{"gene":"PPP3CA","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":1991,"finding":"Yeast CNA1 encodes a calmodulin-regulated phosphoprotein phosphatase (calcineurin/PP2B homolog) whose catalytic subunit is 54% identical to the mammalian counterpart; genetic deletion of both yeast calcineurin catalytic subunit genes (cna1 cna2) causes hypersensitivity to mating pheromone alpha-factor and failure to resume growth during continuous pheromone exposure, placing calcineurin as an antagonist of the mating-pheromone response pathway.","method":"Yeast library screening with calmodulin and human calcineurin cDNA probes; PCR cloning; null mutant construction and pheromone-response epistasis assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with double-mutant rescue phenotype, orthologous to mammalian PPP3CA, replicated with two independent gene deletions","pmids":["1651503"],"is_preprint":false},{"year":2018,"finding":"Mutations in different functional domains of PPP3CA cause distinct clinical disorders: catalytic domain mutations and frameshift (loss-of-function) result in epileptic encephalopathy, while auto-inhibitory (AI) domain mutations cause multiple congenital abnormalities. Yeast model experiments demonstrated that catalytic domain mutations decrease calcineurin signaling whereas AI domain mutations increase calcineurin signaling, establishing a gain-of-function vs. loss-of-function dichotomy.","method":"Clinical exome sequencing; yeast model functional assays for calcineurin signaling activity","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast functional assay with distinct domain mutations, single lab, supported by clinical correlation","pmids":["29432562"],"is_preprint":false},{"year":2019,"finding":"PPP3CA (calcineurin Aα) negatively regulates hypoxia-inducible factor (HIF) transcriptional activity in a phosphatase-activity-dependent manner: a constitutively active PPP3CA inhibited HIF target gene expression without altering HIF-1α or HIF-2α protein levels, while a catalytically inactive mutant had no effect. Ionomycin-activated endogenous PPP3CA phenocopied the effect, and PPP3CA silencing abolished this ionomycin effect.","method":"siRNA screen of 25 phosphatase catalytic subunits; constitutively active vs. catalytically inactive PPP3CA overexpression; ionomycin pharmacological activation; HIF transcriptional reporter assays in HeLa cells","journal":"Archives of biochemistry and biophysics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (active vs. inactive mutant, siRNA, pharmacological activation), single lab","pmids":["30776328"],"is_preprint":false},{"year":2021,"finding":"PPP3CA truncating variants clustered within a 26-amino acid region in the regulatory domain (RD) produce no detectable mutant protein (expression studies showed RNA from mutant allele but absence of mutant protein), causing more severe early-onset seizures than loss-of-function missense variants in the catalytic domain.","method":"Exome sequencing; expression studies (RNA and protein analysis) of truncating variant in patient cells","journal":"Clinical genetics","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct expression studies in patient cells, multiple patients with clustering, single lab","pmids":["33963760"],"is_preprint":false},{"year":2022,"finding":"CAMTA1 and PPP3CA competitively bind to NFATc4; CAMTA1 knockdown promotes PPP3CA-mediated dephosphorylation of NFATc4, upregulating NFATc4 activity and increasing colorectal cancer chemoresistance to oxaliplatin. PPP3CA is essential for the NFATc4 dephosphorylation and chemoresistance phenotype caused by CAMTA1 knockdown.","method":"Co-immunoprecipitation (Co-IP); siRNA knockdown of PPP3CA and CAMTA1; luciferase reporter assays; xenograft tumor model","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP for complex, functional rescue with siRNA, in vitro and in vivo validation, single lab","pmids":["35332122"],"is_preprint":false},{"year":2022,"finding":"TRIM72 interacts with PPP3CA (shown by Co-IP); PPP3CA mediates the effects of TRIM72 on hypoxia-induced lactate production and MCT4 (monocarboxylate transporter 4) promoter activity, as well as PI3K/Akt/mTOR pathway signaling in breast cancer cells.","method":"Co-immunoprecipitation (Co-IP); siRNA knockdown of PPP3CA; luciferase reporter assay; western blot; xenograft model","journal":"Anti-cancer drugs","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP with partial mechanistic follow-up, single lab","pmids":["35324524"],"is_preprint":false},{"year":2020,"finding":"PPP3CA knockdown in metanephric mesenchyme mK4 cells promoted E-cadherin expression, cell apoptosis, and reduced cell proliferation and migration; the underlying mechanism was associated with dephosphorylation of ERK1/2 by PPP3CA.","method":"Lentiviral knockdown; flow cytometry; EdU/CCK-8 assay; wound healing assay; western blot for ERK1/2 phosphorylation","journal":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single KD approach with ERK1/2 phosphorylation readout, limited mechanistic depth","pmids":["33169579"],"is_preprint":false},{"year":2025,"finding":"OR2T6 physically binds PPP3CA (Co-IP and mass spectrometry); OR2T6 promotes PPP3CA protein stability and enzyme activity through the ubiquitin-proteasome system and by promoting calcium ion influx via the Gs/cAMP/PKA channel signaling axis. PPP3CA activity downstream of OR2T6 suppresses the AKT/mTOR pathway, inhibiting tumor proliferation and promoting autophagy initiation; PPP3CA also facilitates nuclear translocation of TFEB, leading to transcriptional inactivation of lysosomal target genes and blocking autophagic flux.","method":"Co-immunoprecipitation