HAI-2/PAR-2, HAI-2/ENaC-α); biochemical analysis of matriptase and prostasin activation in placental tissue","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple double-knockout genetic epistasis experiments combined with biochemical analysis; single lab but highly systematic with multiple orthogonal approaches","pmids":["22952456"],"is_preprint":false},{"year":2013,"finding":"HAI-2 inhibits matriptase-2 at the cell surface and in conditioned medium, forming a stable complex with matriptase-2 (demonstrated by co-immunoprecipitation). HAI-2 thereby indirectly elevates hepcidin expression (HAMP) by blocking matriptase-2-mediated suppression of hepcidin, likely through preventing matriptase-2 from cleaving membrane-bound hemojuvelin.","method":"Co-expression of matriptase-2 and HAI-2; complex isolation by immunoprecipitation from cell lysates and conditioned medium; HAMP mRNA measurement","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP demonstrating complex formation, functional gene expression readout; single lab with two orthogonal methods","pmids":["23293962"],"is_preprint":false},{"year":2013,"finding":"HAI-2 suppresses prostate cancer cell migration, invasion, tumorigenicity and metastasis primarily through regulation of matriptase: HAI-2 knockdown increases matriptase activation, and matriptase knockdown rescues the invasion induced by HAI-2 loss. HAI-2 overexpression in N2 cells reduced tumor growth and metastasis in orthotopic xenografts.","method":"HAI-2 knockdown and overexpression in human PCa cell lines (103E, N1, N2); matriptase knockdown rescue experiment; orthotopic xenograft mouse model; immunohistochemistry","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis by knockdown rescue, in vivo xenograft, single lab multiple methods","pmids":["24121274"],"is_preprint":false},{"year":2014,"finding":"HAI-2, but not HAI-1, regulates prostasin-dependent matriptase zymogen activation. In HAI-2-deficient intestinal epithelial cells, matriptase is lost due to accelerated prostasin-driven activation and subsequent shedding. HAI-1 ablation did not affect matriptase in intestinal cells. Gene silencing in Caco-2 monolayers confirmed that HAI-2 loss causes accelerated matriptase shedding mechanistically through loss of prostasin regulation.","method":"Genetically engineered HAI-1 and HAI-2 knockout mice; gene silencing in Caco-2 intestinal epithelial cell monolayers; immunofluorescence and biochemical fractionation of matriptase localization/activation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vivo knockout plus cell-based gene silencing with mechanistic biochemical analysis; multiple orthogonal methods; single lab but rigorous","pmids":["24962579"],"is_preprint":false},{"year":2014,"finding":"Y163C mutation in Kunitz domain 2 (KD2) of HAI-2 causes complete loss of inhibitory activity on two intestinal serine proteases, prostasin and TMPRSS13, but mutations in KD1 (Y68C) affect a different subset of proteases. HAI-2 wild-type inhibits nine gastrointestinal serine proteases. The thiol group of the introduced cysteine (not loss of Tyr) is responsible for HAI-2 loss of function.","method":"Xenopus laevis oocyte expression system cellular assay; site-directed mutagenesis of KD1 and KD2; functional assay against nine GI serine proteases","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro functional assay with mutagenesis in defined expression system; single lab but multiple mutants and substrates tested","pmids":["24722141"],"is_preprint":false},{"year":2015,"finding":"HAI-2 inhibition of matriptase depends on subcellular co-localization: in mammary epithelial cells, HAI-2 resides in intracellular vesicle/granule structures and does not form matriptase-HAI-2 complexes upon matriptase activation. In breast cancer cells, a proportion of HAI-2 translocates to the cell surface where it inhibits matriptase, forming three distinct matriptase-HAI-2 complexes.","method":"Induction of matriptase zymogen activation in human mammary epithelial vs. breast cancer cells; immunofluorescent staining of HAI-2 localization; immunoprecipitation of matriptase-HAI-2 complexes; cell surface biotinylation","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, immunofluorescence, cell-surface biotinylation; single lab, multiple orthogonal methods","pmids":["25786220"],"is_preprint":false},{"year":2015,"finding":"HAI-2 (SPINT2) inhibits HGF-induced MET-AKT signaling in melanoma cells and decreases cell motility and invasive growth. Epigenetic silencing of SPINT2 by DNA methylation activates the HGF-MET pathway.","method":"Ectopic SPINT2 expression in melanoma cells; MET/AKT phosphorylation assay; cell motility and invasion assay; decitabine-induced reactivation","journal":"The Journal of investigative dermatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain-of-function with defined signaling readout; single lab, multiple functional methods","pmids":["25910030"],"is_preprint":false},{"year":2015,"finding":"The N-glycan on Asn-57 of HAI-2 determines subcellular distribution: oligomannose-type N-glycan (25-kDa form) is largely ER-retained, while complex-type N-glycan (30–40-kDa form) is targeted to vesicles/granules and the cell surface where it can inhibit matriptase. In breast cancer cells, the mature 30–40-kDa HAI-2 translocates to the cell surface to form matriptase-HAI-2 complexes.","method":"Biochemical characterization of two HAI-2 species by size and N-glycan type; subcellular fractionation; immunofluorescence; matriptase-HAI-2 complex detection","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical glycan characterization plus functional localization data; single lab, multiple methods","pmids":["26171609"],"is_preprint":false},{"year":2016,"finding":"HAI-2 inhibits influenza virus H1N1 and H3N2 hemagglutinin cleavage and reduces viral infection in cell culture; HAI-2 administration in a mouse influenza model confers protection, acting by inhibiting host proteases (matriptase, TMPRSS2) that cleave HA.","method":"In vitro HA cleavage inhibition assay; cell culture infection assay; mouse influenza model","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro cleavage assay plus cell culture and mouse model; single lab, multiple orthogonal assays","pmids":["24978308"],"is_preprint":false},{"year":2016,"finding":"HAI-2 forms complexes with prostasin in human milk in addition to matriptase-HAI-2 complexes, providing in vivo evidence that both matriptase and prostasin are HAI-2 target proteases during lactation.","method":"Purification of protease-inhibitor complexes from human milk; immunoaffinity and ion-exchange chromatography; mass spectrometry identification","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo complex detection by biochemical purification and MS; single lab","pmids":["27043831"],"is_preprint":false},{"year":2017,"finding":"HAI-2 Kunitz domain 1 mutations (K42N, C47F, R48L) delay SEA domain cleavage of matriptase, causing accumulation of non-SEA-cleaved matriptase in the ER, thereby regulating matriptase secretory transport and cell-surface localization. Two of the mutants (C47F, R48L) also show reduced ability to inhibit matriptase proteolytic activity. HAI-2 thus separately: (1) stabilizes matriptase, (2) regulates its secretory transport via maturation/oligomerization, and (3) inhibits its proteolytic activity.","method":"Site-directed mutagenesis of HAI-2 KD1 binding loop; expression in cells; analysis of matriptase SEA domain cleavage by immunoblot; subcellular localization by immunofluorescence","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with biochemical and cell-biological functional readouts; single lab, multiple methods","pmids":["28371047"],"is_preprint":false},{"year":2018,"finding":"In human skin, HAI-2 is primarily intracellularly localized in basal and spinous layer keratinocytes, preventing its interaction with active prostasin or matriptase; consequently HAI-1 (not HAI-2) is the prominent inhibitor of prostasin and matriptase in skin. Subcellular localization thus determines tissue-specific protease inhibitor function.","method":"Immunohistochemistry of human foreskin; immunoblot analysis of foreskin lysates; comparison of protease activation status across tissues","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiment with functional inference; single lab, two orthogonal methods","pmids":["29438412"],"is_preprint":false},{"year":2018,"finding":"HAI-2 loss in oral squamous cell carcinoma (OSCC) cells causes a pro-invasive phenotype via excess prostasin activity: SPINT2 knockout suppresses OSCC proliferation and invasion, and this is rescued by HAI-2 re-expression or prostasin knockdown. Prostasin protein levels increase markedly in HAI-2-deficient OSCC cells.","method":"Homozygous SPINT2 CRISPR knockout in OSCC lines; prostasin knockdown rescue; Matrigel invasion assay; western blot","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout with epistasis rescue by prostasin knockdown; single lab","pmids":["29545930"],"is_preprint":false},{"year":2018,"finding":"HAI-2 loss in mice with HAI-2-deficient intestinal epithelial cells causes excessive prostasin proteolysis with prolonged active prostasin and depletion of HAI-1 monomer, but this phenotype is not observed in epidermal (HaCaT) cells, identifying tissue-selective functional relationship between HAI-2 and prostasin in intestinal epithelium.","method":"Targeted HAI-2 deletion in Caco-2 and HaCaT cells; biochemical analysis of prostasin activation, matriptase activation, and HAI-1 levels","journal":"PLoS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — targeted gene deletion in two cell types with biochemical mechanistic analysis; single lab","pmids":["29617460"],"is_preprint":false},{"year":2019,"finding":"SPINT2 missense mutations in KD2 (p.Phe161Val, p.Tyr163Cys, p.Gly168Ser) found in SCSD patients selectively impair inhibition of prostasin-catalyzed cleavage but do not affect matriptase inhibition. Homology modeling suggests these mutations induce KD2 misfolding. This implies prostasin needs to engage an exosite on KD2 in addition to the KD1 binding loop for inhibition.","method":"Functional inhibition assays with wild-type and mutant HAI-2 against prostasin and matriptase; homology modeling","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro functional assay discriminating substrate specificity with structural modeling; single lab, multiple mutants","pmids":["30445423"],"is_preprint":false},{"year":2019,"finding":"HAI-2 functions as a novel inhibitor of plasmin in lung cancer: HAI-2 downregulation increases cell-surface plasmin activity, EMT, and invasion; HAI-2 suppresses plasmin-mediated activations of HGF and TGF-β1. Plasmin was identified as a novel HAI-2 target by pulldown and LC/MS/MS. In xenograft models, HAI-2 loss increased lung cancer metastasis via elevated plasmin activity.","method":"Pulldown and LC/MS/MS identification of plasmin as HAI-2 target; HAI-2 knockdown/overexpression; plasmin activity assay; transwell invasion; in vivo xenograft","journal":"British journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomic identification plus functional validation in vitro and in vivo; single lab, multiple methods","pmids":["30765871"],"is_preprint":false},{"year":2020,"finding":"HAI-2 forms a stable complex with TMPRSS2 (identified by co-immunoprecipitation and LC/MS/MS) and potently inhibits TMPRSS2 proteolytic activity, with both KD1 and KD2 showing comparable inhibitory effects. HAI-2 suppresses TMPRSS2-induced