{"gene":"HPCAL1","run_date":"2026-04-28T18:06:53","timeline":{"discoveries":[{"year":2022,"finding":"HPCAL1 acts as a selective autophagy receptor for CDH2 (cadherin 2), mediating its lysosomal degradation during ferroptosis; PRKCQ (protein kinase C theta)-mediated phosphorylation of HPCAL1 on Thr149 and a non-classical LC3-interacting region (LIR) motif at amino acids 46–51 are required for autophagic CDH2 degradation; HPCAL1-dependent CDH2 depletion reduces membrane tension and promotes lipid peroxidation, driving ferroptotic cell death.","method":"Quantitative proteomics, site-directed mutagenesis, bioinformatic LIR motif analysis, co-immunoprecipitation, in vitro kinase assays, genetic knockdown/overexpression with ferroptosis phenotypic readouts, mouse models of pancreatitis and tumor suppression, drug screening (4208 compounds)","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods (proteomics, mutagenesis, MS, KD/KO, mouse models) in a single rigorous study","pmids":["35403545"],"is_preprint":false},{"year":2013,"finding":"HPCAL1 (VILIP-3) physically interacts with the transcription factor PHOX2B via yeast two-hybrid and co-immunoprecipitation; wild-type PHOX2B and CCHS-associated polyalanine expansion mutants induce Ca2+-independent nuclear translocation of HPCAL1, whereas neuroblastoma-associated frameshift/truncation PHOX2B mutants impair this translocation, keeping HPCAL1 in the cytoplasm; shRNA knockdown of HPCAL1 in neuroblastoma cells expressing PHOX2B impairs neurite outgrowth and inhibits sympathetic neuronal differentiation.","method":"Large-scale yeast two-hybrid screen, co-immunoprecipitation, subcellular localization imaging, shRNA knockdown with neurite outgrowth and transcriptional profiling readouts","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 — reciprocal interaction validated by yeast two-hybrid + Co-IP, functional consequence confirmed by KD with defined cellular phenotype","pmids":["23873030"],"is_preprint":false},{"year":2019,"finding":"HPCAL1 promotes glioblastoma cell proliferation by activating the Wnt/β-catenin signalling pathway: HPCAL1 overexpression stimulates β-catenin nuclear accumulation and reduces GSK3β Ser9 phosphorylation, while HPCAL1 knockdown decreases ERK phosphorylation; ERK activity is required downstream of HPCAL1 to drive CCND1 and c-Myc transcription.","method":"Ectopic overexpression and shRNA knockdown in GBM cell lines and xenograft models, Western blotting for GSK3β phosphorylation, β-catenin localization, ERK phosphorylation, cell proliferation assays","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2–3 — KD/OE with defined pathway readouts (β-catenin, GSK3β, ERK) and in vivo xenograft, single lab study","pmids":["30843345"],"is_preprint":false},{"year":2025,"finding":"HPCAL1 inhibits TGF-β signalling in hepatic stellate cells by directly interacting (via its EF-hand 4 domain) with Smad2 and regulating its ubiquitination; exosomal miR-342-3p from liver macrophages suppresses HPCAL1 expression in stellate cells, thereby activating HSCs and promoting liver fibrosis.","method":"Co-immunoprecipitation, Western blotting, qPCR, luciferase reporter gene assay, cellular immunofluorescence, in vivo and in vitro fibrosis models, miRNA target validation","journal":"Human genomics","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP with domain mapping plus functional in vivo/in vitro validation, single lab study","pmids":["39910671"],"is_preprint":false},{"year":2025,"finding":"HPCAL1 binds the mitophagy receptor BNIP3 in a calcium-dependent manner, stabilising BNIP3 and enhancing its interaction with LC3-II, thereby excessively activating mitophagy; this mitophagy activation drives a ROS burst that promotes ferroptosis, creating a mitophagy–ferroptosis feedback loop exacerbating intestinal ischemia-reperfusion injury.","method":"Co-immunoprecipitation, Western blotting, fluorescent probe-based ROS/lipid peroxidation detection, mitochondrial membrane potential assays, autophagic flux assays, mouse I/R and rat IEC-6 H/R models, genetic disruption