{"gene":"HPCAL1","run_date":"2026-06-10T01:55:22","timeline":{"discoveries":[{"year":2022,"finding":"HPCAL1 acts as a selective autophagy receptor for CDH2 (cadherin 2) during ferroptosis. PRKCQ (protein kinase C theta) phosphorylates HPCAL1 on Thr149, and a non-classical LC3-interacting region (LIR) motif at amino acids 46-51 is required for autophagic degradation of CDH2. HPCAL1-dependent CDH2 depletion reduces membrane tension and favors lipid peroxidation, increasing susceptibility to ferroptotic death.","method":"Quantitative proteomics, site-directed mutagenesis, bioinformatic analyses, drug screening (4208 compounds), genetic/pharmacological inhibition in mouse models","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods including mutagenesis of functional sites, proteomics, in vivo mouse models, and pharmacological validation in a single rigorous study","pmids":["35403545"],"is_preprint":false},{"year":2013,"finding":"HPCAL1 physically binds wild-type PHOX2B (identified by yeast two-hybrid screen); neuroblastoma-associated PHOX2B frameshift and truncation mutants fail to bind HPCAL1 or bind only weakly. WT PHOX2B and CCHS-associated polyalanine expansion mutants induce Ca2+-independent nuclear translocation of HPCAL1, whereas neuroblastoma-associated frameshift (676delG) and truncation (K155X) mutants impair HPCAL1 subcellular localization, keeping it cytoplasmic. shRNA knockdown of HPCAL1 in PHOX2B-expressing neuroblastoma cells impaired neurite outgrowth and produced transcriptional profiles indicative of inhibited sympathetic neuronal differentiation.","method":"Yeast two-hybrid screen (>10,000 human genes), subcellular localization imaging, shRNA knockdown with transcriptional profiling and neurite outgrowth assay","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid with functional follow-up (localization, KD phenotype), multiple orthogonal methods in a single study","pmids":["23873030"],"is_preprint":false},{"year":2003,"finding":"VILIP-3/HPCAL1 undergoes a fast and reversible calcium-myristoyl switch in living cells, leading to calcium-dependent translocation to distinct subcellular compartments. GFP-tagged VILIP-3 shows different calcium-dependent translocation compared to VILIP-1 (e.g., differing behavior at Golgi membranes), and endogenously expressed VILIP-3 and VILIP-1 show different calcium-dependent dendritic localization in hippocampal neurons.","method":"Live-cell imaging with GFP-tagged proteins in cell lines and hippocampal neurons, calcium stimulation experiments","journal":"Molecular and cellular neurosciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live-cell imaging with functional translocation assay, single lab with two protein comparisons and multiple cell types","pmids":["14664824"],"is_preprint":false},{"year":2002,"finding":"VILIP-3/HPCAL1 and VILIP-1 show different calcium-dependent subcellular localization and membrane association in subcellular fractions, activate different cGMP signaling pathways, and have distinct sets of protein interaction partners, indicating cell-type-specific signaling functions.","method":"Subcellular fractionation, calcium-dependent membrane association assay, cGMP signaling assay, protein interaction partner identification","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — multiple biochemical methods in a single lab comparing two proteins; functional pathway activation measured directly","pmids":["12445467"],"is_preprint":false},{"year":2019,"finding":"HPCAL1 promotes glioblastoma cell proliferation by activating the Wnt/β-catenin pathway: HPCAL1 stimulates β-catenin accumulation and nuclear translocation, reduces Ser9 phosphorylation of GSK3β upon knockdown, and promotes ERK phosphorylation. HPCAL1-driven proliferation and transcription of CCND1 and c-Myc depends on ERK activity.","method":"Ectopic expression and shRNA knockdown in GBM cells (in vitro and in vivo xenograft), Western blotting for β-catenin, GSK3β-pSer9, ERK phosphorylation; pharmacological inhibition of ERK; β-catenin silencing rescue experiments","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO/OE with defined cellular phenotype, epistasis-like rescue experiments, multiple signaling readouts, single lab","pmids":["30843345"],"is_preprint":false},{"year":2025,"finding":"HPCAL1 inhibits TGF-β signaling in hepatic stellate cells (HSCs) by