{"gene":"CAPN3","run_date":"2026-06-09T22:57:17","timeline":{"discoveries":[{"year":1993,"finding":"p94/CAPN3 undergoes rapid and exhaustive autocatalytic (autolytic) degradation immediately after translation, with the active-site cysteine (C129) required for this process. Mutation of C129 to serine or alanine, or deletion of the p94-specific IS2 region, prevents autolysis and allows stable accumulation of the protein. p94 also shows nuclear localization when expressed in COS cells.","method":"COS cell and L8 cell expression of wild-type and deletion/active-site mutant p94; antibody detection; in vitro translation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — active-site mutagenesis combined with deletion mutagenesis in multiple cell systems and in vitro translation, replicated across constructs","pmids":["8486713"],"is_preprint":false},{"year":1995,"finding":"p94/CAPN3 binds to connectin/titin through its p94-specific IS2 region (not through the calpain small subunit). The connectin-insoluble fraction of washed myofibrils contains full-length intact p94, suggesting connectin binding stabilizes p94 and regulates its activity.","method":"Yeast two-hybrid screening of skeletal muscle library; biochemical fractionation of myofibrils","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid plus biochemical fractionation; replicated and extended by multiple subsequent studies","pmids":["8537379"],"is_preprint":false},{"year":1997,"finding":"p94/CAPN3 binds to the extreme C-terminus of connectin/titin at a region flanked by two IgC2 motifs and the M-is7 intervening sequence. The C-terminal connectin-binding region is tissue-specifically alternatively spliced, with only the skeletal muscle-type (containing M-is7) able to bind p94.","method":"Yeast two-hybrid system; C-terminal connectin sequence analysis and binding mapping","journal":"Archives of biochemistry and biophysics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid with domain mapping, single lab","pmids":["9185618"],"is_preprint":false},{"year":1998,"finding":"Native p94 purified from rabbit skeletal muscle undergoes autolysis in at least three consecutive steps (producing 60 kDa and 58 kDa intermediates before a stable 55 kDa fragment), and the autolysis is partly an intermolecular reaction. All autolysis cleavage sites are located within the p94-specific IS1 region. p94(C129S) purifies as a homodimer. The autolytic site specificity differs from ubiquitous mu- and m-calpains.","method":"Antibody-affinity purification of native p94; active-site mutant p94(C129S) expressed in COS cells; biochemical characterization of autolysis products","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — native protein purification combined with active-site mutagenesis and biochemical characterization of autolytic products","pmids":["9794799"],"is_preprint":false},{"year":1999,"finding":"Recombinant human p94 expressed in insect (Sf9) cells binds calcium with high affinity and its autolytic activity requires Ca2+; significant autolysis occurs even at submicromolar Ca2+ levels in soluble cell extracts.","method":"Baculovirus expression system; Ca2+ binding assays; autolysis assays","journal":"European journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro characterization with recombinant protein, single lab, single study","pmids":["10504417"],"is_preprint":false},{"year":1999,"finding":"Two autolytic cleavage sites in human CAPN3 were identified by N-terminal sequencing of purified ~55 kDa fragments: cleavage occurs between residues 30–31 (in the NS region) and between residues 412–413 (in the IS1 region).","method":"Recombinant CAPN3 expressed in Sf9 insect cells and E. coli; purification by Ni2+ affinity and immunopurification; N-terminal amino acid sequencing of fragments","journal":"Archives of biochemistry and biophysics","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — direct protein sequencing of autolytic fragments, single lab","pmids":["10068445"],"is_preprint":false},{"year":2000,"finding":"Transgenic mice expressing catalytically inactive p94 (C129S active-site mutant) develop a myopathy phenotype (decreased grip strength, lobulated and split fibers, centrally placed nuclei) in an age- and expression-dependent manner, demonstrating that loss of p94 protease activity (or dominant-negative accumulation of inactive p94) causes muscle pathology.","method":"Transgenic mouse generation; histological analysis of muscle; grip strength measurement","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — active-site mutant transgenic mouse model with defined histological and functional phenotype, single lab","pmids":["10814721"],"is_preprint":false},{"year":2002,"finding":"The protease core of p94 (domains I–II plus IS1 and NS; 47 kDa) undergoes Ca2+-dependent intramolecular autolysis. The inactive C129S mutant core was not cleaved by wild-type p94 core in trans, confirming intramolecularity. Autolysis rate was independent of enzyme concentration.","method":"Recombinant p94 protease core expression; active-site mutant (C129S); in vitro Ca2+-dependent autolysis assay; concentration-dependence analysis","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted in vitro autolysis with active-site mutant controls establishing intramolecular mechanism, single lab","pmids":["12482600"],"is_preprint":false},{"year":2003,"finding":"An alternatively spliced variant of p94 (p94delta, lacking IS1, exons 15 and 16) is stably expressed, shows Ca2+-dependent caseinolytic and autolytic activities, but is NOT inhibited by calpastatin; instead, p94delta cleaves calpastatin as a substrate. This positions CAPN3 as a potential regulator of the conventional calpain system by degrading its endogenous inhibitor.","method":"COS and insect cell expression of p94delta splice variant; in vitro caseinolytic assay; calpastatin inhibition/cleavage assays; FRET-based activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assays with defined substrate and inhibitor, multiple orthogonal methods in one study","pmids":["14594950"],"is_preprint":false},{"year":2004,"finding":"IS1 of p94 acts as an internal autoinhibitory propeptide that blocks substrate and inhibitor access to the active site. Autoproteolytic removal of IS1 activates the enzyme. A helix-disrupting mutation (L286P) in IS1 causes premature autoproteolysis. The two autolyzed portions of the core remain tightly associated after IS1 cleavage.","method":"Recombinant p94 core expression; deletion and point mutagenesis; exogenous substrate hydrolysis assays; circular dichroism; inhibitor titration (E-64, leupeptin); modeling","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal methods (mutagenesis, enzymatic assays, CD, structural modeling) establishing IS1 as internal propeptide","pmids":["15073171"],"is_preprint":false},{"year":2004,"finding":"C3/CAPN3 knockout (C3KO) mice show abnormal sarcomere formation: myogenic cells fuse normally in vitro but fail to form well-organized sarcomeres. In vitro studies demonstrated that CAPN3 can bind and cleave titin, and LGMD2A-pathogenic mutations reduce affinity of CAPN3 for titin.","method":"CAPN3 knockout mouse generation; electron microscopy of myofibers; in vitro titin-binding and cleavage assays; analysis of pathogenic mutants","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse model with EM phenotype plus in vitro binding and cleavage assays, multiple orthogonal methods","pmids":["15138196"],"is_preprint":false},{"year":2006,"finding":"Autolysis of p94/CAPN3 proceeds without immediate disassembly in IS1, but a cryptic autolytic site in IS2 is critical for fragment disassembly. N2A connectin fragment binding to p94 suppresses autolytic disassembly (specifically IS2 autolysis), as shown in a yeast Gal4 transcriptional reporter system. LGMD2A pathogenic missense mutations reduce autolytic activity detectable in this system.","method":"In vitro autolysis analysis; yeast Gal4-p94 hybrid transcriptional reporter (beta-galactosidase readout); active-site and disease-mutant analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro autolysis assay combined with a genetic reporter system and mutagenesis, multiple orthogonal methods","pmids":["16627476"],"is_preprint":false},{"year":2006,"finding":"Strict Ca2+-dependence of p94 protease core activity was established: even substoichiometric Ca2+ promotes intramolecular autoproteolysis (first cleavage), while the second intermolecular cleavage requires higher Ca2+. Na+, K+, and Mg2+ cannot substitute