pro-HGF activation, ECM degradation, and prostate cancer invasion and metastasis in orthotopic xenograft models.","method":"Co-IP and LC/MS/MS; recombinant protein inhibition assay; immunofluorescence co-localization; HAI-2 overexpression in xenograft model","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — biochemical complex identification by Co-IP/MS, in vitro inhibition assay, in vivo xenograft; multiple orthogonal methods in single study","pmids":["32778768"],"is_preprint":false},{"year":2020,"finding":"HAI-2 and HAI-1 inhibit the catalytic activity of the matriptase zymogen toward peptide substrates and natural protein substrates (pro-HGF, pro-prostasin) at comparable concentrations to their inhibition of activated matriptase, indicating that the Kunitz inhibitors interact with the active sites of both zymogen and activated matriptase similarly.","method":"Purified protein in vitro inhibition assay; cell-based assay with HAI-1/HAI-2 and zymogen vs. activated matriptase","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified proteins plus cell-based assay; single lab, two orthogonal systems","pmids":["32338287"],"is_preprint":false},{"year":2020,"finding":"SPINT2 inhibits proteolytic cleavage-activation of influenza A (H1N1, H3N2, H7N9) hemagglutinin and HMPV F protein by trypsin, recombinant matriptase, or KLK5, and reduces viral growth in cell culture by inhibiting matriptase or TMPRSS2. Inhibition is effective whether added at infection or 24 h post-infection.","method":"Cleavage and fusion inhibition assays with recombinant SPINT2; cell culture viral growth assay","journal":"Virology","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro cleavage assay plus cell culture antiviral assay; single lab, multiple virus strains","pmids":["32056846"],"is_preprint":false},{"year":2021,"finding":"SPINT2 acts as a general regulator of CDKN1A transcription via histone acetylation. Loss of SPINT2 improves survival of tetraploid cells (identified by genome-wide RNAi screen) by reducing CDKN1A expression, linking SPINT2 to cell cycle arrest after genome doubling.","method":"Genome-wide RNAi screen in HCT116 colorectal cancer cells; mass spectrometry and immunoprecipitation; chromatin/histone acetylation analysis","journal":"Cellular oncology (Dordrecht, Netherlands)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide screen with mechanistic follow-up by IP and histone acetylation assay; single lab","pmids":["34962618"],"is_preprint":false},{"year":2021,"finding":"In HAI-2-deficient mice expressing only zymogen-locked prostasin (Prss8 R44Q), which does not bind HAI-2, postnatal intestinal failure develops (villous atrophy, tufted villi, loss of goblet cells, loss of colonic crypt structure) with reduced EpCAM, E-cadherin, occludin, and claudins-1/-7, and elevated claudin-4, demonstrating that HAI-2 regulates intestinal epithelial barrier integrity through a prostasin-dependent pathway.","method":"Genetic mouse model (Spint2-/-;Prss8R44Q/R44Q); histology; immunoblot of junctional proteins","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic epistasis model with detailed molecular phenotyping; mechanistic pathway placement established","pmids":["29617460"],"is_preprint":false},{"year":2021,"finding":"In Caco-2 colorectal cells (but not HaCaT keratinocytes), HAI-2 deletion causes constitutive high-level prostasin zymogen activation, prolonged active prostasin half-life, depletion of HAI-1 monomer, and secondary increase in matriptase activation. This cell-type-selective excessive proteolysis explains organ-selective intestinal damage from SPINT2 mutations.","method":"Targeted HAI-2 deletion in Caco-2 vs. HaCaT cells; biochemical assays for prostasin and matriptase activation/inhibition status; half-life measurements","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — comparative cell-type deletion experiments with detailed biochemical analysis; single lab","pmids":["34089062"],"is_preprint":false},{"year":2021,"finding":"Differences in intracellular Arg/Lys-rich and EHLVY motifs between HAI-1 and HAI-2 account for their distinct subcellular distributions (HAI-1 on cell surface and inside; HAI-2 predominantly in intracellular granules). Domain swap and point mutation experiments confirmed these motifs as the key targeting signals.","method":"Domain swap mutagenesis; point mutations; immunocytochemistry; cell surface biotinylation/avidin depletion","journal":"Human cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with direct localization readout; single lab, multiple orthogonal methods","pmids":["34643933"],"is_preprint":false},{"year":2021,"finding":"In zebrafish, Spint2 is required for hatching gland cell cohesion, collective intra-epidermal migration, and survival prior to degranulation. Spint2 acts independently of tested matriptases and prostasins, but displays tight genetic interaction with E-cadherin, promoting hatching gland cell cohesiveness and survival. No genetic interaction with EpCAM was observed.","method":"Zebrafish spint2 mutant analysis; chimera analysis; genetic epistasis with matriptase, prostasin, E-cadherin, EpCAM mutants","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo genetic epistasis in zebrafish with chimera and multiple double-mutant analyses; ortholog study","pmids":["33826923"],"is_preprint":false},{"year":2021,"finding":"SPINT2 knockdown in lung cancer (Calu-3) cells leads to a strong increase in SARS-CoV-2 viral load, while SPINT2 overexpression drastically reduces viral load, demonstrating that SPINT2 restricts SARS-CoV-2 infection, likely through inhibition of TMPRSS2.","method":"SPINT2 siRNA knockdown and overexpression in Calu-3 cells; viral load quantification","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function with direct viral load readout; single lab","pmids":["34181691"],"is_preprint":false},{"year":2023,"finding":"N-glycosylation at Asn-57 (not Asn-94) is required for correct HAI-2 protein folding and protease inhibitory activity. Non-glycosylated HAI-2 (Asn-57 mutant) is synthesized as disulfide-linked oligomers with distorted conformations and lacks protease inhibitory function. The oligomannose-type N-glycan is the precursor of the complex-type N-glycan form.","method":"Point mutations of Asn-57 and Asn-94 in HAI-2; biochemical characterization of oligomerization, glycosylation state, and protease inhibitory activity in HAI-2 knockout Caco-2 cells","journal":"Glycobiology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — site-directed mutagenesis with biochemical functional readout; single lab, multiple orthogonal methods","pmids":["36637420"],"is_preprint":false},{"year":2023,"finding":"Early-onset tufting enteropathy and postnatal lethality in Spint2-deficient mice are driven by matriptase activity but NOT through excessive proteolysis of EpCAM: expression of cleavage-resistant EpCAM failed to rescue intestinal failure. Matriptase inactivation counteracted Spint2 deficiency even with cleavage-resistant EpCAM, indicating matriptase-driven intestinal dysfunction proceeds via substrates other than EpCAM.","method":"Genetic mouse models combining Spint2 deficiency with cleavage-resistant EpCAM knockin and intestinal St14 (matriptase) inactivation; in vitro and in vivo EpCAM cleavage assays","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic epistasis combinations with clear mechanistic negative result; rigorous in vivo study","pmids":["37539662"],"is_preprint":false},{"year":2024,"finding":"SCSD-associated SPINT2 missense mutations in KD2 (e.g., p.Phe161Val, p.Tyr163Cys, p.Gly168Ser) inactivate HAI-2 through two mechanisms: (1) ~50% of protein forms disulfide-linked oligomers due to disarrayed disulfide bonding, losing protease inhibitory activity; (2) remaining monomeric protein is trapped in an immature, lightly glycosylated form and cannot suppress prostasin proteolysis. These mutants cannot rescue excessive prostasin proteolysis caused by HAI-2 knockout in Caco-2 cells.","method":"Doxycycline-inducible expression of HAI-2 KD2 mutants in HAI-2-knockout Caco-2 cells; non-reducing SDS-PAGE for oligomer detection; glycosylation state analysis; prostasin inhibition functional assay","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mechanistic study with inducible expression, mutagenesis, biochemical oligomerization/glycosylation analysis, and functional prostasin inhibition assay in KO rescue system; multiple orthogonal methods","pmids":["38271183"],"is_preprint":false},{"year":2025,"finding":"SPINT2 interacts with ACSL4 and prevents its ubiquitination by the E3 ligase NEDD4L, thereby stabilizing ACSL4 protein and promoting ferroptotic cell death in gallbladder cancer. SPINT2 deficiency alters lipid metabolism and reduces ferroptosis susceptibility.","method":"Co-immunoprecipitation demonstrating SPINT2-ACSL4 interaction; ubiquitination assay; NEDD4L identification as E3 ligase; in vitro and in vivo tumor suppression assays; metabolomic profiling","journal":"International journal of biological macromolecules","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP with ubiquitination assay; single lab, novel mechanism with limited independent replication","pmids":["41067336"],"is_preprint":false},{"year":2025,"finding":"DNMT1 downregulation during senescence causes hypomethylation of SPINT2 promoter CpG sites, leading to SPINT2 upregulation. SPINT2 overexpression alone induces senescence, and SPINT2 knockdown mitigates DNMT1 inhibition-induced senescence. SPINT2 drives senescence by inhibiting c-Met signaling; downstream targets include COL27A1, STAM2, and CBL.","method":"DNMT1 knockdown and pharmacological inhibition; SPINT2 siRNA knockdown and overexpression; methylation-specific sequencing; transcriptomic profiling; senescence marker assays","journal":"Aging","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple complementary genetic and epigenetic approaches with defined c-Met pathway placement; single lab","pmids":["40838961"],"is_preprint":false},{"year":2019,"finding":"STYK1 overexpression significantly decreases SPINT2 protein levels in NSCLC cells, and SPINT2 overexpression reverses STYK1-mediated NSCLC proliferation, migration, invasion, and EMT both in vitro and in vivo, placing SPINT2 downstream of STYK1 in a lung cancer progression pathway.","method":"RNA-seq, qRT-PCR, western blot after STYK1 overexpression; SPINT2 overexpression rescue; in vivo tumor model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis rescue experiment with multiple functional assays; single lab","pmids":["31164631"],"is_preprint":false},{"year":2019,"finding":"SPINT2 loss in glioma promotes cell growth and invasion partly via increased MMP2 expression and activity, as demonstrated by SPINT2 knockdown and knock-in functional assays in adult and pediatric HGG cell lines.","method":"SPINT2 knockdown and knock-in in HGG cell lines; MMP2 activity assay; invasion and viability assays","journal":"Cellular oncology (Dordrecht, Netherlands)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — paired loss- and gain-of-function with defined MMP2 mechanism; single lab","pmids":["31701492"],"is_preprint":false},{"year":2019,"finding":"SPINT2 restores expression reduces c-Met activation in GBM cells and suppresses tumorigenic properties in vitro and in vivo, with SPINT2 downregulation resulting from promoter hypermethylation (confirmed by targeted bisulfite sequencing, 5-aza treatment, DNMT1 knockdown, and luciferase reporter assay).","method":"5-aza treatment, DNMT1 