regulating ubiquitination of Smad2 through direct interactions via its EF-hand 4 domain, acting as a fibrogenesis suppressor. Macrophage-derived exosomal miR-342-3p inhibits HPCAL1 expression in HSCs, thereby activating HSCs and promoting liver fibrosis.","method":"Co-immunoprecipitation (in vivo and in vitro), Western blotting, qPCR, luciferase reporter gene assay, cellular immunofluorescence, exosome extraction and culture experiments","journal":"Human genomics","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — co-IP identifying direct EF-hand 4 domain interaction with Smad2, ubiquitination assay, multiple orthogonal methods, single lab","pmids":["39910671"],"is_preprint":false},{"year":2025,"finding":"HPCAL1 binds to the mitophagy receptor BNIP3 in a calcium-dependent manner, enhancing BNIP3 stability and its interaction with LC3-II, thereby excessively activating mitophagy. This promotes ferroptosis via a ROS burst, independent of GPX4 expression changes. Disrupting HPCAL1 or BNIP3 breaks this cycle and improves cell survival.","method":"Co-immunoprecipitation, Western blotting, qPCR, fluorescent probe-based detection, ROS/lipid peroxidation assays, mitochondrial membrane potential and autophagic flux assays in mouse I/R model and IEC-6 H/R model","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with calcium dependence, multiple orthogonal functional assays, in vivo and in vitro models, single lab","pmids":["41482082"],"is_preprint":false},{"year":2026,"finding":"HPCAL1 forms distinct protein complexes with β-catenin together with TCF7 or p65 transcription factors, differentially transactivating Wnt6, Wnt7A, and Wnt11 ligands in colorectal cancer cells. HPCAL1 augments activation and nuclear localization of β-catenin. Pharmacological inhibition of HPCAL1 by desloratadine curtails Wnt6, Wnt7A, and Wnt11 expression and suppresses Wnt/β-catenin signaling.","method":"Co-immunoprecipitation (biochemical complex identification), RNA sequencing, knockdown/overexpression in CRC cell lines (in vitro and in vivo xenografts), pharmacological inhibition with desloratadine","journal":"Oncogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP identifying complexes with β-catenin/TCF7/p65, RNA-seq pathway validation, multiple orthogonal methods, single lab","pmids":["42091587"],"is_preprint":false},{"year":1999,"finding":"VILIP-3/HPCAL1 immunoreactivity in human brain is restricted primarily to cerebellar Purkinje cells, a subpopulation of granule neurons, brain stem nuclei, and multiple subcortical neurons, with intracellular localization in perikarya, dendrites, and some axons. Glia do not express VILIP-3.","method":"Immunohistochemistry with specific polyclonal antisera on human brain tissue","journal":"Journal of neurocytology","confidence":"Medium","confidence_rationale":"Tier 3 / Strong — specific antibody-based localization across multiple brain regions, replicated across multiple cell types in human tissue","pmids":["10851344"],"is_preprint":false}],"current_model":"HPCAL1 (VILIP-3) is a neuronal calcium sensor protein with an EF-hand calcium-myristoyl switch that mediates calcium-dependent subcellular translocation; it acts as a selective autophagy receptor for CDH2 degradation during ferroptosis (requiring PRKCQ-mediated Thr149 phosphorylation and a non-classical LIR motif), promotes Wnt/β-catenin signaling by forming complexes with β-catenin/TCF7/p65, suppresses TGF-β signaling in hepatic stellate cells by regulating Smad2 ubiquitination via its EF-hand 4 domain, and activates excessive mitophagy through calcium-dependent binding to BNIP3, thereby driving ferroptosis via ROS; it also interacts with PHOX2B to regulate sympathetic neuronal differentiation."