{"gene":"CASP6","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":1995,"finding":"CASP6 (Mch2 alpha) encodes a cysteine protease with activity against the fluorogenic peptide DEVD-AMC and cleaves poly(ADP-ribose) polymerase (PARP) in vitro; overexpression of Mch2 alpha (but not the Mch2 beta isoform) induces apoptosis in Sf9 insect cells.","method":"Recombinant protein expression, fluorogenic peptide cleavage assay, in vitro PARP cleavage, baculovirus overexpression","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 1 — in vitro enzymatic assay with recombinant protein, replicated in multiple readouts","pmids":["7796396"],"is_preprint":false},{"year":1996,"finding":"CASP6 (Mch2 alpha) cleaves nuclear lamin A at the conserved VEID↓NG sequence in the coiled-coil rod domain, producing the apoptotic lamin A fragment; CPP32 (caspase-3) does not cleave lamin A, establishing distinct substrate specificities between these caspases.","method":"In vitro cleavage assay with recombinant IRPs, cell-free apoptosis extracts, Zn2+ inhibition, affinity labeling","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro cleavage, replicated in cell-free system, confirmed with inhibitor","pmids":["8710882"],"is_preprint":false},{"year":1996,"finding":"CASP6 (Mch2) is processed from its zymogen to a proteolytically active dimeric species during apoptosis and by granzyme B; it functions downstream of death inhibitors Bcl-2, Bcl-xL, and CrmA; it cleaves lamin A to its signature apoptotic fragment, whereas caspase-3 (Yama) and caspase-7 (LAP3) do not, identifying CASP6 as an apoptotic laminase.","method":"In vitro cleavage assay, granzyme B processing, epistasis with Bcl-2/Bcl-xL/CrmA overexpression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro reconstitution plus genetic epistasis, independent confirmation of lamin A cleavage","pmids":["8663580"],"is_preprint":false},{"year":1997,"finding":"CASP6 (Mch2/caspase-6) and caspase-3 (CPP32) are the major active caspases present in apoptotic tumor cells in response to diverse apoptosis-inducing stimuli; both are present as multiple active species whose composition varies between cell lines.","method":"Active caspase detection approach using biotinylated caspase probes, multiple cell lines, multiple stimuli","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — orthogonal detection method, replicated across multiple cell lines and stimuli","pmids":["9171342"],"is_preprint":false},{"year":1999,"finding":"CASP6 is activated during serum deprivation-induced apoptosis in primary human neurons; it directly cleaves amyloid precursor protein (APP) at the C-terminus generating Capp3 and Capp6.5 fragments; caspase-6 inhibition prevents the serum deprivation-mediated increase in amyloid beta peptide, establishing a caspase-6-dependent amyloidogenic pathway.","method":"Inhibitor studies (z-VEID-fmk), in vitro cleavage of APP by recombinant caspase-6, pulse-chase metabolic labeling, immunoblot of human AD brain","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — direct in vitro substrate cleavage plus functional loss-of-function in primary neurons with defined biochemical readout","pmids":["10438520"],"is_preprint":false},{"year":2000,"finding":"In cerebellar granule cell apoptosis induced by trophic support withdrawal, active caspase-6 can process and activate procaspase-3 in cellular extracts, whereas caspase-3 does not activate caspase-6; cell-permeable caspase-6 inhibitor prevents caspase-3 activation, placing caspase-6 upstream of caspase-3 in this pathway.","method":"Fluorogenic peptide assays, immunoblot, cell-permeable selective inhibitors (CP-VEID-cho, CP-DEVD-cho), ex vivo cerebellar granule cell system","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1–2 — biochemical reconstitution in cellular extracts plus selective inhibitor epistasis","pmids":["11279545"],"is_preprint":false},{"year":2001,"finding":"CASP6 cleaves SATB1 at amino acid position 254, separating the DNA-binding domains from the PDZ-like dimerization domain (aa 90–204); cleavage abolishes BUR-binding activity and causes rapid dissociation of SATB1 from chromatin in vivo, coinciding with high-molecular-weight chromatin fragmentation in apoptotic T cells.","method":"In vitro cleavage with caspase-6, site-directed mutagenesis, in vivo chromatin dissociation assay, Fas-induced apoptosis in Jurkat cells","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro reconstitution plus in vivo functional consequence confirmed by chromatin dissociation","pmids":["11463840"],"is_preprint":false},{"year":2002,"finding":"Disruption of both CASP6 alleles in chicken DT40 cells reveals that caspase-6 activity is essential for lamin A/C cleavage during apoptosis and for complete chromatin condensation and apoptotic body formation when lamin A is present; lamins A and C are caspase-6-only substrates in this system.","method":"Gene disruption (both alleles), cell-free nuclear disassembly assay, complementation with exogenous caspase-6, caspase-6 inhibitor z-VEID-fmk","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 — genetic knockout with defined biochemical phenotype, rescued by complementation, replicated with inhibitor","pmids":["11953316"],"is_preprint":false},{"year":2002,"finding":"p53 binds the third intron of the caspase-6 gene and transcriptionally activates it; p53-dependent increase in procaspase-6 protein enables increased caspase-6 activity and lamin A cleavage in response to Adriamycin; specific inhibition of caspase-6 blocks p53-mediated cell death.","method":"Chromatin immunoprecipitation (ChIP) of p53 to caspase-6 intron 3, reporter assay, immunoblot of lamin A cleavage, caspase-6 inhibitor","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 — direct DNA binding shown by ChIP, functional validation with inhibitor","pmids":["12089322"],"is_preprint":false},{"year":2003,"finding":"CBP (CREB-binding protein) is a caspase-6 substrate in primary neurons; caspase-6-mediated cleavage of CBP reduces CBP/p300 histone acetyltransferase activity during neuronal apoptosis, linking caspase-6 to transcriptional dysregulation and histone deacetylation in neurodegeneration.","method":"In vitro cleavage assay with recombinant caspase-6, HAT activity assay, primary neuron apoptosis model","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 — direct in vitro substrate identification with functional HAT activity readout","pmids":["14657026"],"is_preprint":false},{"year":2004,"finding":"Active caspase-6 p20 subunit is present in neurofibrillary tangles, neuropil threads, and neuritic plaques in Alzheimer's disease brain; a neoepitope antibody to caspase-6-cleaved Tau detects intracellular and extracellular tangles and pretangles, indicating early caspase-6 activity in AD pathogenesis.","method":"Neoepitope antibodies to active caspase-6 p20 and caspase-6-cleaved Tau, immunohistochemistry of AD brain tissue","journal":"The American journal of pathology","confidence":"High","confidence_rationale":"Tier 2 — neoepitope antibodies in human tissue, multiple lesion types, correlated with disease stage","pmids":["15277226"],"is_preprint":false},{"year":2004,"finding":"Caspase-6 cleaves the N-terminus of tau in vitro at D13 (a semicanonical cleavage site), preventing immunoreactivity with N-terminal tau antibodies; mass spectrometry confirmed this cleavage site, linking caspase-6-mediated N-terminal tau truncation to neurofibrillary tangle evolution in AD.","method":"In vitro cleavage of tau by recombinant caspase-6, mass spectrometry confirmation of cleavage site, immunohistochemistry","journal":"The Journal of neuroscience : the official journal of the Society for Neuroscience","confidence":"High","confidence_rationale":"Tier 1 — in vitro enzymatic cleavage with mass spectrometry identification of cleavage site","pmids":["15356202"],"is_preprint":false},{"year":2004,"finding":"ARK5 kinase phosphorylates caspase-6 at Ser257, preventing its activation; mutant caspase-6 with S257A substitution is not inhibited by ARK5 phosphorylation and induces cell death even in ARK5-expressing cells; active ARK5 phosphorylates wild-type but not S257A caspase-6 in vitro.","method":"In vitro kinase assay with ARK5 and caspase-6, site-directed mutagenesis (S257A), cell death assay, ARK5 antisense","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro kinase assay plus mutagenesis with functional cell death readout","pmids":["15273717"],"is_preprint":false},{"year":2004,"finding":"Caspase-6 VEID-cleaving activity temporally correlates with procaspase-6 processing during lens fiber cell organelle elimination, distinct from caspase-3 activity seen in apoptosis, suggesting caspase-6 has a role in normal lens differentiation separate from classical apoptosis.","method":"Fluorogenic peptide (VEID-AFC) cleavage assay in lens extracts, Western blot for procaspase-6, transgenic mouse comparison","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — correlated biochemical activity with developmental process, but mechanistic details limited to association","pmids":["15161922"],"is_preprint":false},{"year":2006,"finding":"Caspase-1 is an upstream activator of caspase-6 in primary human neurons: recombinant caspase-1 cleaves pro-caspase-6 in vitro to generate caspase-6 activity; caspase-1 inhibitor (Z-YVAD-fmk) prevents caspase-6 activation and cell death during serum deprivation.","method":"In vitro cleavage of pro-caspase-6 by recombinant caspase-1, caspase-1 inhibitor, dominant-negative caspase-1 construct, primary human neuron cultures","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro reconstitution plus genetic dominant-negative and pharmacological inhibition","pmids":["16123779"],"is_preprint":false},{"year":2006,"finding":"Caspase-6 is selectively activated during resveratrol-induced apoptosis in colon cancer cells and its activity is essential for lamin A cleavage; partial knockdown of caspase-6 by siRNA significantly inhibits both lamin A cleavage and apoptosis; caspase-6 activation and lamin A cleavage are reduced in Bax-/- and p53-/- cells.","method":"siRNA knockdown, caspase-6 peptide inhibitors, Western blot for lamin A cleavage, TUNEL, flow cytometry","journal":"Proteomics","confidence":"High","confidence_rationale":"Tier 2 — siRNA knockdown plus pharmacological inhibition with specific biochemical and apoptotic readouts","pmids":["16518869"],"is_preprint":false},{"year":2007,"finding":"p53 transcriptionally activates caspase-6 (and caspase-7) via direct DNA binding to intronic p53 response elements; ChIP, reporter gene, and EMSA assays confirm p53 binding; p53-/- cells fail to upregulate caspase-6/7 or activate them after cisplatin treatment.","method":"ChIP, reporter gene assay, EMSA, real-time PCR, p53-/- cells, p53 overexpression/inhibition","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1–2 — three orthogonal methods (ChIP, reporter, EMSA) confirm p53 transcriptional regulation","pmids":["18064040"],"is_preprint":false},{"year":2008,"finding":"Proteomic analysis identified 24 caspase-6 substrates in human neurons, including alpha-tubulin, alpha-actinin-4, spinophilin, and drebrin; caspase-6 cleavage sites were identified for drebrin, spinophilin, and alpha-tubulin; a neoepitope antibody to caspase-6-cleaved alpha-tubulin co-localizes with active caspase-6 in Alzheimer's disease lesions.","method":"2D gel proteomics, LC/MS/MS, in vitro cleavage confirmation, neoepitope