knockdown, luciferase reporter for methylation-mediated regulation; SPINT2 re-expression; c-Met phosphorylation assay; in vivo GBM model","journal":"Journal of neuro-oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple approaches confirming methylation regulation plus functional c-Met pathway placement; single lab","pmids":["30838489"],"is_preprint":false},{"year":2018,"finding":"SPINT2 promotes SMC proliferation and migration inhibition in aortic smooth muscle cells: SPINT2 overexpression reduces active MMP-2 and MMP-9 expression, suppresses SMC switching from contractile to synthetic phenotype, and inhibits ERK activation. A specific ERK agonist reverses SPINT2-mediated inhibition of SMC migration and phenotypic switching.","method":"SPINT2 overexpression via adenoviral vector in primary mouse aortic SMCs; PDGF-BB induction model; MTT, Ki-67, wound healing, ELISA, western blot; ERK agonist rescue","journal":"Experimental and therapeutic medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain-of-function with pathway rescue by ERK agonist; single lab, multiple methods","pmids":["37928510"],"is_preprint":false},{"year":2025,"finding":"Co-expression of HAI-2 (SPINT2) with TMPRSS2 ectodomain enables efficient production of active wildtype TMPRSS2 in mammalian cells, demonstrating that HAI-2 stabilizes TMPRSS2 zymogen during expression. Purified TMPRSS2 cleaves synthetic and protein substrates efficiently (kcat/KM 10^4–10^6 M-1s-1).","method":"Mammalian cell co-expression of TMPRSS2 ectodomain with HAI-2; purification and kinetic characterization of recombinant TMPRSS2","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution demonstrating stabilization function; single study, novel finding","pmids":["41408854"],"is_preprint":false}],"current_model":"SPINT2 encodes HAI-2, a type I transmembrane, bi-Kunitz-domain serine protease inhibitor that regulates pericellular proteolysis primarily by inhibiting membrane-anchored serine proteases—including matriptase, prostasin, hepsin, TMPRSS2, matriptase-2, and plasmin—through its Kunitz domain 1 (and, for prostasin, requiring an exosite on Kunitz domain 2), thereby controlling HGF activation, EpCAM stability, epithelial barrier integrity, iron homeostasis (via matriptase-2/hepcidin axis), and viral entry (influenza, SARS-CoV-2); its cell-type-selective function is determined by N-glycosylation-dependent subcellular localization (intracellular vs. cell-surface), and loss-of-function via promoter hypermethylation or missense mutations causing protein misfolding/oligomerization underlies syndromic congenital sodium diarrhea/tufting enteropathy and drives tumor progression across multiple cancer types."},"narrative":{"mechanistic_narrative":"SPINT2 encodes HAI-2, a Kunitz-type serine protease inhibitor that governs pericellular proteolysis in epithelia and during development by restraining a network of membrane-anchored and soluble serine proteases [PMID:15792801, PMID:19592578, PMID:22952456]. Its prototypic activity is inhibition of pro-HGF activation, blocking conversion to active HGF via Kunitz domain 1 (KD1) [PMID:15792801], and KD1 is the principal determinant of its anti-invasive, tumor-suppressive function [PMID:19107935]. In vivo, HAI-2 is essential for placental and neural tube development, and its core function is inhibition of matriptase: genetic loss of matriptase rescues the lethality of Spint2-null mice [PMID:19592578]. HAI-2 sits in a proteolytic cascade with prostasin acting downstream of matriptase and upstream of HAI-2 [PMID:22952456], and it regulates prostasin-driven matriptase zymogen activation, controlling matriptase stability, maturation/secretory transport, and catalytic activity as separable activities [PMID:24962579, PMID:28371047, PMID:32338287]. HAI-2 inhibits a broad protease panel including matriptase-2 (elevating hepcidin via the hemojuvelin axis) [PMID:23293962], TMPRSS2 [PMID:32778768], prostasin and TMPRSS13 [PMID:24722141], and plasmin [PMID:30765871], with KD1 and KD2 contributing distinct specificities and prostasin requiring an exosite on KD2 in addition to the KD1 binding loop [PMID:24722141, PMID:30445423]. Cell-type-selective function is set by subcellular localization: complex-type N-glycosylation at Asn-57 and intracellular targeting motifs determine whether HAI-2 is ER-retained, granule-resident, or cell-surface, dictating whether it engages active proteases [PMID:26171609, PMID:29438412, PMID:34643933, PMID:36637420]. In intestinal epithelium, HAI-2 loss causes excessive prostasin proteolysis, disrupting barrier junctional proteins (EpCAM, E-cadherin, claudins) and producing tufting enteropathy through a matriptase/prostasin-dependent, EpCAM-cleavage-independent route [PMID:29617460, PMID:34089062, PMID:37539662]. Loss-of-function SPINT2 mutations cause syndromic congenital sodium diarrhea, acting through loss of protease inhibition; KD2 missense mutations inactivate HAI-2 by inducing disulfide-linked oligomerization and trapping immature protein, selectively abolishing prostasin inhibition [PMID:19185281, PMID:30445423, PMID:38271183]. SPINT2 is epigenetically silenced by promoter hypermethylation across cancers, derepressing HGF-MET signaling and promoting invasion [PMID:25910030, PMID:30838489]. HAI-2 additionally restricts viral entry by blocking host-protease cleavage of influenza hemagglutinin and limiting SARS-CoV-2 infection through TMPRSS2 inhibition [PMID:24978308, PMID:32056846, PMID:34181691].","teleology":[{"year":2005,"claim":"Established that HAI-2 is a potent serine protease inhibitor whose anti-HGF-activation function maps to Kunitz domain 1, defining its core biochemical mechanism.","evidence":"In vitro enzymatic assay with recombinant hepsin and Kunitz-domain mutagenesis","pmids":["15792801"],"confidence":"High","gaps":["Did not establish the in vivo relevance of hepsin as a target","KD2 contribution not addressed"]},{"year":2005,"claim":"Linked HAI-2 inhibitory function to tumor suppression, showing re-expression suppresses colony formation while a patient-derived missense mutant is inactive.","evidence":"Wild-type vs P111S re-expression in RCC cells with colony/motility assays and pathway inhibitors","pmids":["15930277"],"confidence":"Medium","gaps":["Specific protease target driving the phenotype not identified","ERK/PLC-gamma dependence shown pharmacologically only"]},{"year":2009,"claim":"Demonstrated in vivo that the essential developmental function of HAI-2 is matriptase inhibition, via double-knockout rescue of lethality.","evidence":"Spint2/St14 double-knockout genetic epistasis in mice with placental and neural tube phenotyping","pmids":["19592578"],"confidence":"High","gaps":["Identity of the additional protease driving residual neural tube defects unresolved"]},{"year":2009,"claim":"Established SPINT2 as the causative gene for syndromic congenital sodium diarrhea and tied disease to loss of protease-inhibitory activity.","evidence":"Human genetic linkage in families plus in vitro trypsin inhibition assays with mutant proteins","pmids":["19185281"],"confidence":"High","gaps":["Physiological protease responsible for sodium homeostasis not pinpointed at the time"]},{"year":2012,"claim":"Placed HAI-2 within an ordered proteolytic cascade, showing prostasin acts downstream of matriptase and upstream of HAI-2, with prostasin paradoxically required for matriptase activation.","evidence":"Compound knockout mice (HAI-2/Prss8, c-Met, PAR-2, ENaC) and biochemical activation analysis in placenta","pmids":["22952456"],"confidence":"High","gaps":["Molecular basis of prostasin-dependent matriptase activation not fully resolved"]},{"year":2013,"claim":"Expanded the target repertoire to matriptase-2 and connected HAI-2 to iron homeostasis through hepcidin regulation.","evidence":"Co-IP complex isolation and HAMP mRNA measurement after co-expression","pmids":["23293962"],"confidence":"Medium","gaps":["Hemojuvelin cleavage step inferred, not directly demonstrated","Single Co-IP without in vivo confirmation"]},{"year":2014,"claim":"Defined HAI-2 as the selective regulator of prostasin-dependent matriptase zymogen activation in intestine, distinguishing it functionally from HAI-1.","evidence":"HAI-1/HAI-2 knockout mice and Caco-2 silencing with biochemical fractionation of matriptase","pmids":["24962579"],"confidence":"High","gaps":["Why HAI-1 cannot substitute in intestine not fully explained at this stage"]},{"year":2014,"claim":"Mapped domain-specific protease specificity, showing KD1 and KD2 inhibit distinct protease subsets and that a free cysteine thiol drives mutant loss of function.","evidence":"Xenopus oocyte expression with KD1/KD2 mutagenesis against nine GI serine proteases","pmids":["24722141"],"confidence":"Medium","gaps":["Structural basis of KD2-specific recognition not defined","Heterologous oocyte system"]},{"year":2015,"claim":"Established that HAI-2 function is gated by subcellular localization and N-glycan maturation, explaining cell-type-selective activity.","evidence":"Immunofluorescence, surface biotinylation, glycan characterization, and matriptase-HAI-2 complex detection in mammary vs breast cancer cells","pmids":["25786220","26171609"],"confidence":"Medium","gaps":["Trigger for surface translocation in cancer cells unknown","Single-lab observations"]},{"year":2015,"claim":"Connected SPINT2 epigenetic silencing to aberrant HGF-MET-AKT signaling in cancer, providing a mechanism for its tumor-suppressive loss.","evidence":"Ectopic expression and decitabine reactivation in melanoma with MET/AKT phosphorylation readouts","pmids":["25910030"],"confidence":"Medium","gaps":["Protease intermediary between HAI-2 loss and MET activation not specified here"]},{"year":2017,"claim":"Resolved that HAI-2 KD1 separately stabilizes matriptase, regulates its secretory transport via SEA-domain maturation, and inhibits its proteolytic activity.","evidence":"KD1 binding-loop mutagenesis with immunoblot of SEA cleavage and immunofluorescence localization","pmids":["28371047"],"confidence":"Medium","gaps":["Mechanism coupling KD1 binding to SEA-domain processing not defined"]},{"year":2019,"claim":"Identified plasmin as a novel HAI-2 target and linked HAI-2 loss to plasmin-driven EMT, HGF/TGF-beta1 activation, and metastasis.","evidence":"Pulldown/LC-MS/MS identification with knockdown/overexpression and xenograft validation in lung cancer","pmids":["30765871"],"confidence":"Medium","gaps":["Kunitz domain mediating plasmin inhibition not mapped"]},{"year":2019,"claim":"Showed that SCSD-associated KD2 mutations selectively impair prostasin inhibition, implying prostasin engages a KD2 exosite beyond the KD1 active-site loop.","evidence":"Functional inhibition assays of mutants against prostasin and matriptase with homology modeling","pmids":["30445423"],"confidence":"Medium","gaps":["Exosite inferred from modeling, not structurally determined"]},{"year":2020,"claim":"Established HAI-2 as a TMPRSS2 inhibitor and stabilizing partner with anti-tumor consequences in prostate cancer.","evidence":"Co-IP/LC-MS/MS, recombinant inhibition assays, co-localization, and orthotopic xenografts","pmids":["32778768"],"confidence":"High","gaps":["Relative in vivo importance of TMPRSS2 vs matriptase targeting not quantified"]},{"year":2020,"claim":"Demonstrated that HAI-2 inhibits matriptase zymogen as well as activated matriptase, indicating active-site engagement of the zymogen form.","evidence":"Purified-protein in vitro inhibition assays with zymogen vs activated