for Ca2+ in catalysis. A two-stage model of p94 core activation is proposed.","method":"Purified recombinant p94 core and deletion mutant lacking NS and IS1; in vitro Ca2+-titration autolysis assays; metal ion substitution experiments","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — rigorous in vitro reconstitution with purified protein, multiple conditions and controls, single lab","pmids":["16533054"],"is_preprint":false},{"year":2007,"finding":"A comprehensive proteomics survey of p94/CAPN3 substrates in COS7 cells identified novel substrates including components of the protein synthesis system and glycolytic enzymes, in addition to conventional calpain substrates.","method":"iTRAQ labeling and 2-D LC-MALDI comparative proteomics in COS7 cells expressing active vs. inactive p94","journal":"Biotechnology journal","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — mass spectrometry-based substrate survey, single lab, no independent validation of individual substrates","pmids":["17373644"],"is_preprint":false},{"year":2008,"finding":"p94/CAPN3 binds to connectin/titin at multiple sites including the N2A region, the PEVK region, and the M-line region. p94-N2A interaction suppresses p94 autolysis and protects connectin from proteolysis. MARP2/Ankrd2 competes with p94 for N2A binding and is also cleaved by p94 as a substrate. A titin N2A fragment carrying the mdm (muscular dystrophy with myositis) deletion resists proteolysis and shows weakened MARP binding.","method":"COS7 cell co-expression interaction studies; in vitro proteolysis assays; competition binding experiments; analysis of mdm deletion construct","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple binding sites mapped by cell-based assays plus in vitro proteolysis, multiple orthogonal methods","pmids":["18310072"],"is_preprint":false},{"year":2013,"finding":"A novel CAPN3-binding protein PLEIAD (SIMC1/C5orf25) suppresses CAPN3 protease activity (autolysis). PLEIAD also interacts with CTBP1 (a transcriptional co-regulator), and CTBP1 is proteolyzed by CAPN3 in COS7 cells at specific sites. PLEIAD can thus serve both as a CAPN3 suppressor and as a scaffold for CAPN3 substrate recruitment.","method":"Co-immunoprecipitation; COS7 cell expression of CAPN3 and PLEIAD; in vitro proteolysis assays; identification of CTBP1 cleavage sites","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP combined with in vitro proteolysis and cleavage site mapping, single lab","pmids":["23707407"],"is_preprint":false},{"year":2014,"finding":"CAPN3 protease activity can be reconstituted by intermolecular complementation (iMOC) between its N-terminal and C-terminal autolytic fragments. Active-site mutant CAPN3 activity can be rescued by iMOC with wild-type autolytic fragments, demonstrating that two different CAPN3 mutants can complement each other to restore protease function.","method":"Expression and purification of CAPN3 autolytic fragments; in vitro reconstitution of protease activity; complementation assays with active-site mutants","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution demonstrating functional complementation, multiple mutant combinations tested, single lab","pmids":["25512505"],"is_preprint":false},{"year":2015,"finding":"CAPN3 cleaves C-terminal titin (M-band region) at multiple specific sites in vitro, and cleavage products are detectable in normal muscle by western blot and immunofluorescence. The TMD/LGMD2J-causing FINmaj mutation alters this in vitro processing while preserving CAPN3 binding to mutant titin. Pathological loss of M-band titin in TMD/LGMD2J is independent of CAPN3.","method":"In vitro CAPN3 cleavage assays with C-terminal titin; protein sequencing of cleavage products; western blot and immunofluorescence of normal and disease muscle","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro cleavage with site identification by protein sequencing, validated in tissue by multiple methods, single lab","pmids":["25877298"],"is_preprint":false},{"year":2020,"finding":"CAPN3 forms a homotrimer (not merely a homodimer) in native and recombinant preparations, as shown by blue native PAGE, chemical cross-linking, and sequential immunoprecipitation. Trimer formation requires the PEF (penta-EF-hand) domain; the addition of the adjacent CBSW domain to the PEF domain reinforces trimeric assembly. Deletion of the NS, IS1, or IS2 regions does not abolish trimer formation.","method":"Blue native PAGE; chemical cross-linking; sequential immunoprecipitation; domain-deletion analysis of recombinant CAPN3","journal":"Biochimica et biophysica acta. Proteins and proteomics","confidence":"High","confidence_rationale":"Tier 2 / Moderate — three orthogonal methods (BN-PAGE, cross-linking, sequential IP) with domain mapping, single lab","pmids":["32200007"],"is_preprint":false},{"year":2021,"finding":"In zebrafish, CAPN3 (capn3b) is recruited by the nucleolar protein DEF from the cytoplasm to the nucleolus, where it cleaves substrates including p53, Chk1, and Wee1 in a Ca2+-dependent, ubiquitin-proteasome-independent manner. Loss of capn3b causes accumulation of Chk1 and Wee1 in the nucleolus and disrupts synchronized cell cycle re-entry after partial hepatectomy.","method":"Zebrafish capn3b null mutant generation; partial hepatectomy model; mass spectrometry of nuclear proteins; demonstration of Chk1 and Wee1 as CAPN3 substrates; nucleolar localization studies","journal":"Cell regeneration (London, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout with defined substrates identified by MS, functional liver regeneration phenotype, zebrafish model (ortholog)","pmids":["32588143"],"is_preprint":false},{"year":1996,"finding":"Antisense oligonucleotide-mediated reduction of p94 expression in differentiating rat myoblasts causes dramatic ultrastructural perturbations in myotubes, particularly affecting myofibrillar stability and Z-line integrity, implicating p94 in myofibrillar maintenance.","method":"Antisense oligonucleotide treatment of rat primary myoblast cultures; ultrastructural analysis by electron microscopy","journal":"Cell growth & differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — loss-of-function with defined ultrastructural phenotype, single lab","pmids":["8930395"],"is_preprint":false},{"year":2002,"finding":"Reduction of p94 activity in a muscle cell inducible system results in a significant increase in myogenin levels, suggesting p94 participates in a myogenesis regulatory pathway by modulating myogenic regulatory factors.","method":"Inducible expression system in muscle cell line; western blot for myogenin levels","journal":"Journal of biotechnology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single method (western blot), indirect evidence for pathway involvement, single lab","pmids":["12044555"],"is_preprint":false},{"year":2003,"finding":"A p94-like CAPN3 splice variant lacking IS1 and exon 15 (a putative nuclear localization signal) is expressed in human peripheral blood mononuclear cells (PBMCs), particularly B- and T-lymphocytes. This variant activates and inactivates without forming the low-Ca2+-requiring form typical of classical calpains, and shows lower sensitivity to calpastatin than ubiquitous calpains.","method":"Chromatographic fractionation of PBMC lysates; biochemical characterization of activation kinetics and calpastatin sensitivity; cell-type distribution by expression analysis","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical characterization of isolated protein with multiple assays, single lab","pmids":["12882647"],"is_preprint":false},{"year":2005,"finding":"Immunohistochemical analysis of single myofibers shows that p94/CAPN3 localizes at the Z-line and N2-line regions of sarcomeres. M-line localization of p94 is influenced by cellular context including contractile status, fiber type, sarcomere maturation, and composition of signal complexes.","method":"Immunohistochemistry on isolated single myofibers","journal":"Journal of muscle research and cell motility","confidence":"Low","confidence_rationale":"Tier 3 / Weak — immunohistochemistry only, no functional consequence directly tested for localization, single lab","pmids":["16453164"],"is_preprint":false},{"year":2015,"finding":"CAPN3 is the first (and only) intracellular enzyme found to depend on Na+ for its activation (in addition to Ca2+), and it is the only protease (other than certain viral proteases) able to regain protease function after autolytic dissociation via intermolecular complementation (iMOC). CAPN3 has both proteolytic and non-proteolytic functions.","method":"Review/synthesis of experimental data; Na+-dependence originally established by biochemical assays; iMOC from reconstitution experiments (see PMID 25512505)","journal":"Biochimie","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — Na+-dependence finding cited as established experimental result (biochemical assays); iMOC replicated; review paper synthesizing primary data","pmids":["26363099"],"is_preprint":false}],"current_model":"CAPN3/p94 is a skeletal muscle-specific, Ca2+- and Na2+-dependent cysteine protease that undergoes rapid and exhaustive autolysis mediated by its unique IS1 (acting as an internal propeptide) and IS2 insertion sequences; it binds to connectin/titin at multiple sites (N2A, PEVK, M-line, C-terminus) which suppresses its autolysis and stabilizes the enzyme, and this interaction is essential for normal sarcomere assembly and remodeling; CAPN3 cleaves titin, calpastatin, MARP2/Ankrd2, CTBP1, Chk1, and Wee1 as substrates, is negatively regulated by the scaffolding protein PLEIAD, forms a homotrimer via its PEF domain, can restore protease activity after autolytic fragmentation via intermolecular complementation (iMOC), and its loss of proteolytic function causes limb-girdle muscular dystrophy type 2A (LGMD2A)."},"narrative":{"mechanistic_narrative":"CAPN3/p94 is a skeletal muscle-enriched, Ca2+-dependent cysteine protease that governs sarcomere assembly, remodeling, and myofibrillar integrity, with loss of its proteolytic function causing limb-girdle muscular dystrophy type 2A [PMID:8486713, PMID:10814721, PMID:15138196]. Its defining biochemical property is rapid, exhaustive autolysis driven by the active-site cysteine C129 and its calpain-unique insertion sequences: IS1 acts as an internal autoinhibitory propeptide whose autoproteolytic removal activates the enzyme, while a cryptic site in IS2 controls fragment disassembly [PMID:8486713, PMID:15073171, PMID:16627476]. Catalysis is strictly Ca2+-dependent—even substoichiometric Ca2+ drives an initial intramolecular cleavage, with a second intermolecular cleavage requiring higher Ca2+—and uniquely also depends on Na+ [PMID:12482600, PMID:16533054, PMID:26363099]. CAPN3 binds connectin/titin at multiple sites (N2A, PEVK, M-line, and the alternatively spliced skeletal-muscle C-terminus) through its IS2 region; this interaction stabilizes the enzyme by suppressing autolysis and is integral to its sarcomeric role, and LGMD2A mutations weaken titin binding [PMID:8537379, PMID:9185618, PMID:18310072, PMID:15138196]. The enzyme processes titin, calpastatin, MARP2/Ankrd2, and CTBP1 as substrates, is negatively regulated by the scaffold PLEIAD (SIMC1) which also recruits substrates, assembles into a PEF-domain-dependent homotrimer, and can regain activity after autolytic fragmentation via intermolecular complementation [PMID:18310072, PMID:14594950, PMID:23707407, PMID:32200007, PMID:25512505]. A nucleolar role cleaving cell-cycle regulators Chk1 and Wee1 has been defined in a zebrafish ortholog [PMID:32588143].","teleology":[{"year":1993,"claim":"Established the founding paradox of CAPN3 biology—that the protease destroys itself immediately after translation—and pinned this to the active site and the muscle-specific IS2 region.","evidence":"Active-site (C129S/A) and IS2-deletion mutants expressed in COS/L8 cells and in vitro translation","pmids":["8486713"],"confidence":"High","gaps":["Did not resolve whether autolysis is intra- or intermolecular","Functional significance of nuclear localization not established"]},{"year":1995,"claim":"Identified connectin/titin as a binding partner that stabilizes intact CAPN3, answering how the rapidly self-destructing enzyme could persist in muscle.","evidence":"Yeast two-hybrid of skeletal muscle library and myofibril fractionation localizing intact p94 to the connectin-insoluble fraction","pmids":["8537379"],"confidence":"High","gaps":["Binding shown via IS2 but mechanism of autolysis suppression not defined","Number of titin binding sites unknown at this stage"]},{"year":1997,"claim":"Mapped a C-terminal titin binding site whose tissue-specific alternative splicing restricts CAPN3 anchoring to skeletal muscle.","evidence":"Yeast two-hybrid domain mapping of the connectin C-terminus (M-is7 region)","pmids":["9185618"],"confidence":"Medium","gaps":["Single-lab Y2H without biochemical confirmation of the complex","Functional consequence of C-terminal anchoring not tested"]},{"year":1998,"claim":"Defined the multi-step autolytic pathway and localized all cleavage sites to IS1, while showing native CAPN3 forms a homodimer—framing the structural basis of self-processing.","evidence":"Affinity purification of native rabbit p94 and characterization of C129S mutant and autolytic intermediates","pmids":["9794799"],"confidence":"High","gaps":["Relative contribution of intra- vs intermolecular reaction not quantified","Oligomeric state later revised to trimer"]},{"year":1999,"claim":"Demonstrated CAPN3 binds Ca2+ with high affinity and requires it for autolysis even at submicromolar concentrations, establishing Ca2+ as the catalytic trigger.","evidence":"Baculovirus-expressed human p94 with Ca2+ binding and autolysis assays; N-terminal sequencing of fragments (residues 30-31 and 412-413)","pmids":["10504417","10068445"],"confidence":"Medium","gaps":["Single-lab in vitro characterization","Physiological Ca2+ trigger in muscle not directly demonstrated"]},{"year":2000,"claim":"Showed in vivo that loss of catalytic activity (or dominant-negative inactive enzyme) is sufficient to produce dystrophic muscle pathology, linking protease function to muscle integrity.","evidence":"Transgenic mice expressing C129S inactive p94 with histological and grip-strength analysis","pmids":["10814721"],"confidence":"Medium","gaps":["Cannot fully separate dominant-negative accumulation from loss-of-function","Specific in vivo substrate driving pathology not identified"]},{"year":2002,"claim":"Resolved the initiating autolytic cleavage as intramolecular and concentration-independent, refining the activation mechanism of the protease core.","evidence":"Recombinant protease core with C129S trans-cleavage controls and concentration-dependence assays","pmids":["12482600"],"confidence":"High","gaps":["Later intermolecular steps not addressed here","Behavior of full-length enzyme vs isolated core may differ"]},{"year":2003,"claim":"Revealed CAPN3 can both resist and degrade calpastatin, positioning it as a regulator of the conventional calpain system, and documented physiologically active splice variants.","evidence":"COS/insect expression of p94delta variant with caseinolytic, autolytic, calpastatin inhibition/cleavage, and FRET assays; biochemical characterization of a PBMC variant","pmids":["14594950","12882647"],"confidence":"High","gaps":["In vivo relevance of calpastatin cleavage not established","Tissue distribution and function of non-muscle variants unclear"]},{"year":2004,"claim":"Defined IS1 as an internal autoinhibitory propeptide blocking substrate/inhibitor access, explaining how autoproteolysis activates the enzyme; and showed in a KO mouse that CAPN3 is required for organized sarcomere formation and binds/cleaves titin.","evidence":"Recombinant core mutagenesis with CD, inhibitor titration, and substrate assays; CAPN3 KO mouse EM with in vitro titin binding/cleavage and pathogenic mutant analysis","pmids":["15073171","15138196"],"confidence":"High","gaps":["Structural model of IS1 occlusion not crystallographically resolved","Which titin cleavages are functionally critical in vivo unclear"]},{"year":2006,"claim":"Established a two-stage Ca2+-activation model—intramolecular first cleavage at low Ca2+, intermolecular disassembly at higher Ca2+—and showed N2A titin binding suppresses IS2 autolysis and stabilizes fragments.","evidence":"Purified core Ca2+-titration and metal-substitution assays; in vitro autolysis plus yeast Gal4-p94 reporter with disease mutants","pmids":["16533054","16627476"],"confidence":"High","gaps":["Reporter-based activity readout is indirect","How N2A binding mechanically blocks IS2 cleavage not structurally defined"]},{"year":2007,"claim":"Expanded the CAPN3 substrate landscape beyond conventional calpain targets to translation machinery and glycolytic enzymes via unbiased proteomics.","evidence":"iTRAQ/2-D LC-MALDI comparison of active vs inactive p94 in COS7 cells","pmids":["17373644"],"confidence":"Medium","gaps":["Individual