},"narrative":{"mechanistic_narrative":"HPCAL1 (VILIP-3) is a neuronal calcium sensor that uses an EF-hand calcium-myristoyl switch to undergo fast, reversible calcium-dependent translocation between subcellular compartments, distinguishing its behavior and interaction partners from the related VILIP-1 [PMID:14664824, PMID:12445467]. Beyond calcium sensing, HPCAL1 functions as a selective autophagy receptor: during ferroptosis it is phosphorylated on Thr149 by PRKCQ and, through a non-classical LIR motif (residues 46-51), targets CDH2 for autophagic degradation, lowering membrane tension and promoting lipid peroxidation [PMID:35403545]. It also binds the mitophagy receptor BNIP3 in a calcium-dependent manner, stabilizing BNIP3 and its LC3-II interaction to drive excessive mitophagy and a ROS burst that potentiates ferroptosis [PMID:41482082]. HPCAL1 promotes Wnt/β-catenin signaling by augmenting β-catenin accumulation and nuclear localization and by forming β-catenin complexes with TCF7 or p65 to transactivate Wnt ligands, supporting proliferation in glioblastoma and colorectal cancer [PMID:30843345, PMID:42091587]. Conversely, in hepatic stellate cells it suppresses TGF-β signaling by promoting Smad2 ubiquitination through its EF-hand 4 domain, acting as a fibrogenesis suppressor [PMID:39910671]. In the nervous system, HPCAL1 binds PHOX2B and is required for neurite outgrowth and sympathetic neuronal differentiation [PMID:23873030].","teleology":[{"year":1999,"claim":"Establishing where HPCAL1 protein resides was the first step in defining its biological context; immunohistochemistry showed it is a neuron-restricted protein expressed in specific brain populations.","evidence":"Immunohistochemistry with specific polyclonal antisera on human brain tissue","pmids":["10851344"],"confidence":"Medium","gaps":["Does not address molecular function","No mechanism for the cell-type-restricted expression pattern"]},{"year":2002,"claim":"To determine whether HPCAL1 is a functional calcium sensor distinct from related VILIPs, biochemical work showed it has unique calcium-dependent membrane association, distinct interaction partners, and activates a different cGMP pathway than VILIP-1.","evidence":"Subcellular fractionation, calcium-dependent membrane association and cGMP signaling assays comparing two proteins","pmids":["12445467"],"confidence":"Medium","gaps":["Interaction partners not individually identified","cGMP pathway components downstream unresolved"]},{"year":2003,"claim":"The mechanism of calcium-triggered relocalization was resolved by demonstrating a fast reversible calcium-myristoyl switch driving translocation to distinct compartments, differing from VILIP-1.","evidence":"Live-cell imaging of GFP-tagged proteins in cell lines and hippocampal neurons with calcium stimulation","pmids":["14664824"],"confidence":"Medium","gaps":["Functional consequence of translocation not defined","Target compartments and effectors at each site unknown"]},{"year":2013,"claim":"Linking HPCAL1 to a developmental program, a yeast two-hybrid screen identified PHOX2B as a partner whose disease mutants disrupt HPCAL1 localization, and knockdown impaired sympathetic neuronal differentiation.","evidence":"Yeast two-hybrid screen, subcellular localization imaging, shRNA knockdown with transcriptional profiling and neurite outgrowth assay","pmids":["23873030"],"confidence":"High","gaps":["Direct biochemical mechanism by which HPCAL1 drives differentiation not defined","Whether PHOX2B-induced nuclear translocation of HPCAL1 alters transcription directly is unresolved"]},{"year":2019,"claim":"The first oncogenic mechanism placed HPCAL1 upstream of Wnt/β-catenin and ERK signaling, showing it stabilizes β-catenin and drives proliferation in glioblastoma.","evidence":"Ectopic expression and shRNA knockdown in GBM cells in vitro and in xenografts, signaling Western blots, ERK inhibition and β-catenin rescue","pmids":["30843345"],"confidence":"Medium","gaps":["Direct molecular target of HPCAL1 in the pathway not defined","Relationship between calcium-sensing activity and signaling role unaddressed"]},{"year":2022,"claim":"HPCAL1 was redefined as a selective autophagy receptor, establishing a phosphorylation-dependent (PRKCQ/Thr149) and LIR-dependent mechanism that degrades CDH2 to sensitize cells to ferroptosis.","evidence":"Quantitative proteomics, site-directed mutagenesis of Thr149 and the LIR motif, drug screening, and genetic/pharmacological models in mice","pmids":["35403545"],"confidence":"High","gaps":["How calcium sensing relates to receptor function not defined","Generality of HPCAL1 cargo selection beyond CDH2 unknown"]},{"year":2025,"claim":"Two studies extended the mechanism: HPCAL1 suppresses TGF-β fibrogenesis via EF-hand 4-dependent Smad2 ubiquitination, and drives ferroptosis by calcium-dependent stabilization of the mitophagy receptor BNIP3.","evidence":"Co-IP, ubiquitination and luciferase assays in hepatic stellate cells (Smad2); Co-IP, ROS/lipid