of HPCAL1 or BNIP3","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP with functional genetic disruption in two in vivo/in vitro models, single lab study","pmids":["41482082"],"is_preprint":false},{"year":2002,"finding":"VILIP-3/HPCAL1 and VILIP-1 show different calcium-dependent subcellular localisations in intact cells and subcellular fractions, activate different cGMP signalling pathways, and bind distinct protein interaction partners, demonstrating cell-type-specific signalling functions.","method":"Subcellular fractionation with calcium titration, signalling pathway assays (cGMP), co-immunoprecipitation/pull-down for interaction partners","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2–3 — multiple methods (fractionation, signalling assays, interaction screens) in one study, but limited mechanistic depth for HPCAL1 specifically","pmids":["12445467"],"is_preprint":false},{"year":2003,"finding":"VILIP-3/HPCAL1 undergoes a fast and reversible calcium-myristoyl switch in living cells, with calcium-dependent translocation to distinct subcellular compartments (including Golgi membranes) that differs from VILIP-1, as shown in GFP-tagged constructs in cell lines and hippocampal neurons.","method":"GFP-tagged protein live imaging in cell lines and primary hippocampal neurons; endogenous localization in dendrites; calcium-dependent translocation assays","journal":"Molecular and cellular neurosciences","confidence":"Medium","confidence_rationale":"Tier 2 — direct live-cell imaging with GFP constructs and endogenous protein, functional localization comparison, single lab","pmids":["14664824"],"is_preprint":false}],"current_model":"HPCAL1 is a neuronal calcium sensor (NCS/EF-hand) protein that undergoes a calcium-myristoyl switch to translocate to specific membrane compartments; it acts as a selective autophagy receptor mediating PRKCQ-phosphorylation-dependent, LIR motif-driven autophagic degradation of CDH2 to promote ferroptosis, and also binds BNIP3 in a calcium-dependent manner to over-activate mitophagy and amplify ferroptosis; it interacts with PHOX2B to regulate nuclear localisation and sympathetic neuronal differentiation, inhibits TGF-β/Smad2 signalling in hepatic stellate cells via EF-hand 4 domain-mediated ubiquitination control, and activates Wnt/β-catenin–ERK signalling to drive glioblastoma proliferation."},"narrative":{"teleology":[{"year":2002,"claim":"Establishing that HPCAL1 has calcium-dependent signaling properties distinct from its paralog VILIP-1 answered whether NCS family members are functionally redundant, showing they activate different cGMP pathways and bind different partners.","evidence":"Subcellular fractionation with calcium titration, cGMP signaling assays, and pull-down interaction screens in cell lines","pmids":["12445467"],"confidence":"Medium","gaps":["Specific HPCAL1 interaction partners not identified","Downstream effectors of HPCAL1-specific cGMP signaling unknown","In vivo relevance not tested"]},{"year":2003,"claim":"Demonstrating that HPCAL1 undergoes a fast, reversible calcium-myristoyl switch with translocation to Golgi and other compartments resolved how HPCAL1 achieves membrane targeting specificity distinct from VILIP-1.","evidence":"GFP-tagged HPCAL1 live-cell imaging in cell lines and primary hippocampal neurons with calcium-dependent translocation assays","pmids":["14664824"],"confidence":"Medium","gaps":["Membrane receptor or tethering factor at the Golgi not identified","Whether myristoylation is required for all downstream signaling functions untested","Endogenous dynamics in non-neuronal cells uncharacterized"]},{"year":2013,"claim":"Identifying HPCAL1 as a physical interactor of PHOX2B that undergoes calcium-independent nuclear translocation upon PHOX2B binding, and showing that HPCAL1 knockdown impairs neurite outgrowth, established a direct role for HPCAL1 in sympathetic neuronal differentiation.","evidence":"Yeast two-hybrid screen, reciprocal co-immunoprecipitation, subcellular localization imaging, shRNA knockdown with neurite outgrowth