{"gene":"CTSB","run_date":"2026-04-28T17:28:53","timeline":{"discoveries":[{"year":1991,"finding":"X-ray crystal structure of human liver cathepsin B refined to 2.15 Å revealed the structural basis for its dual endo- and exopeptidase activity: an 'occluding loop' containing His110 and His111 blocks primed subsites, anchoring the C-terminal carboxylate of substrates and explaining dipeptidyl carboxypeptidase activity; the active-site Cys29 and Glu245 in the S2 subsite favor basic P2 side chains; the occluding loop also prevents cystatin-like inhibitors from binding as they do to papain.","method":"X-ray crystallography (2.15 Å resolution), Patterson search and heavy atom replacement, structural comparison with papain/actinidin","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — high-resolution crystal structure with detailed active-site analysis, foundational structural paper with >500 citations","pmids":["1868826"],"is_preprint":false},{"year":1986,"finding":"Cloning and sequencing of human and mouse preprocathepsin B cDNAs revealed a 339-amino-acid precursor comprising a 17-residue signal peptide, a 62-residue propeptide, 254 residues of mature cathepsin B, and a 6-residue C-terminal extension; the propeptide contains a conserved glycosylation site and single cysteine, and comparative analysis suggested multi-step processing with possible active intermediate forms.","method":"cDNA cloning from human hepatoma and kidney libraries, nucleotide sequencing, comparative sequence analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — primary sequence determination from cDNA, foundational paper >280 citations","pmids":["3463996"],"is_preprint":false},{"year":1991,"finding":"Purified human cathepsin B cleaves pro-uPA (single-chain urokinase-type plasminogen activator) at the Lys158-Ile159 bond—the same site cleaved by plasmin and kallikrein—generating enzymatically active two-chain uPA; this activation is blocked by the CTSB-specific inhibitor E-64; cathepsin B can also activate receptor-bound pro-uPA on tumor cell surfaces, whereas cathepsin D cannot activate pro-uPA.","method":"In vitro enzymatic assay with purified human cathepsin B, N-terminal amino acid sequencing of cleavage products, inhibitor (E-64) blocking, receptor-binding assay on U937 cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro cleavage with mutagenic/inhibitor controls and sequencing of cleavage site, >300 citations","pmids":["1900515"],"is_preprint":false},{"year":1992,"finding":"Cathepsin B cleaves the cartilage proteoglycan aggrecan at a single Gly-Val bond within the interglobular domain, only three amino acids C-terminal to the metalloproteinase (stromelysin) cleavage site, generating distinct G1 and G2 fragments.","method":"In vitro protease digestion of purified aggrecan G1-G2 domain fragment, SDS-PAGE fragment analysis, comparison with MMP-2, MMP-7, MMP-9 cleavage patterns","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro cleavage with site identification, >250 citations","pmids":["1326552"],"is_preprint":false},{"year":1984,"finding":"Cystatin C (gamma-trace) was identified as the tightest-binding protein inhibitor of cathepsin B discovered at the time, also potently inhibiting cathepsins H and L and the plant cysteine proteinases papain and ficin, establishing a physiological role for extracellular cystatins in regulating cathepsin B activity.","method":"Enzyme inhibition kinetics with purified proteins, determination of inhibition constants","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 — direct in vitro inhibition kinetics with purified proteins, >280 citations","pmids":["6203523"],"is_preprint":false},{"year":2001,"finding":"Cathepsin B (released from lysosomes during necrosis) cleaves PARP-1 to generate a ~50 kDa fragment distinct from the apoptotic 89 kDa caspase-3 fragment; this cleavage is not inhibited by the broad-spectrum caspase inhibitor zVAD-fmk and is reproduced by purified cathepsin B in vitro on affinity-purified bovine PARP-1.","method":"In vitro cleavage assay with purified lysosomal proteases (cathepsins B, D, G) on affinity-purified PARP-1; lysosomal-rich fractions from Jurkat cells; comparison with necrotic cell lysates","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro cleavage with purified enzyme, confirmed in cellular necrosis model, >280 citations","pmids":["11536009"],"is_preprint":false},{"year":2005,"finding":"Cathepsin B (and to a lesser extent cathepsin L) is required for endosomal proteolytic cleavage of Ebola virus glycoprotein GP1, a step essential for viral entry into host cells; selective protease inhibitors and protease-deficient cell lines confirmed that CatB mediates GP1 cleavage enabling membrane fusion, and CatB/CatL inhibitors reduce multiplication of infectious Ebola virus-Zaire in culture.","method":"Selective protease inhibitors, protease-deficient cell lines, VSV pseudotype entry assay, biochemical proteolysis assay of EboV GP, infectious EboV-Zaire multiplication assay","journal":"Science","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods (inhibitors, KO cell lines, biochemical assays, infectious virus), >690 citations","pmids":["15831716"],"is_preprint":false},{"year":1991,"finding":"In Alzheimer disease brains, cathepsin B (along with other lysosomal hydrolases) accumulates abnormally in neuronal perikarya and is found extracellularly in senile plaques, co-localizing with degenerating neuronal processes; in control brains cathepsin B is restricted to intracellular lysosomal compartments, establishing that lysosomal dysfunction and cathepsin B mis-localization occur in AD neurodegeneration.","method":"Immunocytochemistry, immunoelectron microscopy, and enzyme histochemistry in human post-mortem brain tissue from AD and control subjects","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — direct localization by immunoelectron microscopy with functional implication for amyloidogenesis, >260 citations","pmids":["1837142"],"is_preprint":false},{"year":2002,"finding":"Cathepsin B is upregulated and enzymatically active in inflamed atherosclerotic lesions (but not in normal aorta or silent lesions) in apoE-knockout mice; cathepsin B activity was imaged in vivo within active atherosclerotic plaques using intravenously injectable near-infrared cathepsin B-activatable imaging beacons, confirming a role for cathepsin B proteolytic activity in vascular inflammation.","method":"In vivo near-infrared fluorescence tomographic imaging with cathepsin B-specific activatable probes, immunohistochemistry, Western blot in apoE-KO and apoE/eNOS double-KO mouse atherosclerosis models","journal":"Circulation","confidence":"High","confidence_rationale":"Tier 2 — direct in vivo activity imaging corroborated by immunohistochemistry, >265 citations","pmids":["12057992"],"is_preprint":false},{"year":2011,"finding":"Serum amyloid A (SAA) activates the NLRP3 inflammasome in macrophages through a cathepsin B-sensitive pathway (confirmed by cathepsin B inhibitor blocking IL-1β secretion) and via P2X7 receptor; SAA also induces cathepsin B secretion, identifying cathepsin B as a key effector in inflammasome-driven IL-1β maturation.","method":"Cathepsin B inhibitor treatment, siRNA knockdown of NLRP3 and ASC, ASC-KO macrophages, IL-1β ELISA, TLR2/4 blocking antibodies in human/mouse macrophages and THP-1 cells","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (KO, siRNA, inhibitors) in multiple cell types, >230 citations","pmids":["21508263"],"is_preprint":false},{"year":2014,"finding":"In transgenic mouse models of pancreatic and mammary carcinomas, cathepsin B in tumor cells and tumor-associated macrophages causally promotes tumor initiation, growth, angiogenesis, invasion, and metastasis; absence of cathepsin B enhances apoptosis; cathepsin B also associates with the tumor cell plasma membrane at elevated expression levels characteristic of cancer.","method":"Transgenic mouse models (MMTV-PyMT, RIP1-Tag2), genetic knockout of cathepsin B, tumor growth and metastasis assays, membrane fractionation","journal":"Proteomics. Clinical applications","confidence":"High","confidence_rationale":"Tier 2 — genetic loss-of-function in multiple transgenic cancer models, >280 citations","pmids":["24677670"],"is_preprint":false},{"year":2016,"finding":"Under homeostatic conditions, cathepsin B cleaves the lysosomal calcium channel MCOLN1/TRPML1, which suppresses the transcription factor TFEB and reduces expression of lysosomal and autophagy-related genes, thereby controlling the number of lysosomes and autophagosomes in the cell; the cytosolic bacterium Francisella novicida exploits this CTSB activity to suppress lysosome/autophagosome availability and enhance its intracellular survival.","method":"CTSB knockout cells and mice, lysosome/autophagosome quantification, MCOLN1 cleavage assay, TFEB reporter assay, bacterial infection models","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with multiple orthogonal readouts (channel cleavage, transcription factor activity, organelle number), replicated in vivo","pmids":["27786577"],"is_preprint":false},{"year":2016,"finding":"Skeletal muscle-secreted cathepsin B (CTSB) acts as a myokine elevated by exercise (running); recombinant CTSB application enhances BDNF and doublecortin (DCX) expression in adult hippocampal progenitor cells through a mechanism dependent on the multifunctional protein P11; in CTSB knockout mice, running fails to enhance adult hippocampal neurogenesis and spatial memory; plasma CTSB levels correlate with fitness and hippocampus-dependent memory in humans.","method":"CTSB KO mice, recombinant CTSB treatment of hippocampal progenitor cells, P11-dependency assay, hippocampal neurogenesis quantification, plasma CTSB measurement in mice/monkeys/humans, treadmill exercise protocol","journal":"Cell metabolism","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with defined neurogenic phenotype, recombinant protein rescue, translational validation in primates and humans, >440 citations","pmids":["27345423"],"is_preprint":false},{"year":2022,"finding":"Glucose metabolism in M2-like tumor-associated macrophages (TAMs) fuels the hexosamine biosynthetic pathway, leading to O-GlcNAcylation of cathepsin B at serine 210 by lysosome-localized OGT (O-GlcNAc transferase); this modification elevates mature cathepsin B levels in macrophages and promotes its secretion into the tumor microenvironment, thereby driving cancer metastasis and chemoresistance.","method":"Mass spectrometry identification of O-GlcNAcylation site (Ser210), OGT KO in macrophages, glycosylation site mutagenesis, co-immunoprecipitation, in vitro and in vivo metastasis assays, patient TAM correlation analysis","journal":"Cancer cell","confidence":"High","confidence_rationale":"Tier 1–2 — MS-identified PTM site with mutagenesis validation, OGT KO, functional rescue, in vivo metastasis model, >270 citations","pmids":["36084651"],"is_preprint":false},{"year":1992,"finding":"Human CTSB was definitively mapped to chromosome 8p22-p23.1 by three independent methods: PCR analysis of human-hamster somatic cell hybrids, FISH signal comparison in fibroblasts with chromosome 8 deletions, and fluorescence in situ hybridization to metaphase spreads with cathepsin B cosmid clones, resolving a prior ambiguity with a reported 13q14 location.","method":"PCR on somatic cell hybrid DNA, interphase FISH with chromosome 8 deletion fibroblasts, metaphase FISH with cosmid clones","journal":"Human genetics","confidence":"High","confidence_rationale":"Tier 2 — three independent orthogonal mapping methods confirming chromosomal assignment","pmids":["1577456"],"is_preprint":false},{"year":2017,"finding":"Tandem duplications of a 2.62 kb overlapping genomic region upstream of CTSB—containing an active keratinocyte enhancer—segregate with keratolytic winter erythema (KWE) in South African and Norwegian families; the duplication drives increased CTSB expression in keratinocytes of affected individuals, causing erythrokeratolysis, establishing CTSB overexpression as the cause of KWE.","method":"Targeted resequencing, SNP array, whole-genome sequencing, enhancer activity assays in keratinocyte cell lines, qPCR, immunohistochemistry of palmar epidermis, ChIA-PET chromatin interaction analysis","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 — variant segregation with disease in two independent populations combined with functional enhancer validation and expression quantification","pmids":["28457472"],"is_preprint":false},{"year":2020,"finding":"A gain-of-function missense mutation in CTSB, affecting a highly conserved residue, was found in a patient with autosomal dominant diffuse palmoplantar keratoderma; protein modelling predicted increased endopeptidase activity, and a cathepsin B enzymatic assay confirmed elevated proteolytic activity of the mutant, identifying the first gain-of-function CTSB variant.","method":"Whole exome sequencing, direct sequencing, protein modelling, cathepsin B enzymatic activity assay on patient-derived material","journal":"Clinical and experimental dermatology","confidence":"Medium","confidence_rationale":"Tier 1–2 — functional enzymatic assay in a single patient, corroborated by structural modelling; single case","pmids":["32683719"],"is_preprint":false},{"year":2019,"finding":"Cancer cell-secreted CST6 enters osteoclast precursors by endocytosis and suppresses cathepsin B (CTSB) activity; loss of CTSB activity leads to up-regulation of its hydrolytic substrate SPHK1, which then suppresses osteoclast maturation by inhibiting RANKL-induced p38 activation, defining a CST6-CTSB-SPHK1 signaling axis in osteoclastogenesis and bone metastasis.","method":"In vitro osteoclastogenesis assay, in vivo bone metastasis mouse model, CTSB activity assay, SPHK1 substrate identification, p38 signaling analysis, siRNA knockdown, recombinant CST6 protein treatment","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 — multiple methods including substrate identification and in vivo validation, single lab","pmids":["34815788"],"is_preprint":false},{"year":2016,"finding":"HDAC3 deficiency in macrophages increases cathepsin B (CTSB) expression via elevated histone acetylation at the CTSB locus; over-expressed CTSB causes lysosomal degradation of RIP1 (receptor-interacting serine-threonine kinase 1), reducing TNFα-mediated NF-κB activation and impairing inflammatory response to Pseudomonas aeruginosa infection.","method":"HDAC3 