and mass spectrometry; ubiquitin-proteasome pathway analysis; calcium influx assays; AKT/mTOR pathway western blot; TFEB nuclear translocation assay; in vitro and in vivo tumor assays","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP+MS for interaction, multiple orthogonal functional assays, single lab","pmids":["41214150"],"is_preprint":false},{"year":2025,"finding":"MAZ transcriptionally represses PPP3CA by binding to its promoter; reduced PPP3CA leads to activation of the ERK/MAPK pathway, increased NFATc1 and MMP9 levels, and promotion of osteoclast differentiation and osteoporosis progression in vivo.","method":"Bioinformatics; OVX mouse model; RANKL-induced osteoclast differentiation; PPP3CA overexpression; promoter binding assay; ERK1/2 phosphorylation western blot; in vivo animal experiments","journal":"Tissue & cell","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, mechanistic follow-up with PPP3CA overexpression but limited direct PPP3CA biochemistry","pmids":["40602229"],"is_preprint":false},{"year":2026,"finding":"The disease-causing E282K missense variant in PPP3CA/calcineurin alters substrate dephosphorylation by disrupting an acidic substrate recruitment pocket (the 'E282 pocket') adjacent to the active site that normally binds basophilic substrates via arginine residues. The CNE282K structure shows that the pocket transforms from acidic to basic and is blocked by a E282K-E237 salt bridge, shifting substrate preference from basic to acidic substrates as confirmed by in vitro assays and cellular phosphoproteomics.","method":"Crystal/structural analysis of CNE282K; in vitro dephosphorylation assays; active-site mutagenesis; cell phosphoproteomics; molecular/cellular techniques","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — structure determination combined with in vitro reconstitution, active site mutagenesis, and cell-based phosphoproteomics in a single rigorous study","pmids":["41698888"],"is_preprint":false},{"year":1996,"finding":"Calcineurin (PPP3CA/calcineurin Aα) is a heterodimer consisting of a 58-59 kDa calmodulin-binding catalytic subunit (calcineurin A) and a 19 kDa Ca2+-binding regulatory subunit (calcineurin B); it functions in transcriptional regulation in T lymphocytes by dephosphorylating NF-AT, which is essential for interleukin-2 gene transcription. Chromosomal localization of PPP3CA confirmed to chromosome 4.","method":"Chromosome mapping; biochemical characterization of subunit composition; functional literature synthesis with NF-AT dephosphorylation","journal":"Cytogenetics and cell genetics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — chromosomal mapping study; NF-AT dephosphorylation mechanism cited from prior literature, not independently demonstrated in this paper","pmids":["8978785"],"is_preprint":false},{"year":2024,"finding":"PPP3CA pathogenic variants within the calmodulin-binding domain result in childhood-onset epilepsy with focal and generalized seizures, while variants within the regulatory domain lead to early-onset drug-resistant severe epilepsy, indicating that variant location within PPP3CA determines the severity of calcineurin dysfunction and clinical phenotype.","method":"Clinical case series with literature review; electro-clinical characterization; genotype-phenotype correlation analysis","journal":"Seizure","confidence":"Low","confidence_rationale":"Tier 3 / Weak — clinical/phenotypic genotype-phenotype correlation without direct biochemical mechanism experiments","pmids":["39305655"],"is_preprint":false}],"current_model":"PPP3CA encodes the alpha isoform of the calcineurin catalytic subunit (a Ca2+/calmodulin-dependent serine-threonine phosphatase); it dephosphorylates substrates—including NFATc4 and basophilic targets recruited via an acidic E282 pocket adjacent to the active site—to regulate transcription, HIF activity, ERK/MAPK signaling, AKT/mTOR signaling, and autophagy, with catalytic domain loss-of-function causing epileptic encephalopathy and auto-inhibitory domain gain-of-function causing congenital abnormalities, as mechanistically supported by structural studies, in vitro phosphatase assays, mutagenesis, and epistasis experiments."},"narrative":{"mechanistic_narrative":"PPP3CA encodes the catalytic subunit of calcineurin, a Ca2+/calmodulin-regulated serine-threonine phosphatase that functions as a heterodimer with a Ca2+-binding regulatory subunit to couple calcium signals to substrate dephosphorylation [PMID:8978785]. Its evolutionarily conserved role as a calmodulin-regulated phosphatase was established through the yeast ortholog CNA1, where loss of catalytic activity disrupts the regulation of the mating-pheromone response [PMID:1651503]. Substrate selection depends on an acidic recruitment pocket (the E282 pocket) adjacent to the active site that binds basophilic substrates via arginine contacts; the disease-causing E282K variant inverts the charge of this pocket and shifts substrate preference from basic to acidic targets, as shown by structure determination, in vitro dephosphorylation, and cellular phosphoproteomics [PMID:41698888]. Through its phosphatase activity, PPP3CA dephosphorylates NFAT-family transcription factors—including NFATc4, with which it associates in competition with CAMTA1—to regulate downstream transcription [PMID:35332122, PMID:8978785], and it negatively regulates HIF transcriptional activity in an activity-dependent manner without changing HIF-alpha protein levels [PMID:30776328]. PPP3CA further modulates AKT/mTOR signaling and autophagy, including TFEB nuclear translocation, downstream of receptor inputs [PMID:41214150]. Loss-of-function