readouts in neuroblastoma cells","pmids":["23873030"],"confidence":"High","gaps":["Transcriptional targets jointly regulated by HPCAL1-PHOX2B unknown","Whether HPCAL1 modulates PHOX2B DNA-binding activity untested","In vivo neuronal differentiation role not confirmed in animal models"]},{"year":2019,"claim":"Showing that HPCAL1 activates Wnt/β-catenin signaling through GSK3β modulation and requires downstream ERK activity to drive CCND1 and c-Myc transcription revealed a proliferative signaling axis in glioblastoma.","evidence":"Overexpression and shRNA knockdown in GBM cell lines and xenograft models with Western blotting for β-catenin, GSK3β, and ERK phosphorylation","pmids":["30843345"],"confidence":"Medium","gaps":["Direct molecular target linking HPCAL1 to GSK3β or β-catenin not identified","Whether calcium-myristoyl switch is required for Wnt pathway activation unknown","Single-lab finding not independently replicated"]},{"year":2022,"claim":"Identifying HPCAL1 as a selective autophagy receptor that delivers CDH2 for lysosomal degradation via PRKCQ phosphorylation and a non-classical LIR motif resolved the molecular mechanism coupling autophagy to membrane tension reduction during ferroptosis.","evidence":"Quantitative proteomics, site-directed mutagenesis of Thr149 and LIR motif, in vitro kinase assays, co-immunoprecipitation, genetic knockdown/knockout in cell lines and mouse pancreatitis/tumor models, drug screen of 4208 compounds","pmids":["35403545"],"confidence":"High","gaps":["Structural basis of non-classical LIR–LC3 interaction unresolved","How HPCAL1 selectively recognizes CDH2 cargo remains unknown","Whether HPCAL1 mediates autophagy of additional cargo beyond CDH2 untested"]},{"year":2025,"claim":"Demonstrating that HPCAL1 inhibits TGF-β signaling by binding Smad2 through its EF-hand 4 domain and controlling Smad2 ubiquitination established HPCAL1 as a negative regulator of hepatic stellate cell activation and liver fibrosis.","evidence":"Co-immunoprecipitation with domain mapping, luciferase reporter assays, immunofluorescence, in vivo and in vitro fibrosis models, miR-342-3p target validation","pmids":["39910671"],"confidence":"Medium","gaps":["E3 ubiquitin ligase recruited by HPCAL1 to ubiquitinate Smad2 not identified","Whether calcium binding by EF-hand 4 is required for Smad2 interaction untested","Single-lab observation awaiting independent replication"]},{"year":2025,"claim":"Showing that HPCAL1 binds and stabilizes the mitophagy receptor BNIP3 in a calcium-dependent manner, enhancing LC3-II interaction and driving excessive mitophagy-ROS-ferroptosis, established a second autophagy-related mechanism by which HPCAL1 promotes ferroptotic cell death.","evidence":"Co-immunoprecipitation, ROS and lipid peroxidation fluorescent probes, mitochondrial membrane potential and autophagic flux assays, genetic disruption in mouse I/R and rat IEC-6 H/R models","pmids":["41482082"],"confidence":"Medium","gaps":["Whether the HPCAL1-BNIP3 interaction requires myristoylation or specific EF-hand domains not determined","Relationship between CDH2-targeted autophagy and BNIP3-mediated mitophagy in the same cell not clarified","Single-lab finding in ischemia-reperfusion context, generalizability unknown"]},{"year":null,"claim":"A unified structural and regulatory model explaining how HPCAL1 selectively engages diverse cargo (CDH2, BNIP3, Smad2, PHOX2B) and switches between autophagy receptor, signaling modulator, and nuclear functions remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No crystal or cryo-EM structure of HPCAL1 in complex with any partner","How calcium-myristoyl switching versus calcium-independent nuclear translocation are regulated in the same cell is unknown","Whether HPCAL1 autophagy receptor and signaling functions are coordinated or context-exclusive is unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,4]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,3]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[6]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[5,6]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[0,4]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[0,4]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,3]}],"complexes":[],"partners":["CDH2","PHOX2B","BNIP3","SMAD2","PRKCQ","LC3"],"other_free_text":[]},"mechanistic_narrative":"HPCAL1 (also known as VILIP-3) is a neuronal calcium sensor protein that employs a calcium-myristoyl switch for reversible, calcium-dependent translocation to specific membrane compartments including the Golgi, enabling cell-type-specific signaling [PMID:14664824, PMID:12445467]. HPCAL1 functions as a selective autophagy receptor that, upon PRKCQ-mediated phosphorylation at Thr149, engages LC3 via a non-classical LIR motif to target CDH2 for lysosomal degradation, reducing membrane tension and promoting lipid peroxidation during ferroptosis [PMID:35403545]. HPCAL1 also binds the mitophagy receptor BNIP3 in a calcium-dependent manner, stabilizing BNIP3 and amplifying mitophagy-driven ROS production to further potentiate ferroptosis [PMID:41482082]. Beyond autophagy, HPCAL1 interacts with PHOX2B to regulate its nuclear translocation and promote sympathetic neuronal differentiation [PMID:23873030], inhibits TGF-β/Smad2 signaling by controlling Smad2 ubiquitination via its EF-hand 4 domain [PMID:39910671], and activates Wnt/β-catenin–ERK signaling to drive glioblastoma proliferation [PMID:30843345]."},"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":"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":"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":103,"is_preprint":false},{"pmid":"22375104","id":"PMC_22375104","title":"The visinin-like proteins VILIP-1 and VILIP-3 in Alzheimer's disease-old wine in new bottles.","date":"2012","source":"Frontiers in molecular neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/22375104","citation_count":58,"is_preprint":false},{"pmid":"12445467","id":"PMC_12445467","title":"Evidence for different functional properties of the neuronal calcium sensor proteins VILIP-1 and VILIP-3: from subcellular localization to cellular function.","date":"2002","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/12445467","citation_count":39,"is_preprint":false},{"pmid":"14664824","id":"PMC_14664824","title":"Calcium-myristoyl switch, subcellular localization, and calcium-dependent translocation of the neuronal calcium sensor protein VILIP-3, and comparison with VILIP-1 in hippocampal neurons.","date":"2003","source":"Molecular and cellular neurosciences","url":"https://pubmed.ncbi.nlm.nih.gov/14664824","citation_count":36,"is_preprint":false},{"pmid":"30843345","id":"PMC_30843345","title":"HPCAL1 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":"38066636","id":"PMC_38066636","title":"Phosphoregulation in the N-terminus of NRT2.1 affects nitrate uptake by controlling the interaction of NRT2.1 with NAR2.1 and kinase HPCAL1 in Arabidopsis.","date":"2024","source":"Journal of experimental botany","url":"https://pubmed.ncbi.nlm.nih.gov/38066636","citation_count":9,"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":null,"id":"bio_10.1101_2025.09.17.676758","title":"Proteomic and Kinetic Characterization of Prion Seeding in Distinct Human CJD Strains Unveils Early Diagnostic Biomarkers","date":"2025-09-17","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.17.676758","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.01.15.25320619","title":"Comprehensive Analysis of Metabolic Reprogramming-Associated Key Genes and Immune Microenvironment in Heart Failure with Preserved Ejection Fraction","date":"2025-01-16","source":"bioRxiv","url":"https://doi.org/10.1101/2025.01.15.25320619","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10302,"output_tokens":2165,"usd":0.031691},"stage2":{"model":"claude-opus-4-6","input_tokens":5478,"output_tokens":2511,"usd":0.135247},"total_usd":0.166938,"stage1_batch_id":"msgbatch_01DzrsgVgYngY1hvM3DU1yF2","stage2_batch_id":"msgbatch_01KA9b7iRzKFdhoBUT7vCvSe","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2022,\n      \"finding\": \"HPCAL1 acts as a selective autophagy receptor for CDH2 (cadherin 2), mediating its lysosomal degradation during ferroptosis; PRKCQ (protein kinase C theta)-mediated phosphorylation of HPCAL1 on Thr149 and a non-classical LC3-interacting region (LIR) motif at amino acids 46–51 are required for autophagic CDH2 degradation; HPCAL1-dependent CDH2 depletion reduces membrane tension and promotes lipid peroxidation, driving ferroptotic cell death.