macrophage-specific KO mice, RNAseq, CHIPseq, Western blot, immunofluorescence, qRT-PCR, in vivo P. aeruginosa infection model, LPS intratracheal instillation","journal":"Cell & bioscience","confidence":"Medium","confidence_rationale":"Tier 2 — genetic KO with RNAseq/CHIPseq and in vivo validation, single lab","pmids":["35658939"],"is_preprint":false},{"year":2014,"finding":"Cathepsin B promotes porcine preadipocyte differentiation by degrading fibronectin (Fn), a key extracellular matrix component and target gene of Wnt/β-catenin signaling; CTSB treatment relieved the anti-adipogenic effect of the Wnt/β-catenin activator LiCl, indicating that CTSB attenuates Wnt/β-catenin pathway activity through Fn degradation to facilitate adipogenesis.","method":"CTSB treatment of primary preadipocytes, fibronectin degradation assay, LiCl (Wnt activator) co-treatment, lipid accumulation staining, adipogenic gene expression analysis","journal":"Molecular and cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 3 — substrate identification (fibronectin) with pathway functional readout, single lab, primary cells","pmids":["24878992"],"is_preprint":false},{"year":2022,"finding":"CTSB degrades ferroportin (FPN), the main iron export protein in macrophages; oxidized LDL (ox-LDL) upregulates macrophage CTSB, which negatively regulates FPN protein levels by promoting its degradation, disrupting iron homeostasis and inducing ferroptosis, thereby promoting atherosclerotic plaque progression.","method":"Co-immunoprecipitation (CTSB-FPN interaction), CTSB knockdown and pharmacological inhibition, FPN protein stability assay, ferroptosis markers in macrophages, in vivo ApoE-KO and HFD rat AS models, single-cell transcriptome analysis of human AS tissue","journal":"Molecular and cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP binding partner identified, KD/inhibitor functional validation in vitro and in vivo, single lab","pmids":["39960586"],"is_preprint":false},{"year":2023,"finding":"Lysosomal membrane permeabilization (LMP) releases cathepsin B into the cytoplasm where it activates the NLRP3 inflammasome, leading to caspase-1-dependent pyroptosis (GSDMD-N cleavage, IL-1β/IL-18 release); in sepsis-induced acute kidney injury (LPS-treated HK-2 cells), CTSB activity is elevated, CTSB inhibition with CA074 reverses LMP-induced mitochondrial membrane potential loss and apoptosis via the mitochondrial pathway.","method":"CTSB activity assay, CA074 inhibitor, JC-1 mitochondrial membrane potential, Annexin V/PI apoptosis staining, acridine orange lysosomal staining, Western blot, CCK8, CLP mouse model, DIA proteomics","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 — inhibitor-based epistasis with multiple orthogonal cellular readouts, in vivo CLP model, single lab","pmids":["36713420"],"is_preprint":false},{"year":2019,"finding":"In arsenic-induced liver fibrosis, NaAsO2 upregulates autophagy flux, which promotes cytoplasmic cathepsin B (CTSB) release from lysosomes; cytoplasmic CTSB activates the NLRP3 inflammasome, driving hepatic stellate cell (HSC) activation; inhibition of autophagy decreases cytoplasmic CTSB and attenuates NLRP3 inflammasome activation and HSC activation.","method":"Autophagy inhibitor (chloroquine), CTSB activity assay, NLRP3 inhibitor, immunofluorescence, Western blot in HSC-t6 cells and primary rat HSCs, in vivo NaAsO2-treated rat model","journal":"Chemosphere","confidence":"Medium","confidence_rationale":"Tier 2 — pharmacological pathway dissection with multiple inhibitors in vitro and in vivo, single lab","pmids":["31669990"],"is_preprint":false},{"year":2025,"finding":"ZRANB1 mediates K33-linked deubiquitination of CTSB, stabilizing CTSB protein expression; MINPP1 modulates this deubiquitination to regulate CTSB levels and downstream ferroptosis in HBV-positive hepatocellular carcinoma cells; the MINPP1-ZRANB1-CTSB axis is active only in HBV-positive HCC cells and promotes ferroptosis via a glycolytic bypass mechanism.","method":"Immunoprecipitation, immunofluorescence, ubiquitin modification analysis (K33-linkage), CTSB expression/stability assays, in vivo xenograft experiments","journal":"Biology direct","confidence":"Medium","confidence_rationale":"Tier 2 — IP-based PTM identification with in vivo validation, single lab, novel deubiquitination site","pmids":["41035046"],"is_preprint":false},{"year":2025,"finding":"ETS1 transcription factor is expressed in septoclasts (cartilage-resorbing cells at the chondro-osseous junction) and promotes transcription of Ctsb and Mmp13 during differentiation of septoclasts from pericytes; ETS1 siRNA knockdown in primary septoclast cultures significantly reduced Ctsb and Mmp13 expression.","method":"RNA-seq of isolated septoclast and pericyte populations, ETS1 siRNA knockdown in primary septoclast cultures, immunofluorescence localization","journal":"Cell and tissue research","confidence":"Medium","confidence_rationale":"Tier 2–3 — siRNA KD with defined gene expression readout in primary cells, single lab","pmids":["40387924"],"is_preprint":false},{"year":2025,"finding":"OGT-mediated O-GlcNAcylation of cathepsin B in prefrontal astrocytes promotes CTSB maturation; reduced O-GlcNAcylation (via OGT downregulation by BXHPD treatment) lowers ROS, attenuates lysosomal membrane permeabilization and cytoplasmic CTSB leakage, and suppresses NLRP3 inflammasome activation, improving depressive-like behaviors in a corticosterone mouse model.","method":"CO-IP for OGT-CTSB interaction, O-GlcNAcylation analysis, Western blot, immunofluorescence (OGT/S100β and CTSB/LAMP1 colocalization), DHE staining for ROS, Aldh1l1-Cre/ERT2 astrocyte-specific mice, behavioral tests","journal":"Journal of ethnopharmacology","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP interaction, O-GlcNAc modification, cell-type-specific in vivo model, single lab","pmids":["41391522"],"is_preprint":false},{"year":2025,"finding":"METTL3 upregulates CTSB expression in chondrocytes by promoting m6A methylation of CTSB mRNA, as confirmed by m6A RNA immunoprecipitation and dual-luciferase reporter assays; METTL3 silencing protects against IL-1β-induced chondrocyte apoptosis, inflammation, oxidative stress, and ferroptosis, effects that are rescued by CTSB overexpression, establishing a METTL3-m6A-CTSB regulatory axis in osteoarthritis.","method":"m6A RNA immunoprecipitation (MeRIP), dual-luciferase reporter assay, METTL3 and CTSB siRNA knockdown, Western blot, flow cytometry, TUNEL, ELISA, ROS/MDA/Fe2+ assays in human chondrocytes","journal":"Journal of orthopaedic surgery and research","confidence":"Medium","confidence_rationale":"Tier 2 — MeRIP and luciferase confirm m6A regulatory mechanism, functional rescue validates axis, single lab","pmids":["41466292"],"is_preprint":false},{"year":2024,"finding":"Cathepsin B promotes microglial efferocytosis of apoptotic neurons during brain development; CTSB is enriched in microglia in high-neuronal-turnover brain regions; myeloid-specific CTSB knockdown in zebrafish led to dysmorphic microglia containing undigested dead cells and accumulation of apoptotic cells, phenocopied by global Ctsb KO in mice; live imaging revealed deficits in phagolysosomal fusion and acidification, identifying a role in lysosomal digestion rather than initial phagocytic uptake.","method":"Zebrafish myeloid-specific CTSB knockdown, global Ctsb-KO mice, live fluorescence imaging of phagolysosomal fusion and acidification, apoptosis markers (TUNEL), confocal imaging of brain regions, behavioral assessment","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — genetic KO/KD in two model organisms with live imaging of lysosomal mechanism, preprint not yet peer-reviewed","pmids":[],"is_preprint":true},{"year":2025,"finding":"Beauvericin (BEA) acts as an uncompetitive inhibitor of cathepsin B; NMR analyses confirmed direct interaction between BEA and CTSB; enzyme kinetics established uncompetitive inhibition; molecular docking identified a putative BEA binding site in human CTSB distinct from the active site; BEA significantly suppresses CTSB activity in mouse BMDCs and human iDCs.","method":"Enzyme kinetics (uncompetitive inhibitor determination), NMR spectroscopy (direct binding), molecular docking, CTSB activity assay in BMDCs and THP-1-derived iDCs","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 — enzyme kinetics plus NMR direct binding, preprint; mechanism well-defined but not yet peer-reviewed","pmids":[],"is_preprint":true},{"year":2025,"finding":"Elevated lysosomal cathepsin B levels and CTSB leakage to the cytoplasm trigger amyloidogenesis in mucopolysaccharidosis type IIIC and sialidosis mouse models; CTSB-deficient MPS IIIC mice (Hgsnat-P304L/Ctsb-/-) and mice chronically treated with brain-penetrating CTSB inhibitor E64 showed drastic reduction in neuronal Thioflavin-S-positive/APP-positive amyloid deposits and restored autophagy markers; E64 treatment rescued behavioral deficits.","method":"CTSB-deficient double-KO mice, chronic brain-penetrating E64 inhibitor treatment, Thioflavin-S staining, β-amyloid immunostaining, P62/LC3 autophagy markers, behavioral testing (hyperactivity, anxiety), immunofluorescence of cortical neurons","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — genetic KO and pharmacological inhibition with multiple amyloidogenesis readouts in vivo, preprint not peer-reviewed","pmids":[],"is_preprint":true},{"year":2024,"finding":"Extralysosomal cathepsin B in progressive multiple sclerosis brains cleaves the C-terminal domain of TAF1, a core component of the general transcription factor TFIID; loss of C-terminal TAF1 disrupts RNAPII promoter-proximal pausing at oligodendroglial myelination genes; mice lacking the C-terminal TAF1 domain exhibit MS-like brain transcriptomic signature, CNS-resident inflammation, progressive demyelination, and motor disability.","method":"Detection of TAF1 C-terminal underexpression in MS brain tissue, identification of CTSB-mediated endoproteolysis of TAF1, generation of Taf1Δ38 mice, transcriptomic analysis, immunofluorescence, behavioral assessment","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 — mechanistic link between CTSB and TAF1 cleavage shown in tissue but biochemical reconstitution not explicitly detailed; preprint, single study","pmids":[],"is_preprint":true},{"year":2025,"finding":"Cathepsin B (CTSB) cleaves the lysosomal nitrate transporter Sialin to generate a proteolytic fragment called Sialin2 that localizes to mitochondria and acts as a nitrate sensor; Sialin2 scaffolds LKB1-AMPK complexes to drive mitochondrial biogenesis and metabolic adaptation.","method":"Cryo-EM structure of Sialin2, microscale thermophoresis (MST) nitrate-binding assay, fractionation showing CTSB-dependent generation of Sialin2, AMPK complex co-immunoprecipitation, sCiSiNiS biosensor for real-time nitrate signaling","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 2 for Sialin2 characterization, but CTSB's specific role in generating Sialin2 by cleavage is stated without detailed mutagenesis/reconstitution of that specific step; preprint, single study","pmids":[],"is_preprint":true}],"current_model":"Cathepsin B (CTSB) is a lysosomal cysteine endopeptidase and dipeptidyl carboxypeptidase whose 2.15 Å crystal structure reveals an occluding loop (His110/His111) that enables exopeptidase activity and restricts cystatin binding; it cleaves multiple substrates including pro-uPA (activating plasminogen signaling), aggrecan, PARP-1, MCOLN1/TRPML1 (regulating TFEB and lysosome/autophagosome biogenesis), RIP1 (modulating NF-κB), ferroportin (disrupting macrophage iron homeostasis), fibronectin (promoting adipogenesis via Wnt/β-catenin attenuation), and the Ebola virus glycoprotein GP1 (enabling viral entry); upon lysosomal membrane permeabilization, cytoplasmic CTSB activates the NLRP3 inflammasome to drive pyroptosis and IL-1β maturation; muscle-secreted CTSB acts as an exercise-induced myokine that promotes hippocampal BDNF/DCX expression and neurogenesis via P11; its activity and stability are regulated post-translationally by O-GlcNAcylation at Ser210 (by lysosomal OGT), K33-linked deubiquitination (by ZRANB1), and m6A-mediated mRNA upregulation (by METTL3), while gain-of-function mutations cause palmoplantar keratoderma and regulatory enhancer duplications upstream of CTSB cause keratolytic winter erythema."