antibody, immunohistochemistry of AD brain","journal":"Molecular & cellular proteomics : MCP","confidence":"High","confidence_rationale":"Tier 1–2 — MS-based substrate discovery confirmed by in vitro cleavage assay and neoepitope validation in human tissue","pmids":["18487604"],"is_preprint":false},{"year":2008,"finding":"Self-activation of caspase-6: caspase-6 undergoes self-processing in vitro and in vivo; the pro-domain prevents self-activation in vivo but not in vitro; cleavage at either D179 or D193 in the linker is sufficient for activity; caspase-6 activity does not necessarily induce cell death in HEK293T cells.","method":"Site-directed mutagenesis of pro-domain (D23), linker cleavage sites (D179, D193), in vitro and in vivo activity assays","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 1 — reconstituted self-processing with mutagenesis of key cleavage sites","pmids":["19133298"],"is_preprint":false},{"year":2008,"finding":"Active caspase-6 and caspase-6-cleaved huntingtin fragments (at aa 586) co-localize specifically in the nucleus of striatal cells; cell stress (staurosporine) causes nuclear translocation and activation of caspase-6 and increases nuclear 586 aa huntingtin fragments; caspase-2/3-generated 552 aa fragments localize to the perinuclear region.","method":"Neo-epitope antibodies to caspase fragments, subcellular fractionation, immunofluorescence, staurosporine treatment","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 — neo-epitope antibodies with subcellular fractionation and live cell imaging, mechanistic subcellular localization with functional implication","pmids":["18445618"],"is_preprint":false},{"year":2008,"finding":"Caspase-6 plays an important regulatory role in bile acid-induced hepatocyte apoptosis: caspase-6 is activated between caspase-9 and caspase-8 (GCDCA-induced activation of caspases-3/-7 is reduced in caspase-6-deficient cells); GCDCA-induced apoptosis is reduced by 50% in caspase-6-deficient HepG2-Ntcp cells and in primary rat hepatocytes pretreated with caspase-6 inhibitor.","method":"Caspase-6-deficient cells (siRNA/KO), caspase inhibitors (caspase-9, -6, -8), Western blot for caspase activation, primary rat hepatocytes","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — caspase-6-deficient cells plus pharmacological inhibition establishing epistatic position","pmids":["19017654"],"is_preprint":false},{"year":2009,"finding":"Crystal structure of caspase-6 reveals it is a constitutive dimer independent of maturation state; the ligand-free structure shows a partially mature but latent conformation with misaligned catalytic machinery and absent substrate-recognition elements; an elongated central alpha-helix replaces the beta-sheet normally abutting substrate in other caspases.","method":"X-ray crystallography, pre-steady-state kinetics","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with biochemical validation of dimerization state and kinetics","pmids":["19694615"],"is_preprint":false},{"year":2009,"finding":"Caspase-6 cleaves DJ-1 (a Parkinson's disease gene product); the PD-associated D149A mutation renders DJ-1 resistant to caspase-6 proteolysis, abolishing its protective phenotype; the caspase-6-derived C-terminal fragment of DJ-1 accounts for p53-dependent cell death.","method":"In vitro cleavage assay, site-directed mutagenesis (D149A), cell death assay, p53-dependent cell death measurement","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro cleavage confirmed with mutagenesis of cleavage-site and functional consequence","pmids":["19680261"],"is_preprint":false},{"year":2010,"finding":"Valosin-containing protein (p97/VCP) is a caspase-6 substrate; caspase-6 cleaves p97 at VAPD(179) generating 28 and 20 kDa N-terminal fragments (not generated by caspase-3 or -7); the p97(1–179) fragment impairs ubiquitin-fusion degradation and N-end rule pathways and destabilizes endogenous p97; cleavage is detected in MCI and AD hippocampal neurons using a neoepitope antibody.","method":"In vitro cleavage assay, mass spectrometry cleavage site identification, overexpression of cleavage fragment, ubiquitin pathway functional assay, neoepitope immunohistochemistry","journal":"The Journal of neuroscience : the official journal of the Society for Neuroscience","confidence":"High","confidence_rationale":"Tier 1 — in vitro cleavage with MS site identification, functional consequence assay, validated in human tissue","pmids":["20427671"],"is_preprint":false},{"year":2010,"finding":"AMPK family member ARK5 phosphorylates caspase-6 at Ser257 to suppress its activation; active ARK5 phosphorylates wild-type but not S257A caspase-6 in vitro; this phosphorylation prevents procaspase-6 activation and thereby mediates resistance to FasL/Fas-induced cell death in colorectal cancer cells.","method":"In vitro kinase assay, S257A mutagenesis, cell death assays, ARK5 antisense","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro kinase assay confirmed by site-directed mutagenesis with functional readout","pmids":["15273717"],"is_preprint":false},{"year":2010,"finding":"Mutant nucleophosmin (NPMc+) directly binds the cleaved, active forms of caspase-6 and caspase-8 (but not procaspases), inhibiting their activities; this cytoplasmic NPMc+ interaction suppresses apoptosis and caspase-6/-8-mediated myeloid differentiation.","method":"Co-immunoprecipitation, direct binding assay, caspase activity assay, myeloid differentiation assay","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — co-IP of active caspase forms plus functional inhibition assay","pmids":["20606168"],"is_preprint":false},{"year":2010,"finding":"Caspase-9 activation in ischemic brain induces downstream caspase-6 activation, which mediates axonal loss before neuronal death; intranasal delivery of a caspase-9 inhibitor at 4 h post-reperfusion reduces caspase-6 activation and axonal loss, establishing caspase-9 as upstream of caspase-6 in ischemic neuronal death.","method":"Caspase-trapping technique in vivo, selective caspase-9 inhibitor (intranasal delivery), temporal/spatial expression analysis, rat stroke model","journal":"The Journal of neuroscience : the official journal of the Society for Neuroscience","confidence":"High","confidence_rationale":"Tier 2 — in vivo inhibitor epistasis with defined temporal sequence and axonal readout","pmids":["21677173"],"is_preprint":false},{"year":2010,"finding":"RIPK1 is an intrinsic pathway caspase-6 substrate; during intrinsic apoptosis, caspase-6 cleaves RIPK1 to prevent RIPK1-dependent pro-inflammatory cytokine production and inhibit the necroptotic pathway.","method":"In vitro cleavage assay, caspase-6 KO/inhibitor studies, cytokine measurement, necroptosis assay","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1–2 — direct in vitro substrate cleavage plus functional genetic and pharmacological validation","pmids":["22858542"],"is_preprint":false},{"year":2012,"finding":"Phosphorylation of caspase-6 at Ser257 (by ARK5) inhibits its activity by causing a steric clash with Pro201 in the L2' loop, resulting in misalignment of the substrate-binding groove and preventing substrate binding; crystal structure of phosphomimetic S257D mutant reveals the structural basis; removal of the proline side chain (P201A) alleviates the clash and restores near-wild-type activity.","method":"X-ray crystallography of S257D phosphomimetic, site-directed mutagenesis (P201A), biochemical activity assay","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with mutagenesis and biochemical validation","pmids":["22483120"],"is_preprint":false},{"year":2012,"finding":"Crystal structures of ΔproCASP6-S257E (phosphomimetic zymogen) and p20/p10-S257E (active form phosphomimetic) reveal that phosphorylation at Ser257 locks the zymogen in a TEVD(193)-bound inhibited state and causes steric hindrance in the active enzyme, preventing self-activation and substrate binding.","method":"X-ray crystallography of phosphomimetic mutants, molecular dynamics simulations, biochemical assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus MD simulation and biochemical assays, orthogonal methods","pmids":["22433863"],"is_preprint":false},{"year":2012,"finding":"Caspase-6 deficiency protects neurons against excitotoxicity, nerve growth factor deprivation, and myelin-induced axonal degeneration; Casp6-/- mice show age-dependent increases in cortical and striatal volume, hypoactive phenotype, and learning deficits, revealing a physiological role of caspase-6 in neuronal homeostasis.","method":"Constitutive Casp6-/- mice, neuronal culture experiments, volumetric MRI, behavioral testing","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 — genetic knockout with multiple defined cellular and behavioral phenotypes","pmids":["22262731"],"is_preprint":false},{"year":2012,"finding":"Caspase-6 regulates B cell activation and differentiation into plasma cells by modifying cell cycle entry: Casp6 KO B cells enter G1 faster but do not increase S phase entry; instead they preferentially differentiate into syndecan-1+ plasma cells with enhanced antibody production.","method":"Casp6 knockout mice, cell cycle analysis (G0/G1/S), flow cytometry for plasma cell markers, serum Ig measurement","journal":"Journal of immunology (Baltimore, Md. : 1950)","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with defined cell biological phenotype and multiple readouts","pmids":["18981099"],"is_preprint":false},{"year":2013,"finding":"p53 increases caspase-6 mRNA levels and activity in tissues expressing mutant huntingtin; this is blocked by the p53 transcriptional inhibitor pifithrin-alpha but not by inhibition of p53's mitochondrial function, placing p53-dependent transcription upstream of caspase-6 activation in HD.","method":"Mouse embryonic fibroblasts from YAC128 mice, pifithrin-alpha treatment, qRT-PCR, caspase-6 activity assay, lamin A cleavage","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 — pharmacological dissection of p53 pathways with specific readouts in HD model cells","pmids":["24070868"],"is_preprint":false},{"year":2015,"finding":"The NLRP1 inflammasome activates caspase-1, which in turn activates caspase-6 in human neurons; NLRP1 or caspase-1 siRNA abolishes stress-induced caspase-6 activation; this NLRP1/Casp1/Casp6 pathway is confirmed in vivo in Nlrp1-/- and Casp1-/- mice and promotes axonal degeneration and amyloid beta 42 ratio increase.","method":"siRNA knockdown of NLRP1 and caspase-1, cell-free caspase-1 activation assay, Nlrp1-/- and Casp1-/- mice, ASC speck formation assay","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1–2 — genetic epistasis in vitro and in vivo with multiple readouts","pmids":["25744023"],"is_preprint":false},{"year":2018,"finding":"A tri-arginine exosite patch (Arg42–Arg44) at the hinge between the N-terminal domain and core of caspase-6 is required for protein substrate hydrolysis but not short peptide cleavage; mutagenesis of this exosite and the cancer-associated R44K mutation markedly reduce protein substrate turnover; hydrogen-deuterium exchange MS reveals a substrate-binding platform encompassing the NTD and 240's region.","method":"Site-directed mutagenesis of exosite residues, hydrogen-deuterium exchange MS, protein and peptide substrate kinetics","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis confirmed by HDX-MS and kinetic assays","pmids":["30420425"],"is_preprint":false},{"year":2019,"finding":"Thioredoxin-1 (Trx1) acts as a gatekeeper for caspase-6 activation: reduced Trx1 decreases caspase-6 enzymatic activity and lamin-B1 cleavage, while fully oxidized Trx1 enhances caspase-6 activation; inhibition of Trx1 promotes nuclear lamin-B1 cleavage in a caspase-6-dependent manner, upstream of caspase-3/7.","method":"Pharmacological/genetic Trx1 inhibition, cell-free nuclear preparations, purified enzymatic assays with reduced/oxidized Trx1, caspase-6 inhibitor, lamin-B1 cleavage assay","journal":"Free radical biology & medicine","confidence":"High","confidence_rationale":"Tier 1–2 — purified enzymatic assays with defined redox forms, confirmed in cellular and in vivo AD model","pmids":["30769159"],"is_preprint":false},{"year":2020,"finding":"Caspase-6 facilitates RIPK3–ZBP1 interaction during influenza A virus (IAV) infection: caspase-6 interacts with RIPK3 through RHIM-dependent binding and promotes ZBP1–RIPK3 association and PANoptosome assembly; this function is independent of caspase-6 enzymatic activity. Caspase-6 is also required for alternative activation of alveolar macrophages during IAV infection.","method":"Casp6-/- mice, co-immunoprecipitation of RIPK3–ZBP1–caspase-6, catalytic mutant of caspase-6, IAV infection model, macrophage polarization assay","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP, KO mice with defined phenotype, catalytic mutant dissecting enzymatic vs. scaffold function","pmids":["32298652"],"is_preprint":false},{"year":2020,"finding":"AMPK phosphorylates caspase-6 protein to inhibit its activation, keeping hepatocyte apoptosis in check; when AMPK activity is suppressed (as in NASH), caspase-6 is activated by caspase-3 or -7; active caspase-6 cleaves Bid to induce cytochrome c release, generating a feedforward apoptotic loop; liver-specific AMPK knockout aggravates NASH damage.","method":"AMPK phosphorylation of caspase-6 (kinase assay), AMPK KO mice (liver-specific), caspase-6 inhibition in vivo, Bid cleavage assay, human and mouse NASH models","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro kinase assay, genetic KO, pharmacological inhibition in multiple models with defined substrates","pmids":["32029622"],"is_preprint":false},{"year":2010,"finding":"Neutrophil-macrophage contact induces caspase-6 activity in alveolar macrophages; caspase-6 cleaves IRAK-M, relieving its inhibition of TLR signaling; without caspase-6 expression, PMN stimulation fails to cleave IRAK-M, degrade IκBα, or induce TNF-α; Casp6-/- mice subjected to sepsis show impaired TNF-α production and decreased mortality.","method":"Caspase-6 KO mice (cecal ligation and puncture), IRAK-M cleavage-resistant mutant, PMN-macrophage co-culture, TNF-α measurement, IκBα degradation","journal":"Journal of immunology (Baltimore, Md. : 1950)","confidence":"High","confidence_rationale":"Tier 2 — KO mice, cleavage-resistant mutant, and in vitro reconstitution establish mechanism","pmids":["21098228"],"is_preprint":false},{"year":2001,"finding":"Transcription factor AP-2alpha is preferentially cleaved by caspase-6 in vitro at Asp19 (DRHD sequence); this cleavage leads to AP-2alpha degradation by the proteasome and loss of DNA-binding activity; mutation of D19A abolishes cleavage by all three caspases tested; cells expressing mutant AP-2alpha are resistant to TNF-alpha-induced apoptosis.","method":"In vitro cleavage with recombinant caspase-6/1/3, D19A mutagenesis, proteasome inhibitor, DNA-binding EMSA, apoptosis assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro cleavage, mutagenesis of cleavage site, functional consequence confirmed","pmids":["11438643"],"is_preprint":false},{"year":2002,"finding":"Human UFD2 (ubiquitin fusion degradation protein) is efficiently cleaved by caspase-6 and granzyme B at Asp123; caspases-3 and -7 cleave at an upstream site (Asp109) with ~10-fold lower efficiency; truncation at the granzyme B/caspase-6 cleavage site abolishes E3-like ubiquitination activity of UFD2.","method":"In vitro cleavage kinetics with recombinant caspases, granzyme B, mass spectrometry for cleavage sites, ubiquitination activity assay","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with kinetics plus functional ubiquitination assay","pmids":["11802788"],"is_preprint":false},{"year":2022,"finding":"FGF4 activates an FGFR4→CaMKKβ→AMPK→Caspase-6 signaling axis in the liver; AMPK phosphorylation of caspase-6 downstream of FGFR4 activation inhibits hepatocellular apoptosis and reduces liver damage in NAFLD/NASH models.","method":"Recombinant FGF4 administration, FGFR4-selective inhibitor, AMPK activation/inhibition, caspase-6 activity assay, mouse NAFLD/NASH models","journal":"Hepatology (Baltimore, Md.)","confidence":"High","confidence_rationale":"Tier 2 — pharmacological and genetic pathway dissection with specific substrate (caspase-6) readout","pmids":["35152446"],"is_preprint":false}],"current_model":"CASP6 is an executioner caspase that constitutively dimerizes and, upon activation by upstream initiator caspases (caspase-1, -8, -9) or by self-processing at D179/D193, cleaves a distinct set of substrates including lamin A/C (at VEID↓), tau (at D13), huntingtin (at D586), RIPK1, IRAK-M, p97/VCP, APP, CBP, SATB1, AP-2alpha, UFD2, and Bid; its activity is negatively regulated by AMPK/ARK5-mediated phosphorylation at Ser257 (which causes substrate-binding groove misalignment) and by thioredoxin-1; in neurons it drives axonal degeneration and is activated via the NLRP1–caspase-1 axis; it also performs scaffold/non-enzymatic functions by facilitating RIPK3–ZBP1 PANoptosome assembly during viral infection; transcriptionally, CASP6 is a p53 target gene regulated through intronic p53 response elements."},"narrative":{"teleology":[{"year":1995,"claim":"Identification of CASP6 as a novel cysteine protease capable of cleaving PARP and inducing apoptosis established it as a member of the executioner caspase family.","evidence":"Recombinant protein expression, fluorogenic peptide cleavage, PARP cleavage, and baculovirus overexpression in Sf9 cells","pmids":["7796396"],"confidence":"High","gaps":["Physiological substrates unknown","Relationship to other caspases not established","Activation mechanism not defined"]},{"year":1996,"claim":"Discovery that CASP6 is the specific apoptotic laminase — cleaving lamin A at VEID↓NG while caspase-3 cannot — defined its unique substrate specificity and explained how nuclear lamina disassembly is executed during apoptosis.","evidence":"In vitro cleavage assays with recombinant caspases, cell-free apoptosis extracts, granzyme B processing, epistasis with Bcl-2/CrmA","pmids":["8710882","8663580"],"confidence":"High","gaps":["Structural basis for VEID specificity unknown","In vivo genetic confirmation of lamin A as exclusive caspase-6 substrate needed"]},{"year":1999,"claim":"Establishing CASP6 as a neuronal caspase that cleaves APP and drives amyloidogenic processing opened its connection to Alzheimer's disease pathogenesis.","evidence":"Inhibitor studies (z-VEID-fmk), in vitro APP cleavage by recombinant caspase-6, pulse-chase labeling in primary human neurons","pmids":["10438520"],"confidence":"High","gaps":["In vivo relevance to AD amyloid burden not yet confirmed","Upstream activation mechanism in neurons unknown"]},{"year":2000,"claim":"Demonstrating that caspase-6 activates caspase-3 (but not vice versa) in cerebellar neurons placed CASP6 upstream of caspase-3 in neuronal apoptotic cascades, revising its classification as a purely downstream executioner.","evidence":"Selective cell-permeable inhibitors and reconstitution in cerebellar granule cell extracts","pmids":["11279545"],"confidence":"High","gaps":["Whether this hierarchical relationship holds in non-neuronal contexts unclear","Initiator caspase upstream of caspase-6 not identified"]},{"year":2001,"claim":"Identification of SATB1 and AP-2α as caspase-6 substrates expanded the functional repertoire to chromatin organization and transcription factor regulation during apoptosis.","evidence":"In vitro cleavage, site-directed mutagenesis, chromatin dissociation and DNA-binding assays in Jurkat and other cell lines","pmids":["11463840","11438643"],"confidence":"High","gaps":["Whether caspase-6 cleavage of these factors occurs in vivo during physiological apoptosis uncertain"]},{"year":2002,"claim":"Genetic ablation of both CASP6 alleles in DT40 cells provided definitive evidence that caspase-6 is essential for lamin A/C cleavage and complete apoptotic morphology, while p53 was shown to directly transactivate the CASP6 gene through intronic response elements.","evidence":"DT40 knockout with complementation; ChIP, reporter assay, and caspase-6 inhibitor in p53-responsive cells","pmids":["11953316","12089322"],"confidence":"High","gaps":["Mammalian knockout confirmation of lamin cleavage essentiality needed","Other transcription factors regulating CASP6 not explored"]},{"year":2004,"claim":"Discovery that caspase-6 cleaves tau at D13 and that active caspase-6 colocalizes with neurofibrillary tangles in AD brain provided direct biochemical and pathological evidence linking caspase-6 to tauopathy, while ARK5 phosphorylation at Ser257 was identified as a negative regulatory mechanism.","evidence":"In vitro tau cleavage with MS site identification, neoepitope antibody immunohistochemistry of AD brain; ARK5 kinase assay with S257A mutagenesis","pmids":["15356202","15277226","15273717"],"confidence":"High","gaps":["Causal role of tau D13 cleavage in tangle progression unproven","Structural mechanism of Ser257 phosphorylation-mediated inhibition unknown"]},{"year":2006,"claim":"Identification of caspase-1 as a direct upstream activator of caspase-6 in primary human neurons established the inflammasome–caspase-6 connection and explained how neuronal caspase-6 becomes activated during trophic deprivation.","evidence":"Recombinant caspase-1 cleaves pro-caspase-6 in vitro; caspase-1 inhibitor and dominant-negative block caspase-6 activation in primary neurons","pmids":["16123779"],"confidence":"High","gaps":["Inflammasome component responsible for caspase-1 activation in neurons not identified","Whether caspase-1-dependent activation occurs in non-neuronal cells unknown"]},{"year":2008,"claim":"Proteomic discovery of 24 neuronal caspase-6 substrates (including α-tubulin, spinophilin, drebrin) and demonstration that caspase-6 undergoes self-processing at D179/D193 expanded both its substrate network and the understanding of its activation mechanisms.","evidence":"2D gel/LC-MS/MS proteomics in neurons, neoepitope validation in AD brain; site-directed mutagenesis of self-cleavage sites","pmids":["18487604","19133298"],"confidence":"High","gaps":["Biological consequence of most newly identified substrate cleavages not assessed","Physiological context in which self-activation dominates unclear"]},{"year":2009,"claim":"Crystal structure of caspase-6 revealed constitutive dimerization with a unique latent conformation featuring a misaligned catalytic site and an elongated central α-helix replacing the substrate-binding β-sheet, explaining how the enzyme remains inactive until proteolytic maturation.","evidence":"X-ray crystallography with pre-steady-state kinetics","pmids":["19694615"],"confidence":"High","gaps":["No structure of substrate-bound active form","Mechanism of allosteric activation not fully resolved"]},{"year":2010,"claim":"Multiple studies established CASP6 as a key