\",\n      \"method\": \"Quantitative proteomics, site-directed mutagenesis, bioinformatic LIR motif analysis, co-immunoprecipitation, in vitro kinase assays, genetic knockdown/overexpression with ferroptosis phenotypic readouts, mouse models of pancreatitis and tumor suppression, drug screening (4208 compounds)\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods (proteomics, mutagenesis, MS, KD/KO, mouse models) in a single rigorous study\",\n      \"pmids\": [\"35403545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"HPCAL1 (VILIP-3) physically interacts with the transcription factor PHOX2B via yeast two-hybrid and co-immunoprecipitation; wild-type PHOX2B and CCHS-associated polyalanine expansion mutants induce Ca2+-independent nuclear translocation of HPCAL1, whereas neuroblastoma-associated frameshift/truncation PHOX2B mutants impair this translocation, keeping HPCAL1 in the cytoplasm; shRNA knockdown of HPCAL1 in neuroblastoma cells expressing PHOX2B impairs neurite outgrowth and inhibits sympathetic neuronal differentiation.\",\n      \"method\": \"Large-scale yeast two-hybrid screen, co-immunoprecipitation, subcellular localization imaging, shRNA knockdown with neurite outgrowth and transcriptional profiling readouts\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal interaction validated by yeast two-hybrid + Co-IP, functional consequence confirmed by KD with defined cellular phenotype\",\n      \"pmids\": [\"23873030\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HPCAL1 promotes glioblastoma cell proliferation by activating the Wnt/β-catenin signalling pathway: HPCAL1 overexpression stimulates β-catenin nuclear accumulation and reduces GSK3β Ser9 phosphorylation, while HPCAL1 knockdown decreases ERK phosphorylation; ERK activity is required downstream of HPCAL1 to drive CCND1 and c-Myc transcription.\",\n      \"method\": \"Ectopic overexpression and shRNA knockdown in GBM cell lines and xenograft models, Western blotting for GSK3β phosphorylation, β-catenin localization, ERK phosphorylation, cell proliferation assays\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — KD/OE with defined pathway readouts (β-catenin, GSK3β, ERK) and in vivo xenograft, single lab study\",\n      \"pmids\": [\"30843345\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HPCAL1 inhibits TGF-β signalling in hepatic stellate cells by directly interacting (via its EF-hand 4 domain) with Smad2 and regulating its ubiquitination; exosomal miR-342-3p from liver macrophages suppresses HPCAL1 expression in stellate cells, thereby activating HSCs and promoting liver fibrosis.\",\n      \"method\": \"Co-immunoprecipitation, Western blotting, qPCR, luciferase reporter gene assay, cellular immunofluorescence, in vivo and in vitro fibrosis models, miRNA target validation\",\n      \"journal\": \"Human genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP with domain mapping plus functional in vivo/in vitro validation, single lab study\",\n      \"pmids\": [\"39910671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HPCAL1 binds the mitophagy receptor BNIP3 in a calcium-dependent manner, stabilising BNIP3 and enhancing its interaction with LC3-II, thereby excessively activating mitophagy; this mitophagy activation drives a ROS burst that promotes ferroptosis, creating a mitophagy–ferroptosis feedback loop exacerbating intestinal ischemia-reperfusion injury.