matriptase","pmids":["32338287"],"confidence":"Medium","gaps":["Structural basis of zymogen active-site accessibility not resolved"]},{"year":2020,"claim":"Extended HAI-2 antiviral function across multiple respiratory viruses by blocking host-protease cleavage of viral fusion proteins.","evidence":"Cleavage/fusion inhibition assays and cell-culture viral growth assays across influenza strains and HMPV","pmids":["32056846","24978308"],"confidence":"Medium","gaps":["In vivo efficacy beyond influenza mouse model not established"]},{"year":2021,"claim":"Established that intestinal barrier failure from HAI-2 loss proceeds through a prostasin-dependent pathway with disruption of junctional proteins.","evidence":"Spint2-/-;Prss8R44Q mouse model with histology and junctional protein immunoblots","pmids":["29617460"],"confidence":"High","gaps":["Direct prostasin substrate(s) responsible for junction loss not identified"]},{"year":2021,"claim":"Explained organ-selective disease through cell-type-selective excessive prostasin proteolysis, present in intestinal but not keratinocyte cells.","evidence":"Comparative HAI-2 deletion in Caco-2 vs HaCaT with prostasin/matriptase activation and half-life biochemistry","pmids":["34089062"],"confidence":"Medium","gaps":["Molecular determinant of intestinal vs epidermal selectivity not pinpointed"]},{"year":2021,"claim":"Identified the intracellular targeting motifs (Arg/Lys-rich, EHLVY) distinguishing HAI-2 granule retention from HAI-1 surface distribution.","evidence":"Domain-swap and point mutagenesis with immunocytochemistry and surface biotinylation","pmids":["34643933"],"confidence":"Medium","gaps":["Trafficking machinery recognizing these motifs not identified"]},{"year":2021,"claim":"Revealed a protease-independent developmental role in zebrafish, with HAI-2 promoting epithelial cohesion via genetic interaction with E-cadherin.","evidence":"Zebrafish spint2 mutant, chimera, and double-mutant epistasis analyses","pmids":["33826923"],"confidence":"Medium","gaps":["Mechanism of E-cadherin-dependent cohesion not defined","Ortholog system may differ from mammals"]},{"year":2021,"claim":"Connected SPINT2 to cell-cycle control of tetraploid cells through CDKN1A transcription regulation via histone acetylation, distinct from its protease-inhibitor role.","evidence":"Genome-wide RNAi screen in HCT116 with IP/MS and histone acetylation analysis","pmids":["34962618"],"confidence":"Medium","gaps":["Mechanism linking a secreted Kunitz inhibitor to nuclear chromatin regulation unclear"]},{"year":2023,"claim":"Defined that N-glycosylation at Asn-57 is required for correct folding and inhibitory activity, with non-glycosylated HAI-2 forming misfolded oligomers.","evidence":"Asn-57/Asn-94 mutagenesis with oligomerization, glycosylation, and inhibition assays in Caco-2 KO cells","pmids":["36637420"],"confidence":"Medium","gaps":["Folding pathway requirement for the glycan not structurally characterized"]},{"year":2023,"claim":"Showed that matriptase-driven intestinal disease in HAI-2 deficiency proceeds through substrates other than EpCAM, refining the disease mechanism.","evidence":"Spint2-deficient mice with cleavage-resistant EpCAM knockin and intestinal matriptase inactivation","pmids":["37539662"],"confidence":"High","gaps":["Identity of the relevant matriptase substrate(s) remains unknown"]},{"year":2024,"claim":"Established the molecular basis by which SCSD KD2 mutations inactivate HAI-2 through disulfide-linked oligomerization and immature glycoform trapping.","evidence":"Inducible KD2-mutant expression in HAI-2-KO Caco-2 cells with non-reducing PAGE, glycan, and prostasin inhibition assays","pmids":["38271183"],"confidence":"High","gaps":["Whether oligomers exert dominant-negative effects in patients not addressed"]},{"year":2025,"claim":"Uncovered a non-protease mechanism in which SPINT2 stabilizes ACSL4 by blocking NEDD4L-mediated ubiquitination, promoting ferroptosis in cancer.","evidence":"Co-IP, ubiquitination assays, E3-ligase identification, and tumor suppression/metabolomic profiling","pmids":["41067336"],"confidence":"Medium","gaps":["Single-lab Co-IP-based mechanism without independent replication","How a Kunitz inhibitor competes with an E3 ligase unclear"]},{"year":2025,"claim":"Linked SPINT2 to cellular senescence, where promoter hypomethylation upregulates SPINT2 to drive senescence via c-Met inhibition.","evidence":"DNMT1 knockdown/inhibition, SPINT2 knockdown/overexpression, methylation sequencing, and transcriptomics","pmids":["40838961"],"confidence":"Medium","gaps":["Causal role of named downstream targets (COL27A1, STAM2, CBL) not functionally validated"]},{"year":null,"claim":"The structural basis of HAI-2 substrate selectivity (KD1 vs KD2 specificity and the proposed prostasin KD2 exosite) and how a secreted Kunitz inhibitor mediates intracellular roles (CDKN1A chromatin regulation, ACSL4 stabilization) remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No experimental structure of HAI-2 bound to a target protease","Mechanistic basis of non-protease intracellular functions unverified","Identity of matriptase substrate driving intestinal disease unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,6,9,21,22]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,2,8,15,22]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[10,14,21]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[10,12,16,21]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[10,12,27]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[12,15,30]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,8,15,22]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[2,5,28]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[3,19,32]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[13,23,29]}],"complexes":[],"partners":["ST14","PRSS8","TMPRSS2","TMPRSS6","TMPRSS13","PLG","ACSL4","NEDD4L"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O43291","full_name":"Kunitz-type protease inhibitor 2","aliases":["Hepatocyte growth factor activator inhibitor type 2","HAI-2","Placental bikunin"],"length_aa":252,"mass_kda":28.2,"function":"Inhibitor of HGFAC (PubMed:9346890). Also inhibits plasmin, and plasma and tissue kallikrein (PubMed:9115294). Inhibits serine protease activity of TMPRSS13 (PubMed:20977675, PubMed:28710277). Inhibits serine protease activity of ST14/matriptase and PRSS8/prostasin in vitro (PubMed:28710277, PubMed:30445423)","subcellular_location":"Cell membrane; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/O43291/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SPINT2","classification":"Not Classified","n_dependent_lines":42,"n_total_lines":1208,"dependency_fraction":0.0347682119205298},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SPINT2","total_profiled":1310},"omim":[{"mim_id":"619711","title":"CHROMOSOME 19 OPEN READING FRAME 33; C19ORF33","url":"https://www.omim.org/entry/619711"},{"mim_id":"613217","title":"DIARRHEA 5, WITH TUFTING ENTEROPATHY, CONGENITAL; DIAR5","url":"https://www.omim.org/entry/613217"},{"mim_id":"605124","title":"SERINE PEPTIDASE INHIBITOR, KUNITZ-TYPE, 2; SPINT2","url":"https://www.omim.org/entry/605124"},{"mim_id":"270420","title":"DIARRHEA 3, SECRETORY SODIUM, CONGENITAL, WITH OR WITHOUT OTHER CONGENITAL ANOMALIES; DIAR3","url":"https://www.omim.org/entry/270420"},{"mim_id":"214700","title":"DIARRHEA 1, SECRETORY CHLORIDE, CONGENITAL; DIAR1","url":"https://www.omim.org/entry/214700"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Golgi apparatus","reliability":"Enhanced"},{"location":"Vesicles","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SPINT2"},"hgnc":{"alias_symbol":["Kop","HAI-2","HAI2"],"prev_symbol":[]},"alphafold":{"accession":"O43291","domains":[{"cath_id":"4.10.410.10","chopping":"20-90","consensus_level":"high","plddt":87.5304,"start":20,"end":90},{"cath_id":"4.10.410.10","chopping":"130-189","consensus_level":"high","plddt":92.436,"start":130,"end":189},{"cath_id":"1.20.5","chopping":"194-238","consensus_level":"medium","plddt":87.3262,"start":194,"end":238}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O43291","model_url":"https://alphafold.ebi.ac.uk/files/AF-O43291-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O43291-F1-predicted_aligned_error_v6.png","plddt_mean":81.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SPINT2","jax_strain_url":"https://www.jax.org/strain/search?query=SPINT2"},"sequence":{"accession":"O43291","fasta_url":"https://rest.uniprot.org/uniprotkb/O43291.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O43291/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O43291"}},"corpus_meta":[{"pmid":"15792801","id":"PMC_15792801","title":"Hepsin 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Kop. and Antitrichia californica Sull., from southwest ecological region in Turkey.","date":"2021","source":"Natural product research","url":"https://pubmed.ncbi.nlm.nih.gov/33949257","citation_count":1,"is_preprint":false},{"pmid":"30111395","id":"PMC_30111395","title":"[Expression and Clinical Significances of HGFA, Matriptase, HAI-1 and HAI-2 in Acute Myeloid Leukemia].","date":"2018","source":"Zhongguo shi yan xue ye xue za zhi","url":"https://pubmed.ncbi.nlm.nih.gov/30111395","citation_count":1,"is_preprint":false},{"pmid":"41408854","id":"PMC_41408854","title":"Efficient production of fully active, SARS-CoV-2-priming, wildtype TMPRSS2 ectodomain via co-expression of HAI-2 allows for both auto- and cross-activation mechanisms.","date":"2025","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/41408854","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.08.18.670989","title":"Endothelial Colony-Forming Cell Transcriptomic Profiling in CT-defined Coronary Artery Disease from the BioHEART-CT Study Implicate CCBE1 in Mitochondrial Dysfunction-associated Atherosclerosis","date":"2025-08-22","source":"bioRxiv","url":"https://doi.org/10.1101/2025.08.18.670989","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":47268,"output_tokens":10385,"usd":0.148789,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":20861,"output_tokens":6767,"usd":0.13674,"stage2_stop_reason":"end_turn"},"total_usd":0.285529,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"HAI-2 (SPINT2) potently inhibits hepsin-mediated activation of pro-HGF into biologically active HGF, with IC50 of 1.3 nM; inhibition is due to Kunitz domain-1, as shown by HAI-1B Kunitz domain mutants (R260A, K401A) losing inhibitory activity.\",\n      \"method\": \"In vitro enzymatic assay with soluble recombinant hepsin and soluble HAI-2; Kunitz domain mutagenesis\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay with defined IC50, active-site mutagenesis identifying KD-1 as the inhibitory domain; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"15792801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Restoration of HAI-2/SPINT2 expression in a RCC cell line reduced in vitro colony formation, whereas a missense mutant (P111S) had no significant effect, placing HAI-2 as a functional tumor suppressor in RCC. Increased cell motility from HAI-2 inactivation was abrogated by ERK/MAPK and PLC-γ inhibitors.