variants in the catalytic domain cause epileptic encephalopathy whereas auto-inhibitory domain gain-of-function variants cause multiple congenital abnormalities, defining a domain-dependent functional dichotomy [PMID:29432562].","teleology":[{"year":1991,"claim":"Established that the calcineurin catalytic subunit is a conserved calmodulin-regulated phosphatase with a defined biological role, providing the foundational identity for mammalian PPP3CA.","evidence":"Yeast CNA1/CNA2 cloning with calmodulin and human calcineurin probes and double-null pheromone-response epistasis","pmids":["1651503"],"confidence":"High","gaps":["Does not identify mammalian substrates","Mechanism inferred from yeast orthologs rather than human protein"]},{"year":1996,"claim":"Defined PPP3CA's subunit architecture and linked it to NF-AT dephosphorylation in transcriptional regulation, framing its role in immune gene expression.","evidence":"Chromosome mapping and biochemical subunit characterization with literature synthesis of NF-AT dephosphorylation","pmids":["8978785"],"confidence":"Low","gaps":["NF-AT dephosphorylation cited from prior literature, not demonstrated here","No direct enzymatic assay in this study"]},{"year":2018,"claim":"Resolved how distinct PPP3CA domain mutations produce opposite clinical outcomes, establishing a gain-of-function versus loss-of-function dichotomy.","evidence":"Clinical exome sequencing with yeast functional assays of catalytic versus auto-inhibitory domain mutations","pmids":["29432562"],"confidence":"Medium","gaps":["Functional readouts in yeast, not human neurons","Does not define the affected substrates per disorder"]},{"year":2019,"claim":"Showed that PPP3CA acts as a phosphatase-dependent negative regulator of HIF transcriptional output independent of HIF-alpha stability, expanding its transcriptional reach beyond NFAT.","evidence":"siRNA phosphatase screen, active versus catalytically inactive overexpression, ionomycin activation, and HIF reporter assays in HeLa cells","pmids":["30776328"],"confidence":"Medium","gaps":["Direct HIF-pathway substrate not identified","Single cell-line system"]},{"year":2020,"claim":"Linked PPP3CA to ERK1/2 dephosphorylation and control of proliferation, migration, and apoptosis in metanephric mesenchyme cells.","evidence":"Lentiviral knockdown with proliferation/migration/apoptosis assays and ERK1/2 phospho-western blot in mK4 cells","pmids":["33169579"],"confidence":"Low","gaps":["Single knockdown approach","Direct dephosphorylation of ERK1/2 not biochemically demonstrated"]},{"year":2021,"claim":"Demonstrated that truncating variants in a regulatory-domain hotspot abolish mutant protein and cause more severe seizures than catalytic missense variants, refining genotype-severity relationships.","evidence":"Exome sequencing with RNA and protein expression studies in patient cells","pmids":["33963760"],"confidence":"Medium","gaps":["Mechanism of protein loss not defined","Single lab"]},{"year":2022,"claim":"Identified competitive NFATc4 binding between CAMTA1 and PPP3CA as a switch controlling NFATc4 dephosphorylation and chemoresistance, placing PPP3CA in a tunable transcriptional module.","evidence":"Co-IP, siRNA knockdown rescue, luciferase reporters, and xenograft model in colorectal cancer","pmids":["35332122"],"confidence":"Medium","gaps":["Competitive binding inferred, not structurally resolved","Single lab"]},{"year":2022,"claim":"Connected PPP3CA to TRIM72-driven glycolytic and PI3K/Akt/mTOR signaling in breast cancer.","evidence":"Co-IP, PPP3CA siRNA, MCT4 promoter luciferase assay, and xenograft model","pmids":["35324524"],"confidence":"Low","gaps":["Single Co-IP without reciprocal validation","Direct phosphatase substrate in this axis unknown"]},{"year":2025,"claim":"Showed PPP3CA stability and activity are regulated by OR2T6 via calcium influx, with downstream suppression of AKT/mTOR and dual control of autophagy through TFEB translocation.","evidence":"Co-IP/MS, ubiquitin-proteasome and calcium influx assays, AKT/mTOR western blot, and TFEB translocation assays with tumor models","pmids":["41214150"],"confidence":"Medium","gaps":["Whether TFEB is a direct PPP3CA substrate not established","Single lab"]},{"year":2025,"claim":"Placed PPP3CA under transcriptional repression by MAZ, with loss activating ERK/MAPK and NFATc1/MMP9 to drive osteoclast differentiation.","evidence":"Promoter binding assay, OVX mouse model, RANKL osteoclast differentiation, and PPP3CA overexpression with phospho-ERK western blot","pmids":["40602229"],"confidence":"Low","gaps":["Limited direct PPP3CA biochemistry","Single lab"]},{"year":2026,"claim":"Provided the structural basis for substrate recruitment, showing an acidic E282 pocket selects basophilic substrates and that the E282K disease variant inverts this selectivity.","evidence":"CNE282K structure determination, in vitro dephosphorylation, active-site mutagenesis, and cellular phosphoproteomics","pmids":["41698888"],"confidence":"High","gaps":["Full repertoire of acidic substrates gained by E282K not catalogued","Link to specific disease phenotype at organ level not established"]},{"year":null,"claim":"How calcineurin substrate selection, domain-specific dysfunction, and the diverse downstream pathways (HIF, ERK, AKT/mTOR, autophagy) integrate into the distinct disease phenotypes remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified substrate map across tissues","Mechanistic basis distinguishing epileptic encephalopathy from congenital abnormalities at substrate level unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,9,10]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,9,10]}],"localization":[],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[2,4,10]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[6,7]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[7]}],"complexes":["calcineurin"],"partners":["PPP3R1","NFATC4","CAMTA1","TRIM72","OR2T6"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q08209","full_name":"Protein phosphatase 3 catalytic subunit alpha","aliases":["CAM-PRP catalytic subunit","Calcineurin A alpha","Calmodulin-dependent calcineurin A subunit alpha isoform","CNA alpha","Serine/threonine-protein phosphatase 2B catalytic subunit alpha isoform"],"length_aa":521,"mass_kda":58.7,"function":"Calcium-dependent, calmodulin-stimulated protein phosphatase which plays an essential role in the transduction of intracellular Ca(2+)-mediated signals (PubMed:15671020, PubMed:18838687, PubMed:19154138, PubMed:23468591, PubMed:30254215). Many of the substrates contain a PxIxIT motif and/or a LxVP motif (PubMed:17498738, PubMed:17502104, PubMed:22343722, PubMed:23468591, PubMed:27974827). In response to increased Ca(2+) levels, dephosphorylates and activates phosphatase SSH1 which results in cofilin dephosphorylation (PubMed:15671020). In response to increased Ca(2+) levels following mitochondrial depolarization, dephosphorylates DNM1L inducing DNM1L translocation to the mitochondrion (PubMed:18838687). Positively regulates the CACNA1B/CAV2.2-mediated Ca(2+) release probability at hippocampal neuronal soma and synaptic terminals (By similarity). Dephosphorylates heat shock protein HSPB1 (By similarity). Dephosphorylates and activates transcription factor NFATC1 (PubMed:19154138). In response to increased Ca(2+) levels, regulates NFAT-mediated transcription probably by dephosphorylating NFAT and promoting its nuclear translocation (PubMed:26248042). Dephosphorylates and inactivates transcription factor ELK1 (PubMed:19154138). Dephosphorylates DARPP32 (PubMed:19154138). May dephosphorylate CRTC2 at 'Ser-171' resulting in CRTC2 dissociation from 14-3-3 proteins (PubMed:30611118). Dephosphorylates transcription factor TFEB at 'Ser-211' following Coxsackievirus B3 infection, promoting nuclear translocation (PubMed:33691586). Required for postnatal development of the nephrogenic zone and superficial glomeruli in the kidneys, cell cycle homeostasis in the nephrogenic zone, and ultimately normal kidney function (By similarity). Plays a role in intracellular AQP2 processing and localization to the apical membrane in the kidney, may thereby be required for efficient kidney filtration (By similarity). Required for secretion of salivary enzymes amylase, peroxidase, lysozyme and sialic acid via formation of secretory vesicles in the submandibular glands (By similarity). Required for calcineurin activity and homosynaptic depotentiation in the hippocampus (By similarity). Required for normal differentiation and survival of keratinocytes and therefore required for epidermis superstructure formation (By similarity). Positively regulates osteoblastic bone formation, via promotion of osteoblast differentiation (By similarity). Positively regulates osteoclast differentiation, potentially via NFATC1 signaling (By similarity). May play a role in skeletal muscle fiber type specification, potentially via NFATC1 signaling (By similarity). Negatively regulates MAP3K14/NIK signaling via inhibition of nuclear translocation of the transcription factors RELA and RELB (By similarity). Required for antigen-specific T-cell proliferation response (By similarity). Dephosphorylates KLHL3, promoting the interaction between KLHL3 and WNK4 and subsequent degradation of WNK4 (PubMed:30718414). Negatively regulates SLC9A1 activity (PubMed:31375679)","subcellular_location":"Cytoplasm; Cell membrane; Cell membrane, sarcolemma; Cytoplasm, myofibril, sarcomere, Z line; Cell projection, dendritic spine","url":"https://www.uniprot.org/uniprotkb/Q08209/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PPP3CA","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CALM1","stoichiometry":0.2},{"gene":"CALM2","stoichiometry":0.2},{"gene":"CALM3","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/PPP3CA","total_profiled":1310},"omim":[{"mim_id":"618265","title":"ARTHROGRYPOSIS, CLEFT PALATE, CRANIOSYNOSTOSIS, AND IMPAIRED INTELLECTUAL DEVELOPMENT; ACCIID","url":"https://www.omim.org/entry/618265"},{"mim_id":"617711","title":"DEVELOPMENTAL AND EPILEPTIC ENCEPHALOPATHY 91; DEE91","url":"https://www.omim.org/entry/617711"},{"mim_id":"615409","title":"SPERMATOGENESIS-ASSOCIATED PROTEIN 33; SPATA33","url":"https://www.omim.org/entry/615409"},{"mim_id":"613821","title":"PROTEIN PHOSPHATASE 3, REGULATORY SUBUNIT B, BETA; PPP3R2","url":"https://www.omim.org/entry/613821"},{"mim_id":"613614","title":"MICRO RNA 499; MIR499","url":"https://www.omim.org/entry/613614"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Calyx","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"brain","ntpm":144.7}],"url":"https://www.proteinatlas.org/search/PPP3CA"},"hgnc":{"alias_symbol":["CNA1","PPP2B"],"prev_symbol":["CALN","CALNA"]},"alphafold":{"accession":"Q08209","domains":[{"cath_id":"3.60.21.10","chopping":"27-333","consensus_level":"high","plddt":97.5052,"start":27,"end":333},{"cath_id":"-","chopping":"346-378_385-484","consensus_level":"high","plddt":75.1947,"start":346,"end":484}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q08209","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q08209-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q08209-F1-predicted_aligned_error_v6.png","plddt_mean":85.