substrates not independently validated","Non-muscle cell context may not reflect physiological cleavage"]},{"year":2008,"claim":"Mapped CAPN3 to multiple titin sites (N2A, PEVK, M-line) and uncovered a competitive substrate relationship with MARP2/Ankrd2 at N2A, integrating CAPN3 into a titin-based mechanosignaling node.","evidence":"COS7 co-expression, in vitro proteolysis, competition binding, and mdm-deletion analysis","pmids":["18310072"],"confidence":"High","gaps":["In vivo stoichiometry of competing partners unknown","Signaling output downstream of N2A complex not defined"]},{"year":2013,"claim":"Identified PLEIAD/SIMC1 as a dual regulator that both suppresses CAPN3 autolysis and scaffolds substrate (CTBP1) delivery, adding a non-titin regulatory layer.","evidence":"Reciprocal co-IP, COS7 co-expression, in vitro proteolysis, and CTBP1 cleavage-site mapping","pmids":["23707407"],"confidence":"Medium","gaps":["Single-lab evidence","Physiological setting where PLEIAD-CAPN3-CTBP1 axis operates not established"]},{"year":2014,"claim":"Demonstrated intermolecular complementation (iMOC): autolytic N- and C-terminal fragments and even two different inactive mutants can reconstitute active protease, redefining post-autolytic CAPN3 as functional rather than spent.","evidence":"In vitro reconstitution of purified autolytic fragments and active-site mutant complementation","pmids":["25512505"],"confidence":"High","gaps":["In vivo occurrence and prevalence of iMOC unknown","Implications for genotype-phenotype in LGMD2A compound heterozygotes untested"]},{"year":2015,"claim":"Resolved CAPN3 cleavage of M-band titin at defined sites in normal muscle and showed the FINmaj/LGMD2J mutation alters processing without disrupting binding, and synthesized the unique Na+-dependence of CAPN3 activation.","evidence":"In vitro titin cleavage with product sequencing plus tissue western/IF; review synthesis of Na+-dependence and iMOC","pmids":["25877298","26363099"],"confidence":"High","gaps":["M-band titin loss in TMD/LGMD2J shown independent of CAPN3","Molecular basis of Na+-dependence not structurally defined"]},{"year":2020,"claim":"Revised the oligomeric state to a PEF-domain-dependent homotrimer (reinforced by the CBSW domain), correcting the earlier dimer model and locating the assembly determinant.","evidence":"Blue native PAGE, chemical cross-linking, sequential IP, and domain-deletion analysis of recombinant CAPN3","pmids":["32200007"],"confidence":"High","gaps":["Functional consequence of trimerization for catalysis not defined","Whether trimer assembles on titin in situ unknown"]},{"year":2021,"claim":"Uncovered a nucleolar, cell-cycle role: DEF recruits CAPN3 to the nucleolus to cleave Chk1 and Wee1 (and p53) and license synchronized cell-cycle re-entry, extending CAPN3 function beyond the sarcomere.","evidence":"Zebrafish capn3b null mutant, partial hepatectomy regeneration model, nuclear-protein MS, and substrate cleavage assays","pmids":["32588143"],"confidence":"Medium","gaps":["Demonstrated in zebrafish ortholog, not human CAPN3","Whether muscle CAPN3 performs an analogous nucleolar function unknown"]},{"year":null,"claim":"A high-resolution structure of full-length CAPN3 explaining how IS1 autoinhibition, Na+/Ca2+ dependence, titin anchoring, and PEF-domain trimerization are integrated remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No experimental atomic structure of intact CAPN3 in the timeline","In vivo substrate hierarchy driving LGMD2A pathology not defined","Physiological trigger and tissue context of Na+ co-dependence unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,3,8,9,14,15,17,19]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,3,12]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[1,2,14]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[10,20,23]},{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[19]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-397014","term_label":"Muscle contraction","supporting_discovery_ids":[10,20]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,8,14,15]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[6,10,17]}],"complexes":["CAPN3 homotrimer","CAPN3-titin/connectin complex"],"partners":["TTN","ANKRD2","SIMC1","CTBP1","CAST","CHEK1","WEE1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P20807","full_name":"Calpain-3","aliases":["Calcium-activated neutral proteinase 3","CANP 3","Calpain L3","Calpain p94","Muscle-specific calcium-activated neutral protease 3","New calpain 1","nCL-1"],"length_aa":821,"mass_kda":94.3,"function":"Calcium-regulated non-lysosomal thiol-protease. Proteolytically cleaves CTBP1 at 'His-409'. 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England)","url":"https://pubmed.ncbi.nlm.nih.gov/32588143","citation_count":14,"is_preprint":false},{"pmid":"29456684","id":"PMC_29456684","title":"CAPN3, DCT, MLANA and TYRP1 are overexpressed in skin of vitiligo vulgaris Mexican patients.","date":"2018","source":"Experimental and therapeutic medicine","url":"https://pubmed.ncbi.nlm.nih.gov/29456684","citation_count":14,"is_preprint":false},{"pmid":"35309930","id":"PMC_35309930","title":"Targeting the Ubiquitin-Proteasome System in Limb-Girdle Muscular Dystrophy With CAPN3 Mutations.","date":"2022","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/35309930","citation_count":13,"is_preprint":false},{"pmid":"29685414","id":"PMC_29685414","title":"A novel CAPN3 mutation in late-onset limb-girdle muscular dystrophy with early respiratory insufficiency.","date":"2018","source":"Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia","url":"https://pubmed.ncbi.nlm.nih.gov/29685414","citation_count":13,"is_preprint":false},{"pmid":"22006685","id":"PMC_22006685","title":"Does the severity of the LGMD2A phenotype in compound heterozygotes depend on the combination of mutations?","date":"2011","source":"Muscle & nerve","url":"https://pubmed.ncbi.nlm.nih.gov/22006685","citation_count":13,"is_preprint":false},{"pmid":"27011640","id":"PMC_27011640","title":"Limb-girdle muscular dystrophy in the Agarwals: Utility of founder mutations in CAPN3 gene.","date":"2016","source":"Annals of Indian Academy of Neurology","url":"https://pubmed.ncbi.nlm.nih.gov/27011640","citation_count":13,"is_preprint":false},{"pmid":"10068445","id":"PMC_10068445","title":"Purification and identification of two putative autolytic sites in human calpain 3 (p94) expressed in heterologous systems.","date":"1999","source":"Archives of biochemistry and biophysics","url":"https://pubmed.ncbi.nlm.nih.gov/10068445","citation_count":12,"is_preprint":false},{"pmid":"28300015","id":"PMC_28300015","title":"FRZB and melusin, overexpressed in LGMD2A, regulate integrin β1D isoform replacement altering myoblast fusion and the integrin-signalling pathway.","date":"2017","source":"Expert reviews in molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/28300015","citation_count":12,"is_preprint":false},{"pmid":"12044555","id":"PMC_12044555","title":"Development of an inducible system to assess p94 (CAPN3) function in cultured muscle cells.","date":"2002","source":"Journal of biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/12044555","citation_count":11,"is_preprint":false},{"pmid":"21172462","id":"PMC_21172462","title":"SNP-array based whole genome homozygosity mapping: a quick and powerful tool to achieve an accurate diagnosis in LGMD2 patients.","date":"2010","source":"European journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/21172462","citation_count":11,"is_preprint":false},{"pmid":"29599076","id":"PMC_29599076","title":"Characteristics of PPT1 and TPP1 enzymes in neuronal ceroid lipofuscinosis (NCL) 1 and 2 by dried blood spots (DBS) and leukocytes and their application to newborn screening.","date":"2018","source":"Molecular genetics and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/29599076","citation_count":11,"is_preprint":false},{"pmid":"30415788","id":"PMC_30415788","title":"Severe limb-girdle muscular dystrophy 2A in two young siblings from Guinea-Bissau associated with a novel null homozygous mutation in CAPN3 gene.","date":"2018","source":"Neuromuscular disorders : NMD","url":"https://pubmed.ncbi.nlm.nih.gov/30415788","citation_count":10,"is_preprint":false},{"pmid":"34452850","id":"PMC_34452850","title":"Nucleolus-localized Def-CAPN3 protein degradation pathway and its role in cell cycle control and ribosome biogenesis.","date":"2021","source":"Journal of genetics and genomics = Yi chuan xue