mediator of neuronal axonal degeneration downstream of caspase-9 in ischemia, identified RIPK1 and IRAK-M as immunologically important substrates, and confirmed AMPK-dependent Ser257 phosphorylation as a regulatory mechanism, collectively broadening caspase-6's roles beyond classical apoptosis.","evidence":"Caspase-9 inhibitor in rat stroke model; RIPK1 cleavage with caspase-6 KO; IRAK-M cleavage-resistant mutant in Casp6−/− mice with sepsis; ARK5/AMPK kinase assays","pmids":["21677173","22858542","21098228","15273717"],"confidence":"High","gaps":["How caspase-6 selects between pro-apoptotic and immunomodulatory substrate cleavage in vivo unclear","Structural basis for RIPK1 and IRAK-M recognition not determined"]},{"year":2012,"claim":"Crystal structures of Ser257 phosphomimetic mutants revealed the structural mechanism of AMPK-mediated inhibition — phospho-Ser257 clashes with Pro201 in the L2' loop, misaligning the substrate-binding groove — while Casp6−/− mice showed neuroprotection yet age-dependent brain volume increases and behavioral deficits, demonstrating a physiological homeostatic role.","evidence":"X-ray crystallography of S257D/S257E mutants with P201A rescue; Casp6−/− mice with MRI, behavioral testing, and neuronal culture neuroprotection","pmids":["22483120","22433863","22262731"],"confidence":"High","gaps":["Whether Ser257 phosphorylation is dynamically regulated in neurons in vivo unconfirmed","Mechanism of caspase-6-dependent brain volume regulation unknown"]},{"year":2015,"claim":"Elucidation of the NLRP1→caspase-1→caspase-6 pathway in human neurons, validated in Nlrp1−/− and Casp1−/− mice, defined the complete inflammasome-to-executioner cascade driving axonal degeneration and Aβ42 ratio increase.","evidence":"siRNA of NLRP1 and caspase-1, Nlrp1−/− and Casp1−/− mice, ASC speck formation assay","pmids":["25744023"],"confidence":"High","gaps":["Triggers of NLRP1 activation in aging/AD neurons not defined","Whether NLRP1–caspase-6 axis is druggable not tested"]},{"year":2018,"claim":"Identification of a tri-arginine exosite (R42–R44) required for protein (but not peptide) substrate hydrolysis, with HDX-MS revealing a substrate-binding platform spanning the NTD and 240's loop, established that caspase-6 uses an exosite mechanism for macromolecular substrate recognition.","evidence":"Site-directed mutagenesis of exosite residues, HDX-MS, protein vs. peptide kinetics","pmids":["30420425"],"confidence":"High","gaps":["No co-crystal structure with protein substrate","How exosite contributes to substrate selectivity among different protein substrates not mapped"]},{"year":2020,"claim":"Discovery that caspase-6 performs a non-enzymatic scaffold function by facilitating RIPK3–ZBP1 PANoptosome assembly via RHIM-dependent binding during influenza infection, independent of catalytic activity, fundamentally expanded the gene's functional repertoire beyond proteolysis, while AMPK-mediated inhibition of caspase-6 was placed in a hepatoprotective FGF4→FGFR4→CaMKKβ→AMPK axis in NASH.","evidence":"Casp6−/− mice with IAV, co-IP of RIPK3–ZBP1–caspase-6, catalytic mutant dissection; AMPK KO mice, Bid cleavage assay, NASH models","pmids":["32298652","32029622"],"confidence":"High","gaps":["RHIM-binding interface of caspase-6 not structurally characterized","Whether scaffold function extends to other PANoptosome-inducing stimuli unknown","In vivo relevance of the AMPK–caspase-6–Bid feedforward loop in human NASH not confirmed"]},{"year":null,"claim":"Key unresolved questions include: the structure of active caspase-6 bound to a protein substrate, how caspase-6 switches between enzymatic and scaffold functions in different cellular contexts, and the therapeutic potential of caspase-6 modulation in neurodegeneration and liver disease.","evidence":"","pmids":[],"confidence":"Low","gaps":["No co-crystal structure with protein substrate exists","Context-dependent regulatory switches between scaffold and enzymatic roles undefined","No clinical data on caspase-6-targeted interventions"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,4,6,7,11,17,22,23,27,37,38,39,40]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[36]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[19,7]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3,25]}],"pathway":[{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[0,1,2,3,7,15,20,30,37]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[36,38,31]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[4,10,11,19,32,37,41]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[12,24,28,29,37,41]}],"complexes":["PANoptosome (RIPK3–ZBP1–CASP6)"],"partners":["RIPK3","ZBP1","CASP1","CASP9","CASP3","NUAK1","TXN","TP53"],"other_free_text":[]},"mechanistic_narrative":"CASP6 is an executioner cysteine protease that cleaves a distinct repertoire of protein substrates during apoptosis and fulfills non-enzymatic scaffold functions in innate immune signaling. As the sole apoptotic laminase, CASP6 cleaves lamin A/C at the VEID↓NG site to drive nuclear envelope disassembly and chromatin condensation, and it processes diverse substrates including tau (at D13), APP, huntingtin (at D586), p97/VCP, SATB1, CBP, RIPK1, IRAK-M, DJ-1, AP-2α, and UFD2, linking it to neurodegeneration, ubiquitin-proteasome disruption, and inflammatory signaling [PMID:8710882, PMID:11953316, PMID:15356202, PMID:10438520, PMID:20427671, PMID:22858542, PMID:21098228]. CASP6 is activated by upstream caspase-1 (via the NLRP1 inflammasome), caspase-9, or through self-processing at D179/D193, and its activity is negatively regulated by AMPK/ARK5-mediated phosphorylation at Ser257, which causes substrate-binding groove misalignment as revealed by crystal structures, and by thioredoxin-1 redox state; it is a direct p53 transcriptional target induced through intronic p53 response elements [PMID:25744023, PMID:22483120, PMID:22433863, PMID:30769159, PMID:12089322]. Independent of its catalytic activity, CASP6 facilitates RIPK3–ZBP1 PANoptosome assembly through RHIM-dependent binding during influenza A virus infection, demonstrating a scaffold function in innate immunity [PMID:32298652]."},"prefetch_data":{"uniprot":{"accession":"P55212","full_name":"Caspase-6","aliases":["Apoptotic protease Mch-2"],"length_aa":293,"mass_kda":33.3,"function":"Cysteine protease that plays essential roles in programmed cell death, axonal degeneration, development and innate immunity (PubMed:19133298, PubMed:22858542, PubMed:27032039, PubMed:28864531, PubMed:30420425, PubMed:32298652, PubMed:8663580). Acts as a non-canonical executioner caspase during apoptosis: localizes in the nucleus and cleaves the nuclear structural protein NUMA1 and lamin A/LMNA thereby inducing nuclear shrinkage and fragmentation (PubMed:11953316, PubMed:17401638, PubMed:8663580, PubMed:9463409). Lamin-A/LMNA cleavage is required for chromatin condensation and nuclear disassembly during apoptotic execution (PubMed:11953316). Acts as a regulator of liver damage by promoting hepatocyte apoptosis: in absence of phosphorylation by AMP-activated protein kinase (AMPK), catalyzes cleavage of BID, leading to cytochrome c release, thereby participating in nonalcoholic steatohepatitis (PubMed:32029622). Cleaves PARK7/DJ-1 in cells undergoing apoptosis (By similarity). Involved in intrinsic apoptosis by mediating cleavage of RIPK1 (PubMed:22858542). Furthermore, cleaves many transcription factors such as NF-kappa-B and cAMP response element-binding protein/CREBBP (PubMed:10559921, PubMed:14657026). Cleaves phospholipid scramblase proteins XKR4 and XKR9 (By similarity). In addition to apoptosis, involved in different forms of programmed cell death (PubMed:32298652). Plays an essential role in defense against viruses by acting as a central mediator of the ZBP1-mediated pyroptosis, apoptosis, and necroptosis (PANoptosis), independently of its cysteine protease activity (PubMed:32298652). PANoptosis is a unique inflammatory programmed cell death, which provides a molecular scaffold that allows the interactions and activation of machinery required for inflammasome/pyroptosis, apoptosis and necroptosis (PubMed:32298652). Mechanistically, interacts with RIPK3 and enhances the interaction between RIPK3 and ZBP1, leading to ZBP1-mediated inflammasome activation and cell death (PubMed:32298652). Plays an essential role in axon degeneration during axon pruning which is the remodeling of axons during neurogenesis but not apoptosis (By similarity). Regulates B-cell programs both during early development and after antigen stimulation (By similarity) (Microbial infection) Proteolytically cleaves the N protein of coronaviruses such as MERS-CoV and SARS-CoV (PubMed:18155731, PubMed:35922005). The cleavage of MERS-CoV N-protein leads to two fragments and modulates coronavirus replication by regulating IFN signaling. The two fragments produced by the cleavage interact with IRF3 inhibiting its nuclear translocation after activation and reduce the expression of IFNB and IFN-stimulated genes (PubMed:35922005). The same mechanism seems to be used by other coronaviruses such as SARS-CoV and SARS-CoV-2 to enhance their replication (PubMed:35922005)","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/P55212/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CASP6","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000138794","cell_line_id":"CID001707","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"nucleoplasm","grade":3}],"interactors":[],"url":"https://opencell.sf.czbiohub.org/target/CID001707","total_profiled":1310},"omim":[{"mim_id":"618852","title":"AUTOINFLAMMATION WITH EPISODIC FEVER AND LYMPHADENOPATHY; AIEFL","url":"https://www.omim.org/entry/618852"},{"mim_id":"613004","title":"HUNTINGTIN; HTT","url":"https://www.omim.org/entry/613004"},{"mim_id":"606418","title":"24-@DEHYDROCHOLESTEROL REDUCTASE; DHCR24","url":"https://www.omim.org/entry/606418"},{"mim_id":"603453","title":"RECEPTOR-INTERACTING SERINE/THREONINE KINASE 1; RIPK1","url":"https://www.omim.org/entry/603453"},{"mim_id":"603385","title":"NEDD8-ACTIVATING ENZYME E1, SUBUNIT 1; NAE1","url":"https://www.omim.org/entry/603385"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Nucleoli fibrillar center","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"intestine","ntpm":38.1}],"url":"https://www.proteinatlas.org/search/CASP6"},"hgnc":{"alias_symbol":["MCH2","caspase-6","CSP-6"],"prev_symbol":[]},"alphafold":{"accession":"P55212","domains":[{"cath_id":"3.40.50.1460","chopping":"44-172_185-282","consensus_level":"high","plddt":90.9801,"start":44,"end":282}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P55212","model_url":"https://alphafold.ebi.ac.uk/files/AF-P55212-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P55212-F1-predicted_aligned_error_v6.png","plddt_mean":84.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CASP6","jax_strain_url":"https://www.jax.org/strain/search?query=CASP6"},"sequence":{"accession":"P55212","fasta_url":"https://rest.uniprot.org/uniprotkb/P55212.