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PPP3CA","jax_strain_url":"https://www.jax.org/strain/search?query=PPP3CA"},"sequence":{"accession":"Q08209","fasta_url":"https://rest.uniprot.org/uniprotkb/Q08209.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q08209/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q08209"}},"corpus_meta":[{"pmid":"1651503","id":"PMC_1651503","title":"Yeast has homologs (CNA1 and CNA2 gene products) of mammalian calcineurin, a calmodulin-regulated phosphoprotein phosphatase.","date":"1991","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/1651503","citation_count":280,"is_preprint":false},{"pmid":"16251352","id":"PMC_16251352","title":"The CNA1 histone of the ciliate Tetrahymena thermophila is essential for chromosome segregation in the germline micronucleus.","date":"2005","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/16251352","citation_count":51,"is_preprint":false},{"pmid":"28942967","id":"PMC_28942967","title":"De Novo Mutations in PPP3CA Cause Severe Neurodevelopmental Disease with Seizures.","date":"2017","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/28942967","citation_count":49,"is_preprint":false},{"pmid":"29432562","id":"PMC_29432562","title":"Loss-of-function and 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neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/40548073","citation_count":1,"is_preprint":false},{"pmid":"40796177","id":"PMC_40796177","title":"Circular RNA hsa_circ_0099188 regulates inducible nitric oxide synthase and chemokine transcription in macrophages by targeting the hsa-miR-381-3p/PPP3CA and hsa-miR-381-3p/KLF4 pathways in response to 4,4'-methylene diphenyl diisocyanate-glutathione conjugate exposure.","date":"2025","source":"Toxicological sciences : an official journal of the Society of Toxicology","url":"https://pubmed.ncbi.nlm.nih.gov/40796177","citation_count":1,"is_preprint":false},{"pmid":"41214150","id":"PMC_41214150","title":"OR2T6 modulates autophagy through the PPP3CA-mediated pathways to suppress gastric cancer.","date":"2025","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/41214150","citation_count":0,"is_preprint":false},{"pmid":"33169579","id":"PMC_33169579","title":"[PPP3CA silence regulates MET process, cell apoptosis, proliferation and migration in metanephric mesenchyme cells].","date":"2020","source":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/33169579","citation_count":0,"is_preprint":false},{"pmid":"40602229","id":"PMC_40602229","title":"Activation of ERK/MAPK signaling by MAZ through transcriptional repression of PPP3CA to promote osteoclastogenesis and osteoporosis progression: Functions and mechanisms.","date":"2025","source":"Tissue & cell","url":"https://pubmed.ncbi.nlm.nih.gov/40602229","citation_count":0,"is_preprint":false},{"pmid":"41698888","id":"PMC_41698888","title":"The clinical missense variant E282K in PPP3CA/calcineurin shifts substrate dephosphorylation by altering active site recruitment.","date":"2026","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/41698888","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.01.02.631159","title":"The calcineurin pathway regulates extreme thermotolerance, cell membrane and wall integrity, antifungal resistance, and virulence in  <i>Candida auris</i>","date":"2025-01-03","source":"bioRxiv","url":"https://doi.org/10.1101/2025.01.02.631159","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.07.02.662784","title":"Calcineurin-responsive zinc finger 1 (Crz1) contributes to stress tolerance and virulence in the pathogenic fungus  <i>Trichosporon asahii</i>","date":"2025-07-02","source":"bioRxiv","url":"https://doi.org/10.1101/2025.07.02.662784","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.09.06.611525","title":"The penta-EF-hand protein Pef1 of  <i>Candida albicans</i>  functions at sites of membrane perturbation to support polarized growth and membrane integrity","date":"2024-09-07","source":"bioRxiv","url":"https://doi.org/10.1101/2024.09.06.611525","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16504,"output_tokens":3222,"usd":0.048921,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10811,"output_tokens":3354,"usd":0.068952,"stage2_stop_reason":"end_turn"},"total_usd":0.117873,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1991,\n      \"finding\": \"Yeast CNA1 encodes a calmodulin-regulated phosphoprotein phosphatase (calcineurin/PP2B homolog) whose catalytic subunit is 54% identical to the mammalian counterpart; genetic deletion of both yeast calcineurin catalytic subunit genes (cna1 cna2) causes hypersensitivity to mating pheromone alpha-factor and failure to resume growth during continuous pheromone exposure, placing calcineurin as an antagonist of the mating-pheromone response pathway.\",\n      \"method\": \"Yeast library screening with calmodulin and human calcineurin cDNA probes; PCR cloning; null mutant construction and pheromone-response epistasis assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with double-mutant rescue phenotype, orthologous to mammalian PPP3CA, replicated with two independent gene deletions\",\n      \"pmids\": [\"1651503\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Mutations in different functional domains of PPP3CA cause distinct clinical disorders: catalytic domain mutations and frameshift (loss-of-function) result in epileptic encephalopathy, while auto-inhibitory (AI) domain mutations cause multiple congenital abnormalities. Yeast model experiments demonstrated that catalytic domain mutations decrease calcineurin signaling whereas AI domain mutations increase calcineurin signaling, establishing a gain-of-function vs. loss-of-function dichotomy.