\",\n      \"method\": \"Co-immunoprecipitation, Western blotting, fluorescent probe-based ROS/lipid peroxidation detection, mitochondrial membrane potential assays, autophagic flux assays, mouse I/R and rat IEC-6 H/R models, genetic disruption of HPCAL1 or BNIP3\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with functional genetic disruption in two in vivo/in vitro models, single lab study\",\n      \"pmids\": [\"41482082\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"VILIP-3/HPCAL1 and VILIP-1 show different calcium-dependent subcellular localisations in intact cells and subcellular fractions, activate different cGMP signalling pathways, and bind distinct protein interaction partners, demonstrating cell-type-specific signalling functions.\",\n      \"method\": \"Subcellular fractionation with calcium titration, signalling pathway assays (cGMP), co-immunoprecipitation/pull-down for interaction partners\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — multiple methods (fractionation, signalling assays, interaction screens) in one study, but limited mechanistic depth for HPCAL1 specifically\",\n      \"pmids\": [\"12445467\"],\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, with calcium-dependent translocation to distinct subcellular compartments (including Golgi membranes) that differs from VILIP-1, as shown in GFP-tagged constructs in cell lines and hippocampal neurons.\",\n      \"method\": \"GFP-tagged protein live imaging in cell lines and primary hippocampal neurons; endogenous localization in dendrites; calcium-dependent translocation assays\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct live-cell imaging with GFP constructs and endogenous protein, functional localization comparison, single lab\",\n      \"pmids\": [\"14664824\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HPCAL1 is a neuronal calcium sensor (NCS/EF-hand) protein that undergoes a calcium-myristoyl switch to translocate to specific membrane compartments; it acts as a selective autophagy receptor mediating PRKCQ-phosphorylation-dependent, LIR motif-driven autophagic degradation of CDH2 to promote ferroptosis, and also binds BNIP3 in a calcium-dependent manner to over-activate mitophagy and amplify ferroptosis; it interacts with PHOX2B to regulate nuclear localisation and sympathetic neuronal differentiation, inhibits TGF-β/Smad2 signalling in hepatic stellate cells via EF-hand 4 domain-mediated ubiquitination control, and activates Wnt/β-catenin–ERK signalling to drive glioblastoma proliferation.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"HPCAL1 (also known as VILIP-3) is a neuronal calcium sensor protein that employs a calcium-myristoyl switch for reversible, calcium-dependent translocation to specific membrane compartments including the Golgi, enabling cell-type-specific signaling [PMID:14664824, PMID:12445467]. HPCAL1 functions as a selective autophagy receptor that, upon PRKCQ-mediated phosphorylation at Thr149, engages LC3 via a non-classical LIR motif to target CDH2 for lysosomal degradation, reducing membrane tension and promoting lipid peroxidation during ferroptosis [PMID:35403545]. HPCAL1 also binds the mitophagy receptor BNIP3 in a calcium-dependent manner, stabilizing BNIP3 and amplifying mitophagy-driven ROS production to further potentiate ferroptosis [PMID:41482082]. Beyond autophagy, HPCAL1 interacts with PHOX2B to regulate its nuclear translocation and promote sympathetic neuronal differentiation [PMID:23873030], inhibits TGF-β/Smad2 signaling by controlling Smad2 ubiquitination via its EF-hand 4 domain [PMID:39910671], and activates Wnt/β-catenin–ERK signaling to drive glioblastoma proliferation [PMID:30843345].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Establishing that HPCAL1 has calcium-dependent signaling properties distinct from its paralog VILIP-1 answered whether NCS family members are functionally redundant, showing they activate different cGMP pathways and bind different partners.