},"narrative":{"teleology":[{"year":1984,"claim":"Before physiological regulators of cathepsin B were known, identification of cystatin C as a potent endogenous inhibitor established that cathepsin B activity is tightly controlled extracellularly by cystatin-family proteins.","evidence":"Enzyme inhibition kinetics with purified cystatin C against cathepsin B and related cysteine proteases","pmids":["6203523"],"confidence":"High","gaps":["Intracellular regulation of CTSB activity by endogenous inhibitors not addressed","Tissue-specific relevance of cystatin C–CTSB interaction not explored"]},{"year":1986,"claim":"Cloning of preprocathepsin B cDNA revealed the full precursor architecture—signal peptide, propeptide, mature chain, and C-terminal extension—establishing the multi-step processing pathway required for enzyme maturation.","evidence":"cDNA cloning and sequencing from human hepatoma and kidney libraries","pmids":["3463996"],"confidence":"High","gaps":["Precise sites and proteases responsible for each processing step not identified","Regulation of propeptide removal in different tissues unknown"]},{"year":1991,"claim":"The 2.15 Å crystal structure resolved the long-standing question of how cathepsin B achieves both endo- and exopeptidase activity: an occluding loop (His110/His111) blocks the primed subsites, anchoring substrate C-termini for dipeptidyl carboxypeptidase activity and sterically preventing cystatin binding.","evidence":"X-ray crystallography at 2.15 Å resolution with structural comparison to papain/actinidin","pmids":["1868826"],"confidence":"High","gaps":["Dynamic behavior of the occluding loop and endo/exo switching mechanism not captured by static structure","Structural basis for substrate selectivity at the S2 subsite incompletely characterized"]},{"year":1991,"claim":"Demonstration that cathepsin B activates pro-uPA at the same Lys158-Ile159 bond as plasmin established CTSB as a direct activator of pericellular plasminogen signaling, linking it to extracellular matrix remodeling and tumor invasion.","evidence":"In vitro cleavage of purified pro-uPA with N-terminal sequencing and E-64 inhibitor blocking; cell surface activation on U937 cells","pmids":["1900515"],"confidence":"High","gaps":["In vivo relevance of CTSB-mediated pro-uPA activation not demonstrated","Relative contribution versus other activating proteases unclear"]},{"year":1991,"claim":"Immunoelectron microscopy of Alzheimer disease brains revealed cathepsin B mislocalization from lysosomes to neuronal perikarya and senile plaques, establishing lysosomal dysfunction and CTSB redistribution as features of AD neurodegeneration.","evidence":"Immunocytochemistry and immunoelectron microscopy on human post-mortem AD vs. control brain tissue","pmids":["1837142"],"confidence":"High","gaps":["Whether CTSB mislocalization is causative or consequential in AD pathology not resolved","Specific CTSB substrates contributing to amyloidogenesis not identified in this study"]},{"year":1992,"claim":"Identification of CTSB cleavage of aggrecan at a specific Gly-Val bond in the interglobular domain, near the MMP cleavage site, established cathepsin B as a cartilage-degrading protease relevant to joint pathology.","evidence":"In vitro proteolysis of purified aggrecan G1-G2 domain with fragment analysis by SDS-PAGE","pmids":["1326552"],"confidence":"High","gaps":["In vivo contribution of CTSB to aggrecanolysis versus MMPs not quantified"]},{"year":2001,"claim":"Discovery that cathepsin B (released during necrosis) generates a distinct ~50 kDa PARP-1 fragment not produced by caspases revealed a necrosis-specific proteolytic signature and distinguished cathepsin-mediated cell death from apoptosis.","evidence":"In vitro cleavage of purified PARP-1 by cathepsin B; comparison with necrotic cell lysates; caspase inhibitor (zVAD-fmk) insensitivity","pmids":["11536009"],"confidence":"High","gaps":["PARP-1 cleavage site by CTSB not mapped","Functional consequences of the ~50 kDa PARP-1 fragment not characterized"]},{"year":2005,"claim":"Cathepsin B was shown to be required for endosomal cleavage of Ebola virus GP1 to enable membrane fusion and viral entry, defining CTSB as a host-factor dependency for filovirus infection.","evidence":"Selective protease inhibitors, protease-deficient cell lines, VSV pseudotype entry assays, and infectious Ebola virus multiplication assays","pmids":["15831716"],"confidence":"High","gaps":["Precise cleavage sites on GP1 by CTSB not fully mapped","Extent of cathepsin L redundancy in vivo not settled"]},{"year":2011,"claim":"Demonstration that cathepsin B mediates SAA-induced NLRP3 inflammasome activation in macrophages established the paradigm that lysosomal membrane permeabilization and cytoplasmic CTSB release are upstream triggers of inflammasome-driven IL-1β maturation.","evidence":"Cathepsin B inhibitor, siRNA knockdown of NLRP3/ASC, ASC-KO macrophages, IL-1β ELISA in human and mouse macrophages","pmids":["21508263"],"confidence":"High","gaps":["Whether CTSB directly cleaves an NLRP3 component or acts indirectly not resolved","Relative contribution of CTSB versus other lysosomal cathepsins to inflammasome activation unclear"]},{"year":2014,"claim":"Genetic deletion of cathepsin B in transgenic mouse cancer models demonstrated a causal role in tumor initiation, growth, angiogenesis, and metastasis, with CTSB associating with the tumor cell plasma membrane at elevated expression, establishing its non-redundant function in cancer progression.","evidence":"CTSB knockout in MMTV-PyMT and RIP1-Tag2 transgenic mouse models with tumor growth, metastasis, and apoptosis quantification","pmids":["24677670"],"confidence":"High","gaps":["Specific tumor-promoting substrates cleaved by membrane-associated CTSB not identified","Cell-type-specific contributions (tumor vs. stroma) not fully dissected"]},{"year":2016,"claim":"CTSB was found to cleave the lysosomal calcium channel MCOLN1/TRPML1, thereby suppressing TFEB activation and limiting lysosome/autophagosome biogenesis under homeostatic conditions—a mechanism co-opted by intracellular bacteria to enhance survival.","evidence":"CTSB KO cells and mice, MCOLN1 cleavage assay, TFEB reporter, lysosome/autophagosome quantification, Francisella novicida infection model","pmids":["27786577"],"confidence":"High","gaps":["Cleavage site on MCOLN1 not mapped","Whether CTSB regulation of TFEB is a general homeostatic mechanism or context-specific not fully established"]},{"year":2016,"claim":"Identification of cathepsin B as an exercise-induced myokine that promotes hippocampal BDNF/DCX expression and neurogenesis through P11 provided the first direct mechanistic link between a muscle-secreted protease and exercise-dependent cognitive enhancement.","evidence":"CTSB KO mice fail to show running-induced neurogenesis; recombinant CTSB rescues hippocampal progenitor differentiation via P11; plasma CTSB correlates with fitness in humans","pmids":["27345423"],"confidence":"High","gaps":["Mechanism by which circulating CTSB crosses the blood-brain barrier not established","Direct proteolytic target of CTSB that engages P11 signaling not identified"]},{"year":2017,"claim":"Tandem duplications of a keratinocyte enhancer upstream of CTSB were shown to segregate with keratolytic winter erythema in two independent populations, establishing CTSB overexpression as the genetic cause of KWE.","evidence":"Targeted resequencing, WGS, enhancer activity assays in keratinocytes, qPCR, ChIA-PET chromatin interaction analysis in South African and Norwegian families","pmids":["28457472"],"confidence":"High","gaps":["Downstream keratinocyte substrates whose excessive cleavage causes erythrokeratolysis not identified","Whether enhancer duplication alters CTSB expression in non-epidermal tissues unknown"]},{"year":2020,"claim":"A gain-of-function missense mutation in CTSB was identified as causing autosomal dominant diffuse palmoplantar keratoderma, confirmed by elevated enzymatic activity, representing the first gain-of-function CTSB point mutation linked to Mendelian disease.","evidence":"Whole exome sequencing, protein modeling, and cathepsin B enzymatic activity assay on patient-derived material (single case)","pmids":["32683719"],"confidence":"Medium","gaps":["Single patient without independent replication","Precise mechanism by which increased CTSB activity causes keratoderma not determined","The specific residue affected and structural consequence not fully validated by crystal structure"]},{"year":2022,"claim":"Discovery that O-GlcNAcylation of cathepsin B at Ser210 by lysosome-localized OGT stabilizes and promotes CTSB secretion in tumor-associated macrophages revealed a metabolic post-translational control mechanism linking glucose metabolism to metastasis.","evidence":"Mass spectrometry identification of Ser210 O-GlcNAcylation, site-directed mutagenesis, OGT KO macrophages, in vivo metastasis assays","pmids":["36084651"],"confidence":"High","gaps":["Whether O-GlcNAcylation at Ser210 affects catalytic activity directly or only protein stability not distinguished","Generalizability beyond tumor-associated macrophages not tested"]},{"year":2022,"claim":"CTSB was shown to degrade ferroportin in ox-LDL-stimulated macrophages, disrupting iron export and inducing ferroptosis, providing a direct mechanistic link between CTSB and iron-dependent cell death in atherosclerosis.","evidence":"Co-immunoprecipitation of CTSB-FPN, CTSB knockdown/inhibition rescuing FPN levels, ferroptosis markers, ApoE-KO mouse and rat atherosclerosis models","pmids":["39960586"],"confidence":"Medium","gaps":["Ferroportin cleavage site by CTSB not mapped","Co-IP without reciprocal validation reported","Single lab study"]},{"year":2025,"claim":"Identification of K33-linked deubiquitination of CTSB by ZRANB1, modulated by MINPP1, revealed a previously unknown ubiquitin-dependent regulatory layer controlling CTSB protein stability and ferroptosis in HBV-positive hepatocellular carcinoma.","evidence":"Immunoprecipitation, K33-linkage ubiquitin analysis, CTSB stability assays, in vivo xenograft experiments","pmids":["41035046"],"confidence":"Medium","gaps":["Specific ubiquitination sites on CTSB not mapped","Whether K33-linked regulation operates beyond HBV-positive HCC context unknown","Single lab study"]},{"year":2025,"claim":"METTL3-mediated m6A methylation of CTSB mRNA was shown to upregulate CTSB expression in chondrocytes, with CTSB overexpression rescuing ferroptosis and inflammatory phenotypes upon METTL3 silencing, establishing an epitranscriptomic regulatory axis for CTSB in osteoarthritis.","evidence":"m6A RIP, dual-luciferase reporter assay, METTL3/CTSB siRNA knockdown with rescue, ferroptosis and apoptosis readouts in human chondrocytes","pmids":["41466292"],"confidence":"Medium","gaps":["Specific m6A site(s) on CTSB mRNA not mapped at nucleotide resolution","In vivo validation in OA models not yet reported","Single lab study"]},{"year":null,"claim":"Key unresolved questions include: (1) the direct molecular mechanism by which cytoplasmic CTSB activates the NLRP3 inflammasome (whether via direct NLRP3 cleavage or indirect signaling), (2) the structural basis for the occluding loop's dynamic endo-to-exopeptidase switching in physiological contexts, and (3) the identity of blood-brain barrier transit mechanisms enabling circulating CTSB to promote hippocampal neurogenesis.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural study of the occluding loop in a dynamic or membrane-associated context","Direct NLRP3 cleavage by CTSB never reconstituted with purified components","BBB crossing mechanism for myokine CTSB undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,3,5,6,11,13,17,19,20]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,2,3,5,6,11]}],"localization":[{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[0,7,11,13,21,22,25]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[5,9,21,22]},{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[12,13]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[10]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[9,21,22]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,13]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[11,22]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[5,10,21]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[10,15,16]},{"term_id":"R-HSA-1474244","term_label":"Extracellular matrix organization","supporting_discovery_ids":[3,19]}],"complexes":[],"partners":["CST3","MCOLN1","OGT","ZRANB1","FPN1","NLRP3","P11"],"other_free_text":[]},"mechanistic_narrative":"Cathepsin B is a lysosomal cysteine protease with dual endopeptidase and dipeptidyl carboxypeptidase activity, conferred by a unique occluding loop (His110/His111) that anchors substrate C-termini and restricts cystatin-family inhibitor access [PMID:1868826, PMID:6203523]. It processes a broad spectrum of substrates including pro-uPA, aggrecan, PARP-1, MCOLN1/TRPML1 (thereby controlling TFEB-dependent lysosome/autophagosome biogenesis), ferroportin, fibronectin, and Ebola virus glycoprotein GP1 [PMID:1900515, PMID:1326552, PMID:11536009, PMID:27786577, PMID:15831716, PMID:39960586]; upon lysosomal membrane permeabilization it activates the NLRP3 inflammasome to drive pyroptosis and IL-1β maturation [PMID:21508263, PMID:36713420]. Beyond its intracellular roles, muscle-secreted CTSB functions as an exercise-induced myokine that promotes hippocampal BDNF expression and neurogenesis via P11 [PMID:27345423]; its activity is regulated post-translationally by O-GlcNAcylation at Ser210 and by K33-linked deubiquitination [PMID:36084651, PMID:41035046], and gain-of-function mutations or upstream enhancer duplications cause palmoplantar keratoderma and keratolytic winter erythema, respectively [PMID:32683719, PMID:28457472]."},"prefetch_data":{"uniprot":{"accession":"P07858","full_name":"Cathepsin B","aliases":["APP secretase","APPS","Cathepsin B1"],"length_aa":339,"mass_kda":37.8,"function":"Thiol protease which is believed to participate in intracellular degradation and turnover of proteins (PubMed:12220505). Cleaves matrix extracellular phosphoglycoprotein MEPE (PubMed:12220505). Involved in the solubilization of cross-linked TG/thyroglobulin in the thyroid follicle lumen (By similarity). Has also been implicated in tumor invasion and metastasis (PubMed:3972105)","subcellular_location":"Lysosome; Melanosome; Secreted, extracellular space; Apical cell membrane","url":"https://www.uniprot.org/uniprotkb/P07858/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CTSB","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CTSB","total_profiled":1310},"omim":[{"mim_id":"619389","title":"SPINOCEREBELLAR ATAXIA, AUTOSOMAL RECESSIVE 29; SCAR29","url":"https://www.omim.org/entry/619389"},{"mim_id":"617322","title":"SH3KBP1-BINDING PROTEIN 1; SHKBP1","url":"https://www.omim.org/entry/617322"},{"mim_id":"616967","title":"THIOREDOXIN DOMAIN-CONTAINING PROTEIN 17; TXNDC17","url":"https://www.omim.org/entry/616967"},{"mim_id":"616064","title":"TUBULOINTERSTITIAL NEPHRITIS ANTIGEN-LIKE PROTEIN 1; TINAGL1","url":"https://www.omim.org/entry/616064"},{"mim_id":"610034","title":"VPS33A CORE SUBUNIT OF CORVET AND HOPS COMPLEXES; VPS33A","url":"https://www.omim.org/entry/610034"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Vesicles","reliability":"Supported"},{"location":"Nucleoli","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"thyroid gland","ntpm":991.2}],"url":"https://www.proteinatlas.org/search/CTSB"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P07858","domains":[{"cath_id":"3.90.70.10","chopping":"27-336","consensus_level":"high","plddt":95.8157,"start":27,"end":336}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P07858","model_url":"https://alphafold.ebi.ac.uk/files/AF-P07858-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P07858-F1-predicted_aligned_error_v6.png","plddt_mean":92.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CTSB","jax_strain_url":"https://www.jax.org/strain/search?query=CTSB"},"sequence":{"accession":"P07858","fasta_url":"https://rest.uniprot.org/uniprotkb/P07858.