bao","url":"https://pubmed.ncbi.nlm.nih.gov/34452850","citation_count":9,"is_preprint":false},{"pmid":"26632398","id":"PMC_26632398","title":"Clinical and Pathological Heterogeneity of Korean Patients with CAPN3 Mutations.","date":"2016","source":"Yonsei medical journal","url":"https://pubmed.ncbi.nlm.nih.gov/26632398","citation_count":9,"is_preprint":false},{"pmid":"28535990","id":"PMC_28535990","title":"Intravenous and intratumoral injection of Pluronic P94: The effect of administration route on biodistribution and tumor retention.","date":"2017","source":"Nanomedicine : nanotechnology, biology, and medicine","url":"https://pubmed.ncbi.nlm.nih.gov/28535990","citation_count":9,"is_preprint":false},{"pmid":"23597518","id":"PMC_23597518","title":"Limb-girdle muscular dystrophy type 2a with mutation in CAPN3: the first report in Taiwan.","date":"2013","source":"Pediatrics and neonatology","url":"https://pubmed.ncbi.nlm.nih.gov/23597518","citation_count":9,"is_preprint":false},{"pmid":"30500922","id":"PMC_30500922","title":"Divergent Features of Mitochondrial Deficiencies in LGMD2A Associated With Novel Calpain-3 Mutations.","date":"2019","source":"Journal of neuropathology and experimental neurology","url":"https://pubmed.ncbi.nlm.nih.gov/30500922","citation_count":8,"is_preprint":false},{"pmid":"15498466","id":"PMC_15498466","title":"Bidirectional transcriptional activity of the Pgk1 promoter and transmission ratio distortion in Capn3-deficient mice.","date":"2004","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/15498466","citation_count":8,"is_preprint":false},{"pmid":"30323756","id":"PMC_30323756","title":"Limb Girdle Muscular Dystrophy due to Digenic Inheritance of DES and CAPN3 Mutations.","date":"2018","source":"Case reports in neurology","url":"https://pubmed.ncbi.nlm.nih.gov/30323756","citation_count":8,"is_preprint":false},{"pmid":"25200581","id":"PMC_25200581","title":"[A clinicopathological investigation of two autopsy cases of calpainopathy (LGMD2A)].","date":"2014","source":"Brain and nerve = Shinkei kenkyu no shinpo","url":"https://pubmed.ncbi.nlm.nih.gov/25200581","citation_count":8,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":48897,"output_tokens":6142,"usd":0.119411,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":14822,"output_tokens":5220,"usd":0.102305,"stage2_stop_reason":"end_turn"},"total_usd":0.221716,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1993,\n      \"finding\": \"p94/CAPN3 undergoes rapid and exhaustive autocatalytic (autolytic) degradation immediately after translation, with the active-site cysteine (C129) required for this process. Mutation of C129 to serine or alanine, or deletion of the p94-specific IS2 region, prevents autolysis and allows stable accumulation of the protein. p94 also shows nuclear localization when expressed in COS cells.\",\n      \"method\": \"COS cell and L8 cell expression of wild-type and deletion/active-site mutant p94; antibody detection; in vitro translation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — active-site mutagenesis combined with deletion mutagenesis in multiple cell systems and in vitro translation, replicated across constructs\",\n      \"pmids\": [\"8486713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"p94/CAPN3 binds to connectin/titin through its p94-specific IS2 region (not through the calpain small subunit). The connectin-insoluble fraction of washed myofibrils contains full-length intact p94, suggesting connectin binding stabilizes p94 and regulates its activity.\",\n      \"method\": \"Yeast two-hybrid screening of skeletal muscle library; biochemical fractionation of myofibrils\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast two-hybrid plus biochemical fractionation; replicated and extended by multiple subsequent studies\",\n      \"pmids\": [\"8537379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"p94/CAPN3 binds to the extreme C-terminus of connectin/titin at a region flanked by two IgC2 motifs and the M-is7 intervening sequence. The C-terminal connectin-binding region is tissue-specifically alternatively spliced, with only the skeletal muscle-type (containing M-is7) able to bind p94.\",\n      \"method\": \"Yeast two-hybrid system; C-terminal connectin sequence analysis and binding mapping\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid with domain mapping, single lab\",\n      \"pmids\": [\"9185618\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Native p94 purified from rabbit skeletal muscle undergoes autolysis in at least three consecutive steps (producing 60 kDa and 58 kDa intermediates before a stable 55 kDa fragment), and the autolysis is partly an intermolecular reaction. All autolysis cleavage sites are located within the p94-specific IS1 region. p94(C129S) purifies as a homodimer. The autolytic site specificity differs from ubiquitous mu- and m-calpains.\",\n      \"method\": \"Antibody-affinity purification of native p94; active-site mutant p94(C129S) expressed in COS cells; biochemical characterization of autolysis products\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — native protein purification combined with active-site mutagenesis and biochemical characterization of autolytic products\",\n      \"pmids\": [\"9794799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Recombinant human p94 expressed in insect (Sf9) cells binds calcium with high affinity and its autolytic activity requires Ca2+; significant autolysis occurs even at submicromolar Ca2+ levels in soluble cell extracts.\",\n      \"method\": \"Baculovirus expression system; Ca2+ binding assays; autolysis assays\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro characterization with recombinant protein, single lab, single study\",\n      \"pmids\": [\"10504417\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Two autolytic cleavage sites in human CAPN3 were identified by N-terminal sequencing of purified ~55 kDa fragments: cleavage occurs between residues 30–31 (in the NS region) and between residues 412–413 (in the IS1 region).\",\n      \"method\": \"Recombinant CAPN3 expressed in Sf9 insect cells and E. coli; purification by Ni2+ affinity and immunopurification; N-terminal amino acid sequencing of fragments\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — direct protein sequencing of autolytic fragments, single lab\",\n      \"pmids\": [\"10068445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Transgenic mice expressing catalytically inactive p94 (C129S active-site mutant) develop a myopathy phenotype (decreased grip strength, lobulated and split fibers, centrally placed nuclei) in an age- and expression-dependent manner, demonstrating that loss of p94 protease activity (or dominant-negative accumulation of inactive p94) causes muscle pathology.\",\n      \"method\": \"Transgenic mouse generation; histological analysis of muscle; grip strength measurement\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — active-site mutant transgenic mouse model with defined histological and functional phenotype, single lab\",\n      \"pmids\": [\"10814721\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The protease core of p94 (domains I–II plus IS1 and NS; 47 kDa) undergoes Ca2+-dependent intramolecular autolysis. The inactive C129S mutant core was not cleaved by wild-type p94 core in trans, confirming intramolecularity. Autolysis rate was independent of enzyme concentration.\",\n      \"method\": \"Recombinant p94 protease core expression; active-site mutant (C129S); in vitro Ca2+-dependent autolysis assay; concentration-dependence analysis\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted in vitro autolysis with active-site mutant controls establishing intramolecular mechanism, single lab\",\n      \"pmids\": [\"12482600\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"An alternatively spliced variant of p94 (p94delta, lacking IS1, exons 15 and 16) is stably expressed, shows Ca2+-dependent caseinolytic and autolytic activities, but is NOT inhibited by calpastatin; instead, p94delta cleaves calpastatin as a substrate. This positions CAPN3 as a potential regulator of the conventional calpain system by degrading its endogenous inhibitor.