peroxidation, mitochondrial and autophagic flux assays in I/R and H/R models (BNIP3)","pmids":["39910671","41482082"],"confidence":"Medium","gaps":["Whether HPCAL1 directly recruits a ubiquitin ligase to Smad2 is unresolved","Structural basis of calcium-dependent BNIP3 binding undefined"]},{"year":2026,"claim":"The Wnt mechanism was refined by showing HPCAL1 forms distinct β-catenin complexes with TCF7 or p65 to differentially transactivate specific Wnt ligands in colorectal cancer.","evidence":"Reciprocal co-IP, RNA sequencing, knockdown/overexpression in CRC cells and xenografts, pharmacological inhibition with desloratadine","pmids":["42091587"],"confidence":"Medium","gaps":["Whether HPCAL1 binds β-catenin directly or via an adaptor is unresolved","How complex choice (TCF7 vs p65) is regulated unknown"]},{"year":null,"claim":"How HPCAL1's core calcium-myristoyl sensor activity mechanistically governs its diverse roles as an autophagy receptor, ubiquitination regulator, and transcriptional cofactor remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model linking calcium sensing to receptor and signaling functions","No structural data on the relevant complexes","Tissue-specific selection between opposing pro-ferroptotic and anti-fibrotic roles undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[2,3]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,6]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[4,7]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,2]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,4,7]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,3]}],"pathway":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[6]}],"complexes":[],"partners":["CDH2","PHOX2B","BNIP3","SMAD2","CTNNB1","TCF7","RELA","PRKCQ"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P37235","full_name":"Hippocalcin-like protein 1","aliases":["Calcium-binding protein BDR-1","HLP2","Visinin-like protein 3","VILIP-3"],"length_aa":193,"mass_kda":22.3,"function":"May be involved in the calcium-dependent regulation of rhodopsin phosphorylation","subcellular_location":"Membrane","url":"https://www.uniprot.org/uniprotkb/P37235/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/HPCAL1","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":[],"url":"https://opencell.sf.czbiohub.org/search/HPCAL1","total_profiled":1310},"omim":[{"mim_id":"600207","title":"HIPPOCALCIN-LIKE 1; HPCAL1","url":"https://www.omim.org/entry/600207"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"brain","ntpm":410.9}],"url":"https://www.proteinatlas.org/search/HPCAL1"},"hgnc":{"alias_symbol":["BDR1","HLP2","VILIP-3"],"prev_symbol":[]},"alphafold":{"accession":"P37235","domains":[{"cath_id":"1.10.238.10","chopping":"11-92","consensus_level":"high","plddt":92.2644,"start":11,"end":92},{"cath_id":"1.10.238.10","chopping":"97-186","consensus_level":"high","plddt":87.8704,"start":97,"end":186}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P37235","model_url":"https://alphafold.ebi.ac.uk/files/AF-P37235-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P37235-F1-predicted_aligned_error_v6.png","plddt_mean":86.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=HPCAL1","jax_strain_url":"https://www.jax.org/strain/search?query=HPCAL1"},"sequence":{"accession":"P37235","fasta_url":"https://rest.uniprot.org/uniprotkb/P37235.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P37235/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P37235"}},"corpus_meta":[{"pmid":"22109888","id":"PMC_22109888","title":"Neonatal exposure to estradiol/bisphenol A alters promoter methylation and expression of Nsbp1 and Hpcal1 genes and transcriptional programs of Dnmt3a/b and Mbd2/4 in the rat prostate gland throughout life.","date":"2011","source":"Endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/22109888","citation_count":121,"is_preprint":false},{"pmid":"35403545","id":"PMC_35403545","title":"Identification of HPCAL1 as a specific autophagy receptor involved in ferroptosis.","date":"2022","source":"Autophagy","url":"https://pubmed.ncbi.nlm.nih.gov/35403545","citation_count":107,"is_preprint":false},{"pmid":"10851344","id":"PMC_10851344","title":"Regional and cellular distribution of neural visinin-like protein immunoreactivities (VILIP-1 and VILIP-3) in human brain.","date":"1999","source":"Journal of