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P07858/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P07858"}},"corpus_meta":[{"pmid":"27786577","id":"PMC_27786577","title":"Regulation of lysosomal dynamics and autophagy by 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published in high-impact journal\",\n      \"pmids\": [\"27786577\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cancer cell-derived CST6 (cystatin 6) enters osteoclasts by endocytosis and suppresses CTSB cysteine protease activity, leading to upregulation of the CTSB hydrolytic substrate SPHK1; SPHK1 in turn suppresses osteoclast maturation by inhibiting RANKL-induced p38 activation, defining a CST6-CTSB-SPHK1 signaling axis in osteoclast differentiation.\",\n      \"method\": \"In vitro osteoclastogenesis assay, in vivo metastasis model, CTSB inhibition, substrate identification (SPHK1), p38 signaling readout\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (in vitro and in vivo) with specific substrate and downstream pathway identified\",\n      \"pmids\": [\"34815788\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In hepatocytes, ROS-induced lysosomal membrane permeabilization (LMP) causes CTSB leakage into the cytoplasm, where it activates the NLRP3 inflammasome leading to pyroptosis (caspase-1 cleavage, GSDMD-N, IL-1β, IL-18 production); this pathway is upstream of NLRP3 and downstream of mitochondrial damage/ROS.\",\n      \"method\": \"CTSB inhibitor (CA-074 Me), NLRP3 inhibitor (MCC950), mitophagy inhibitor, siRNA knockdown, acridine orange staining for LMP, Western blot for NLRP3/GSDMD/caspase-1\",\n      \"journal\": \"Journal of agricultural and food chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple inhibitors and siRNA with specific pathway dissection; replicated across in vitro and in vivo models\",\n      \"pmids\": [\"37141464\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HDAC3 deficiency in macrophages leads to elevated CTSB expression (through elevated histone acetylation), and over-expressed CTSB degrades RIP1 (TNFRSF-interacting serine-threonine kinase 1), thereby reducing TNFα-mediated NF-κB activation and inflammatory responses.\",\n      \"method\": \"HDAC3 knockout macrophages, RNAseq, Western blot for RIP1 degradation, NF-κB activity assay, in vivo mouse infection model\",\n      \"journal\": \"Cell & Bioscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — specific substrate (RIP1) identified, mechanism linked to NF-κB pathway, validated in vivo\",\n      \"pmids\": [\"35658939\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"LPS-induced lysosomal membrane permeabilization in renal tubular epithelial (HK-2) cells causes CTSB release into the cytoplasm, which activates the mitochondrial apoptosis pathway (inhibition of mitochondrial membrane potential, activation of intrinsic apoptosis); CTSB inhibitor CA074 reverses this.\",\n      \"method\": \"CTSB activity assay, CA074 inhibitor, acridine orange staining for LMP, JC-1 for mitochondrial membrane potential, Annexin V/PI for apoptosis, CCK8\",\n      \"journal\": \"Frontiers in Immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple assays linking CTSB activity to mitochondrial apoptosis; single lab study\",\n      \"pmids\": [\"36713420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CTSB cleaves the nitrate transporter Sialin to generate a proteolytic fragment (Sialin2) that localizes to mitochondria and acts as a nitrate sensor, scaffolding LKB1-AMPK complexes to drive metabolic adaptation.\",\n      \"method\": \"Cryo-EM structural analysis, microscale thermophoresis (MST) binding assay, cell fractionation/localization, AMPK activation readout\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure and in vitro binding; preprint, not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CTSB degrades ferroportin (FPN) in macrophages, disrupting iron homeostasis and inducing ferroptosis; this mechanism promotes atherosclerosis plaque progression.\",\n      \"method\": \"Co-IP/binding target identification (FPN as CTSB substrate), CTSB knockdown and pharmacological inhibition, Western blot for FPN protein levels, ferroptosis assays in macrophages, ApoE KO and HFD rat in vivo models\",\n      \"journal\": \"Molecular and cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — substrate (FPN) identified, degradation mechanism shown, validated in multiple in vivo models; single lab\",\n      \"pmids\": [\"39960586\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CTSB promotes lysosomal NLRP3 inflammasome-mediated pyroptosis in vascular endothelial cells via NF-κB activation; ox-LDL-induced Na+/Ca2+ overload causes lysosomal damage and CTSB release into cytosol, which activates NF-κB/NLRP3 to mediate pyroptosis.\",\n      \"method\": \"Lentiviral CTSB overexpression and silencing, NF-κB activation assay, Western blot for NLRP3/caspase-1/GSDMD, ion flux measurements\",\n      \"journal\": \"Biochemical pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — specific mechanism (NF-κB activation downstream of cytosolic CTSB) with gain- and loss-of-function; single lab\",\n      \"pmids\": [\"40058708\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CTSB-mediated NLRP3 inflammasome activation (through lysosomal membrane permeabilization and CTSB cytoplasmic leakage) drives hepatic stellate cell activation in arsenic-induced liver fibrosis; inhibition of autophagy decreases cytoplasmic CTSB and attenuates NLRP3 inflammasome activation.\",\n      \"method\": \"NaAsO2 treatment in vivo/in vitro, autophagy inhibitor, CTSB activity measurement, NLRP3 inflammasome assay, collagen deposition quantification\",\n      \"journal\": \"Chemosphere\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — autophagic-CTSB-NLRP3 pathway dissected with multiple inhibitors; single lab, both in vivo and in vitro\",\n      \"pmids\": [\"31669990\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In cardiomyocytes, nicotine decreases lysosomal CTSB activity, impairing autophagy flux (evidenced by LC3-II and p62 accumulation), and CTSB activity reduction connects to increased ROS and activation of p38MAPK and JNK pathways, forming a feedback loop regulating autophagy and cardiac hypertrophy.\",\n      \"method\": \"CTSB activity assay in lysosomes, ROS scavenger (NAC), specific p38MAPK and JNK inhibitors, LC3-II/p62 Western blot, NRVM hypertrophy model\",\n      \"journal\": \"International Journal of Biological Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — lysosomal CTSB activity linked to ROS/MAPK/autophagy pathway with pharmacological dissection; single lab\",\n      \"pmids\": [\"32398966\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CTSB secreted from lysosomes promotes preadipocyte differentiation by degrading fibronectin, a key ECM component that normally activates Wnt/β-catenin signaling; CTSB degrades fibronectin to attenuate Wnt/β-catenin anti-adipogenic signaling.\",\n      \"method\": \"Fibronectin degradation assay, Wnt/β-catenin activation (LiCl treatment), lipid accumulation and adipogenic gene expression in porcine preadipocytes\",\n      \"journal\": \"Molecular and cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — substrate (fibronectin) identified, pathway (Wnt/β-catenin) linked; single lab with moderate mechanistic follow-up\",\n      \"pmids\": [\"24878992\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Nuclear translocation of CTSB in retinoblastoma cells inhibits BRCA1 expression (promoting DNA damage and cell cycle arrest) and activates the STAT3/STING1 pathway to induce lysosomal stress, leading to ferroptosis and autophagy.\",\n      \"method\": \"CTSB nuclear translocation (Western blot, immunofluorescence), comet assay for DNA damage, BRCA1 expression analysis, STAT3/STING1 pathway assessment, flow cytometry for cell cycle/apoptosis\",\n      \"journal\": \"Molecular biotechnology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — multiple readouts but mechanistic chain partially indirect; single lab, no in vitro reconstitution\",\n      \"pmids\": [\"38159170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The transcription factor ETS1 directly promotes expression of CTSB (and MMP13) during differentiation of septoclasts from pericytes; siRNA knockdown of ETS1 in primary septoclast cultures significantly reduced CTSB and MMP13 expression.\",\n      \"method\": \"ETS1 siRNA knockdown, RNA-seq, RT-PCR, immunofluorescence in tibial growth plate\",\n      \"journal\": \"Cell and tissue research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — specific transcription factor (ETS1) identified as regulator of CTSB expression with loss-of-function validation\",\n      \"pmids\": [\"40387924\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MINPP1 stabilizes CTSB protein by modulating K33-linked deubiquitination of CTSB through the deubiquitinase ZRANB1, thereby regulating ferroptosis in HBV-positive hepatocellular carcinoma cells.\",\n      \"method\": \"Immunoprecipitation, ubiquitin modification analysis, ZRANB1 deubiquitinase identification, in vivo tumor models\",\n      \"journal\": \"Biology direct\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — specific ubiquitination site and writer/eraser (ZRANB1) identified by IP and modification analysis; single lab\",\n      \"pmids\": [\"41035046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"METTL3 upregulates CTSB expression by promoting m6A methylation of CTSB mRNA, contributing to chondrocyte injury in osteoarthritis; this was confirmed by m6A RNA immunoprecipitation and dual-luciferase reporter assay.\",\n      \"method\": \"m6A RNA immunoprecipitation, dual-luciferase reporter assay, METTL3 and CTSB siRNA knockdown, Western blot, flow cytometry\",\n      \"journal\": \"Journal of orthopaedic surgery and research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — epigenetic modification (m6A) of CTSB mRNA identified with writer (METTL3) using direct biochemical assays\",\n      \"pmids\": [\"41466292\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OGT (O-GlcNAc transferase) interacts with CTSB and O-GlcNAcylates it; this modification impairs CTSB maturation, and reducing O-GlcNAcylation decreases ROS and LMP, thereby reducing CTSB cytoplasmic leakage and NLRP3 inflammasome activation in astrocytes.\",\n      \"method\": \"Co-IP for OGT-CTSB interaction, O-GlcNAcylation assay, immunofluorescence for CTSB/LAMP1 colocalization, NLRP3 inflammasome activation readout\",\n      \"journal\": \"Journal of ethnopharmacology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP and modification assay for OGT-CTSB interaction; single lab, limited mechanistic depth\",\n      \"pmids\": [\"41391522\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Alcohol-induced phosphorylation of MLKL causes P-MLKL to translocate to the lysosomal membrane, inducing LMP and CTSB cytoplasmic leakage, which then activates the NLRP3 inflammasome and pyroptosis in hepatocytes; OPCs inhibit CTSB leakage by preventing MLKL phosphorylation via ROS scavenging.\",\n      \"method\": \"Western blot for P-MLKL, acridine orange staining for LMP, CTSB activity assay, NLRP3 inhibitor MCC950, CTSB inhibitor CA-074 Me, in vivo mouse model\",\n      \"journal\": \"Phytomedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — specific upstream regulator (P-MLKL → LMP → CTSB) identified with pharmacological dissection in vitro and in vivo\",\n      \"pmids\": [\"39612889\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cathepsin B is a microglial-specific lysosomal protease essential for neuronal efferocytosis during brain development; myeloid-specific CTSB knockdown in zebrafish and global Ctsb KO in mice leads to dysmorphic microglia with undigested apoptotic cells, accumulation of apoptotic cells in brain tissue, and behavioral impairments, with deficits in phagolysosomal fusion and acidification.\",\n      \"method\": \"Myeloid-specific CTSB knockdown (zebrafish siRNA), Ctsb global KO mouse, live imaging for phagolysosomal fusion/acidification, apoptosis markers, behavioral testing\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-type specific KD and KO in two model organisms, live imaging with functional readout; orthogonal methods across species\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CTSB and CTSL (cathepsin L) are essential for survival of Purkinje cells in the cerebellum; nervous system-specific double knockout mice show selective Purkinje cell loss, accumulation of ubiquitin-positive structures reflecting impaired autophagy-lysosomal degradation, motor dysfunction, and activation of astrocytes/microglia in affected regions.\",\n      \"method\": \"Conditional double KO mouse (CTSBflox/flox; CTSLflox/flox; Nestin-Cre), electron microscopy, ubiquitin immunostaining, behavioral testing, autophagosome quantification\",\n      \"journal\": \"The American Journal of Pathology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — nervous system-specific conditional double KO with multiple orthogonal readouts (EM, IHC, behavior); rigorous mechanistic characterization\",\n      \"pmids\": [\"40320169\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Extralysosomal CTSB cleaves the C-terminal region of TAF1 (a core TFIID component), contributing to TAF1 underdetection in progressive multiple sclerosis brains; loss of C-terminal TAF1 impairs RNAPII promoter-proximal pausing and regulation of oligodendroglial myelination genes.\",\n      \"method\": \"Endoproteolysis assay in MS brain samples, Taf1 C-terminal deletion mouse model (Taf1d38), brain transcriptomic profiling, RNAPII pausing analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — specific substrate (TAF1) cleavage by CTSB identified with in vivo model showing mechanistic consequence; preprint only\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Elevated lysosomal CTSB levels and CTSB leakage into the cytoplasm trigger amyloidogenesis in neurons of mucopolysaccharidosis type IIIC and sialidosis mouse models; CTSB-deficient MPS IIIC mice and mice chronically treated with the irreversible brain-penetrant CTSB inhibitor E64 showed drastic reduction of Thioflavin-S-positive/APP-positive neuronal deposits and restored autophagy.\",\n      \"method\": \"Ctsb-/- genetic cross, chronic E64 (irreversible CTSB inhibitor) treatment, Thioflavin-S staining, beta-amyloid immunostaining, P62/LC3 autophagy markers, behavioral rescue\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — irreversible pharmacological inhibition plus genetic KO (two orthogonal approaches) with specific amyloid readout; replicated across two LSD models\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Beauvericin (BEA) directly inhibits CTSB activity through an uncompetitive inhibition mechanism; NMR analysis shows direct BEA-CTSB interaction, enzyme kinetics confirm uncompetitive inhibition, and molecular docking identifies a putative binding site in human CTSB.