\",\n      \"method\": \"COS and insect cell expression of p94delta splice variant; in vitro caseinolytic assay; calpastatin inhibition/cleavage assays; FRET-based activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assays with defined substrate and inhibitor, multiple orthogonal methods in one study\",\n      \"pmids\": [\"14594950\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"IS1 of p94 acts as an internal autoinhibitory propeptide that blocks substrate and inhibitor access to the active site. Autoproteolytic removal of IS1 activates the enzyme. A helix-disrupting mutation (L286P) in IS1 causes premature autoproteolysis. The two autolyzed portions of the core remain tightly associated after IS1 cleavage.\",\n      \"method\": \"Recombinant p94 core expression; deletion and point mutagenesis; exogenous substrate hydrolysis assays; circular dichroism; inhibitor titration (E-64, leupeptin); modeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal methods (mutagenesis, enzymatic assays, CD, structural modeling) establishing IS1 as internal propeptide\",\n      \"pmids\": [\"15073171\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"C3/CAPN3 knockout (C3KO) mice show abnormal sarcomere formation: myogenic cells fuse normally in vitro but fail to form well-organized sarcomeres. In vitro studies demonstrated that CAPN3 can bind and cleave titin, and LGMD2A-pathogenic mutations reduce affinity of CAPN3 for titin.\",\n      \"method\": \"CAPN3 knockout mouse generation; electron microscopy of myofibers; in vitro titin-binding and cleavage assays; analysis of pathogenic mutants\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse model with EM phenotype plus in vitro binding and cleavage assays, multiple orthogonal methods\",\n      \"pmids\": [\"15138196\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Autolysis of p94/CAPN3 proceeds without immediate disassembly in IS1, but a cryptic autolytic site in IS2 is critical for fragment disassembly. N2A connectin fragment binding to p94 suppresses autolytic disassembly (specifically IS2 autolysis), as shown in a yeast Gal4 transcriptional reporter system. LGMD2A pathogenic missense mutations reduce autolytic activity detectable in this system.\",\n      \"method\": \"In vitro autolysis analysis; yeast Gal4-p94 hybrid transcriptional reporter (beta-galactosidase readout); active-site and disease-mutant analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro autolysis assay combined with a genetic reporter system and mutagenesis, multiple orthogonal methods\",\n      \"pmids\": [\"16627476\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Strict Ca2+-dependence of p94 protease core activity was established: even substoichiometric Ca2+ promotes intramolecular autoproteolysis (first cleavage), while the second intermolecular cleavage requires higher Ca2+. Na+, K+, and Mg2+ cannot substitute for Ca2+ in catalysis. A two-stage model of p94 core activation is proposed.\",\n      \"method\": \"Purified recombinant p94 core and deletion mutant lacking NS and IS1; in vitro Ca2+-titration autolysis assays; metal ion substitution experiments\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — rigorous in vitro reconstitution with purified protein, multiple conditions and controls, single lab\",\n      \"pmids\": [\"16533054\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"A comprehensive proteomics survey of p94/CAPN3 substrates in COS7 cells identified novel substrates including components of the protein synthesis system and glycolytic enzymes, in addition to conventional calpain substrates.\",\n      \"method\": \"iTRAQ labeling and 2-D LC-MALDI comparative proteomics in COS7 cells expressing active vs. inactive p94\",\n      \"journal\": \"Biotechnology journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — mass spectrometry-based substrate survey, single lab, no independent validation of individual substrates\",\n      \"pmids\": [\"17373644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"p94/CAPN3 binds to connectin/titin at multiple sites including the N2A region, the PEVK region, and the M-line region. p94-N2A interaction suppresses p94 autolysis and protects connectin from proteolysis. MARP2/Ankrd2 competes with p94 for N2A binding and is also cleaved by p94 as a substrate. A titin N2A fragment carrying the mdm (muscular dystrophy with myositis) deletion resists proteolysis and shows weakened MARP binding.\",\n      \"method\": \"COS7 cell co-expression interaction studies; in vitro proteolysis assays; competition binding experiments; analysis of mdm deletion construct\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple binding sites mapped by cell-based assays plus in vitro proteolysis, multiple orthogonal methods\",\n      \"pmids\": [\"18310072\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"A novel CAPN3-binding protein PLEIAD (SIMC1/C5orf25) suppresses CAPN3 protease activity (autolysis). PLEIAD also interacts with CTBP1 (a transcriptional co-regulator), and CTBP1 is proteolyzed by CAPN3 in COS7 cells at specific sites. PLEIAD can thus serve both as a CAPN3 suppressor and as a scaffold for CAPN3 substrate recruitment.\",\n      \"method\": \"Co-immunoprecipitation; COS7 cell expression of CAPN3 and PLEIAD; in vitro proteolysis assays; identification of CTBP1 cleavage sites\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP combined with in vitro proteolysis and cleavage site mapping, single lab\",\n      \"pmids\": [\"23707407\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CAPN3 protease activity can be reconstituted by intermolecular complementation (iMOC) between its N-terminal and C-terminal autolytic fragments. Active-site mutant CAPN3 activity can be rescued by iMOC with wild-type autolytic fragments, demonstrating that two different CAPN3 mutants can complement each other to restore protease function.\",\n      \"method\": \"Expression and purification of CAPN3 autolytic fragments; in vitro reconstitution of protease activity; complementation assays with active-site mutants\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution demonstrating functional complementation, multiple mutant combinations tested, single lab\",\n      \"pmids\": [\"25512505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CAPN3 cleaves C-terminal titin (M-band region) at multiple specific sites in vitro, and cleavage products are detectable in normal muscle by western blot and immunofluorescence. The TMD/LGMD2J-causing FINmaj mutation alters this in vitro processing while preserving CAPN3 binding to mutant titin. Pathological loss of M-band titin in TMD/LGMD2J is independent of CAPN3.\",\n      \"method\": \"In vitro CAPN3 cleavage assays with C-terminal titin; protein sequencing of cleavage products; western blot and immunofluorescence of normal and disease muscle\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro cleavage with site identification by protein sequencing, validated in tissue by multiple methods, single lab\",\n      \"pmids\": [\"25877298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CAPN3 forms a homotrimer (not merely a homodimer) in native and recombinant preparations, as shown by blue native PAGE, chemical cross-linking, and sequential immunoprecipitation. Trimer formation requires the PEF (penta-EF-hand) domain; the addition of the adjacent CBSW domain to the PEF domain reinforces trimeric assembly. Deletion of the NS, IS1, or IS2 regions does not abolish trimer formation.\",\n      \"method\": \"Blue native PAGE; chemical cross-linking; sequential immunoprecipitation; domain-deletion analysis of recombinant CAPN3\",\n      \"journal\": \"Biochimica et biophysica acta. Proteins and proteomics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — three orthogonal methods (BN-PAGE, cross-linking, sequential IP) with domain mapping, single lab\",\n      \"pmids\": [\"32200007\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In zebrafish, CAPN3 (capn3b) is recruited by the nucleolar protein DEF from the cytoplasm to the nucleolus, where it cleaves substrates including p53, Chk1, and Wee1 in a Ca2+-dependent, ubiquitin-proteasome-independent manner. Loss of capn3b causes accumulation of Chk1 and Wee1 in the nucleolus and disrupts synchronized cell cycle re-entry after partial hepatectomy.\",\n      \"method\": \"Zebrafish capn3b null mutant generation; partial hepatectomy model; mass spectrometry of nuclear proteins; demonstration of Chk1 and Wee1 as CAPN3 substrates; nucleolar localization studies\",\n      \"journal\": \"Cell regeneration (London, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout with defined substrates identified by MS, functional liver regeneration phenotype, zebrafish model (ortholog)\",\n      \"pmids\": [\"32588143\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Antisense oligonucleotide-mediated reduction of p94 expression in differentiating rat myoblasts causes dramatic ultrastructural perturbations in myotubes, particularly affecting myofibrillar stability and Z-line integrity, implicating p94 in myofibrillar maintenance.