\",\n      \"method\": \"Re-expression of wild-type vs. P111S mutant SPINT2 in RCC cell line; pharmacological inhibitor treatment; colony formation assay; cell motility assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function rescue experiment with pathway inhibitors; single lab with multiple functional assays\",\n      \"pmids\": [\"15930277\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Genetic inactivation of Spint2 in mice causes defects in neural tube closure and placental labyrinth development with loss of epithelial polarity, leading to embryonic death. Simultaneous inactivation of matriptase (St14) fully restores placental development and embryonic survival, establishing matriptase inhibition as an essential HAI-2 function. Neural tube defects persisted at lower frequency even without matriptase, indicating HAI-2 also inhibits additional serine proteases required for neural development.\",\n      \"method\": \"Genetic knockout (Spint2-/- mice); genetic epistasis (Spint2/St14 double knockout); phenotypic analysis of placentation and neural tube\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — genetic epistasis in vivo with double-knockout rescue; replicated across multiple genetic combinations\",\n      \"pmids\": [\"19592578\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Loss-of-function mutations in SPINT2 cause syndromic congenital sodium diarrhea. SPINT2 mutations (splice, missense, nonsense) were associated with loss of protein synthesis or failure to inhibit the serine protease trypsin in vitro, demonstrating that HAI-2 protease inhibitory activity is required for normal intestinal sodium homeostasis.\",\n      \"method\": \"Genetic linkage/SNP scan identifying SPINT2 mutations in families with syndromic CSD; in vitro trypsin inhibition assay with patient-derived mutant proteins\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — human genetics combined with functional in vitro assay confirming loss of inhibitory activity; multiple mutations in multiple families\",\n      \"pmids\": [\"19185281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"HAI-2/PB Kunitz domain 1 (KD-1) is critical for anti-invasive and anti-tumorigenic function in hepatocellular carcinoma cells: KD-1 inactivating mutant abolished anti-invasion activity in vitro and tumor suppression in vivo, while KD-2 inactivating mutant did not.\",\n      \"method\": \"Ectopic expression of wild-type and KD-1 vs. KD-2 mutant HAI-2 in HCC cell lines; Matrigel invasion assay; in vivo tumorigenicity assay\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-specific mutagenesis with functional readout in vitro and in vivo; single lab\",\n      \"pmids\": [\"19107935\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Genetic epistasis establishes that prostasin (PRSS8) acts downstream of matriptase and upstream of HAI-2 in a proteolytic cascade: hypomorphic Prss8 mutations rescue early embryonic lethality, mid-gestation placental failure, and neural tube defects in HAI-2-deficient mice. Biochemical analysis revealed prostasin is required for conversion of matriptase zymogen to active matriptase (paradoxically upstream), while prostasin zymogen activation is matriptase-independent. Inactivation of c-Met, PAR-2, or ENaC alpha did not rescue lethality.\",\n      \"method\": \"In vivo genetic epistasis (compound knockout mice: HAI-2/Prss8, HAI-2/c-Met, HAI-2/PAR-2, HAI-2/ENaC-α); biochemical analysis of matriptase and prostasin activation in placental tissue\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple double-knockout genetic epistasis experiments combined with biochemical analysis; single lab but highly systematic with multiple orthogonal approaches\",\n      \"pmids\": [\"22952456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"HAI-2 inhibits matriptase-2 at the cell surface and in conditioned medium, forming a stable complex with matriptase-2 (demonstrated by co-immunoprecipitation). HAI-2 thereby indirectly elevates hepcidin expression (HAMP) by blocking matriptase-2-mediated suppression of hepcidin, likely through preventing matriptase-2 from cleaving membrane-bound hemojuvelin.\",\n      \"method\": \"Co-expression of matriptase-2 and HAI-2; complex isolation by immunoprecipitation from cell lysates and conditioned medium; HAMP mRNA measurement\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP demonstrating complex formation, functional gene expression readout; single lab with two orthogonal methods\",\n      \"pmids\": [\"23293962\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"HAI-2 suppresses prostate cancer cell migration, invasion, tumorigenicity and metastasis primarily through regulation of matriptase: HAI-2 knockdown increases matriptase activation, and matriptase knockdown rescues the invasion induced by HAI-2 loss. HAI-2 overexpression in N2 cells reduced tumor growth and metastasis in orthotopic xenografts.\",\n      \"method\": \"HAI-2 knockdown and overexpression in human PCa cell lines (103E, N1, N2); matriptase knockdown rescue experiment; orthotopic xenograft mouse model; immunohistochemistry\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis by knockdown rescue, in vivo xenograft, single lab multiple methods\",\n      \"pmids\": [\"24121274\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HAI-2, but not HAI-1, regulates prostasin-dependent matriptase zymogen activation. In HAI-2-deficient intestinal epithelial cells, matriptase is lost due to accelerated prostasin-driven activation and subsequent shedding. HAI-1 ablation did not affect matriptase in intestinal cells. Gene silencing in Caco-2 monolayers confirmed that HAI-2 loss causes accelerated matriptase shedding mechanistically through loss of prostasin regulation.\",\n      \"method\": \"Genetically engineered HAI-1 and HAI-2 knockout mice; gene silencing in Caco-2 intestinal epithelial cell monolayers; immunofluorescence and biochemical fractionation of matriptase localization/activation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vivo knockout plus cell-based gene silencing with mechanistic biochemical analysis; multiple orthogonal methods; single lab but rigorous\",\n      \"pmids\": [\"24962579\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Y163C mutation in Kunitz domain 2 (KD2) of HAI-2 causes complete loss of inhibitory activity on two intestinal serine proteases, prostasin and TMPRSS13, but mutations in KD1 (Y68C) affect a different subset of proteases. HAI-2 wild-type inhibits nine gastrointestinal serine proteases. The thiol group of the introduced cysteine (not loss of Tyr) is responsible for HAI-2 loss of function.\",\n      \"method\": \"Xenopus laevis oocyte expression system cellular assay; site-directed mutagenesis of KD1 and KD2; functional assay against nine GI serine proteases\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro functional assay with mutagenesis in defined expression system; single lab but multiple mutants and substrates tested\",\n      \"pmids\": [\"24722141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"HAI-2 inhibition of matriptase depends on subcellular co-localization: in mammary epithelial cells, HAI-2 resides in intracellular vesicle/granule structures and does not form matriptase-HAI-2 complexes upon matriptase activation. In breast cancer cells, a proportion of HAI-2 translocates to the cell surface where it inhibits matriptase, forming three distinct matriptase-HAI-2 complexes.\",\n      \"method\": \"Induction of matriptase zymogen activation in human mammary epithelial vs. breast cancer cells; immunofluorescent staining of HAI-2 localization; immunoprecipitation of matriptase-HAI-2 complexes; cell surface biotinylation\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, immunofluorescence, cell-surface biotinylation; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"25786220\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"HAI-2 (SPINT2) inhibits HGF-induced MET-AKT signaling in melanoma cells and decreases cell motility and invasive growth. Epigenetic silencing of SPINT2 by DNA methylation activates the HGF-MET pathway.\",\n      \"method\": \"Ectopic SPINT2 expression in melanoma cells; MET/AKT phosphorylation assay; cell motility and invasion assay; decitabine-induced reactivation\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain-of-function with defined signaling readout; single lab, multiple functional methods\",\n      \"pmids\": [\"25910030\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The N-glycan on Asn-57 of HAI-2 determines subcellular distribution: oligomannose-type N-glycan (25-kDa form) is largely ER-retained, while complex-type N-glycan (30–40-kDa form) is targeted to vesicles/granules and the cell surface where it can inhibit matriptase. In breast cancer cells, the mature 30–40-kDa HAI-2 translocates to the cell surface to form matriptase-HAI-2 complexes.\",\n      \"method\": \"Biochemical characterization of two HAI-2 species by size and N-glycan type; subcellular fractionation; immunofluorescence; matriptase-HAI-2 complex detection\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical glycan characterization plus functional localization data; single lab, multiple methods\",\n      \"pmids\": [\"26171609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HAI-2 inhibits influenza virus H1N1 and H3N2 hemagglutinin cleavage and reduces viral infection in cell culture; HAI-2 administration in a mouse influenza model confers protection, acting by inhibiting host proteases (matriptase, TMPRSS2) that cleave HA.\",\n      \"method\": \"In vitro HA cleavage inhibition assay; cell culture infection assay; mouse influenza model\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro cleavage assay plus cell culture and mouse model; single lab, multiple orthogonal assays\",\n      \"pmids\": [\"24978308\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HAI-2 forms complexes with prostasin in human milk in addition to matriptase-HAI-2 complexes, providing in vivo evidence that both matriptase and prostasin are HAI-2 target proteases during lactation.\",\n      \"method\": \"Purification of protease-inhibitor complexes from human milk; immunoaffinity and ion-exchange chromatography; mass spectrometry identification\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo complex detection by biochemical purification and MS; single lab\",\n      \"pmids\": [\"27043831\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"HAI-2 Kunitz domain 1 mutations (K42N, C47F, R48L) delay SEA domain cleavage of matriptase, causing accumulation of non-SEA-cleaved matriptase in the ER, thereby regulating matriptase secretory transport and cell-surface localization. Two of the mutants (C47F, R48L) also show reduced ability to inhibit matriptase proteolytic activity. HAI-2 thus separately: (1) stabilizes matriptase, (2) regulates its secretory transport via maturation/oligomerization, and (3) inhibits its proteolytic activity.\",\n      \"method\": \"Site-directed mutagenesis of HAI-2 KD1 binding loop; expression in cells; analysis of matriptase SEA domain cleavage by immunoblot; subcellular localization by immunofluorescence\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with biochemical and cell-biological functional readouts; single lab, multiple methods\",\n      \"pmids\": [\"28371047\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In human skin, HAI-2 is primarily intracellularly localized in basal and spinous layer keratinocytes, preventing its interaction with active prostasin or matriptase; consequently HAI-1 (not HAI-2) is the prominent inhibitor of prostasin and matriptase in skin. Subcellular localization thus determines tissue-specific protease inhibitor function.