\",\n      \"method\": \"Enzyme kinetics (uncompetitive inhibition analysis), NMR for direct protein-ligand interaction, molecular docking, cell-based CTSB activity assay in BMDCs and iDCs\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzyme kinetics with NMR binding confirmation; rigorous mechanistic characterization of inhibition mode\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"A gain-of-function missense mutation in CTSB results in increased cathepsin B endopeptidase proteolytic activity, causing a dominant form of diffuse palmoplantar keratoderma; protein modelling and a direct cathepsin B enzymatic assay confirmed the increased activity.\",\n      \"method\": \"Whole exome sequencing, protein structural modelling, cathepsin B enzymatic activity assay on patient-derived material\",\n      \"journal\": \"Clinical and experimental dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — direct enzymatic assay confirming gain-of-function; single patient/single lab\",\n      \"pmids\": [\"32683719\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Tandem duplications of an enhancer element upstream of CTSB in keratinocytes cause increased CTSB expression in the stratum granulosum, resulting in keratolytic winter erythema (KWE); the enhancer activity correlated with CTSB expression in differentiating keratinocytes.\",\n      \"method\": \"Targeted resequencing, SNP array, whole-genome sequencing, enhancer activity assays in keratinocyte cell lines, qPCR, immunohistochemistry\",\n      \"journal\": \"American Journal of Human Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — enhancer duplication mechanistically linked to CTSB overexpression with functional validation across two independent patient cohorts (South African and Norwegian)\",\n      \"pmids\": [\"28457472\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In late-onset Krabbe disease, GALC malfunction (reduced ceramide levels) leads to decreased activity (not expression) of CTSB and CTSD in lysosomal fractions of patient fibroblasts, suggesting a functional interplay between lysosomal enzymes GALC, CTSB, CTSD, and GCase.\",\n      \"method\": \"Lysosomal fractionation, CTSB/CTSD enzymatic activity assays, expression level analysis (Western blot), patient fibroblasts\",\n      \"journal\": \"Annals of clinical and translational neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct enzymatic activity measurement in lysosomal fractions with expression controls; single case study\",\n      \"pmids\": [\"38837642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In TNBC cell lines, CTSB is not secreted but functions in a cell-intrinsic lysosomal role; CRISPR knockout of CTSB reveals cell-line specific effects on invasion and chemotherapy sensitivity, with differential downstream mTOR and Akt activation explaining the phenotypic differences.\",\n      \"method\": \"CRISPR knockout, 3D invasion assay, cell viability assays with chemotherapy drugs, mTOR/Akt pathway Western blot, secretion assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR KO in two cell lines with multiple orthogonal readouts; preprint, single lab\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"CTSB is a lysosomal cysteine protease that, under homeostatic conditions, cleaves substrates including MCOLN1/TRPML1 (suppressing TFEB-mediated lysosome biogenesis) and fibronectin (attenuating Wnt/β-catenin signaling); upon lysosomal membrane permeabilization—triggered by ROS, LMP, or cellular stress—CTSB leaks into the cytoplasm where it activates the NLRP3 inflammasome (promoting pyroptosis), degrades substrates such as RIP1 (dampening NF-κB signaling), FPN (disrupting iron homeostasis and inducing ferroptosis), TAF1 (impairing transcription at myelination genes), and promotes amyloidogenesis; in specialized contexts CTSB is regulated transcriptionally by ETS1, post-translationally via METTL3-mediated m6A modification and ZRANB1-mediated K33-linked deubiquitination, and its proteolytic activity is governed by endogenous inhibitors (cystatins such as CST6) and can be gain-of-function mutated to cause skin disease; in microglia, CTSB is essential for phagolysosomal fusion and efficient neuronal efferocytosis during brain development.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1991,\n      \"finding\": \"X-ray crystal structure of human liver cathepsin B refined to 2.15 Å revealed the structural basis for its dual endo- and exopeptidase activity: an 'occluding loop' containing His110 and His111 blocks primed subsites, anchoring the C-terminal carboxylate of substrates and explaining dipeptidyl carboxypeptidase activity; the active-site Cys29 and Glu245 in the S2 subsite favor basic P2 side chains; the occluding loop also prevents cystatin-like inhibitors from binding as they do to papain.\",\n      \"method\": \"X-ray crystallography (2.15 Å resolution), Patterson search and heavy atom replacement, structural comparison with papain/actinidin\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution crystal structure with detailed active-site analysis, foundational structural paper with >500 citations\",\n      \"pmids\": [\"1868826\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1986,\n      \"finding\": \"Cloning and sequencing of human and mouse preprocathepsin B cDNAs revealed a 339-amino-acid precursor comprising a 17-residue signal peptide, a 62-residue propeptide, 254 residues of mature cathepsin B, and a 6-residue C-terminal extension; the propeptide contains a conserved glycosylation site and single cysteine, and comparative analysis suggested multi-step processing with possible active intermediate forms.\",\n      \"method\": \"cDNA cloning from human hepatoma and kidney libraries, nucleotide sequencing, comparative sequence analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — primary sequence determination from cDNA, foundational paper >280 citations\",\n      \"pmids\": [\"3463996\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"Purified human cathepsin B cleaves pro-uPA (single-chain urokinase-type plasminogen activator) at the Lys158-Ile159 bond—the same site cleaved by plasmin and kallikrein—generating enzymatically active two-chain uPA; this activation is blocked by the CTSB-specific inhibitor E-64; cathepsin B can also activate receptor-bound pro-uPA on tumor cell surfaces, whereas cathepsin D cannot activate pro-uPA.\",\n      \"method\": \"In vitro enzymatic assay with purified human cathepsin B, N-terminal amino acid sequencing of cleavage products, inhibitor (E-64) blocking, receptor-binding assay on U937 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro cleavage with mutagenic/inhibitor controls and sequencing of cleavage site, >300 citations\",\n      \"pmids\": [\"1900515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"Cathepsin B cleaves the cartilage proteoglycan aggrecan at a single Gly-Val bond within the interglobular domain, only three amino acids C-terminal to the metalloproteinase (stromelysin) cleavage site, generating distinct G1 and G2 fragments.\",\n      \"method\": \"In vitro protease digestion of purified aggrecan G1-G2 domain fragment, SDS-PAGE fragment analysis, comparison with MMP-2, MMP-7, MMP-9 cleavage patterns\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro cleavage with site identification, >250 citations\",\n      \"pmids\": [\"1326552\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1984,\n      \"finding\": \"Cystatin C (gamma-trace) was identified as the tightest-binding protein inhibitor of cathepsin B discovered at the time, also potently inhibiting cathepsins H and L and the plant cysteine proteinases papain and ficin, establishing a physiological role for extracellular cystatins in regulating cathepsin B activity.\",\n      \"method\": \"Enzyme inhibition kinetics with purified proteins, determination of inhibition constants\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct in vitro inhibition kinetics with purified proteins, >280 citations\",\n      \"pmids\": [\"6203523\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Cathepsin B (released from lysosomes during necrosis) cleaves PARP-1 to generate a ~50 kDa fragment distinct from the apoptotic 89 kDa caspase-3 fragment; this cleavage is not inhibited by the broad-spectrum caspase inhibitor zVAD-fmk and is reproduced by purified cathepsin B in vitro on affinity-purified bovine PARP-1.\",\n      \"method\": \"In vitro cleavage assay with purified lysosomal proteases (cathepsins B, D, G) on affinity-purified PARP-1; lysosomal-rich fractions from Jurkat cells; comparison with necrotic cell lysates\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro cleavage with purified enzyme, confirmed in cellular necrosis model, >280 citations\",\n      \"pmids\": [\"11536009\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Cathepsin B (and to a lesser extent cathepsin L) is required for endosomal proteolytic cleavage of Ebola virus glycoprotein GP1, a step essential for viral entry into host cells; selective protease inhibitors and protease-deficient cell lines confirmed that CatB mediates GP1 cleavage enabling membrane fusion, and CatB/CatL inhibitors reduce multiplication of infectious Ebola virus-Zaire in culture.\",\n      \"method\": \"Selective protease inhibitors, protease-deficient cell lines, VSV pseudotype entry assay, biochemical proteolysis assay of EboV GP, infectious EboV-Zaire multiplication assay\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods (inhibitors, KO cell lines, biochemical assays, infectious virus), >690 citations\",\n      \"pmids\": [\"15831716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"In Alzheimer disease brains, cathepsin B (along with other lysosomal hydrolases) accumulates abnormally in neuronal perikarya and is found extracellularly in senile plaques, co-localizing with degenerating neuronal processes; in control brains cathepsin B is restricted to intracellular lysosomal compartments, establishing that lysosomal dysfunction and cathepsin B mis-localization occur in AD neurodegeneration.\",\n      \"method\": \"Immunocytochemistry, immunoelectron microscopy, and enzyme histochemistry in human post-mortem brain tissue from AD and control subjects\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct localization by immunoelectron microscopy with functional implication for amyloidogenesis, >260 citations\",\n      \"pmids\": [\"1837142\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Cathepsin B is upregulated and enzymatically active in inflamed atherosclerotic lesions (but not in normal aorta or silent lesions) in apoE-knockout mice; cathepsin B activity was imaged in vivo within active atherosclerotic plaques using intravenously injectable near-infrared cathepsin B-activatable imaging beacons, confirming a role for cathepsin B proteolytic activity in vascular inflammation.\",\n      \"method\": \"In vivo near-infrared fluorescence tomographic imaging with cathepsin B-specific activatable probes, immunohistochemistry, Western blot in apoE-KO and apoE/eNOS double-KO mouse atherosclerosis models\",\n      \"journal\": \"Circulation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct in vivo activity imaging corroborated by immunohistochemistry, >265 citations\",\n      \"pmids\": [\"12057992\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Serum amyloid A (SAA) activates the NLRP3 inflammasome in macrophages through a cathepsin B-sensitive pathway (confirmed by cathepsin B inhibitor blocking IL-1β secretion) and via P2X7 receptor; SAA also induces cathepsin B secretion, identifying cathepsin B as a key effector in inflammasome-driven IL-1β maturation.\",\n      \"method\": \"Cathepsin B inhibitor treatment, siRNA knockdown of NLRP3 and ASC, ASC-KO macrophages, IL-1β ELISA, TLR2/4 blocking antibodies in human/mouse macrophages and THP-1 cells\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (KO, siRNA, inhibitors) in multiple cell types, >230 citations\",\n      \"pmids\": [\"21508263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In transgenic mouse models of pancreatic and mammary carcinomas, cathepsin B in tumor cells and tumor-associated macrophages causally promotes tumor initiation, growth, angiogenesis, invasion, and metastasis; absence of cathepsin B enhances apoptosis; cathepsin B also associates with the tumor cell plasma membrane at elevated expression levels characteristic of cancer.\",\n      \"method\": \"Transgenic mouse models (MMTV-PyMT, RIP1-Tag2), genetic knockout of cathepsin B, tumor growth and metastasis assays, membrane fractionation\",\n      \"journal\": \"Proteomics. Clinical applications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic loss-of-function in multiple transgenic cancer models, >280 citations\",\n      \"pmids\": [\"24677670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Under homeostatic conditions, cathepsin B cleaves the lysosomal calcium channel MCOLN1/TRPML1, which suppresses the transcription factor TFEB and reduces expression of lysosomal and autophagy-related genes, thereby controlling the number of lysosomes and autophagosomes in the cell; the cytosolic bacterium Francisella novicida exploits this CTSB activity to suppress lysosome/autophagosome availability and enhance its intracellular survival.\",\n      \"method\": \"CTSB knockout cells and mice, lysosome/autophagosome quantification, MCOLN1 cleavage assay, TFEB reporter assay, bacterial infection models\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with multiple orthogonal readouts (channel cleavage, transcription factor activity, organelle number), replicated in vivo\",\n      \"pmids\": [\"27786577\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Skeletal muscle-secreted cathepsin B (CTSB) acts as a myokine elevated by exercise (running); recombinant CTSB application enhances BDNF and doublecortin (DCX) expression in adult hippocampal progenitor cells through a mechanism dependent on the multifunctional protein P11; in CTSB knockout mice, running fails to enhance adult hippocampal neurogenesis and spatial memory; plasma CTSB levels correlate with fitness and hippocampus-dependent memory in humans.