\",\n      \"method\": \"Antisense oligonucleotide treatment of rat primary myoblast cultures; ultrastructural analysis by electron microscopy\",\n      \"journal\": \"Cell growth & differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — loss-of-function with defined ultrastructural phenotype, single lab\",\n      \"pmids\": [\"8930395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Reduction of p94 activity in a muscle cell inducible system results in a significant increase in myogenin levels, suggesting p94 participates in a myogenesis regulatory pathway by modulating myogenic regulatory factors.\",\n      \"method\": \"Inducible expression system in muscle cell line; western blot for myogenin levels\",\n      \"journal\": \"Journal of biotechnology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single method (western blot), indirect evidence for pathway involvement, single lab\",\n      \"pmids\": [\"12044555\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"A p94-like CAPN3 splice variant lacking IS1 and exon 15 (a putative nuclear localization signal) is expressed in human peripheral blood mononuclear cells (PBMCs), particularly B- and T-lymphocytes. This variant activates and inactivates without forming the low-Ca2+-requiring form typical of classical calpains, and shows lower sensitivity to calpastatin than ubiquitous calpains.\",\n      \"method\": \"Chromatographic fractionation of PBMC lysates; biochemical characterization of activation kinetics and calpastatin sensitivity; cell-type distribution by expression analysis\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical characterization of isolated protein with multiple assays, single lab\",\n      \"pmids\": [\"12882647\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Immunohistochemical analysis of single myofibers shows that p94/CAPN3 localizes at the Z-line and N2-line regions of sarcomeres. M-line localization of p94 is influenced by cellular context including contractile status, fiber type, sarcomere maturation, and composition of signal complexes.\",\n      \"method\": \"Immunohistochemistry on isolated single myofibers\",\n      \"journal\": \"Journal of muscle research and cell motility\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — immunohistochemistry only, no functional consequence directly tested for localization, single lab\",\n      \"pmids\": [\"16453164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CAPN3 is the first (and only) intracellular enzyme found to depend on Na+ for its activation (in addition to Ca2+), and it is the only protease (other than certain viral proteases) able to regain protease function after autolytic dissociation via intermolecular complementation (iMOC). CAPN3 has both proteolytic and non-proteolytic functions.\",\n      \"method\": \"Review/synthesis of experimental data; Na+-dependence originally established by biochemical assays; iMOC from reconstitution experiments (see PMID 25512505)\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — Na+-dependence finding cited as established experimental result (biochemical assays); iMOC replicated; review paper synthesizing primary data\",\n      \"pmids\": [\"26363099\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CAPN3/p94 is a skeletal muscle-specific, Ca2+- and Na2+-dependent cysteine protease that undergoes rapid and exhaustive autolysis mediated by its unique IS1 (acting as an internal propeptide) and IS2 insertion sequences; it binds to connectin/titin at multiple sites (N2A, PEVK, M-line, C-terminus) which suppresses its autolysis and stabilizes the enzyme, and this interaction is essential for normal sarcomere assembly and remodeling; CAPN3 cleaves titin, calpastatin, MARP2/Ankrd2, CTBP1, Chk1, and Wee1 as substrates, is negatively regulated by the scaffolding protein PLEIAD, forms a homotrimer via its PEF domain, can restore protease activity after autolytic fragmentation via intermolecular complementation (iMOC), and its loss of proteolytic function causes limb-girdle muscular dystrophy type 2A (LGMD2A).\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CAPN3/p94 is a skeletal muscle-enriched, Ca2+-dependent cysteine protease that governs sarcomere assembly, remodeling, and myofibrillar integrity, with loss of its proteolytic function causing limb-girdle muscular dystrophy type 2A [#0, #6, #10]. Its defining biochemical property is rapid, exhaustive autolysis driven by the active-site cysteine C129 and its calpain-unique insertion sequences: IS1 acts as an internal autoinhibitory propeptide whose autoproteolytic removal activates the enzyme, while a cryptic site in IS2 controls fragment disassembly [#0, #9, #11]. Catalysis is strictly Ca2+-dependent—even substoichiometric Ca2+ drives an initial intramolecular cleavage, with a second intermolecular cleavage requiring higher Ca2+—and uniquely also depends on Na+ [#7, #12, #24]. CAPN3 binds connectin/titin at multiple sites (N2A, PEVK, M-line, and the alternatively spliced skeletal-muscle C-terminus) through its IS2 region; this interaction stabilizes the enzyme by suppressing autolysis and is integral to its sarcomeric role, and LGMD2A mutations weaken titin binding [#1, #2, #14, #10]. The enzyme processes titin, calpastatin, MARP2/Ankrd2, and CTBP1 as substrates, is negatively regulated by the scaffold PLEIAD (SIMC1) which also recruits substrates, assembles into a PEF-domain-dependent homotrimer, and can regain activity after autolytic fragmentation via intermolecular complementation [#14, #8, #15, #18, #16]. A nucleolar role cleaving cell-cycle regulators Chk1 and Wee1 has been defined in a zebrafish ortholog [#19].\",\n  \"teleology\": [\n    {\n      \"year\": 1993,\n      \"claim\": \"Established the founding paradox of CAPN3 biology—that the protease destroys itself immediately after translation—and pinned this to the active site and the muscle-specific IS2 region.\",\n      \"evidence\": \"Active-site (C129S/A) and IS2-deletion mutants expressed in COS/L8 cells and in vitro translation\",\n      \"pmids\": [\"8486713\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve whether autolysis is intra- or intermolecular\", \"Functional significance of nuclear localization not established\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Identified connectin/titin as a binding partner that stabilizes intact CAPN3, answering how the rapidly self-destructing enzyme could persist in muscle.\",\n      \"evidence\": \"Yeast two-hybrid of skeletal muscle library and myofibril fractionation localizing intact p94 to the connectin-insoluble fraction\",\n      \"pmids\": [\"8537379\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding shown via IS2 but mechanism of autolysis suppression not defined\", \"Number of titin binding sites unknown at this stage\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Mapped a C-terminal titin binding site whose tissue-specific alternative splicing restricts CAPN3 anchoring to skeletal muscle.\",\n      \"evidence\": \"Yeast two-hybrid domain mapping of the connectin C-terminus (M-is7 region)\",\n      \"pmids\": [\"9185618\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab Y2H without biochemical confirmation of the complex\", \"Functional consequence of C-terminal anchoring not tested\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Defined the multi-step autolytic pathway and localized all cleavage sites to IS1, while showing native CAPN3 forms a homodimer—framing the structural basis of self-processing.\",\n      \"evidence\": \"Affinity purification of native rabbit p94 and characterization of C129S mutant and autolytic intermediates\",\n      \"pmids\": [\"9794799\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of intra- vs intermolecular reaction not quantified\", \"Oligomeric state later revised to trimer\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Demonstrated CAPN3 binds Ca2+ with high affinity and requires it for autolysis even at submicromolar concentrations, establishing Ca2+ as the catalytic trigger.