\",\n      \"method\": \"Clinical exome sequencing; yeast model functional assays for calcineurin signaling activity\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast functional assay with distinct domain mutations, single lab, supported by clinical correlation\",\n      \"pmids\": [\"29432562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PPP3CA (calcineurin Aα) negatively regulates hypoxia-inducible factor (HIF) transcriptional activity in a phosphatase-activity-dependent manner: a constitutively active PPP3CA inhibited HIF target gene expression without altering HIF-1α or HIF-2α protein levels, while a catalytically inactive mutant had no effect. Ionomycin-activated endogenous PPP3CA phenocopied the effect, and PPP3CA silencing abolished this ionomycin effect.\",\n      \"method\": \"siRNA screen of 25 phosphatase catalytic subunits; constitutively active vs. catalytically inactive PPP3CA overexpression; ionomycin pharmacological activation; HIF transcriptional reporter assays in HeLa cells\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (active vs. inactive mutant, siRNA, pharmacological activation), single lab\",\n      \"pmids\": [\"30776328\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PPP3CA truncating variants clustered within a 26-amino acid region in the regulatory domain (RD) produce no detectable mutant protein (expression studies showed RNA from mutant allele but absence of mutant protein), causing more severe early-onset seizures than loss-of-function missense variants in the catalytic domain.\",\n      \"method\": \"Exome sequencing; expression studies (RNA and protein analysis) of truncating variant in patient cells\",\n      \"journal\": \"Clinical genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct expression studies in patient cells, multiple patients with clustering, single lab\",\n      \"pmids\": [\"33963760\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CAMTA1 and PPP3CA competitively bind to NFATc4; CAMTA1 knockdown promotes PPP3CA-mediated dephosphorylation of NFATc4, upregulating NFATc4 activity and increasing colorectal cancer chemoresistance to oxaliplatin. PPP3CA is essential for the NFATc4 dephosphorylation and chemoresistance phenotype caused by CAMTA1 knockdown.\",\n      \"method\": \"Co-immunoprecipitation (Co-IP); siRNA knockdown of PPP3CA and CAMTA1; luciferase reporter assays; xenograft tumor model\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP for complex, functional rescue with siRNA, in vitro and in vivo validation, single lab\",\n      \"pmids\": [\"35332122\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TRIM72 interacts with PPP3CA (shown by Co-IP); PPP3CA mediates the effects of TRIM72 on hypoxia-induced lactate production and MCT4 (monocarboxylate transporter 4) promoter activity, as well as PI3K/Akt/mTOR pathway signaling in breast cancer cells.\",\n      \"method\": \"Co-immunoprecipitation (Co-IP); siRNA knockdown of PPP3CA; luciferase reporter assay; western blot; xenograft model\",\n      \"journal\": \"Anti-cancer drugs\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP with partial mechanistic follow-up, single lab\",\n      \"pmids\": [\"35324524\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PPP3CA knockdown in metanephric mesenchyme mK4 cells promoted E-cadherin expression, cell apoptosis, and reduced cell proliferation and migration; the underlying mechanism was associated with dephosphorylation of ERK1/2 by PPP3CA.\",\n      \"method\": \"Lentiviral knockdown; flow cytometry; EdU/CCK-8 assay; wound healing assay; western blot for ERK1/2 phosphorylation\",\n      \"journal\": \"Sheng wu gong cheng xue bao = Chinese journal of biotechnology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single KD approach with ERK1/2 phosphorylation readout, limited mechanistic depth\",\n      \"pmids\": [\"33169579\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OR2T6 physically binds PPP3CA (Co-IP and mass spectrometry); OR2T6 promotes PPP3CA protein stability and enzyme activity through the ubiquitin-proteasome system and by promoting calcium ion influx via the Gs/cAMP/PKA channel signaling axis. PPP3CA activity downstream of OR2T6 suppresses the AKT/mTOR pathway, inhibiting tumor proliferation and promoting autophagy initiation; PPP3CA also facilitates nuclear translocation of TFEB, leading to transcriptional inactivation of lysosomal target genes and blocking autophagic flux.\",\n      \"method\": \"Co-immunoprecipitation and mass spectrometry; ubiquitin-proteasome pathway analysis; calcium influx assays; AKT/mTOR pathway western blot; TFEB nuclear translocation assay; in vitro and in vivo tumor assays\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP+MS for interaction, multiple orthogonal functional assays, single lab\",\n      \"pmids\": [\"41214150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MAZ transcriptionally represses PPP3CA by binding to its promoter; reduced PPP3CA leads to activation of the ERK/MAPK pathway, increased NFATc1 and MMP9 levels, and promotion of osteoclast differentiation and osteoporosis progression in vivo.\",\n      \"method\": \"Bioinformatics; OVX mouse model; RANKL-induced osteoclast differentiation; PPP3CA overexpression; promoter binding assay; ERK1/2 phosphorylation western blot; in vivo animal experiments\",\n      \"journal\": \"Tissue & cell\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, mechanistic follow-up with PPP3CA overexpression but limited direct PPP3CA biochemistry\",\n      \"pmids\": [\"40602229\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"The disease-causing E282K missense variant in PPP3CA/calcineurin alters substrate dephosphorylation by disrupting an acidic substrate recruitment pocket (the 'E282 pocket') adjacent to the active site that normally binds basophilic substrates via arginine residues. The CNE282K structure shows that the pocket transforms from acidic to basic and is blocked by a E282K-E237 salt bridge, shifting substrate preference from basic to acidic substrates as confirmed by in vitro assays and cellular phosphoproteomics.