\",\n      \"method\": \"CTSB KO mice, recombinant CTSB treatment of hippocampal progenitor cells, P11-dependency assay, hippocampal neurogenesis quantification, plasma CTSB measurement in mice/monkeys/humans, treadmill exercise protocol\",\n      \"journal\": \"Cell metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with defined neurogenic phenotype, recombinant protein rescue, translational validation in primates and humans, >440 citations\",\n      \"pmids\": [\"27345423\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Glucose metabolism in M2-like tumor-associated macrophages (TAMs) fuels the hexosamine biosynthetic pathway, leading to O-GlcNAcylation of cathepsin B at serine 210 by lysosome-localized OGT (O-GlcNAc transferase); this modification elevates mature cathepsin B levels in macrophages and promotes its secretion into the tumor microenvironment, thereby driving cancer metastasis and chemoresistance.\",\n      \"method\": \"Mass spectrometry identification of O-GlcNAcylation site (Ser210), OGT KO in macrophages, glycosylation site mutagenesis, co-immunoprecipitation, in vitro and in vivo metastasis assays, patient TAM correlation analysis\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — MS-identified PTM site with mutagenesis validation, OGT KO, functional rescue, in vivo metastasis model, >270 citations\",\n      \"pmids\": [\"36084651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"Human CTSB was definitively mapped to chromosome 8p22-p23.1 by three independent methods: PCR analysis of human-hamster somatic cell hybrids, FISH signal comparison in fibroblasts with chromosome 8 deletions, and fluorescence in situ hybridization to metaphase spreads with cathepsin B cosmid clones, resolving a prior ambiguity with a reported 13q14 location.\",\n      \"method\": \"PCR on somatic cell hybrid DNA, interphase FISH with chromosome 8 deletion fibroblasts, metaphase FISH with cosmid clones\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — three independent orthogonal mapping methods confirming chromosomal assignment\",\n      \"pmids\": [\"1577456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Tandem duplications of a 2.62 kb overlapping genomic region upstream of CTSB—containing an active keratinocyte enhancer—segregate with keratolytic winter erythema (KWE) in South African and Norwegian families; the duplication drives increased CTSB expression in keratinocytes of affected individuals, causing erythrokeratolysis, establishing CTSB overexpression as the cause of KWE.\",\n      \"method\": \"Targeted resequencing, SNP array, whole-genome sequencing, enhancer activity assays in keratinocyte cell lines, qPCR, immunohistochemistry of palmar epidermis, ChIA-PET chromatin interaction analysis\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — variant segregation with disease in two independent populations combined with functional enhancer validation and expression quantification\",\n      \"pmids\": [\"28457472\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"A gain-of-function missense mutation in CTSB, affecting a highly conserved residue, was found in a patient with autosomal dominant diffuse palmoplantar keratoderma; protein modelling predicted increased endopeptidase activity, and a cathepsin B enzymatic assay confirmed elevated proteolytic activity of the mutant, identifying the first gain-of-function CTSB variant.\",\n      \"method\": \"Whole exome sequencing, direct sequencing, protein modelling, cathepsin B enzymatic activity assay on patient-derived material\",\n      \"journal\": \"Clinical and experimental dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 — functional enzymatic assay in a single patient, corroborated by structural modelling; single case\",\n      \"pmids\": [\"32683719\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Cancer cell-secreted CST6 enters osteoclast precursors by endocytosis and suppresses cathepsin B (CTSB) activity; loss of CTSB activity leads to up-regulation of its hydrolytic substrate SPHK1, which then suppresses osteoclast maturation by inhibiting RANKL-induced p38 activation, defining a CST6-CTSB-SPHK1 signaling axis in osteoclastogenesis and bone metastasis.\",\n      \"method\": \"In vitro osteoclastogenesis assay, in vivo bone metastasis mouse model, CTSB activity assay, SPHK1 substrate identification, p38 signaling analysis, siRNA knockdown, recombinant CST6 protein treatment\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple methods including substrate identification and in vivo validation, single lab\",\n      \"pmids\": [\"34815788\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HDAC3 deficiency in macrophages increases cathepsin B (CTSB) expression via elevated histone acetylation at the CTSB locus; over-expressed CTSB causes lysosomal degradation of RIP1 (receptor-interacting serine-threonine kinase 1), reducing TNFα-mediated NF-κB activation and impairing inflammatory response to Pseudomonas aeruginosa infection.\",\n      \"method\": \"HDAC3 macrophage-specific KO mice, RNAseq, CHIPseq, Western blot, immunofluorescence, qRT-PCR, in vivo P. aeruginosa infection model, LPS intratracheal instillation\",\n      \"journal\": \"Cell & bioscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with RNAseq/CHIPseq and in vivo validation, single lab\",\n      \"pmids\": [\"35658939\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Cathepsin B promotes porcine preadipocyte differentiation by degrading fibronectin (Fn), a key extracellular matrix component and target gene of Wnt/β-catenin signaling; CTSB treatment relieved the anti-adipogenic effect of the Wnt/β-catenin activator LiCl, indicating that CTSB attenuates Wnt/β-catenin pathway activity through Fn degradation to facilitate adipogenesis.\",\n      \"method\": \"CTSB treatment of primary preadipocytes, fibronectin degradation assay, LiCl (Wnt activator) co-treatment, lipid accumulation staining, adipogenic gene expression analysis\",\n      \"journal\": \"Molecular and cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — substrate identification (fibronectin) with pathway functional readout, single lab, primary cells\",\n      \"pmids\": [\"24878992\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CTSB degrades ferroportin (FPN), the main iron export protein in macrophages; oxidized LDL (ox-LDL) upregulates macrophage CTSB, which negatively regulates FPN protein levels by promoting its degradation, disrupting iron homeostasis and inducing ferroptosis, thereby promoting atherosclerotic plaque progression.\",\n      \"method\": \"Co-immunoprecipitation (CTSB-FPN interaction), CTSB knockdown and pharmacological inhibition, FPN protein stability assay, ferroptosis markers in macrophages, in vivo ApoE-KO and HFD rat AS models, single-cell transcriptome analysis of human AS tissue\",\n      \"journal\": \"Molecular and cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP binding partner identified, KD/inhibitor functional validation in vitro and in vivo, single lab\",\n      \"pmids\": [\"39960586\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Lysosomal membrane permeabilization (LMP) releases cathepsin B into the cytoplasm where it activates the NLRP3 inflammasome, leading to caspase-1-dependent pyroptosis (GSDMD-N cleavage, IL-1β/IL-18 release); in sepsis-induced acute kidney injury (LPS-treated HK-2 cells), CTSB activity is elevated, CTSB inhibition with CA074 reverses LMP-induced mitochondrial membrane potential loss and apoptosis via the mitochondrial pathway.\",\n      \"method\": \"CTSB activity assay, CA074 inhibitor, JC-1 mitochondrial membrane potential, Annexin V/PI apoptosis staining, acridine orange lysosomal staining, Western blot, CCK8, CLP mouse model, DIA proteomics\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — inhibitor-based epistasis with multiple orthogonal cellular readouts, in vivo CLP model, single lab\",\n      \"pmids\": [\"36713420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In arsenic-induced liver fibrosis, NaAsO2 upregulates autophagy flux, which promotes cytoplasmic cathepsin B (CTSB) release from lysosomes; cytoplasmic CTSB activates the NLRP3 inflammasome, driving hepatic stellate cell (HSC) activation; inhibition of autophagy decreases cytoplasmic CTSB and attenuates NLRP3 inflammasome activation and HSC activation.\",\n      \"method\": \"Autophagy inhibitor (chloroquine), CTSB activity assay, NLRP3 inhibitor, immunofluorescence, Western blot in HSC-t6 cells and primary rat HSCs, in vivo NaAsO2-treated rat model\",\n      \"journal\": \"Chemosphere\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological pathway dissection with multiple inhibitors in vitro and in vivo, single lab\",\n      \"pmids\": [\"31669990\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ZRANB1 mediates K33-linked deubiquitination of CTSB, stabilizing CTSB protein expression; MINPP1 modulates this deubiquitination to regulate CTSB levels and downstream ferroptosis in HBV-positive hepatocellular carcinoma cells; the MINPP1-ZRANB1-CTSB axis is active only in HBV-positive HCC cells and promotes ferroptosis via a glycolytic bypass mechanism.\",\n      \"method\": \"Immunoprecipitation, immunofluorescence, ubiquitin modification analysis (K33-linkage), CTSB expression/stability assays, in vivo xenograft experiments\",\n      \"journal\": \"Biology direct\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — IP-based PTM identification with in vivo validation, single lab, novel deubiquitination site\",\n      \"pmids\": [\"41035046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ETS1 transcription factor is expressed in septoclasts (cartilage-resorbing cells at the chondro-osseous junction) and promotes transcription of Ctsb and Mmp13 during differentiation of septoclasts from pericytes; ETS1 siRNA knockdown in primary septoclast cultures significantly reduced Ctsb and Mmp13 expression.\",\n      \"method\": \"RNA-seq of isolated septoclast and pericyte populations, ETS1 siRNA knockdown in primary septoclast cultures, immunofluorescence localization\",\n      \"journal\": \"Cell and tissue research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — siRNA KD with defined gene expression readout in primary cells, single lab\",\n      \"pmids\": [\"40387924\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OGT-mediated O-GlcNAcylation of cathepsin B in prefrontal astrocytes promotes CTSB maturation; reduced O-GlcNAcylation (via OGT downregulation by BXHPD treatment) lowers ROS, attenuates lysosomal membrane permeabilization and cytoplasmic CTSB leakage, and suppresses NLRP3 inflammasome activation, improving depressive-like behaviors in a corticosterone mouse model.\",\n      \"method\": \"CO-IP for OGT-CTSB interaction, O-GlcNAcylation analysis, Western blot, immunofluorescence (OGT/S100β and CTSB/LAMP1 colocalization), DHE staining for ROS, Aldh1l1-Cre/ERT2 astrocyte-specific mice, behavioral tests\",\n      \"journal\": \"Journal of ethnopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP interaction, O-GlcNAc modification, cell-type-specific in vivo model, single lab\",\n      \"pmids\": [\"41391522\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"METTL3 upregulates CTSB expression in chondrocytes by promoting m6A methylation of CTSB mRNA, as confirmed by m6A RNA immunoprecipitation and dual-luciferase reporter assays; METTL3 silencing protects against IL-1β-induced chondrocyte apoptosis, inflammation, oxidative stress, and ferroptosis, effects that are rescued by CTSB overexpression, establishing a METTL3-m6A-CTSB regulatory axis in osteoarthritis.\",\n      \"method\": \"m6A RNA immunoprecipitation (MeRIP), dual-luciferase reporter assay, METTL3 and CTSB siRNA knockdown, Western blot, flow cytometry, TUNEL, ELISA, ROS/MDA/Fe2+ assays in human chondrocytes\",\n      \"journal\": \"Journal of orthopaedic surgery and research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — MeRIP and luciferase confirm m6A regulatory mechanism, functional rescue validates axis, single lab\",\n      \"pmids\": [\"41466292\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cathepsin B promotes microglial efferocytosis of apoptotic neurons during brain development; CTSB is enriched in microglia in high-neuronal-turnover brain regions; myeloid-specific CTSB knockdown in zebrafish led to dysmorphic microglia containing undigested dead cells and accumulation of apoptotic cells, phenocopied by global Ctsb KO in mice; live imaging revealed deficits in phagolysosomal fusion and acidification, identifying a role in lysosomal digestion rather than initial phagocytic uptake.\",\n      \"method\": \"Zebrafish myeloid-specific CTSB knockdown, global Ctsb-KO mice, live fluorescence imaging of phagolysosomal fusion and acidification, apoptosis markers (TUNEL), confocal imaging of brain regions, behavioral assessment\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO/KD in two model organisms with live imaging of lysosomal mechanism, preprint not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Beauvericin (BEA) acts as an uncompetitive inhibitor of cathepsin B; NMR analyses confirmed direct interaction between BEA and CTSB; enzyme kinetics established uncompetitive inhibition; molecular docking identified a putative BEA binding site in human CTSB distinct from the active site; BEA significantly suppresses CTSB activity in mouse BMDCs and human iDCs.\",\n      \"method\": \"Enzyme kinetics (uncompetitive inhibitor determination), NMR spectroscopy (direct binding), molecular docking, CTSB activity assay in BMDCs and THP-1-derived iDCs\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — enzyme kinetics plus NMR direct binding, preprint; mechanism well-defined but not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Elevated lysosomal cathepsin B levels and CTSB leakage to the cytoplasm trigger amyloidogenesis in mucopolysaccharidosis type IIIC and sialidosis mouse models; CTSB-deficient MPS IIIC mice (Hgsnat-P304L/Ctsb-/-) and mice chronically treated with brain-penetrating CTSB inhibitor E64 showed drastic reduction in neuronal Thioflavin-S-positive/APP-positive amyloid deposits and restored autophagy markers; E64 treatment rescued behavioral deficits.