\",\n      \"method\": \"Immunohistochemistry of human foreskin; immunoblot analysis of foreskin lysates; comparison of protease activation status across tissues\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiment with functional inference; single lab, two orthogonal methods\",\n      \"pmids\": [\"29438412\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HAI-2 loss in oral squamous cell carcinoma (OSCC) cells causes a pro-invasive phenotype via excess prostasin activity: SPINT2 knockout suppresses OSCC proliferation and invasion, and this is rescued by HAI-2 re-expression or prostasin knockdown. Prostasin protein levels increase markedly in HAI-2-deficient OSCC cells.\",\n      \"method\": \"Homozygous SPINT2 CRISPR knockout in OSCC lines; prostasin knockdown rescue; Matrigel invasion assay; western blot\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout with epistasis rescue by prostasin knockdown; single lab\",\n      \"pmids\": [\"29545930\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HAI-2 loss in mice with HAI-2-deficient intestinal epithelial cells causes excessive prostasin proteolysis with prolonged active prostasin and depletion of HAI-1 monomer, but this phenotype is not observed in epidermal (HaCaT) cells, identifying tissue-selective functional relationship between HAI-2 and prostasin in intestinal epithelium.\",\n      \"method\": \"Targeted HAI-2 deletion in Caco-2 and HaCaT cells; biochemical analysis of prostasin activation, matriptase activation, and HAI-1 levels\",\n      \"journal\": \"PLoS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — targeted gene deletion in two cell types with biochemical mechanistic analysis; single lab\",\n      \"pmids\": [\"29617460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SPINT2 missense mutations in KD2 (p.Phe161Val, p.Tyr163Cys, p.Gly168Ser) found in SCSD patients selectively impair inhibition of prostasin-catalyzed cleavage but do not affect matriptase inhibition. Homology modeling suggests these mutations induce KD2 misfolding. This implies prostasin needs to engage an exosite on KD2 in addition to the KD1 binding loop for inhibition.\",\n      \"method\": \"Functional inhibition assays with wild-type and mutant HAI-2 against prostasin and matriptase; homology modeling\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro functional assay discriminating substrate specificity with structural modeling; single lab, multiple mutants\",\n      \"pmids\": [\"30445423\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HAI-2 functions as a novel inhibitor of plasmin in lung cancer: HAI-2 downregulation increases cell-surface plasmin activity, EMT, and invasion; HAI-2 suppresses plasmin-mediated activations of HGF and TGF-β1. Plasmin was identified as a novel HAI-2 target by pulldown and LC/MS/MS. In xenograft models, HAI-2 loss increased lung cancer metastasis via elevated plasmin activity.\",\n      \"method\": \"Pulldown and LC/MS/MS identification of plasmin as HAI-2 target; HAI-2 knockdown/overexpression; plasmin activity assay; transwell invasion; in vivo xenograft\",\n      \"journal\": \"British journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomic identification plus functional validation in vitro and in vivo; single lab, multiple methods\",\n      \"pmids\": [\"30765871\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"HAI-2 forms a stable complex with TMPRSS2 (identified by co-immunoprecipitation and LC/MS/MS) and potently inhibits TMPRSS2 proteolytic activity, with both KD1 and KD2 showing comparable inhibitory effects. HAI-2 suppresses TMPRSS2-induced pro-HGF activation, ECM degradation, and prostate cancer invasion and metastasis in orthotopic xenograft models.\",\n      \"method\": \"Co-IP and LC/MS/MS; recombinant protein inhibition assay; immunofluorescence co-localization; HAI-2 overexpression in xenograft model\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — biochemical complex identification by Co-IP/MS, in vitro inhibition assay, in vivo xenograft; multiple orthogonal methods in single study\",\n      \"pmids\": [\"32778768\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"HAI-2 and HAI-1 inhibit the catalytic activity of the matriptase zymogen toward peptide substrates and natural protein substrates (pro-HGF, pro-prostasin) at comparable concentrations to their inhibition of activated matriptase, indicating that the Kunitz inhibitors interact with the active sites of both zymogen and activated matriptase similarly.\",\n      \"method\": \"Purified protein in vitro inhibition assay; cell-based assay with HAI-1/HAI-2 and zymogen vs. activated matriptase\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified proteins plus cell-based assay; single lab, two orthogonal systems\",\n      \"pmids\": [\"32338287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SPINT2 inhibits proteolytic cleavage-activation of influenza A (H1N1, H3N2, H7N9) hemagglutinin and HMPV F protein by trypsin, recombinant matriptase, or KLK5, and reduces viral growth in cell culture by inhibiting matriptase or TMPRSS2. Inhibition is effective whether added at infection or 24 h post-infection.\",\n      \"method\": \"Cleavage and fusion inhibition assays with recombinant SPINT2; cell culture viral growth assay\",\n      \"journal\": \"Virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro cleavage assay plus cell culture antiviral assay; single lab, multiple virus strains\",\n      \"pmids\": [\"32056846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SPINT2 acts as a general regulator of CDKN1A transcription via histone acetylation. Loss of SPINT2 improves survival of tetraploid cells (identified by genome-wide RNAi screen) by reducing CDKN1A expression, linking SPINT2 to cell cycle arrest after genome doubling.\",\n      \"method\": \"Genome-wide RNAi screen in HCT116 colorectal cancer cells; mass spectrometry and immunoprecipitation; chromatin/histone acetylation analysis\",\n      \"journal\": \"Cellular oncology (Dordrecht, Netherlands)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide screen with mechanistic follow-up by IP and histone acetylation assay; single lab\",\n      \"pmids\": [\"34962618\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In HAI-2-deficient mice expressing only zymogen-locked prostasin (Prss8 R44Q), which does not bind HAI-2, postnatal intestinal failure develops (villous atrophy, tufted villi, loss of goblet cells, loss of colonic crypt structure) with reduced EpCAM, E-cadherin, occludin, and claudins-1/-7, and elevated claudin-4, demonstrating that HAI-2 regulates intestinal epithelial barrier integrity through a prostasin-dependent pathway.\",\n      \"method\": \"Genetic mouse model (Spint2-/-;Prss8R44Q/R44Q); histology; immunoblot of junctional proteins\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic epistasis model with detailed molecular phenotyping; mechanistic pathway placement established\",\n      \"pmids\": [\"29617460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In Caco-2 colorectal cells (but not HaCaT keratinocytes), HAI-2 deletion causes constitutive high-level prostasin zymogen activation, prolonged active prostasin half-life, depletion of HAI-1 monomer, and secondary increase in matriptase activation. This cell-type-selective excessive proteolysis explains organ-selective intestinal damage from SPINT2 mutations.\",\n      \"method\": \"Targeted HAI-2 deletion in Caco-2 vs. HaCaT cells; biochemical assays for prostasin and matriptase activation/inhibition status; half-life measurements\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — comparative cell-type deletion experiments with detailed biochemical analysis; single lab\",\n      \"pmids\": [\"34089062\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Differences in intracellular Arg/Lys-rich and EHLVY motifs between HAI-1 and HAI-2 account for their distinct subcellular distributions (HAI-1 on cell surface and inside; HAI-2 predominantly in intracellular granules). Domain swap and point mutation experiments confirmed these motifs as the key targeting signals.\",\n      \"method\": \"Domain swap mutagenesis; point mutations; immunocytochemistry; cell surface biotinylation/avidin depletion\",\n      \"journal\": \"Human cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with direct localization readout; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"34643933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In zebrafish, Spint2 is required for hatching gland cell cohesion, collective intra-epidermal migration, and survival prior to degranulation. Spint2 acts independently of tested matriptases and prostasins, but displays tight genetic interaction with E-cadherin, promoting hatching gland cell cohesiveness and survival. No genetic interaction with EpCAM was observed.\",\n      \"method\": \"Zebrafish spint2 mutant analysis; chimera analysis; genetic epistasis with matriptase, prostasin, E-cadherin, EpCAM mutants\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic epistasis in zebrafish with chimera and multiple double-mutant analyses; ortholog study\",\n      \"pmids\": [\"33826923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SPINT2 knockdown in lung cancer (Calu-3) cells leads to a strong increase in SARS-CoV-2 viral load, while SPINT2 overexpression drastically reduces viral load, demonstrating that SPINT2 restricts SARS-CoV-2 infection, likely through inhibition of TMPRSS2.\",\n      \"method\": \"SPINT2 siRNA knockdown and overexpression in Calu-3 cells; viral load quantification\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function with direct viral load readout; single lab\",\n      \"pmids\": [\"34181691\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"N-glycosylation at Asn-57 (not Asn-94) is required for correct HAI-2 protein folding and protease inhibitory activity. Non-glycosylated HAI-2 (Asn-57 mutant) is synthesized as disulfide-linked oligomers with distorted conformations and lacks protease inhibitory function. The oligomannose-type N-glycan is the precursor of the complex-type N-glycan form.\",\n      \"method\": \"Point mutations of Asn-57 and Asn-94 in HAI-2; biochemical characterization of oligomerization, glycosylation state, and protease inhibitory activity in HAI-2 knockout Caco-2 cells\",\n      \"journal\": \"Glycobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — site-directed mutagenesis with biochemical functional readout; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"36637420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Early-onset tufting enteropathy and postnatal lethality in Spint2-deficient mice are driven by matriptase activity but NOT through excessive proteolysis of EpCAM: expression of cleavage-resistant EpCAM failed to rescue intestinal failure. Matriptase inactivation counteracted Spint2 deficiency even with cleavage-resistant EpCAM, indicating matriptase-driven intestinal dysfunction proceeds via substrates other than EpCAM.\",\n      \"method\": \"Genetic mouse models combining Spint2 deficiency with cleavage-resistant EpCAM knockin and intestinal St14 (matriptase) inactivation; in vitro and in vivo EpCAM cleavage assays\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic epistasis combinations with clear mechanistic negative result; rigorous in vivo study\",\n      \"pmids\": [\"37539662\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SCSD-associated SPINT2 missense mutations in KD2 (e.g., p.Phe161Val, p.Tyr163Cys, p.Gly168Ser) inactivate HAI-2 through two mechanisms: (1) ~50% of protein forms disulfide-linked oligomers due to disarrayed disulfide bonding, losing protease inhibitory activity; (2) remaining monomeric protein is trapped in an immature, lightly glycosylated form and cannot suppress prostasin proteolysis. These mutants cannot rescue excessive prostasin proteolysis caused by HAI-2 knockout in Caco-2 cells.