\",\n      \"method\": \"Crystal/structural analysis of CNE282K; in vitro dephosphorylation assays; active-site mutagenesis; cell phosphoproteomics; molecular/cellular techniques\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — structure determination combined with in vitro reconstitution, active site mutagenesis, and cell-based phosphoproteomics in a single rigorous study\",\n      \"pmids\": [\"41698888\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Calcineurin (PPP3CA/calcineurin Aα) is a heterodimer consisting of a 58-59 kDa calmodulin-binding catalytic subunit (calcineurin A) and a 19 kDa Ca2+-binding regulatory subunit (calcineurin B); it functions in transcriptional regulation in T lymphocytes by dephosphorylating NF-AT, which is essential for interleukin-2 gene transcription. Chromosomal localization of PPP3CA confirmed to chromosome 4.\",\n      \"method\": \"Chromosome mapping; biochemical characterization of subunit composition; functional literature synthesis with NF-AT dephosphorylation\",\n      \"journal\": \"Cytogenetics and cell genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — chromosomal mapping study; NF-AT dephosphorylation mechanism cited from prior literature, not independently demonstrated in this paper\",\n      \"pmids\": [\"8978785\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PPP3CA pathogenic variants within the calmodulin-binding domain result in childhood-onset epilepsy with focal and generalized seizures, while variants within the regulatory domain lead to early-onset drug-resistant severe epilepsy, indicating that variant location within PPP3CA determines the severity of calcineurin dysfunction and clinical phenotype.\",\n      \"method\": \"Clinical case series with literature review; electro-clinical characterization; genotype-phenotype correlation analysis\",\n      \"journal\": \"Seizure\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — clinical/phenotypic genotype-phenotype correlation without direct biochemical mechanism experiments\",\n      \"pmids\": [\"39305655\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PPP3CA encodes the alpha isoform of the calcineurin catalytic subunit (a Ca2+/calmodulin-dependent serine-threonine phosphatase); it dephosphorylates substrates—including NFATc4 and basophilic targets recruited via an acidic E282 pocket adjacent to the active site—to regulate transcription, HIF activity, ERK/MAPK signaling, AKT/mTOR signaling, and autophagy, with catalytic domain loss-of-function causing epileptic encephalopathy and auto-inhibitory domain gain-of-function causing congenital abnormalities, as mechanistically supported by structural studies, in vitro phosphatase assays, mutagenesis, and epistasis experiments.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PPP3CA encodes the catalytic subunit of calcineurin, a Ca2+/calmodulin-regulated serine-threonine phosphatase that functions as a heterodimer with a Ca2+-binding regulatory subunit to couple calcium signals to substrate dephosphorylation [#10]. Its evolutionarily conserved role as a calmodulin-regulated phosphatase was established through the yeast ortholog CNA1, where loss of catalytic activity disrupts the regulation of the mating-pheromone response [#0]. Substrate selection depends on an acidic recruitment pocket (the E282 pocket) adjacent to the active site that binds basophilic substrates via arginine contacts; the disease-causing E282K variant inverts the charge of this pocket and shifts substrate preference from basic to acidic targets, as shown by structure determination, in vitro dephosphorylation, and cellular phosphoproteomics [#9]. Through its phosphatase activity, PPP3CA dephosphorylates NFAT-family transcription factors—including NFATc4, with which it associates in competition with CAMTA1—to regulate downstream transcription [#4, #10], and it negatively regulates HIF transcriptional activity in an activity-dependent manner without changing HIF-alpha protein levels [#2]. PPP3CA further modulates AKT/mTOR signaling and autophagy, including TFEB nuclear translocation, downstream of receptor inputs [#7]. Loss-of-function variants in the catalytic domain cause epileptic encephalopathy whereas auto-inhibitory domain gain-of-function variants cause multiple congenital abnormalities, defining a domain-dependent functional dichotomy [#1].\",\n  \"teleology\": [\n    {\n      \"year\": 1991,\n      \"claim\": \"Established that the calcineurin catalytic subunit is a conserved calmodulin-regulated phosphatase with a defined biological role, providing the foundational identity for mammalian PPP3CA.\",\n      \"evidence\": \"Yeast CNA1/CNA2 cloning with calmodulin and human calcineurin probes and double-null pheromone-response epistasis\",\n      \"pmids\": [\"1651503\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not identify mammalian substrates\", \"Mechanism inferred from yeast orthologs rather than human protein\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Defined PPP3CA's subunit architecture and linked it to NF-AT dephosphorylation in transcriptional regulation, framing its role in immune gene expression.