\",\n      \"method\": \"CTSB-deficient double-KO mice, chronic brain-penetrating E64 inhibitor treatment, Thioflavin-S staining, β-amyloid immunostaining, P62/LC3 autophagy markers, behavioral testing (hyperactivity, anxiety), immunofluorescence of cortical neurons\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO and pharmacological inhibition with multiple amyloidogenesis readouts in vivo, preprint not peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Extralysosomal cathepsin B in progressive multiple sclerosis brains cleaves the C-terminal domain of TAF1, a core component of the general transcription factor TFIID; loss of C-terminal TAF1 disrupts RNAPII promoter-proximal pausing at oligodendroglial myelination genes; mice lacking the C-terminal TAF1 domain exhibit MS-like brain transcriptomic signature, CNS-resident inflammation, progressive demyelination, and motor disability.\",\n      \"method\": \"Detection of TAF1 C-terminal underexpression in MS brain tissue, identification of CTSB-mediated endoproteolysis of TAF1, generation of Taf1Δ38 mice, transcriptomic analysis, immunofluorescence, behavioral assessment\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — mechanistic link between CTSB and TAF1 cleavage shown in tissue but biochemical reconstitution not explicitly detailed; preprint, single study\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cathepsin B (CTSB) cleaves the lysosomal nitrate transporter Sialin to generate a proteolytic fragment called Sialin2 that localizes to mitochondria and acts as a nitrate sensor; Sialin2 scaffolds LKB1-AMPK complexes to drive mitochondrial biogenesis and metabolic adaptation.\",\n      \"method\": \"Cryo-EM structure of Sialin2, microscale thermophoresis (MST) nitrate-binding assay, fractionation showing CTSB-dependent generation of Sialin2, AMPK complex co-immunoprecipitation, sCiSiNiS biosensor for real-time nitrate signaling\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 2 for Sialin2 characterization, but CTSB's specific role in generating Sialin2 by cleavage is stated without detailed mutagenesis/reconstitution of that specific step; preprint, single study\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"Cathepsin B (CTSB) is a lysosomal cysteine endopeptidase and dipeptidyl carboxypeptidase whose 2.15 Å crystal structure reveals an occluding loop (His110/His111) that enables exopeptidase activity and restricts cystatin binding; it cleaves multiple substrates including pro-uPA (activating plasminogen signaling), aggrecan, PARP-1, MCOLN1/TRPML1 (regulating TFEB and lysosome/autophagosome biogenesis), RIP1 (modulating NF-κB), ferroportin (disrupting macrophage iron homeostasis), fibronectin (promoting adipogenesis via Wnt/β-catenin attenuation), and the Ebola virus glycoprotein GP1 (enabling viral entry); upon lysosomal membrane permeabilization, cytoplasmic CTSB activates the NLRP3 inflammasome to drive pyroptosis and IL-1β maturation; muscle-secreted CTSB acts as an exercise-induced myokine that promotes hippocampal BDNF/DCX expression and neurogenesis via P11; its activity and stability are regulated post-translationally by O-GlcNAcylation at Ser210 (by lysosomal OGT), K33-linked deubiquitination (by ZRANB1), and m6A-mediated mRNA upregulation (by METTL3), while gain-of-function mutations cause palmoplantar keratoderma and regulatory enhancer duplications upstream of CTSB cause keratolytic winter erythema.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CTSB encodes cathepsin B, a lysosomal cysteine endopeptidase that functions as a broad-specificity protease controlling lysosome homeostasis, inflammasome activation, cell death, and extracellular matrix remodeling. Within lysosomes, CTSB cleaves the calcium channel MCOLN1/TRPML1 to suppress TFEB-driven lysosome biogenesis [PMID:27786577], degrades fibronectin to attenuate Wnt/β-catenin signaling [PMID:24878992], and hydrolyzes SPHK1 to regulate osteoclast differentiation [PMID:34815788]; upon lysosomal membrane permeabilization, cytoplasmic CTSB activates the NLRP3 inflammasome to drive pyroptosis [PMID:37141464, PMID:31669990], degrades RIP1 to dampen NF-κB signaling [PMID:35658939], and cleaves ferroportin to induce ferroptosis [PMID:39960586]. CTSB is transcriptionally regulated by ETS1 [PMID:40387924], post-transcriptionally upregulated via METTL3-mediated m6A modification [PMID:41466292], and stabilized by ZRANB1-mediated K33-linked deubiquitination [PMID:41035046]; gain-of-function mutations or enhancer duplications at the CTSB locus cause dominant skin diseases including diffuse palmoplantar keratoderma and keratolytic winter erythema [PMID:32683719, PMID:28457472]. In the nervous system, CTSB (together with CTSL) is essential for Purkinje cell survival and autophagy-lysosomal clearance [PMID:40320169], and is required in microglia for phagolysosomal fusion during developmental neuronal efferocytosis.\",\n  \"teleology\": [\n    {\n      \"year\": 2014,\n      \"claim\": \"Identifying fibronectin as a CTSB substrate established that secreted lysosomal CTSB modulates Wnt/β-catenin signaling through ECM degradation, connecting a lysosomal protease to an extracellular signaling pathway in adipogenesis.\",\n      \"evidence\": \"Fibronectin degradation assay and Wnt/β-catenin readout in porcine preadipocytes\",\n      \"pmids\": [\"24878992\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct cleavage-site mapping on fibronectin\", \"Specificity of CTSB versus other cathepsins for fibronectin unclear\", \"In vivo adipogenesis phenotype not tested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstration that CTSB cleaves the lysosomal calcium channel MCOLN1/TRPML1, thereby suppressing TFEB nuclear translocation, revealed a protease-dependent feedback mechanism controlling lysosome and autophagosome biogenesis.\",\n      \"evidence\": \"CTSB inhibition/knockout with TFEB activity readout and lysosome/autophagosome quantification\",\n      \"pmids\": [\"27786577\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cleavage site on MCOLN1 not mapped\", \"Whether other cathepsins contribute redundantly is unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Discovery that tandem enhancer duplications upstream of CTSB cause keratolytic winter erythema (KWE) established the first Mendelian disease mechanism directly attributable to CTSB overexpression in differentiating keratinocytes.\",\n      \"evidence\": \"Whole-genome sequencing and enhancer activity assays in two independent patient cohorts (South African and Norwegian)\",\n      \"pmids\": [\"28457472\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream substrates mediating the skin phenotype not identified\", \"Whether CTSB inhibition reverses KWE untested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Linking lysosomal membrane permeabilization to cytoplasmic CTSB release and NLRP3 inflammasome activation in hepatic stellate cells established the LMP→CTSB→NLRP3 pyroptotic axis, a pathway subsequently replicated across multiple cell types and stimuli.\",\n      \"evidence\": \"CTSB activity measurement, NLRP3 inflammasome assay, and autophagy inhibitor in arsenic-exposed cells and mice\",\n      \"pmids\": [\"31669990\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CTSB directly cleaves an NLRP3 component or acts indirectly was unresolved\", \"Relative contribution of CTSB versus CTSD/CTSL to inflammasome activation not delineated\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Two advances refined understanding of CTSB in disease: (1) a gain-of-function CTSB missense mutation was shown by enzymatic assay to cause dominant palmoplantar keratoderma, proving that increased CTSB activity is pathogenic; (2) reduced lysosomal CTSB activity in cardiomyocytes was linked to impaired autophagy flux and ROS/p38MAPK/JNK-driven cardiac hypertrophy.\",\n      \"evidence\": \"Patient WES with recombinant enzyme kinetics (keratoderma); CTSB activity assay, ROS scavenger, and MAPK inhibitors in neonatal rat ventricular myocytes (hypertrophy)\",\n      \"pmids\": [\"32683719\", \"32398966\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Keratoderma finding from single family—independent replication needed\", \"Whether the MAPK-CTSB feedback loop operates in vivo is untested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identification of CST6 as an endogenous extracellular CTSB inhibitor that stabilizes SPHK1 and suppresses osteoclast differentiation via p38 defined a tumor-microenvironment axis where cancer-secreted cystatin remotely controls bone remodeling through CTSB.\",\n      \"evidence\": \"In vitro osteoclastogenesis and in vivo metastasis model with CTSB inhibition and SPHK1 substrate identification\",\n      \"pmids\": [\"34815788\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct cleavage of SPHK1 by CTSB not reconstituted in vitro\", \"Whether other cystatins compensate in vivo is unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showing that CTSB degrades RIP1 to suppress TNFα/NF-κB signaling in HDAC3-deficient macrophages revealed CTSB as an anti-inflammatory protease when overexpressed, complicating the simpler view that cytoplasmic CTSB is strictly pro-inflammatory.\",\n      \"evidence\": \"HDAC3 KO macrophages, RIP1 degradation by Western blot, NF-κB activity, in vivo mouse infection model\",\n      \"pmids\": [\"35658939\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RIP1 cleavage site not mapped\", \"Whether RIP1 degradation occurs in lysosomes or cytoplasm was not dissected\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Multiple studies consolidated the LMP→CTSB→NLRP3 inflammasome axis across hepatocytes (ROS-driven) and renal epithelial cells (LPS-driven), while also extending CTSB to mitochondrial apoptosis, establishing cytoplasmic CTSB as a general stress effector linking lysosomal damage to diverse cell death modalities.\",\n      \"evidence\": \"Pharmacological inhibition (CA-074 Me, MCC950), siRNA, LMP staining, mitochondrial membrane potential assays across cell types\",\n      \"pmids\": [\"37141464\", \"36713420\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct CTSB substrate in the mitochondrial apoptosis pathway not identified\", \"Whether CTSB cleaves NLRP3 directly or acts through an intermediate remains open\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Discovery that phospho-MLKL translocation to lysosomes causes LMP-dependent CTSB release positioned necroptotic signaling upstream of the CTSB–NLRP3 inflammasome cascade, linking the necroptotic and pyroptotic pathways through lysosomal CTSB.\",\n      \"evidence\": \"P-MLKL Western blot, LMP staining, CTSB/NLRP3 inhibitors, in vivo alcohol-injury mouse model\",\n      \"pmids\": [\"39612889\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether MLKL directly permeabilizes lysosomes or acts through ROS is not resolved\", \"Single stimulus (alcohol); generalizability unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Several parallel discoveries expanded CTSB's substrate repertoire and regulatory inputs: CTSB degrades ferroportin to induce ferroptosis in macrophages; CTSB is stabilized by ZRANB1-mediated K33-linked deubiquitination via MINPP1; and METTL3-dependent m6A methylation upregulates CTSB mRNA, adding epitranscriptomic and ubiquitin-based control layers.\",\n      \"evidence\": \"Co-IP and ubiquitin chain analysis (ZRANB1/MINPP1); m6A-RIP and dual-luciferase (METTL3); FPN degradation assays and ferroptosis readouts in macrophages\",\n      \"pmids\": [\"39960586\", \"41035046\", \"41466292\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"FPN cleavage site unknown\", \"ZRANB1-CTSB interaction awaits independent confirmation\", \"Whether m6A regulation of CTSB is cell-type specific is untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"In the nervous system, combined CTSB/CTSL conditional knockout revealed selective Purkinje cell vulnerability to autophagy-lysosomal failure, and ETS1 was identified as a transcriptional activator of CTSB in septoclast differentiation, broadening CTSB's roles in neural and skeletal development.\",\n      \"evidence\": \"Conditional double KO (Nestin-Cre) with EM and behavioral analysis; ETS1 siRNA in primary septoclasts\",\n      \"pmids\": [\"40320169\", \"40387924\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Individual contributions of CTSB versus CTSL to Purkinje cell survival not resolved\", \"ETS1-CTSB axis not validated in vivo\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key mechanistic gaps remain: the direct substrates through which cytoplasmic CTSB activates NLRP3 are unidentified; cleavage sites on most reported substrates (MCOLN1, RIP1, FPN, TAF1) are unmapped; and how CTSB activity is differentially regulated across lysosomal, cytoplasmic, and nuclear compartments to yield pro-inflammatory versus anti-inflammatory outcomes is not understood.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No direct NLRP3 cleavage or adaptor substrate identified\", \"Cleavage-site mapping lacking for most reported substrates\", \"Compartment-specific regulation of CTSB activity unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 3, 6, 10, 19, 22]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 1, 3, 6, 10, 21, 22]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [0, 1, 9, 18, 24]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 4, 7, 8, 16]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [0, 9, 18, 20]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [2, 4, 7, 8, 16]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2, 3, 7, 8]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 13, 14]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [22, 23]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"MCOLN1\",\n      \"NLRP3\",\n      \"RIP1\",\n      \"FPN\",\n      \"SPHK1\",\n      \"ZRANB1\",\n      \"OGT\",\n      \"CST6\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"Cathepsin B is a lysosomal cysteine protease with dual endopeptidase and dipeptidyl carboxypeptidase activity, conferred by a unique occluding loop (His110/His111) that anchors substrate C-termini and restricts cystatin-family inhibitor access [PMID:1868826, PMID:6203523]. It processes a broad spectrum of substrates including pro-uPA, aggrecan, PARP-1, MCOLN1/TRPML1 (thereby controlling TFEB-dependent lysosome/autophagosome biogenesis), ferroportin, fibronectin, and Ebola virus glycoprotein GP1 [PMID:1900515, PMID:1326552, PMID:11536009, PMID:27786577, PMID:15831716, PMID:39960586]; upon lysosomal membrane permeabilization it activates the NLRP3 inflammasome to drive pyroptosis and IL-1β maturation [PMID:21508263, PMID:36713420]. Beyond its intracellular roles, muscle-secreted CTSB functions as an exercise-induced myokine that promotes hippocampal BDNF expression and neurogenesis via P11 [PMID:27345423]; its activity is regulated post-translationally by O-GlcNAcylation at Ser210 and by K33-linked deubiquitination [PMID:36084651, PMID:41035046], and gain-of-function mutations or upstream enhancer duplications cause palmoplantar keratoderma and keratolytic winter erythema, respectively [PMID:32683719, PMID:28457472].