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P55212/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P55212"}},"corpus_meta":[{"pmid":"8710882","id":"PMC_8710882","title":"Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis.","date":"1996","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/8710882","citation_count":464,"is_preprint":false},{"pmid":"32298652","id":"PMC_32298652","title":"Caspase-6 Is a Key Regulator of Innate Immunity, Inflammasome Activation, and Host Defense.","date":"2020","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/32298652","citation_count":419,"is_preprint":false},{"pmid":"7796396","id":"PMC_7796396","title":"Mch2, a new member of the apoptotic Ced-3/Ice cysteine protease gene family.","date":"1995","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/7796396","citation_count":405,"is_preprint":false},{"pmid":"8663580","id":"PMC_8663580","title":"The CED-3/ICE-like protease Mch2 is activated during apoptosis and cleaves the death substrate lamin A.","date":"1996","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8663580","citation_count":385,"is_preprint":false},{"pmid":"9171342","id":"PMC_9171342","title":"Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells.","date":"1997","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/9171342","citation_count":327,"is_preprint":false},{"pmid":"32029622","id":"PMC_32029622","title":"An AMPK-caspase-6 axis controls liver damage in nonalcoholic steatohepatitis.","date":"2020","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/32029622","citation_count":259,"is_preprint":false},{"pmid":"14657026","id":"PMC_14657026","title":"Critical loss of CBP/p300 histone acetylase activity by caspase-6 during neurodegeneration.","date":"2003","source":"The EMBO 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CASP6.","date":"2005","source":"Proteins","url":"https://pubmed.ncbi.nlm.nih.gov/16187349","citation_count":127,"is_preprint":false},{"pmid":"11279545","id":"PMC_11279545","title":"Caspase 6 activity initiates caspase 3 activation in cerebellar granule cell apoptosis.","date":"2000","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/11279545","citation_count":118,"is_preprint":false},{"pmid":"11479218","id":"PMC_11479218","title":"Treatment of malignant glioma cells with the transfer of constitutively active caspase-6 using the human telomerase catalytic subunit (human telomerase reverse transcriptase) gene promoter.","date":"2001","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/11479218","citation_count":115,"is_preprint":false},{"pmid":"18487604","id":"PMC_18487604","title":"Targets of caspase-6 activity in human neurons and Alzheimer disease.","date":"2008","source":"Molecular & cellular proteomics : 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specifically colocalize in the nucleus.","date":"2008","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/18445618","citation_count":100,"is_preprint":false},{"pmid":"16187345","id":"PMC_16187345","title":"Assessment of predictions submitted for the CASP6 comparative modeling category.","date":"2005","source":"Proteins","url":"https://pubmed.ncbi.nlm.nih.gov/16187345","citation_count":90,"is_preprint":false},{"pmid":"11562423","id":"PMC_11562423","title":"Cloning and molecular characterization of the novel human melanin-concentrating hormone receptor MCH2.","date":"2001","source":"Molecular pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/11562423","citation_count":86,"is_preprint":false},{"pmid":"25340928","id":"PMC_25340928","title":"Activation and regulation of caspase-6 and its role in neurodegenerative diseases.","date":"2014","source":"Annual review of pharmacology and 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baculovirus overexpression\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic assay with recombinant protein, replicated in multiple readouts\",\n      \"pmids\": [\"7796396\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"CASP6 (Mch2 alpha) cleaves nuclear lamin A at the conserved VEID↓NG sequence in the coiled-coil rod domain, producing the apoptotic lamin A fragment; CPP32 (caspase-3) does not cleave lamin A, establishing distinct substrate specificities between these caspases.\",\n      \"method\": \"In vitro cleavage assay with recombinant IRPs, cell-free apoptosis extracts, Zn2+ inhibition, affinity labeling\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro cleavage, replicated in cell-free system, confirmed with inhibitor\",\n      \"pmids\": [\"8710882\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"CASP6 (Mch2) is processed from its zymogen to a proteolytically active dimeric species during apoptosis and by granzyme B; it functions downstream of death inhibitors Bcl-2, Bcl-xL, and CrmA; it cleaves lamin A to its signature apoptotic fragment, whereas caspase-3 (Yama) and caspase-7 (LAP3) do not, identifying CASP6 as an apoptotic laminase.\",\n      \"method\": \"In vitro cleavage assay, granzyme B processing, epistasis with Bcl-2/Bcl-xL/CrmA overexpression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro reconstitution plus genetic epistasis, independent confirmation of lamin A cleavage\",\n      \"pmids\": [\"8663580\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"CASP6 (Mch2/caspase-6) and caspase-3 (CPP32) are the major active caspases present in apoptotic tumor cells in response to diverse apoptosis-inducing stimuli; both are present as multiple active species whose composition varies between cell lines.\",\n      \"method\": \"Active caspase detection approach using biotinylated caspase probes, multiple cell lines, multiple stimuli\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — orthogonal detection method, replicated across multiple cell lines and stimuli\",\n      \"pmids\": [\"9171342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"CASP6 is activated during serum deprivation-induced apoptosis in primary human neurons; it directly cleaves amyloid precursor protein (APP) at the C-terminus generating Capp3 and Capp6.5 fragments; caspase-6 inhibition prevents the serum deprivation-mediated increase in amyloid beta peptide, establishing a caspase-6-dependent amyloidogenic pathway.\",\n      \"method\": \"Inhibitor studies (z-VEID-fmk), in vitro cleavage of APP by recombinant caspase-6, pulse-chase metabolic labeling, immunoblot of human AD brain\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct in vitro substrate cleavage plus functional loss-of-function in primary neurons with defined biochemical readout\",\n      \"pmids\": [\"10438520\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"In cerebellar granule cell apoptosis induced by trophic support withdrawal, active caspase-6 can process and activate procaspase-3 in cellular extracts, whereas caspase-3 does not activate caspase-6; cell-permeable caspase-6 inhibitor prevents caspase-3 activation, placing caspase-6 upstream of caspase-3 in this pathway.\",\n      \"method\": \"Fluorogenic peptide assays, immunoblot, cell-permeable selective inhibitors (CP-VEID-cho, CP-DEVD-cho), ex vivo cerebellar granule cell system\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — biochemical reconstitution in cellular extracts plus selective inhibitor epistasis\",\n      \"pmids\": [\"11279545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CASP6 cleaves SATB1 at amino acid position 254, separating the DNA-binding domains from the PDZ-like dimerization domain (aa 90–204); cleavage abolishes BUR-binding activity and causes rapid dissociation of SATB1 from chromatin in vivo, coinciding with high-molecular-weight chromatin fragmentation in apoptotic T cells.\",\n      \"method\": \"In vitro cleavage with caspase-6, site-directed mutagenesis, in vivo chromatin dissociation assay, Fas-induced apoptosis in Jurkat cells\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro reconstitution plus in vivo functional consequence confirmed by chromatin dissociation\",\n      \"pmids\": [\"11463840\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Disruption of both CASP6 alleles in chicken DT40 cells reveals that caspase-6 activity is essential for lamin A/C cleavage during apoptosis and for complete chromatin condensation and apoptotic body formation when lamin A is present; lamins A and C are caspase-6-only substrates in this system.\",\n      \"method\": \"Gene disruption (both alleles), cell-free nuclear disassembly assay, complementation with exogenous caspase-6, caspase-6 inhibitor z-VEID-fmk\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — genetic knockout with defined biochemical phenotype, rescued by complementation, replicated with inhibitor\",\n      \"pmids\": [\"11953316\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"p53 binds the third intron of the caspase-6 gene and transcriptionally activates it; p53-dependent increase in procaspase-6 protein enables increased caspase-6 activity and lamin A cleavage in response to Adriamycin; specific inhibition of caspase-6 blocks p53-mediated cell death.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP) of p53 to caspase-6 intron 3, reporter assay, immunoblot of lamin A cleavage, caspase-6 inhibitor\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct DNA binding shown by ChIP, functional validation with inhibitor\",\n      \"pmids\": [\"12089322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CBP (CREB-binding protein) is a caspase-6 substrate in primary neurons; caspase-6-mediated cleavage of CBP reduces CBP/p300 histone acetyltransferase activity during neuronal apoptosis, linking caspase-6 to transcriptional dysregulation and histone deacetylation in neurodegeneration.\",\n      \"method\": \"In vitro cleavage assay with recombinant caspase-6, HAT activity assay, primary neuron apoptosis model\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct in vitro substrate identification with functional HAT activity readout\",\n      \"pmids\": [\"14657026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Active caspase-6 p20 subunit is present in neurofibrillary tangles, neuropil threads, and neuritic plaques in Alzheimer's disease brain; a neoepitope antibody to caspase-6-cleaved Tau detects intracellular and extracellular tangles and pretangles, indicating early caspase-6 activity in AD pathogenesis.\",\n      \"method\": \"Neoepitope antibodies to active caspase-6 p20 and caspase-6-cleaved Tau, immunohistochemistry of AD brain tissue\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — neoepitope antibodies in human tissue, multiple lesion types, correlated with disease stage\",\n      \"pmids\": [\"15277226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Caspase-6 cleaves the N-terminus of tau in vitro at D13 (a semicanonical cleavage site), preventing immunoreactivity with N-terminal tau antibodies; mass spectrometry confirmed this cleavage site, linking caspase-6-mediated N-terminal tau truncation to neurofibrillary tangle evolution in AD.\",\n      \"method\": \"In vitro cleavage of tau by recombinant caspase-6, mass spectrometry confirmation of cleavage site, immunohistochemistry\",\n      \"journal\": \"The Journal of neuroscience : the official journal of the Society for Neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic cleavage with mass spectrometry identification of cleavage site\",\n      \"pmids\": [\"15356202\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ARK5 kinase phosphorylates caspase-6 at Ser257, preventing its activation; mutant caspase-6 with S257A substitution is not inhibited by ARK5 phosphorylation and induces cell death even in ARK5-expressing cells; active ARK5 phosphorylates wild-type but not S257A caspase-6 in vitro.