\",\n      \"method\": \"Doxycycline-inducible expression of HAI-2 KD2 mutants in HAI-2-knockout Caco-2 cells; non-reducing SDS-PAGE for oligomer detection; glycosylation state analysis; prostasin inhibition functional assay\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mechanistic study with inducible expression, mutagenesis, biochemical oligomerization/glycosylation analysis, and functional prostasin inhibition assay in KO rescue system; multiple orthogonal methods\",\n      \"pmids\": [\"38271183\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SPINT2 interacts with ACSL4 and prevents its ubiquitination by the E3 ligase NEDD4L, thereby stabilizing ACSL4 protein and promoting ferroptotic cell death in gallbladder cancer. SPINT2 deficiency alters lipid metabolism and reduces ferroptosis susceptibility.\",\n      \"method\": \"Co-immunoprecipitation demonstrating SPINT2-ACSL4 interaction; ubiquitination assay; NEDD4L identification as E3 ligase; in vitro and in vivo tumor suppression assays; metabolomic profiling\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP with ubiquitination assay; single lab, novel mechanism with limited independent replication\",\n      \"pmids\": [\"41067336\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DNMT1 downregulation during senescence causes hypomethylation of SPINT2 promoter CpG sites, leading to SPINT2 upregulation. SPINT2 overexpression alone induces senescence, and SPINT2 knockdown mitigates DNMT1 inhibition-induced senescence. SPINT2 drives senescence by inhibiting c-Met signaling; downstream targets include COL27A1, STAM2, and CBL.\",\n      \"method\": \"DNMT1 knockdown and pharmacological inhibition; SPINT2 siRNA knockdown and overexpression; methylation-specific sequencing; transcriptomic profiling; senescence marker assays\",\n      \"journal\": \"Aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple complementary genetic and epigenetic approaches with defined c-Met pathway placement; single lab\",\n      \"pmids\": [\"40838961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"STYK1 overexpression significantly decreases SPINT2 protein levels in NSCLC cells, and SPINT2 overexpression reverses STYK1-mediated NSCLC proliferation, migration, invasion, and EMT both in vitro and in vivo, placing SPINT2 downstream of STYK1 in a lung cancer progression pathway.\",\n      \"method\": \"RNA-seq, qRT-PCR, western blot after STYK1 overexpression; SPINT2 overexpression rescue; in vivo tumor model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis rescue experiment with multiple functional assays; single lab\",\n      \"pmids\": [\"31164631\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SPINT2 loss in glioma promotes cell growth and invasion partly via increased MMP2 expression and activity, as demonstrated by SPINT2 knockdown and knock-in functional assays in adult and pediatric HGG cell lines.\",\n      \"method\": \"SPINT2 knockdown and knock-in in HGG cell lines; MMP2 activity assay; invasion and viability assays\",\n      \"journal\": \"Cellular oncology (Dordrecht, Netherlands)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — paired loss- and gain-of-function with defined MMP2 mechanism; single lab\",\n      \"pmids\": [\"31701492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SPINT2 restores expression reduces c-Met activation in GBM cells and suppresses tumorigenic properties in vitro and in vivo, with SPINT2 downregulation resulting from promoter hypermethylation (confirmed by targeted bisulfite sequencing, 5-aza treatment, DNMT1 knockdown, and luciferase reporter assay).\",\n      \"method\": \"5-aza treatment, DNMT1 knockdown, luciferase reporter for methylation-mediated regulation; SPINT2 re-expression; c-Met phosphorylation assay; in vivo GBM model\",\n      \"journal\": \"Journal of neuro-oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple approaches confirming methylation regulation plus functional c-Met pathway placement; single lab\",\n      \"pmids\": [\"30838489\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SPINT2 promotes SMC proliferation and migration inhibition in aortic smooth muscle cells: SPINT2 overexpression reduces active MMP-2 and MMP-9 expression, suppresses SMC switching from contractile to synthetic phenotype, and inhibits ERK activation. A specific ERK agonist reverses SPINT2-mediated inhibition of SMC migration and phenotypic switching.\",\n      \"method\": \"SPINT2 overexpression via adenoviral vector in primary mouse aortic SMCs; PDGF-BB induction model; MTT, Ki-67, wound healing, ELISA, western blot; ERK agonist rescue\",\n      \"journal\": \"Experimental and therapeutic medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain-of-function with pathway rescue by ERK agonist; single lab, multiple methods\",\n      \"pmids\": [\"37928510\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Co-expression of HAI-2 (SPINT2) with TMPRSS2 ectodomain enables efficient production of active wildtype TMPRSS2 in mammalian cells, demonstrating that HAI-2 stabilizes TMPRSS2 zymogen during expression. Purified TMPRSS2 cleaves synthetic and protein substrates efficiently (kcat/KM 10^4–10^6 M-1s-1).\",\n      \"method\": \"Mammalian cell co-expression of TMPRSS2 ectodomain with HAI-2; purification and kinetic characterization of recombinant TMPRSS2\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution demonstrating stabilization function; single study, novel finding\",\n      \"pmids\": [\"41408854\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SPINT2 encodes HAI-2, a type I transmembrane, bi-Kunitz-domain serine protease inhibitor that regulates pericellular proteolysis primarily by inhibiting membrane-anchored serine proteases—including matriptase, prostasin, hepsin, TMPRSS2, matriptase-2, and plasmin—through its Kunitz domain 1 (and, for prostasin, requiring an exosite on Kunitz domain 2), thereby controlling HGF activation, EpCAM stability, epithelial barrier integrity, iron homeostasis (via matriptase-2/hepcidin axis), and viral entry (influenza, SARS-CoV-2); its cell-type-selective function is determined by N-glycosylation-dependent subcellular localization (intracellular vs. cell-surface), and loss-of-function via promoter hypermethylation or missense mutations causing protein misfolding/oligomerization underlies syndromic congenital sodium diarrhea/tufting enteropathy and drives tumor progression across multiple cancer types.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SPINT2 encodes HAI-2, a Kunitz-type serine protease inhibitor that governs pericellular proteolysis in epithelia and during development by restraining a network of membrane-anchored and soluble serine proteases [#0, #2, #5]. Its prototypic activity is inhibition of pro-HGF activation, blocking conversion to active HGF via Kunitz domain 1 (KD1) [#0], and KD1 is the principal determinant of its anti-invasive, tumor-suppressive function [#4]. In vivo, HAI-2 is essential for placental and neural tube development, and its core function is inhibition of matriptase: genetic loss of matriptase rescues the lethality of Spint2-null mice [#2]. HAI-2 sits in a proteolytic cascade with prostasin acting downstream of matriptase and upstream of HAI-2 [#5], and it regulates prostasin-driven matriptase zymogen activation, controlling matriptase stability, maturation/secretory transport, and catalytic activity as separable activities [#8, #15, #22]. HAI-2 inhibits a broad protease panel including matriptase-2 (elevating hepcidin via the hemojuvelin axis) [#6], TMPRSS2 [#21], prostasin and TMPRSS13 [#9], and plasmin [#20], with KD1 and KD2 contributing distinct specificities and prostasin requiring an exosite on KD2 in addition to the KD1 binding loop [#9, #19]. Cell-type-selective function is set by subcellular localization: complex-type N-glycosylation at Asn-57 and intracellular targeting motifs determine whether HAI-2 is ER-retained, granule-resident, or cell-surface, dictating whether it engages active proteases [#12, #16, #27, #30]. In intestinal epithelium, HAI-2 loss causes excessive prostasin proteolysis, disrupting barrier junctional proteins (EpCAM, E-cadherin, claudins) and producing tufting enteropathy through a matriptase/prostasin-dependent, EpCAM-cleavage-independent route [#25, #26, #31]. Loss-of-function SPINT2 mutations cause syndromic congenital sodium diarrhea, acting through loss of protease inhibition; KD2 missense mutations inactivate HAI-2 by inducing disulfide-linked oligomerization and trapping immature protein, selectively abolishing prostasin inhibition [#3, #19, #32]. SPINT2 is epigenetically silenced by promoter hypermethylation across cancers, derepressing HGF-MET signaling and promoting invasion [#11, #37]. HAI-2 additionally restricts viral entry by blocking host-protease cleavage of influenza hemagglutinin and limiting SARS-CoV-2 infection through TMPRSS2 inhibition [#13, #23, #29].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Established that HAI-2 is a potent serine protease inhibitor whose anti-HGF-activation function maps to Kunitz domain 1, defining its core biochemical mechanism.\",\n      \"evidence\": \"In vitro enzymatic assay with recombinant hepsin and Kunitz-domain mutagenesis\",\n      \"pmids\": [\"15792801\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish the in vivo relevance of hepsin as a target\", \"KD2 contribution not addressed\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Linked HAI-2 inhibitory function to tumor suppression, showing re-expression suppresses colony formation while a patient-derived missense mutant is inactive.\",\n      \"evidence\": \"Wild-type vs P111S re-expression in RCC cells with colony/motility assays and pathway inhibitors\",\n      \"pmids\": [\"15930277\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific protease target driving the phenotype not identified\", \"ERK/PLC-gamma dependence shown pharmacologically only\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Demonstrated in vivo that the essential developmental function of HAI-2 is matriptase inhibition, via double-knockout rescue of lethality.\",\n      \"evidence\": \"Spint2/St14 double-knockout genetic epistasis in mice with placental and neural tube phenotyping\",\n      \"pmids\": [\"19592578\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the additional protease driving residual neural tube defects unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Established SPINT2 as the causative gene for syndromic congenital sodium diarrhea and tied disease to loss of protease-inhibitory activity.\",\n      \"evidence\": \"Human genetic linkage in families plus in vitro trypsin inhibition assays with mutant proteins\",\n      \"pmids\": [\"19185281\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological protease responsible for sodium homeostasis not pinpointed at the time\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Placed HAI-2 within an ordered proteolytic cascade, showing prostasin acts downstream of matriptase and upstream of HAI-2, with prostasin paradoxically required for matriptase activation.