\",\n      \"evidence\": \"Chromosome mapping and biochemical subunit characterization with literature synthesis of NF-AT dephosphorylation\",\n      \"pmids\": [\"8978785\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"NF-AT dephosphorylation cited from prior literature, not demonstrated here\", \"No direct enzymatic assay in this study\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Resolved how distinct PPP3CA domain mutations produce opposite clinical outcomes, establishing a gain-of-function versus loss-of-function dichotomy.\",\n      \"evidence\": \"Clinical exome sequencing with yeast functional assays of catalytic versus auto-inhibitory domain mutations\",\n      \"pmids\": [\"29432562\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional readouts in yeast, not human neurons\", \"Does not define the affected substrates per disorder\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed that PPP3CA acts as a phosphatase-dependent negative regulator of HIF transcriptional output independent of HIF-alpha stability, expanding its transcriptional reach beyond NFAT.\",\n      \"evidence\": \"siRNA phosphatase screen, active versus catalytically inactive overexpression, ionomycin activation, and HIF reporter assays in HeLa cells\",\n      \"pmids\": [\"30776328\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct HIF-pathway substrate not identified\", \"Single cell-line system\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Linked PPP3CA to ERK1/2 dephosphorylation and control of proliferation, migration, and apoptosis in metanephric mesenchyme cells.\",\n      \"evidence\": \"Lentiviral knockdown with proliferation/migration/apoptosis assays and ERK1/2 phospho-western blot in mK4 cells\",\n      \"pmids\": [\"33169579\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single knockdown approach\", \"Direct dephosphorylation of ERK1/2 not biochemically demonstrated\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrated that truncating variants in a regulatory-domain hotspot abolish mutant protein and cause more severe seizures than catalytic missense variants, refining genotype-severity relationships.\",\n      \"evidence\": \"Exome sequencing with RNA and protein expression studies in patient cells\",\n      \"pmids\": [\"33963760\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of protein loss not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified competitive NFATc4 binding between CAMTA1 and PPP3CA as a switch controlling NFATc4 dephosphorylation and chemoresistance, placing PPP3CA in a tunable transcriptional module.\",\n      \"evidence\": \"Co-IP, siRNA knockdown rescue, luciferase reporters, and xenograft model in colorectal cancer\",\n      \"pmids\": [\"35332122\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Competitive binding inferred, not structurally resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected PPP3CA to TRIM72-driven glycolytic and PI3K/Akt/mTOR signaling in breast cancer.\",\n      \"evidence\": \"Co-IP, PPP3CA siRNA, MCT4 promoter luciferase assay, and xenograft model\",\n      \"pmids\": [\"35324524\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP without reciprocal validation\", \"Direct phosphatase substrate in this axis unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Showed PPP3CA stability and activity are regulated by OR2T6 via calcium influx, with downstream suppression of AKT/mTOR and dual control of autophagy through TFEB translocation.\",\n      \"evidence\": \"Co-IP/MS, ubiquitin-proteasome and calcium influx assays, AKT/mTOR western blot, and TFEB translocation assays with tumor models\",\n      \"pmids\": [\"41214150\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether TFEB is a direct PPP3CA substrate not established\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Placed PPP3CA under transcriptional repression by MAZ, with loss activating ERK/MAPK and NFATc1/MMP9 to drive osteoclast differentiation.\",\n      \"evidence\": \"Promoter binding assay, OVX mouse model, RANKL osteoclast differentiation, and PPP3CA overexpression with phospho-ERK western blot\",\n      \"pmids\": [\"40602229\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Limited direct PPP3CA biochemistry\", \"Single lab\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Provided the structural basis for substrate recruitment, showing an acidic E282 pocket selects basophilic substrates and that the E282K disease variant inverts this selectivity.\",\n      \"evidence\": \"CNE282K structure determination, in vitro dephosphorylation, active-site mutagenesis, and cellular phosphoproteomics\",\n      \"pmids\": [\"41698888\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full repertoire of acidic substrates gained by E282K not catalogued\", \"Link to specific disease phenotype at organ level not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How calcineurin substrate selection, domain-specific dysfunction, and the diverse downstream pathways (HIF, ERK, AKT/mTOR, autophagy) integrate into the distinct disease phenotypes remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified substrate map across tissues\", \"Mechanistic basis distinguishing epileptic encephalopathy from congenital abnormalities at substrate level unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 9, 10]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 9, 10]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [2, 4, 10]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [6, 7]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"complexes\": [\"calcineurin\"],\n    \"partners\": [\"PPP3R1\", \"NFATC4\", \"CAMTA1\", \"TRIM72\", \"OR2T6\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}