\",\n      \"method\": \"In vitro kinase assay with ARK5 and caspase-6, site-directed mutagenesis (S257A), cell death assay, ARK5 antisense\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro kinase assay plus mutagenesis with functional cell death readout\",\n      \"pmids\": [\"15273717\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Caspase-6 VEID-cleaving activity temporally correlates with procaspase-6 processing during lens fiber cell organelle elimination, distinct from caspase-3 activity seen in apoptosis, suggesting caspase-6 has a role in normal lens differentiation separate from classical apoptosis.\",\n      \"method\": \"Fluorogenic peptide (VEID-AFC) cleavage assay in lens extracts, Western blot for procaspase-6, transgenic mouse comparison\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — correlated biochemical activity with developmental process, but mechanistic details limited to association\",\n      \"pmids\": [\"15161922\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Caspase-1 is an upstream activator of caspase-6 in primary human neurons: recombinant caspase-1 cleaves pro-caspase-6 in vitro to generate caspase-6 activity; caspase-1 inhibitor (Z-YVAD-fmk) prevents caspase-6 activation and cell death during serum deprivation.\",\n      \"method\": \"In vitro cleavage of pro-caspase-6 by recombinant caspase-1, caspase-1 inhibitor, dominant-negative caspase-1 construct, primary human neuron cultures\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro reconstitution plus genetic dominant-negative and pharmacological inhibition\",\n      \"pmids\": [\"16123779\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Caspase-6 is selectively activated during resveratrol-induced apoptosis in colon cancer cells and its activity is essential for lamin A cleavage; partial knockdown of caspase-6 by siRNA significantly inhibits both lamin A cleavage and apoptosis; caspase-6 activation and lamin A cleavage are reduced in Bax-/- and p53-/- cells.\",\n      \"method\": \"siRNA knockdown, caspase-6 peptide inhibitors, Western blot for lamin A cleavage, TUNEL, flow cytometry\",\n      \"journal\": \"Proteomics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — siRNA knockdown plus pharmacological inhibition with specific biochemical and apoptotic readouts\",\n      \"pmids\": [\"16518869\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"p53 transcriptionally activates caspase-6 (and caspase-7) via direct DNA binding to intronic p53 response elements; ChIP, reporter gene, and EMSA assays confirm p53 binding; p53-/- cells fail to upregulate caspase-6/7 or activate them after cisplatin treatment.\",\n      \"method\": \"ChIP, reporter gene assay, EMSA, real-time PCR, p53-/- cells, p53 overexpression/inhibition\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — three orthogonal methods (ChIP, reporter, EMSA) confirm p53 transcriptional regulation\",\n      \"pmids\": [\"18064040\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Proteomic analysis identified 24 caspase-6 substrates in human neurons, including alpha-tubulin, alpha-actinin-4, spinophilin, and drebrin; caspase-6 cleavage sites were identified for drebrin, spinophilin, and alpha-tubulin; a neoepitope antibody to caspase-6-cleaved alpha-tubulin co-localizes with active caspase-6 in Alzheimer's disease lesions.\",\n      \"method\": \"2D gel proteomics, LC/MS/MS, in vitro cleavage confirmation, neoepitope antibody, immunohistochemistry of AD brain\",\n      \"journal\": \"Molecular & cellular proteomics : MCP\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — MS-based substrate discovery confirmed by in vitro cleavage assay and neoepitope validation in human tissue\",\n      \"pmids\": [\"18487604\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Self-activation of caspase-6: caspase-6 undergoes self-processing in vitro and in vivo; the pro-domain prevents self-activation in vivo but not in vitro; cleavage at either D179 or D193 in the linker is sufficient for activity; caspase-6 activity does not necessarily induce cell death in HEK293T cells.\",\n      \"method\": \"Site-directed mutagenesis of pro-domain (D23), linker cleavage sites (D179, D193), in vitro and in vivo activity assays\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted self-processing with mutagenesis of key cleavage sites\",\n      \"pmids\": [\"19133298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Active caspase-6 and caspase-6-cleaved huntingtin fragments (at aa 586) co-localize specifically in the nucleus of striatal cells; cell stress (staurosporine) causes nuclear translocation and activation of caspase-6 and increases nuclear 586 aa huntingtin fragments; caspase-2/3-generated 552 aa fragments localize to the perinuclear region.\",\n      \"method\": \"Neo-epitope antibodies to caspase fragments, subcellular fractionation, immunofluorescence, staurosporine treatment\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — neo-epitope antibodies with subcellular fractionation and live cell imaging, mechanistic subcellular localization with functional implication\",\n      \"pmids\": [\"18445618\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Caspase-6 plays an important regulatory role in bile acid-induced hepatocyte apoptosis: caspase-6 is activated between caspase-9 and caspase-8 (GCDCA-induced activation of caspases-3/-7 is reduced in caspase-6-deficient cells); GCDCA-induced apoptosis is reduced by 50% in caspase-6-deficient HepG2-Ntcp cells and in primary rat hepatocytes pretreated with caspase-6 inhibitor.\",\n      \"method\": \"Caspase-6-deficient cells (siRNA/KO), caspase inhibitors (caspase-9, -6, -8), Western blot for caspase activation, primary rat hepatocytes\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — caspase-6-deficient cells plus pharmacological inhibition establishing epistatic position\",\n      \"pmids\": [\"19017654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Crystal structure of caspase-6 reveals it is a constitutive dimer independent of maturation state; the ligand-free structure shows a partially mature but latent conformation with misaligned catalytic machinery and absent substrate-recognition elements; an elongated central alpha-helix replaces the beta-sheet normally abutting substrate in other caspases.\",\n      \"method\": \"X-ray crystallography, pre-steady-state kinetics\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with biochemical validation of dimerization state and kinetics\",\n      \"pmids\": [\"19694615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Caspase-6 cleaves DJ-1 (a Parkinson's disease gene product); the PD-associated D149A mutation renders DJ-1 resistant to caspase-6 proteolysis, abolishing its protective phenotype; the caspase-6-derived C-terminal fragment of DJ-1 accounts for p53-dependent cell death.\",\n      \"method\": \"In vitro cleavage assay, site-directed mutagenesis (D149A), cell death assay, p53-dependent cell death measurement\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro cleavage confirmed with mutagenesis of cleavage-site and functional consequence\",\n      \"pmids\": [\"19680261\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Valosin-containing protein (p97/VCP) is a caspase-6 substrate; caspase-6 cleaves p97 at VAPD(179) generating 28 and 20 kDa N-terminal fragments (not generated by caspase-3 or -7); the p97(1–179) fragment impairs ubiquitin-fusion degradation and N-end rule pathways and destabilizes endogenous p97; cleavage is detected in MCI and AD hippocampal neurons using a neoepitope antibody.\",\n      \"method\": \"In vitro cleavage assay, mass spectrometry cleavage site identification, overexpression of cleavage fragment, ubiquitin pathway functional assay, neoepitope immunohistochemistry\",\n      \"journal\": \"The Journal of neuroscience : the official journal of the Society for Neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro cleavage with MS site identification, functional consequence assay, validated in human tissue\",\n      \"pmids\": [\"20427671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"AMPK family member ARK5 phosphorylates caspase-6 at Ser257 to suppress its activation; active ARK5 phosphorylates wild-type but not S257A caspase-6 in vitro; this phosphorylation prevents procaspase-6 activation and thereby mediates resistance to FasL/Fas-induced cell death in colorectal cancer cells.\",\n      \"method\": \"In vitro kinase assay, S257A mutagenesis, cell death assays, ARK5 antisense\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro kinase assay confirmed by site-directed mutagenesis with functional readout\",\n      \"pmids\": [\"15273717\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Mutant nucleophosmin (NPMc+) directly binds the cleaved, active forms of caspase-6 and caspase-8 (but not procaspases), inhibiting their activities; this cytoplasmic NPMc+ interaction suppresses apoptosis and caspase-6/-8-mediated myeloid differentiation.\",\n      \"method\": \"Co-immunoprecipitation, direct binding assay, caspase activity assay, myeloid differentiation assay\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — co-IP of active caspase forms plus functional inhibition assay\",\n      \"pmids\": [\"20606168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Caspase-9 activation in ischemic brain induces downstream caspase-6 activation, which mediates axonal loss before neuronal death; intranasal delivery of a caspase-9 inhibitor at 4 h post-reperfusion reduces caspase-6 activation and axonal loss, establishing caspase-9 as upstream of caspase-6 in ischemic neuronal death.\",\n      \"method\": \"Caspase-trapping technique in vivo, selective caspase-9 inhibitor (intranasal delivery), temporal/spatial expression analysis, rat stroke model\",\n      \"journal\": \"The Journal of neuroscience : the official journal of the Society for Neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo inhibitor epistasis with defined temporal sequence and axonal readout\",\n      \"pmids\": [\"21677173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"RIPK1 is an intrinsic pathway caspase-6 substrate; during intrinsic apoptosis, caspase-6 cleaves RIPK1 to prevent RIPK1-dependent pro-inflammatory cytokine production and inhibit the necroptotic pathway.\",\n      \"method\": \"In vitro cleavage assay, caspase-6 KO/inhibitor studies, cytokine measurement, necroptosis assay\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct in vitro substrate cleavage plus functional genetic and pharmacological validation\",\n      \"pmids\": [\"22858542\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Phosphorylation of caspase-6 at Ser257 (by ARK5) inhibits its activity by causing a steric clash with Pro201 in the L2' loop, resulting in misalignment of the substrate-binding groove and preventing substrate binding; crystal structure of phosphomimetic S257D mutant reveals the structural basis; removal of the proline side chain (P201A) alleviates the clash and restores near-wild-type activity.\",\n      \"method\": \"X-ray crystallography of S257D phosphomimetic, site-directed mutagenesis (P201A), biochemical activity assay\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with mutagenesis and biochemical validation\",\n      \"pmids\": [\"22483120\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Crystal structures of ΔproCASP6-S257E (phosphomimetic zymogen) and p20/p10-S257E (active form phosphomimetic) reveal that phosphorylation at Ser257 locks the zymogen in a TEVD(193)-bound inhibited state and causes steric hindrance in the active enzyme, preventing self-activation and substrate binding.