\",\n  \"teleology\": [\n    {\n      \"year\": 1984,\n      \"claim\": \"Before physiological regulators of cathepsin B were known, identification of cystatin C as a potent endogenous inhibitor established that cathepsin B activity is tightly controlled extracellularly by cystatin-family proteins.\",\n      \"evidence\": \"Enzyme inhibition kinetics with purified cystatin C against cathepsin B and related cysteine proteases\",\n      \"pmids\": [\"6203523\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Intracellular regulation of CTSB activity by endogenous inhibitors not addressed\", \"Tissue-specific relevance of cystatin C–CTSB interaction not explored\"]\n    },\n    {\n      \"year\": 1986,\n      \"claim\": \"Cloning of preprocathepsin B cDNA revealed the full precursor architecture—signal peptide, propeptide, mature chain, and C-terminal extension—establishing the multi-step processing pathway required for enzyme maturation.\",\n      \"evidence\": \"cDNA cloning and sequencing from human hepatoma and kidney libraries\",\n      \"pmids\": [\"3463996\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise sites and proteases responsible for each processing step not identified\", \"Regulation of propeptide removal in different tissues unknown\"]\n    },\n    {\n      \"year\": 1991,\n      \"claim\": \"The 2.15 Å crystal structure resolved the long-standing question of how cathepsin B achieves both endo- and exopeptidase activity: an occluding loop (His110/His111) blocks the primed subsites, anchoring substrate C-termini for dipeptidyl carboxypeptidase activity and sterically preventing cystatin binding.\",\n      \"evidence\": \"X-ray crystallography at 2.15 Å resolution with structural comparison to papain/actinidin\",\n      \"pmids\": [\"1868826\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamic behavior of the occluding loop and endo/exo switching mechanism not captured by static structure\", \"Structural basis for substrate selectivity at the S2 subsite incompletely characterized\"]\n    },\n    {\n      \"year\": 1991,\n      \"claim\": \"Demonstration that cathepsin B activates pro-uPA at the same Lys158-Ile159 bond as plasmin established CTSB as a direct activator of pericellular plasminogen signaling, linking it to extracellular matrix remodeling and tumor invasion.\",\n      \"evidence\": \"In vitro cleavage of purified pro-uPA with N-terminal sequencing and E-64 inhibitor blocking; cell surface activation on U937 cells\",\n      \"pmids\": [\"1900515\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of CTSB-mediated pro-uPA activation not demonstrated\", \"Relative contribution versus other activating proteases unclear\"]\n    },\n    {\n      \"year\": 1991,\n      \"claim\": \"Immunoelectron microscopy of Alzheimer disease brains revealed cathepsin B mislocalization from lysosomes to neuronal perikarya and senile plaques, establishing lysosomal dysfunction and CTSB redistribution as features of AD neurodegeneration.\",\n      \"evidence\": \"Immunocytochemistry and immunoelectron microscopy on human post-mortem AD vs. control brain tissue\",\n      \"pmids\": [\"1837142\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CTSB mislocalization is causative or consequential in AD pathology not resolved\", \"Specific CTSB substrates contributing to amyloidogenesis not identified in this study\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Identification of CTSB cleavage of aggrecan at a specific Gly-Val bond in the interglobular domain, near the MMP cleavage site, established cathepsin B as a cartilage-degrading protease relevant to joint pathology.\",\n      \"evidence\": \"In vitro proteolysis of purified aggrecan G1-G2 domain with fragment analysis by SDS-PAGE\",\n      \"pmids\": [\"1326552\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo contribution of CTSB to aggrecanolysis versus MMPs not quantified\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Discovery that cathepsin B (released during necrosis) generates a distinct ~50 kDa PARP-1 fragment not produced by caspases revealed a necrosis-specific proteolytic signature and distinguished cathepsin-mediated cell death from apoptosis.\",\n      \"evidence\": \"In vitro cleavage of purified PARP-1 by cathepsin B; comparison with necrotic cell lysates; caspase inhibitor (zVAD-fmk) insensitivity\",\n      \"pmids\": [\"11536009\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"PARP-1 cleavage site by CTSB not mapped\", \"Functional consequences of the ~50 kDa PARP-1 fragment not characterized\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Cathepsin B was shown to be required for endosomal cleavage of Ebola virus GP1 to enable membrane fusion and viral entry, defining CTSB as a host-factor dependency for filovirus infection.\",\n      \"evidence\": \"Selective protease inhibitors, protease-deficient cell lines, VSV pseudotype entry assays, and infectious Ebola virus multiplication assays\",\n      \"pmids\": [\"15831716\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise cleavage sites on GP1 by CTSB not fully mapped\", \"Extent of cathepsin L redundancy in vivo not settled\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstration that cathepsin B mediates SAA-induced NLRP3 inflammasome activation in macrophages established the paradigm that lysosomal membrane permeabilization and cytoplasmic CTSB release are upstream triggers of inflammasome-driven IL-1β maturation.\",\n      \"evidence\": \"Cathepsin B inhibitor, siRNA knockdown of NLRP3/ASC, ASC-KO macrophages, IL-1β ELISA in human and mouse macrophages\",\n      \"pmids\": [\"21508263\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CTSB directly cleaves an NLRP3 component or acts indirectly not resolved\", \"Relative contribution of CTSB versus other lysosomal cathepsins to inflammasome activation unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Genetic deletion of cathepsin B in transgenic mouse cancer models demonstrated a causal role in tumor initiation, growth, angiogenesis, and metastasis, with CTSB associating with the tumor cell plasma membrane at elevated expression, establishing its non-redundant function in cancer progression.\",\n      \"evidence\": \"CTSB knockout in MMTV-PyMT and RIP1-Tag2 transgenic mouse models with tumor growth, metastasis, and apoptosis quantification\",\n      \"pmids\": [\"24677670\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific tumor-promoting substrates cleaved by membrane-associated CTSB not identified\", \"Cell-type-specific contributions (tumor vs. stroma) not fully dissected\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"CTSB was found to cleave the lysosomal calcium channel MCOLN1/TRPML1, thereby suppressing TFEB activation and limiting lysosome/autophagosome biogenesis under homeostatic conditions—a mechanism co-opted by intracellular bacteria to enhance survival.\",\n      \"evidence\": \"CTSB KO cells and mice, MCOLN1 cleavage assay, TFEB reporter, lysosome/autophagosome quantification, Francisella novicida infection model\",\n      \"pmids\": [\"27786577\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cleavage site on MCOLN1 not mapped\", \"Whether CTSB regulation of TFEB is a general homeostatic mechanism or context-specific not fully established\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identification of cathepsin B as an exercise-induced myokine that promotes hippocampal BDNF/DCX expression and neurogenesis through P11 provided the first direct mechanistic link between a muscle-secreted protease and exercise-dependent cognitive enhancement.\",\n      \"evidence\": \"CTSB KO mice fail to show running-induced neurogenesis; recombinant CTSB rescues hippocampal progenitor differentiation via P11; plasma CTSB correlates with fitness in humans\",\n      \"pmids\": [\"27345423\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which circulating CTSB crosses the blood-brain barrier not established\", \"Direct proteolytic target of CTSB that engages P11 signaling not identified\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Tandem duplications of a keratinocyte enhancer upstream of CTSB were shown to segregate with keratolytic winter erythema in two independent populations, establishing CTSB overexpression as the genetic cause of KWE.\",\n      \"evidence\": \"Targeted resequencing, WGS, enhancer activity assays in keratinocytes, qPCR, ChIA-PET chromatin interaction analysis in South African and Norwegian families\",\n      \"pmids\": [\"28457472\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream keratinocyte substrates whose excessive cleavage causes erythrokeratolysis not identified\", \"Whether enhancer duplication alters CTSB expression in non-epidermal tissues unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"A gain-of-function missense mutation in CTSB was identified as causing autosomal dominant diffuse palmoplantar keratoderma, confirmed by elevated enzymatic activity, representing the first gain-of-function CTSB point mutation linked to Mendelian disease.\",\n      \"evidence\": \"Whole exome sequencing, protein modeling, and cathepsin B enzymatic activity assay on patient-derived material (single case)\",\n      \"pmids\": [\"32683719\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single patient without independent replication\", \"Precise mechanism by which increased CTSB activity causes keratoderma not determined\", \"The specific residue affected and structural consequence not fully validated by crystal structure\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Discovery that O-GlcNAcylation of cathepsin B at Ser210 by lysosome-localized OGT stabilizes and promotes CTSB secretion in tumor-associated macrophages revealed a metabolic post-translational control mechanism linking glucose metabolism to metastasis.\",\n      \"evidence\": \"Mass spectrometry identification of Ser210 O-GlcNAcylation, site-directed mutagenesis, OGT KO macrophages, in vivo metastasis assays\",\n      \"pmids\": [\"36084651\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether O-GlcNAcylation at Ser210 affects catalytic activity directly or only protein stability not distinguished\", \"Generalizability beyond tumor-associated macrophages not tested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"CTSB was shown to degrade ferroportin in ox-LDL-stimulated macrophages, disrupting iron export and inducing ferroptosis, providing a direct mechanistic link between CTSB and iron-dependent cell death in atherosclerosis.\",\n      \"evidence\": \"Co-immunoprecipitation of CTSB-FPN, CTSB knockdown/inhibition rescuing FPN levels, ferroptosis markers, ApoE-KO mouse and rat atherosclerosis models\",\n      \"pmids\": [\"39960586\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ferroportin cleavage site by CTSB not mapped\", \"Co-IP without reciprocal validation reported\", \"Single lab study\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identification of K33-linked deubiquitination of CTSB by ZRANB1, modulated by MINPP1, revealed a previously unknown ubiquitin-dependent regulatory layer controlling CTSB protein stability and ferroptosis in HBV-positive hepatocellular carcinoma.\",\n      \"evidence\": \"Immunoprecipitation, K33-linkage ubiquitin analysis, CTSB stability assays, in vivo xenograft experiments\",\n      \"pmids\": [\"41035046\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific ubiquitination sites on CTSB not mapped\", \"Whether K33-linked regulation operates beyond HBV-positive HCC context unknown\", \"Single lab study\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"METTL3-mediated m6A methylation of CTSB mRNA was shown to upregulate CTSB expression in chondrocytes, with CTSB overexpression rescuing ferroptosis and inflammatory phenotypes upon METTL3 silencing, establishing an epitranscriptomic regulatory axis for CTSB in osteoarthritis.\",\n      \"evidence\": \"m6A RIP, dual-luciferase reporter assay, METTL3/CTSB siRNA knockdown with rescue, ferroptosis and apoptosis readouts in human chondrocytes\",\n      \"pmids\": [\"41466292\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific m6A site(s) on CTSB mRNA not mapped at nucleotide resolution\", \"In vivo validation in OA models not yet reported\", \"Single lab study\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: (1) the direct molecular mechanism by which cytoplasmic CTSB activates the NLRP3 inflammasome (whether via direct NLRP3 cleavage or indirect signaling), (2) the structural basis for the occluding loop's dynamic endo-to-exopeptidase switching in physiological contexts, and (3) the identity of blood-brain barrier transit mechanisms enabling circulating CTSB to promote hippocampal neurogenesis.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural study of the occluding loop in a dynamic or membrane-associated context\", \"Direct NLRP3 cleavage by CTSB never reconstituted with purified components\", \"BBB crossing mechanism for myokine CTSB undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 3, 5, 6, 11, 13, 17, 19, 20]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 2, 3, 5, 6, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [0, 7, 11, 13, 21, 22, 25]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [5, 9, 21, 22]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [12, 13]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [9, 21, 22]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 13]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [11, 22]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [5, 10, 21]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [10, 15, 16]},\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [3, 19]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"CST3\",\n      \"MCOLN1\",\n      \"OGT\",\n      \"ZRANB1\",\n      \"FPN1\",\n      \"NLRP3\",\n      \"P11\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}