\",\n      \"evidence\": \"Compound knockout mice (HAI-2/Prss8, c-Met, PAR-2, ENaC) and biochemical activation analysis in placenta\",\n      \"pmids\": [\"22952456\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of prostasin-dependent matriptase activation not fully resolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Expanded the target repertoire to matriptase-2 and connected HAI-2 to iron homeostasis through hepcidin regulation.\",\n      \"evidence\": \"Co-IP complex isolation and HAMP mRNA measurement after co-expression\",\n      \"pmids\": [\"23293962\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Hemojuvelin cleavage step inferred, not directly demonstrated\", \"Single Co-IP without in vivo confirmation\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined HAI-2 as the selective regulator of prostasin-dependent matriptase zymogen activation in intestine, distinguishing it functionally from HAI-1.\",\n      \"evidence\": \"HAI-1/HAI-2 knockout mice and Caco-2 silencing with biochemical fractionation of matriptase\",\n      \"pmids\": [\"24962579\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why HAI-1 cannot substitute in intestine not fully explained at this stage\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Mapped domain-specific protease specificity, showing KD1 and KD2 inhibit distinct protease subsets and that a free cysteine thiol drives mutant loss of function.\",\n      \"evidence\": \"Xenopus oocyte expression with KD1/KD2 mutagenesis against nine GI serine proteases\",\n      \"pmids\": [\"24722141\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of KD2-specific recognition not defined\", \"Heterologous oocyte system\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Established that HAI-2 function is gated by subcellular localization and N-glycan maturation, explaining cell-type-selective activity.\",\n      \"evidence\": \"Immunofluorescence, surface biotinylation, glycan characterization, and matriptase-HAI-2 complex detection in mammary vs breast cancer cells\",\n      \"pmids\": [\"25786220\", \"26171609\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Trigger for surface translocation in cancer cells unknown\", \"Single-lab observations\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Connected SPINT2 epigenetic silencing to aberrant HGF-MET-AKT signaling in cancer, providing a mechanism for its tumor-suppressive loss.\",\n      \"evidence\": \"Ectopic expression and decitabine reactivation in melanoma with MET/AKT phosphorylation readouts\",\n      \"pmids\": [\"25910030\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Protease intermediary between HAI-2 loss and MET activation not specified here\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Resolved that HAI-2 KD1 separately stabilizes matriptase, regulates its secretory transport via SEA-domain maturation, and inhibits its proteolytic activity.\",\n      \"evidence\": \"KD1 binding-loop mutagenesis with immunoblot of SEA cleavage and immunofluorescence localization\",\n      \"pmids\": [\"28371047\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism coupling KD1 binding to SEA-domain processing not defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified plasmin as a novel HAI-2 target and linked HAI-2 loss to plasmin-driven EMT, HGF/TGF-beta1 activation, and metastasis.\",\n      \"evidence\": \"Pulldown/LC-MS/MS identification with knockdown/overexpression and xenograft validation in lung cancer\",\n      \"pmids\": [\"30765871\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Kunitz domain mediating plasmin inhibition not mapped\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed that SCSD-associated KD2 mutations selectively impair prostasin inhibition, implying prostasin engages a KD2 exosite beyond the KD1 active-site loop.\",\n      \"evidence\": \"Functional inhibition assays of mutants against prostasin and matriptase with homology modeling\",\n      \"pmids\": [\"30445423\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Exosite inferred from modeling, not structurally determined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established HAI-2 as a TMPRSS2 inhibitor and stabilizing partner with anti-tumor consequences in prostate cancer.\",\n      \"evidence\": \"Co-IP/LC-MS/MS, recombinant inhibition assays, co-localization, and orthotopic xenografts\",\n      \"pmids\": [\"32778768\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative in vivo importance of TMPRSS2 vs matriptase targeting not quantified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrated that HAI-2 inhibits matriptase zymogen as well as activated matriptase, indicating active-site engagement of the zymogen form.\",\n      \"evidence\": \"Purified-protein in vitro inhibition assays with zymogen vs activated matriptase\",\n      \"pmids\": [\"32338287\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of zymogen active-site accessibility not resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended HAI-2 antiviral function across multiple respiratory viruses by blocking host-protease cleavage of viral fusion proteins.\",\n      \"evidence\": \"Cleavage/fusion inhibition assays and cell-culture viral growth assays across influenza strains and HMPV\",\n      \"pmids\": [\"32056846\", \"24978308\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo efficacy beyond influenza mouse model not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established that intestinal barrier failure from HAI-2 loss proceeds through a prostasin-dependent pathway with disruption of junctional proteins.\",\n      \"evidence\": \"Spint2-/-;Prss8R44Q mouse model with histology and junctional protein immunoblots\",\n      \"pmids\": [\"29617460\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct prostasin substrate(s) responsible for junction loss not identified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Explained organ-selective disease through cell-type-selective excessive prostasin proteolysis, present in intestinal but not keratinocyte cells.\",\n      \"evidence\": \"Comparative HAI-2 deletion in Caco-2 vs HaCaT with prostasin/matriptase activation and half-life biochemistry\",\n      \"pmids\": [\"34089062\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular determinant of intestinal vs epidermal selectivity not pinpointed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified the intracellular targeting motifs (Arg/Lys-rich, EHLVY) distinguishing HAI-2 granule retention from HAI-1 surface distribution.\",\n      \"evidence\": \"Domain-swap and point mutagenesis with immunocytochemistry and surface biotinylation\",\n      \"pmids\": [\"34643933\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Trafficking machinery recognizing these motifs not identified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Revealed a protease-independent developmental role in zebrafish, with HAI-2 promoting epithelial cohesion via genetic interaction with E-cadherin.\",\n      \"evidence\": \"Zebrafish spint2 mutant, chimera, and double-mutant epistasis analyses\",\n      \"pmids\": [\"33826923\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of E-cadherin-dependent cohesion not defined\", \"Ortholog system may differ from mammals\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Connected SPINT2 to cell-cycle control of tetraploid cells through CDKN1A transcription regulation via histone acetylation, distinct from its protease-inhibitor role.\",\n      \"evidence\": \"Genome-wide RNAi screen in HCT116 with IP/MS and histone acetylation analysis\",\n      \"pmids\": [\"34962618\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking a secreted Kunitz inhibitor to nuclear chromatin regulation unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined that N-glycosylation at Asn-57 is required for correct folding and inhibitory activity, with non-glycosylated HAI-2 forming misfolded oligomers.\",\n      \"evidence\": \"Asn-57/Asn-94 mutagenesis with oligomerization, glycosylation, and inhibition assays in Caco-2 KO cells\",\n      \"pmids\": [\"36637420\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Folding pathway requirement for the glycan not structurally characterized\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed that matriptase-driven intestinal disease in HAI-2 deficiency proceeds through substrates other than EpCAM, refining the disease mechanism.\",\n      \"evidence\": \"Spint2-deficient mice with cleavage-resistant EpCAM knockin and intestinal matriptase inactivation\",\n      \"pmids\": [\"37539662\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the relevant matriptase substrate(s) remains unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established the molecular basis by which SCSD KD2 mutations inactivate HAI-2 through disulfide-linked oligomerization and immature glycoform trapping.\",\n      \"evidence\": \"Inducible KD2-mutant expression in HAI-2-KO Caco-2 cells with non-reducing PAGE, glycan, and prostasin inhibition assays\",\n      \"pmids\": [\"38271183\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether oligomers exert dominant-negative effects in patients not addressed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Uncovered a non-protease mechanism in which SPINT2 stabilizes ACSL4 by blocking NEDD4L-mediated ubiquitination, promoting ferroptosis in cancer.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, E3-ligase identification, and tumor suppression/metabolomic profiling\",\n      \"pmids\": [\"41067336\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab Co-IP-based mechanism without independent replication\", \"How a Kunitz inhibitor competes with an E3 ligase unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linked SPINT2 to cellular senescence, where promoter hypomethylation upregulates SPINT2 to drive senescence via c-Met inhibition.\",\n      \"evidence\": \"DNMT1 knockdown/inhibition, SPINT2 knockdown/overexpression, methylation sequencing, and transcriptomics\",\n      \"pmids\": [\"40838961\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal role of named downstream targets (COL27A1, STAM2, CBL) not functionally validated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The structural basis of HAI-2 substrate selectivity (KD1 vs KD2 specificity and the proposed prostasin KD2 exosite) and how a secreted Kunitz inhibitor mediates intracellular roles (CDKN1A chromatin regulation, ACSL4 stabilization) remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No experimental structure of HAI-2 bound to a target protease\", \"Mechanistic basis of non-protease intracellular functions unverified\", \"Identity of matriptase substrate driving intestinal disease unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 6, 9, 21, 22]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 2, 8, 15, 22]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [10, 14, 21]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [10, 12, 16, 21]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [10, 12, 27]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [12, 15, 30]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 8, 15, 22]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2, 5, 28]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [3, 19, 32]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [13, 23, 29]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ST14\", \"PRSS8\", \"TMPRSS2\", \"TMPRSS6\", \"TMPRSS13\", \"PLG\", \"ACSL4\", \"NEDD4L\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":10,"faith_total":10,"faith_pct":100.0}}