\",\n      \"method\": \"X-ray crystallography of phosphomimetic mutants, molecular dynamics simulations, biochemical assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus MD simulation and biochemical assays, orthogonal methods\",\n      \"pmids\": [\"22433863\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Caspase-6 deficiency protects neurons against excitotoxicity, nerve growth factor deprivation, and myelin-induced axonal degeneration; Casp6-/- mice show age-dependent increases in cortical and striatal volume, hypoactive phenotype, and learning deficits, revealing a physiological role of caspase-6 in neuronal homeostasis.\",\n      \"method\": \"Constitutive Casp6-/- mice, neuronal culture experiments, volumetric MRI, behavioral testing\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic knockout with multiple defined cellular and behavioral phenotypes\",\n      \"pmids\": [\"22262731\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Caspase-6 regulates B cell activation and differentiation into plasma cells by modifying cell cycle entry: Casp6 KO B cells enter G1 faster but do not increase S phase entry; instead they preferentially differentiate into syndecan-1+ plasma cells with enhanced antibody production.\",\n      \"method\": \"Casp6 knockout mice, cell cycle analysis (G0/G1/S), flow cytometry for plasma cell markers, serum Ig measurement\",\n      \"journal\": \"Journal of immunology (Baltimore, Md. : 1950)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with defined cell biological phenotype and multiple readouts\",\n      \"pmids\": [\"18981099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"p53 increases caspase-6 mRNA levels and activity in tissues expressing mutant huntingtin; this is blocked by the p53 transcriptional inhibitor pifithrin-alpha but not by inhibition of p53's mitochondrial function, placing p53-dependent transcription upstream of caspase-6 activation in HD.\",\n      \"method\": \"Mouse embryonic fibroblasts from YAC128 mice, pifithrin-alpha treatment, qRT-PCR, caspase-6 activity assay, lamin A cleavage\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological dissection of p53 pathways with specific readouts in HD model cells\",\n      \"pmids\": [\"24070868\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The NLRP1 inflammasome activates caspase-1, which in turn activates caspase-6 in human neurons; NLRP1 or caspase-1 siRNA abolishes stress-induced caspase-6 activation; this NLRP1/Casp1/Casp6 pathway is confirmed in vivo in Nlrp1-/- and Casp1-/- mice and promotes axonal degeneration and amyloid beta 42 ratio increase.\",\n      \"method\": \"siRNA knockdown of NLRP1 and caspase-1, cell-free caspase-1 activation assay, Nlrp1-/- and Casp1-/- mice, ASC speck formation assay\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — genetic epistasis in vitro and in vivo with multiple readouts\",\n      \"pmids\": [\"25744023\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"A tri-arginine exosite patch (Arg42–Arg44) at the hinge between the N-terminal domain and core of caspase-6 is required for protein substrate hydrolysis but not short peptide cleavage; mutagenesis of this exosite and the cancer-associated R44K mutation markedly reduce protein substrate turnover; hydrogen-deuterium exchange MS reveals a substrate-binding platform encompassing the NTD and 240's region.\",\n      \"method\": \"Site-directed mutagenesis of exosite residues, hydrogen-deuterium exchange MS, protein and peptide substrate kinetics\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis confirmed by HDX-MS and kinetic assays\",\n      \"pmids\": [\"30420425\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Thioredoxin-1 (Trx1) acts as a gatekeeper for caspase-6 activation: reduced Trx1 decreases caspase-6 enzymatic activity and lamin-B1 cleavage, while fully oxidized Trx1 enhances caspase-6 activation; inhibition of Trx1 promotes nuclear lamin-B1 cleavage in a caspase-6-dependent manner, upstream of caspase-3/7.\",\n      \"method\": \"Pharmacological/genetic Trx1 inhibition, cell-free nuclear preparations, purified enzymatic assays with reduced/oxidized Trx1, caspase-6 inhibitor, lamin-B1 cleavage assay\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — purified enzymatic assays with defined redox forms, confirmed in cellular and in vivo AD model\",\n      \"pmids\": [\"30769159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Caspase-6 facilitates RIPK3–ZBP1 interaction during influenza A virus (IAV) infection: caspase-6 interacts with RIPK3 through RHIM-dependent binding and promotes ZBP1–RIPK3 association and PANoptosome assembly; this function is independent of caspase-6 enzymatic activity. Caspase-6 is also required for alternative activation of alveolar macrophages during IAV infection.\",\n      \"method\": \"Casp6-/- mice, co-immunoprecipitation of RIPK3–ZBP1–caspase-6, catalytic mutant of caspase-6, IAV infection model, macrophage polarization assay\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP, KO mice with defined phenotype, catalytic mutant dissecting enzymatic vs. scaffold function\",\n      \"pmids\": [\"32298652\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"AMPK phosphorylates caspase-6 protein to inhibit its activation, keeping hepatocyte apoptosis in check; when AMPK activity is suppressed (as in NASH), caspase-6 is activated by caspase-3 or -7; active caspase-6 cleaves Bid to induce cytochrome c release, generating a feedforward apoptotic loop; liver-specific AMPK knockout aggravates NASH damage.\",\n      \"method\": \"AMPK phosphorylation of caspase-6 (kinase assay), AMPK KO mice (liver-specific), caspase-6 inhibition in vivo, Bid cleavage assay, human and mouse NASH models\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro kinase assay, genetic KO, pharmacological inhibition in multiple models with defined substrates\",\n      \"pmids\": [\"32029622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Neutrophil-macrophage contact induces caspase-6 activity in alveolar macrophages; caspase-6 cleaves IRAK-M, relieving its inhibition of TLR signaling; without caspase-6 expression, PMN stimulation fails to cleave IRAK-M, degrade IκBα, or induce TNF-α; Casp6-/- mice subjected to sepsis show impaired TNF-α production and decreased mortality.\",\n      \"method\": \"Caspase-6 KO mice (cecal ligation and puncture), IRAK-M cleavage-resistant mutant, PMN-macrophage co-culture, TNF-α measurement, IκBα degradation\",\n      \"journal\": \"Journal of immunology (Baltimore, Md. : 1950)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mice, cleavage-resistant mutant, and in vitro reconstitution establish mechanism\",\n      \"pmids\": [\"21098228\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Transcription factor AP-2alpha is preferentially cleaved by caspase-6 in vitro at Asp19 (DRHD sequence); this cleavage leads to AP-2alpha degradation by the proteasome and loss of DNA-binding activity; mutation of D19A abolishes cleavage by all three caspases tested; cells expressing mutant AP-2alpha are resistant to TNF-alpha-induced apoptosis.\",\n      \"method\": \"In vitro cleavage with recombinant caspase-6/1/3, D19A mutagenesis, proteasome inhibitor, DNA-binding EMSA, apoptosis assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro cleavage, mutagenesis of cleavage site, functional consequence confirmed\",\n      \"pmids\": [\"11438643\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Human UFD2 (ubiquitin fusion degradation protein) is efficiently cleaved by caspase-6 and granzyme B at Asp123; caspases-3 and -7 cleave at an upstream site (Asp109) with ~10-fold lower efficiency; truncation at the granzyme B/caspase-6 cleavage site abolishes E3-like ubiquitination activity of UFD2.\",\n      \"method\": \"In vitro cleavage kinetics with recombinant caspases, granzyme B, mass spectrometry for cleavage sites, ubiquitination activity assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with kinetics plus functional ubiquitination assay\",\n      \"pmids\": [\"11802788\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FGF4 activates an FGFR4→CaMKKβ→AMPK→Caspase-6 signaling axis in the liver; AMPK phosphorylation of caspase-6 downstream of FGFR4 activation inhibits hepatocellular apoptosis and reduces liver damage in NAFLD/NASH models.\",\n      \"method\": \"Recombinant FGF4 administration, FGFR4-selective inhibitor, AMPK activation/inhibition, caspase-6 activity assay, mouse NAFLD/NASH models\",\n      \"journal\": \"Hepatology (Baltimore, Md.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological and genetic pathway dissection with specific substrate (caspase-6) readout\",\n      \"pmids\": [\"35152446\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CASP6 is an executioner caspase that constitutively dimerizes and, upon activation by upstream initiator caspases (caspase-1, -8, -9) or by self-processing at D179/D193, cleaves a distinct set of substrates including lamin A/C (at VEID↓), tau (at D13), huntingtin (at D586), RIPK1, IRAK-M, p97/VCP, APP, CBP, SATB1, AP-2alpha, UFD2, and Bid; its activity is negatively regulated by AMPK/ARK5-mediated phosphorylation at Ser257 (which causes substrate-binding groove misalignment) and by thioredoxin-1; in neurons it drives axonal degeneration and is activated via the NLRP1–caspase-1 axis; it also performs scaffold/non-enzymatic functions by facilitating RIPK3–ZBP1 PANoptosome assembly during viral infection; transcriptionally, CASP6 is a p53 target gene regulated through intronic p53 response elements.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CASP6 is an executioner cysteine protease that cleaves a distinct repertoire of protein substrates during apoptosis and fulfills non-enzymatic scaffold functions in innate immune signaling. As the sole apoptotic laminase, CASP6 cleaves lamin A/C at the VEID↓NG site to drive nuclear envelope disassembly and chromatin condensation, and it processes diverse substrates including tau (at D13), APP, huntingtin (at D586), p97/VCP, SATB1, CBP, RIPK1, IRAK-M, DJ-1, AP-2α, and UFD2, linking it to neurodegeneration, ubiquitin-proteasome disruption, and inflammatory signaling [PMID:8710882, PMID:11953316, PMID:15356202, PMID:10438520, PMID:20427671, PMID:22858542, PMID:21098228]. CASP6 is activated by upstream caspase-1 (via the NLRP1 inflammasome), caspase-9, or through self-processing at D179/D193, and its activity is negatively regulated by AMPK/ARK5-mediated phosphorylation at Ser257, which causes substrate-binding groove misalignment as revealed by crystal structures, and by thioredoxin-1 redox state; it is a direct p53 transcriptional target induced through intronic p53 response elements [PMID:25744023, PMID:22483120, PMID:22433863, PMID:30769159, PMID:12089322]. Independent of its catalytic activity, CASP6 facilitates RIPK3–ZBP1 PANoptosome assembly through RHIM-dependent binding during influenza A virus infection, demonstrating a scaffold function in innate immunity [PMID:32298652].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Identification of CASP6 as a novel cysteine protease capable of cleaving PARP and inducing apoptosis established it as a member of the executioner caspase family.