\",\n      \"evidence\": \"Baculovirus-expressed human p94 with Ca2+ binding and autolysis assays; N-terminal sequencing of fragments (residues 30-31 and 412-413)\",\n      \"pmids\": [\"10504417\", \"10068445\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab in vitro characterization\", \"Physiological Ca2+ trigger in muscle not directly demonstrated\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Showed in vivo that loss of catalytic activity (or dominant-negative inactive enzyme) is sufficient to produce dystrophic muscle pathology, linking protease function to muscle integrity.\",\n      \"evidence\": \"Transgenic mice expressing C129S inactive p94 with histological and grip-strength analysis\",\n      \"pmids\": [\"10814721\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cannot fully separate dominant-negative accumulation from loss-of-function\", \"Specific in vivo substrate driving pathology not identified\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Resolved the initiating autolytic cleavage as intramolecular and concentration-independent, refining the activation mechanism of the protease core.\",\n      \"evidence\": \"Recombinant protease core with C129S trans-cleavage controls and concentration-dependence assays\",\n      \"pmids\": [\"12482600\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Later intermolecular steps not addressed here\", \"Behavior of full-length enzyme vs isolated core may differ\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Revealed CAPN3 can both resist and degrade calpastatin, positioning it as a regulator of the conventional calpain system, and documented physiologically active splice variants.\",\n      \"evidence\": \"COS/insect expression of p94delta variant with caseinolytic, autolytic, calpastatin inhibition/cleavage, and FRET assays; biochemical characterization of a PBMC variant\",\n      \"pmids\": [\"14594950\", \"12882647\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of calpastatin cleavage not established\", \"Tissue distribution and function of non-muscle variants unclear\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined IS1 as an internal autoinhibitory propeptide blocking substrate/inhibitor access, explaining how autoproteolysis activates the enzyme; and showed in a KO mouse that CAPN3 is required for organized sarcomere formation and binds/cleaves titin.\",\n      \"evidence\": \"Recombinant core mutagenesis with CD, inhibitor titration, and substrate assays; CAPN3 KO mouse EM with in vitro titin binding/cleavage and pathogenic mutant analysis\",\n      \"pmids\": [\"15073171\", \"15138196\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural model of IS1 occlusion not crystallographically resolved\", \"Which titin cleavages are functionally critical in vivo unclear\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Established a two-stage Ca2+-activation model—intramolecular first cleavage at low Ca2+, intermolecular disassembly at higher Ca2+—and showed N2A titin binding suppresses IS2 autolysis and stabilizes fragments.\",\n      \"evidence\": \"Purified core Ca2+-titration and metal-substitution assays; in vitro autolysis plus yeast Gal4-p94 reporter with disease mutants\",\n      \"pmids\": [\"16533054\", \"16627476\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reporter-based activity readout is indirect\", \"How N2A binding mechanically blocks IS2 cleavage not structurally defined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Expanded the CAPN3 substrate landscape beyond conventional calpain targets to translation machinery and glycolytic enzymes via unbiased proteomics.\",\n      \"evidence\": \"iTRAQ/2-D LC-MALDI comparison of active vs inactive p94 in COS7 cells\",\n      \"pmids\": [\"17373644\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Individual substrates not independently validated\", \"Non-muscle cell context may not reflect physiological cleavage\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Mapped CAPN3 to multiple titin sites (N2A, PEVK, M-line) and uncovered a competitive substrate relationship with MARP2/Ankrd2 at N2A, integrating CAPN3 into a titin-based mechanosignaling node.\",\n      \"evidence\": \"COS7 co-expression, in vitro proteolysis, competition binding, and mdm-deletion analysis\",\n      \"pmids\": [\"18310072\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo stoichiometry of competing partners unknown\", \"Signaling output downstream of N2A complex not defined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identified PLEIAD/SIMC1 as a dual regulator that both suppresses CAPN3 autolysis and scaffolds substrate (CTBP1) delivery, adding a non-titin regulatory layer.\",\n      \"evidence\": \"Reciprocal co-IP, COS7 co-expression, in vitro proteolysis, and CTBP1 cleavage-site mapping\",\n      \"pmids\": [\"23707407\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab evidence\", \"Physiological setting where PLEIAD-CAPN3-CTBP1 axis operates not established\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrated intermolecular complementation (iMOC): autolytic N- and C-terminal fragments and even two different inactive mutants can reconstitute active protease, redefining post-autolytic CAPN3 as functional rather than spent.\",\n      \"evidence\": \"In vitro reconstitution of purified autolytic fragments and active-site mutant complementation\",\n      \"pmids\": [\"25512505\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo occurrence and prevalence of iMOC unknown\", \"Implications for genotype-phenotype in LGMD2A compound heterozygotes untested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Resolved CAPN3 cleavage of M-band titin at defined sites in normal muscle and showed the FINmaj/LGMD2J mutation alters processing without disrupting binding, and synthesized the unique Na+-dependence of CAPN3 activation.\",\n      \"evidence\": \"In vitro titin cleavage with product sequencing plus tissue western/IF; review synthesis of Na+-dependence and iMOC\",\n      \"pmids\": [\"25877298\", \"26363099\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"M-band titin loss in TMD/LGMD2J shown independent of CAPN3\", \"Molecular basis of Na+-dependence not structurally defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revised the oligomeric state to a PEF-domain-dependent homotrimer (reinforced by the CBSW domain), correcting the earlier dimer model and locating the assembly determinant.\",\n      \"evidence\": \"Blue native PAGE, chemical cross-linking, sequential IP, and domain-deletion analysis of recombinant CAPN3\",\n      \"pmids\": [\"32200007\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of trimerization for catalysis not defined\", \"Whether trimer assembles on titin in situ unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Uncovered a nucleolar, cell-cycle role: DEF recruits CAPN3 to the nucleolus to cleave Chk1 and Wee1 (and p53) and license synchronized cell-cycle re-entry, extending CAPN3 function beyond the sarcomere.\",\n      \"evidence\": \"Zebrafish capn3b null mutant, partial hepatectomy regeneration model, nuclear-protein MS, and substrate cleavage assays\",\n      \"pmids\": [\"32588143\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Demonstrated in zebrafish ortholog, not human CAPN3\", \"Whether muscle CAPN3 performs an analogous nucleolar function unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A high-resolution structure of full-length CAPN3 explaining how IS1 autoinhibition, Na+/Ca2+ dependence, titin anchoring, and PEF-domain trimerization are integrated remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No experimental atomic structure of intact CAPN3 in the timeline\", \"In vivo substrate hierarchy driving LGMD2A pathology not defined\", \"Physiological trigger and tissue context of Na+ co-dependence unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 3, 8, 9, 14, 15, 17, 19]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 3, 12]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [1, 2, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [10, 20, 23]},\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [19]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-397014\", \"supporting_discovery_ids\": [10, 20]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 8, 14, 15]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [6, 10, 17]}\n    ],\n    \"complexes\": [\"CAPN3 homotrimer\", \"CAPN3-titin/connectin complex\"],\n    \"partners\": [\"TTN\", \"ANKRD2\", \"SIMC1\", \"CTBP1\", \"CAST\", \"CHEK1\", \"WEE1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":6,"faith_total":6,"faith_pct":100.0}}