neurocytology","url":"https://pubmed.ncbi.nlm.nih.gov/10851344","citation_count":105,"is_preprint":false},{"pmid":"22375104","id":"PMC_22375104","title":"The visinin-like proteins VILIP-1 and VILIP-3 in 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promotes glioblastoma proliferation via activation of Wnt/β-catenin signalling pathway.","date":"2019","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/30843345","citation_count":32,"is_preprint":false},{"pmid":"23873030","id":"PMC_23873030","title":"Mutations that disrupt PHOXB interaction with the neuronal calcium sensor HPCAL1 impede cellular differentiation in neuroblastoma.","date":"2013","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/23873030","citation_count":25,"is_preprint":false},{"pmid":"38510151","id":"PMC_38510151","title":"N6-methyladenosine-associated genetic variants in NECTIN2 and HPCAL1 are risk factors for abdominal aortic aneurysm.","date":"2024","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/38510151","citation_count":7,"is_preprint":false},{"pmid":"39910671","id":"PMC_39910671","title":"Liver macrophage-derived exosomal miRNA-342-3p promotes liver fibrosis by inhibiting HPCAL1 in stellate cells.","date":"2025","source":"Human genomics","url":"https://pubmed.ncbi.nlm.nih.gov/39910671","citation_count":4,"is_preprint":false},{"pmid":"41482082","id":"PMC_41482082","title":"HPCAL1-BNIP3 axis promotes mitophagy-ferroptosis feedback loop that exacerbates intestinal ischemia-reperfusion injury.","date":"2025","source":"Free radical biology & medicine","url":"https://pubmed.ncbi.nlm.nih.gov/41482082","citation_count":1,"is_preprint":false},{"pmid":"32523354","id":"PMC_32523354","title":"Responses to ALK Inhibitor Treatments in a Patient with Non-Small Cell Lung Cancer Harboring a Novel HPCAL1-ALK Fusion Variant: A Case Report.","date":"2020","source":"OncoTargets and therapy","url":"https://pubmed.ncbi.nlm.nih.gov/32523354","citation_count":1,"is_preprint":false},{"pmid":"42091587","id":"PMC_42091587","title":"HPCAL1 promotes colorectal cancer progression via TCF7/p65-mediated Wnt ligand upregulation and Wnt/β-catenin pathway 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PRKCQ (protein kinase C theta) phosphorylates HPCAL1 on Thr149, and a non-classical LC3-interacting region (LIR) motif at amino acids 46-51 is required for autophagic degradation of CDH2. HPCAL1-dependent CDH2 depletion reduces membrane tension and favors lipid peroxidation, increasing susceptibility to ferroptotic death.\",\n      \"method\": \"Quantitative proteomics, site-directed mutagenesis, bioinformatic analyses, drug screening (4208 compounds), genetic/pharmacological inhibition in mouse models\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods including mutagenesis of functional sites, proteomics, in vivo mouse models, and pharmacological validation in a single rigorous study\",\n      \"pmids\": [\"35403545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"HPCAL1 physically binds wild-type PHOX2B (identified by yeast two-hybrid screen); neuroblastoma-associated PHOX2B frameshift and truncation mutants fail to bind HPCAL1 or bind only weakly. WT PHOX2B and CCHS-associated polyalanine expansion mutants induce Ca2+-independent nuclear translocation of HPCAL1, whereas neuroblastoma-associated frameshift (676delG) and truncation (K155X) mutants impair HPCAL1 subcellular localization, keeping it cytoplasmic. shRNA knockdown of HPCAL1 in PHOX2B-expressing neuroblastoma cells impaired neurite outgrowth and produced transcriptional profiles indicative of inhibited sympathetic neuronal differentiation.\",\n      \"method\": \"Yeast two-hybrid screen (>10,000 human genes), subcellular localization imaging, shRNA knockdown with transcriptional profiling and neurite outgrowth assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid with functional follow-up (localization, KD phenotype), multiple orthogonal methods in a single study\",\n      \"pmids\": [\"23873030\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"VILIP-3/HPCAL1 undergoes a fast and reversible calcium-myristoyl switch in living cells, leading to calcium-dependent translocation to distinct subcellular compartments. GFP-tagged VILIP-3 shows different calcium-dependent translocation compared to VILIP-1 (e.g., differing behavior at Golgi membranes), and endogenously expressed VILIP-3 and VILIP-1 show different calcium-dependent dendritic localization in hippocampal neurons.