\",\n      \"evidence\": \"Recombinant protein expression, fluorogenic peptide cleavage, PARP cleavage, and baculovirus overexpression in Sf9 cells\",\n      \"pmids\": [\"7796396\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological substrates unknown\", \"Relationship to other caspases not established\", \"Activation mechanism not defined\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Discovery that CASP6 is the specific apoptotic laminase — cleaving lamin A at VEID↓NG while caspase-3 cannot — defined its unique substrate specificity and explained how nuclear lamina disassembly is executed during apoptosis.\",\n      \"evidence\": \"In vitro cleavage assays with recombinant caspases, cell-free apoptosis extracts, granzyme B processing, epistasis with Bcl-2/CrmA\",\n      \"pmids\": [\"8710882\", \"8663580\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for VEID specificity unknown\", \"In vivo genetic confirmation of lamin A as exclusive caspase-6 substrate needed\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Establishing CASP6 as a neuronal caspase that cleaves APP and drives amyloidogenic processing opened its connection to Alzheimer's disease pathogenesis.\",\n      \"evidence\": \"Inhibitor studies (z-VEID-fmk), in vitro APP cleavage by recombinant caspase-6, pulse-chase labeling in primary human neurons\",\n      \"pmids\": [\"10438520\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance to AD amyloid burden not yet confirmed\", \"Upstream activation mechanism in neurons unknown\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Demonstrating that caspase-6 activates caspase-3 (but not vice versa) in cerebellar neurons placed CASP6 upstream of caspase-3 in neuronal apoptotic cascades, revising its classification as a purely downstream executioner.\",\n      \"evidence\": \"Selective cell-permeable inhibitors and reconstitution in cerebellar granule cell extracts\",\n      \"pmids\": [\"11279545\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this hierarchical relationship holds in non-neuronal contexts unclear\", \"Initiator caspase upstream of caspase-6 not identified\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identification of SATB1 and AP-2α as caspase-6 substrates expanded the functional repertoire to chromatin organization and transcription factor regulation during apoptosis.\",\n      \"evidence\": \"In vitro cleavage, site-directed mutagenesis, chromatin dissociation and DNA-binding assays in Jurkat and other cell lines\",\n      \"pmids\": [\"11463840\", \"11438643\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether caspase-6 cleavage of these factors occurs in vivo during physiological apoptosis uncertain\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Genetic ablation of both CASP6 alleles in DT40 cells provided definitive evidence that caspase-6 is essential for lamin A/C cleavage and complete apoptotic morphology, while p53 was shown to directly transactivate the CASP6 gene through intronic response elements.\",\n      \"evidence\": \"DT40 knockout with complementation; ChIP, reporter assay, and caspase-6 inhibitor in p53-responsive cells\",\n      \"pmids\": [\"11953316\", \"12089322\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mammalian knockout confirmation of lamin cleavage essentiality needed\", \"Other transcription factors regulating CASP6 not explored\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Discovery that caspase-6 cleaves tau at D13 and that active caspase-6 colocalizes with neurofibrillary tangles in AD brain provided direct biochemical and pathological evidence linking caspase-6 to tauopathy, while ARK5 phosphorylation at Ser257 was identified as a negative regulatory mechanism.\",\n      \"evidence\": \"In vitro tau cleavage with MS site identification, neoepitope antibody immunohistochemistry of AD brain; ARK5 kinase assay with S257A mutagenesis\",\n      \"pmids\": [\"15356202\", \"15277226\", \"15273717\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Causal role of tau D13 cleavage in tangle progression unproven\", \"Structural mechanism of Ser257 phosphorylation-mediated inhibition unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identification of caspase-1 as a direct upstream activator of caspase-6 in primary human neurons established the inflammasome–caspase-6 connection and explained how neuronal caspase-6 becomes activated during trophic deprivation.\",\n      \"evidence\": \"Recombinant caspase-1 cleaves pro-caspase-6 in vitro; caspase-1 inhibitor and dominant-negative block caspase-6 activation in primary neurons\",\n      \"pmids\": [\"16123779\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Inflammasome component responsible for caspase-1 activation in neurons not identified\", \"Whether caspase-1-dependent activation occurs in non-neuronal cells unknown\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Proteomic discovery of 24 neuronal caspase-6 substrates (including α-tubulin, spinophilin, drebrin) and demonstration that caspase-6 undergoes self-processing at D179/D193 expanded both its substrate network and the understanding of its activation mechanisms.\",\n      \"evidence\": \"2D gel/LC-MS/MS proteomics in neurons, neoepitope validation in AD brain; site-directed mutagenesis of self-cleavage sites\",\n      \"pmids\": [\"18487604\", \"19133298\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biological consequence of most newly identified substrate cleavages not assessed\", \"Physiological context in which self-activation dominates unclear\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Crystal structure of caspase-6 revealed constitutive dimerization with a unique latent conformation featuring a misaligned catalytic site and an elongated central α-helix replacing the substrate-binding β-sheet, explaining how the enzyme remains inactive until proteolytic maturation.\",\n      \"evidence\": \"X-ray crystallography with pre-steady-state kinetics\",\n      \"pmids\": [\"19694615\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of substrate-bound active form\", \"Mechanism of allosteric activation not fully resolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Multiple studies established CASP6 as a key mediator of neuronal axonal degeneration downstream of caspase-9 in ischemia, identified RIPK1 and IRAK-M as immunologically important substrates, and confirmed AMPK-dependent Ser257 phosphorylation as a regulatory mechanism, collectively broadening caspase-6's roles beyond classical apoptosis.\",\n      \"evidence\": \"Caspase-9 inhibitor in rat stroke model; RIPK1 cleavage with caspase-6 KO; IRAK-M cleavage-resistant mutant in Casp6−/− mice with sepsis; ARK5/AMPK kinase assays\",\n      \"pmids\": [\"21677173\", \"22858542\", \"21098228\", \"15273717\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How caspase-6 selects between pro-apoptotic and immunomodulatory substrate cleavage in vivo unclear\", \"Structural basis for RIPK1 and IRAK-M recognition not determined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Crystal structures of Ser257 phosphomimetic mutants revealed the structural mechanism of AMPK-mediated inhibition — phospho-Ser257 clashes with Pro201 in the L2' loop, misaligning the substrate-binding groove — while Casp6−/− mice showed neuroprotection yet age-dependent brain volume increases and behavioral deficits, demonstrating a physiological homeostatic role.\",\n      \"evidence\": \"X-ray crystallography of S257D/S257E mutants with P201A rescue; Casp6−/− mice with MRI, behavioral testing, and neuronal culture neuroprotection\",\n      \"pmids\": [\"22483120\", \"22433863\", \"22262731\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Ser257 phosphorylation is dynamically regulated in neurons in vivo unconfirmed\", \"Mechanism of caspase-6-dependent brain volume regulation unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Elucidation of the NLRP1→caspase-1→caspase-6 pathway in human neurons, validated in Nlrp1−/− and Casp1−/− mice, defined the complete inflammasome-to-executioner cascade driving axonal degeneration and Aβ42 ratio increase.\",\n      \"evidence\": \"siRNA of NLRP1 and caspase-1, Nlrp1−/− and Casp1−/− mice, ASC speck formation assay\",\n      \"pmids\": [\"25744023\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Triggers of NLRP1 activation in aging/AD neurons not defined\", \"Whether NLRP1–caspase-6 axis is druggable not tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identification of a tri-arginine exosite (R42–R44) required for protein (but not peptide) substrate hydrolysis, with HDX-MS revealing a substrate-binding platform spanning the NTD and 240's loop, established that caspase-6 uses an exosite mechanism for macromolecular substrate recognition.\",\n      \"evidence\": \"Site-directed mutagenesis of exosite residues, HDX-MS, protein vs. peptide kinetics\",\n      \"pmids\": [\"30420425\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No co-crystal structure with protein substrate\", \"How exosite contributes to substrate selectivity among different protein substrates not mapped\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Discovery that caspase-6 performs a non-enzymatic scaffold function by facilitating RIPK3–ZBP1 PANoptosome assembly via RHIM-dependent binding during influenza infection, independent of catalytic activity, fundamentally expanded the gene's functional repertoire beyond proteolysis, while AMPK-mediated inhibition of caspase-6 was placed in a hepatoprotective FGF4→FGFR4→CaMKKβ→AMPK axis in NASH.\",\n      \"evidence\": \"Casp6−/− mice with IAV, co-IP of RIPK3–ZBP1–caspase-6, catalytic mutant dissection; AMPK KO mice, Bid cleavage assay, NASH models\",\n      \"pmids\": [\"32298652\", \"32029622\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RHIM-binding interface of caspase-6 not structurally characterized\", \"Whether scaffold function extends to other PANoptosome-inducing stimuli unknown\", \"In vivo relevance of the AMPK–caspase-6–Bid feedforward loop in human NASH not confirmed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: the structure of active caspase-6 bound to a protein substrate, how caspase-6 switches between enzymatic and scaffold functions in different cellular contexts, and the therapeutic potential of caspase-6 modulation in neurodegeneration and liver disease.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No co-crystal structure with protein substrate exists\", \"Context-dependent regulatory switches between scaffold and enzymatic roles undefined\", \"No clinical data on caspase-6-targeted interventions\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 4, 6, 7, 11, 17, 22, 23, 27, 37, 38, 39, 40]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [36]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [19, 7]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3, 25]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [0, 1, 2, 3, 7, 15, 20, 30, 37]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [36, 38, 31]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [4, 10, 11, 19, 32, 37, 41]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [12, 24, 28, 29, 37, 41]}\n    ],\n    \"complexes\": [\n      \"PANoptosome (RIPK3–ZBP1–CASP6)\"\n    ],\n    \"partners\": [\n      \"RIPK3\",\n      \"ZBP1\",\n      \"CASP1\",\n      \"CASP9\",\n      \"CASP3\",\n      \"NUAK1\",\n      \"TXN\",\n      \"TP53\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}