\",\n      \"method\": \"Live-cell imaging with GFP-tagged proteins in cell lines and hippocampal neurons, calcium stimulation experiments\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live-cell imaging with functional translocation assay, single lab with two protein comparisons and multiple cell types\",\n      \"pmids\": [\"14664824\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"VILIP-3/HPCAL1 and VILIP-1 show different calcium-dependent subcellular localization and membrane association in subcellular fractions, activate different cGMP signaling pathways, and have distinct sets of protein interaction partners, indicating cell-type-specific signaling functions.\",\n      \"method\": \"Subcellular fractionation, calcium-dependent membrane association assay, cGMP signaling assay, protein interaction partner identification\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — multiple biochemical methods in a single lab comparing two proteins; functional pathway activation measured directly\",\n      \"pmids\": [\"12445467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HPCAL1 promotes glioblastoma cell proliferation by activating the Wnt/β-catenin pathway: HPCAL1 stimulates β-catenin accumulation and nuclear translocation, reduces Ser9 phosphorylation of GSK3β upon knockdown, and promotes ERK phosphorylation. HPCAL1-driven proliferation and transcription of CCND1 and c-Myc depends on ERK activity.\",\n      \"method\": \"Ectopic expression and shRNA knockdown in GBM cells (in vitro and in vivo xenograft), Western blotting for β-catenin, GSK3β-pSer9, ERK phosphorylation; pharmacological inhibition of ERK; β-catenin silencing rescue experiments\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO/OE with defined cellular phenotype, epistasis-like rescue experiments, multiple signaling readouts, single lab\",\n      \"pmids\": [\"30843345\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HPCAL1 inhibits TGF-β signaling in hepatic stellate cells (HSCs) by regulating ubiquitination of Smad2 through direct interactions via its EF-hand 4 domain, acting as a fibrogenesis suppressor. Macrophage-derived exosomal miR-342-3p inhibits HPCAL1 expression in HSCs, thereby activating HSCs and promoting liver fibrosis.\",\n      \"method\": \"Co-immunoprecipitation (in vivo and in vitro), Western blotting, qPCR, luciferase reporter gene assay, cellular immunofluorescence, exosome extraction and culture experiments\",\n      \"journal\": \"Human genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — co-IP identifying direct EF-hand 4 domain interaction with Smad2, ubiquitination assay, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"39910671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HPCAL1 binds to the mitophagy receptor BNIP3 in a calcium-dependent manner, enhancing BNIP3 stability and its interaction with LC3-II, thereby excessively activating mitophagy. This promotes ferroptosis via a ROS burst, independent of GPX4 expression changes. Disrupting HPCAL1 or BNIP3 breaks this cycle and improves cell survival.\",\n      \"method\": \"Co-immunoprecipitation, Western blotting, qPCR, fluorescent probe-based detection, ROS/lipid peroxidation assays, mitochondrial membrane potential and autophagic flux assays in mouse I/R model and IEC-6 H/R model\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with calcium dependence, multiple orthogonal functional assays, in vivo and in vitro models, single lab\",\n      \"pmids\": [\"41482082\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"HPCAL1 forms distinct protein complexes with β-catenin together with TCF7 or p65 transcription factors, differentially transactivating Wnt6, Wnt7A, and Wnt11 ligands in colorectal cancer cells. HPCAL1 augments activation and nuclear localization of β-catenin. Pharmacological inhibition of HPCAL1 by desloratadine curtails Wnt6, Wnt7A, and Wnt11 expression and suppresses Wnt/β-catenin signaling.\",\n      \"method\": \"Co-immunoprecipitation (biochemical complex identification), RNA sequencing, knockdown/overexpression in CRC cell lines (in vitro and in vivo xenografts), pharmacological inhibition with desloratadine\",\n      \"journal\": \"Oncogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP identifying complexes with β-catenin/TCF7/p65, RNA-seq pathway validation, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"42091587\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"VILIP-3/HPCAL1 immunoreactivity in human brain is restricted primarily to cerebellar Purkinje cells, a subpopulation of granule neurons, brain stem nuclei, and multiple subcortical neurons, with intracellular localization in perikarya, dendrites, and some axons. Glia do not express VILIP-3.\",\n      \"method\": \"Immunohistochemistry with specific polyclonal antisera on human brain tissue\",\n      \"journal\": \"Journal of neurocytology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Strong — specific antibody-based localization across multiple brain regions, replicated across multiple cell types in human tissue\",\n      \"pmids\": [\"10851344\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HPCAL1 (VILIP-3) is a neuronal calcium sensor protein with an EF-hand calcium-myristoyl switch that mediates calcium-dependent subcellular translocation; it acts as a selective autophagy receptor for CDH2 degradation during ferroptosis (requiring PRKCQ-mediated Thr149 phosphorylation and a non-classical LIR motif), promotes Wnt/β-catenin signaling by forming complexes with β-catenin/TCF7/p65, suppresses TGF-β signaling in hepatic stellate cells by regulating Smad2 ubiquitination via its EF-hand 4 domain, and activates excessive mitophagy through calcium-dependent binding to BNIP3, thereby driving ferroptosis via ROS; it also interacts with PHOX2B to regulate sympathetic neuronal differentiation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"HPCAL1 (VILIP-3) is a neuronal calcium sensor that uses an EF-hand calcium-myristoyl switch to undergo fast, reversible calcium-dependent translocation between subcellular compartments, distinguishing its behavior and interaction partners from the related VILIP-1 [#2, #3]. Beyond calcium sensing, HPCAL1 functions as a selective autophagy receptor: during ferroptosis it is phosphorylated on Thr149 by PRKCQ and, through a non-classical LIR motif (residues 46-51), targets CDH2 for autophagic degradation, lowering membrane tension and promoting lipid peroxidation [#0]. It also binds the mitophagy receptor BNIP3 in a calcium-dependent manner, stabilizing BNIP3 and its LC3-II interaction to drive excessive mitophagy and a ROS burst that potentiates ferroptosis [#6]. HPCAL1 promotes Wnt/\\u03b2-catenin signaling by augmenting \\u03b2-catenin accumulation and nuclear localization and by forming \\u03b2-catenin complexes with TCF7 or p65 to transactivate Wnt ligands, supporting proliferation in glioblastoma and colorectal cancer [#4, #7]. Conversely, in hepatic stellate cells it suppresses TGF-\\u03b2 signaling by promoting Smad2 ubiquitination through its EF-hand 4 domain, acting as a fibrogenesis suppressor [#5]. In the nervous system, HPCAL1 binds PHOX2B and is required for neurite outgrowth and sympathetic neuronal differentiation [#1].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Establishing where HPCAL1 protein resides was the first step in defining its biological context; immunohistochemistry showed it is a neuron-restricted protein expressed in specific brain populations.\",\n      \"evidence\": \"Immunohistochemistry with specific polyclonal antisera on human brain tissue\",\n      \"pmids\": [\"10851344\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not address molecular function\", \"No mechanism for the cell-type-restricted expression pattern\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"To determine whether HPCAL1 is a functional calcium sensor distinct from related VILIPs, biochemical work showed it has unique calcium-dependent membrane association, distinct interaction partners, and activates a different cGMP pathway than VILIP-1.\",\n      \"evidence\": \"Subcellular fractionation, calcium-dependent membrane association and cGMP signaling assays comparing two proteins\",\n      \"pmids\": [\"12445467\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Interaction partners not individually identified\", \"cGMP pathway components downstream unresolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"The mechanism of calcium-triggered relocalization was resolved by demonstrating a fast reversible calcium-myristoyl switch driving translocation to distinct compartments, differing from VILIP-1.\",\n      \"evidence\": \"Live-cell imaging of GFP-tagged proteins in cell lines and hippocampal neurons with calcium stimulation\",\n      \"pmids\": [\"14664824\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of translocation not defined\", \"Target compartments and effectors at each site unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Linking HPCAL1 to a developmental program, a yeast two-hybrid screen identified PHOX2B as a partner whose disease mutants disrupt HPCAL1 localization, and knockdown impaired sympathetic neuronal differentiation.\",\n      \"evidence\": \"Yeast two-hybrid screen, subcellular localization imaging, shRNA knockdown with transcriptional profiling and neurite outgrowth assay\",\n      \"pmids\": [\"23873030\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct biochemical mechanism by which HPCAL1 drives differentiation not defined\", \"Whether PHOX2B-induced nuclear translocation of HPCAL1 alters transcription directly is unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The first oncogenic mechanism placed HPCAL1 upstream of Wnt/\\u03b2-catenin and ERK signaling, showing it stabilizes \\u03b2-catenin and drives proliferation in glioblastoma.\",\n      \"evidence\": \"Ectopic expression and shRNA knockdown in GBM cells in vitro and in xenografts, signaling Western blots, ERK inhibition and \\u03b2-catenin rescue\",\n      \"pmids\": [\"30843345\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular target of HPCAL1 in the pathway not defined\", \"Relationship between calcium-sensing activity and signaling role unaddressed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"HPCAL1 was redefined as a selective autophagy receptor, establishing a phosphorylation-dependent (PRKCQ/Thr149) and LIR-dependent mechanism that degrades CDH2 to sensitize cells to ferroptosis.\",\n      \"evidence\": \"Quantitative proteomics, site-directed mutagenesis of Thr149 and the LIR motif, drug screening, and genetic/pharmacological models in mice\",\n      \"pmids\": [\"35403545\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How calcium sensing relates to receptor function not defined\", \"Generality of HPCAL1 cargo selection beyond CDH2 unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Two studies extended the mechanism: HPCAL1 suppresses TGF-\\u03b2 fibrogenesis via EF-hand 4-dependent Smad2 ubiquitination, and drives ferroptosis by calcium-dependent stabilization of the mitophagy receptor BNIP3.\",\n      \"evidence\": \"Co-IP, ubiquitination and luciferase assays in hepatic stellate cells (Smad2); Co-IP, ROS/lipid peroxidation, mitochondrial and autophagic flux assays in I/R and H/R models (BNIP3)\",\n      \"pmids\": [\"39910671\", \"41482082\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether HPCAL1 directly recruits a ubiquitin ligase to Smad2 is unresolved\", \"Structural basis of calcium-dependent BNIP3 binding undefined\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"The Wnt mechanism was refined by showing HPCAL1 forms distinct \\u03b2-catenin complexes with TCF7 or p65 to differentially transactivate specific Wnt ligands in colorectal cancer.\",\n      \"evidence\": \"Reciprocal co-IP, RNA sequencing, knockdown/overexpression in CRC cells and xenografts, pharmacological inhibition with desloratadine\",\n      \"pmids\": [\"42091587\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether HPCAL1 binds \\u03b2-catenin directly or via an adaptor is unresolved\", \"How complex choice (TCF7 vs p65) is regulated unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How HPCAL1's core calcium-myristoyl sensor activity mechanistically governs its diverse roles as an autophagy receptor, ubiquitination regulator, and transcriptional cofactor remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model linking calcium sensing to receptor and signaling functions\", \"No structural data on the relevant complexes\", \"Tissue-specific selection between opposing pro-ferroptotic and anti-fibrotic roles undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [4, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 4, 7]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CDH2\", \"PHOX2B\", \"BNIP3\", \"SMAD2\", \"CTNNB1\", \"TCF7\", \"RELA\", \"PRKCQ\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}