\",\n      \"evidence\": \"Subcellular fractionation with calcium titration, cGMP signaling assays, and pull-down interaction screens in cell lines\",\n      \"pmids\": [\"12445467\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific HPCAL1 interaction partners not identified\", \"Downstream effectors of HPCAL1-specific cGMP signaling unknown\", \"In vivo relevance not tested\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Demonstrating that HPCAL1 undergoes a fast, reversible calcium-myristoyl switch with translocation to Golgi and other compartments resolved how HPCAL1 achieves membrane targeting specificity distinct from VILIP-1.\",\n      \"evidence\": \"GFP-tagged HPCAL1 live-cell imaging in cell lines and primary hippocampal neurons with calcium-dependent translocation assays\",\n      \"pmids\": [\"14664824\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Membrane receptor or tethering factor at the Golgi not identified\", \"Whether myristoylation is required for all downstream signaling functions untested\", \"Endogenous dynamics in non-neuronal cells uncharacterized\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identifying HPCAL1 as a physical interactor of PHOX2B that undergoes calcium-independent nuclear translocation upon PHOX2B binding, and showing that HPCAL1 knockdown impairs neurite outgrowth, established a direct role for HPCAL1 in sympathetic neuronal differentiation.\",\n      \"evidence\": \"Yeast two-hybrid screen, reciprocal co-immunoprecipitation, subcellular localization imaging, shRNA knockdown with neurite outgrowth readouts in neuroblastoma cells\",\n      \"pmids\": [\"23873030\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Transcriptional targets jointly regulated by HPCAL1-PHOX2B unknown\", \"Whether HPCAL1 modulates PHOX2B DNA-binding activity untested\", \"In vivo neuronal differentiation role not confirmed in animal models\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showing that HPCAL1 activates Wnt/β-catenin signaling through GSK3β modulation and requires downstream ERK activity to drive CCND1 and c-Myc transcription revealed a proliferative signaling axis in glioblastoma.\",\n      \"evidence\": \"Overexpression and shRNA knockdown in GBM cell lines and xenograft models with Western blotting for β-catenin, GSK3β, and ERK phosphorylation\",\n      \"pmids\": [\"30843345\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular target linking HPCAL1 to GSK3β or β-catenin not identified\", \"Whether calcium-myristoyl switch is required for Wnt pathway activation unknown\", \"Single-lab finding not independently replicated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identifying HPCAL1 as a selective autophagy receptor that delivers CDH2 for lysosomal degradation via PRKCQ phosphorylation and a non-classical LIR motif resolved the molecular mechanism coupling autophagy to membrane tension reduction during ferroptosis.\",\n      \"evidence\": \"Quantitative proteomics, site-directed mutagenesis of Thr149 and LIR motif, in vitro kinase assays, co-immunoprecipitation, genetic knockdown/knockout in cell lines and mouse pancreatitis/tumor models, drug screen of 4208 compounds\",\n      \"pmids\": [\"35403545\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of non-classical LIR–LC3 interaction unresolved\", \"How HPCAL1 selectively recognizes CDH2 cargo remains unknown\", \"Whether HPCAL1 mediates autophagy of additional cargo beyond CDH2 untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrating that HPCAL1 inhibits TGF-β signaling by binding Smad2 through its EF-hand 4 domain and controlling Smad2 ubiquitination established HPCAL1 as a negative regulator of hepatic stellate cell activation and liver fibrosis.\",\n      \"evidence\": \"Co-immunoprecipitation with domain mapping, luciferase reporter assays, immunofluorescence, in vivo and in vitro fibrosis models, miR-342-3p target validation\",\n      \"pmids\": [\"39910671\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ubiquitin ligase recruited by HPCAL1 to ubiquitinate Smad2 not identified\", \"Whether calcium binding by EF-hand 4 is required for Smad2 interaction untested\", \"Single-lab observation awaiting independent replication\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Showing that HPCAL1 binds and stabilizes the mitophagy receptor BNIP3 in a calcium-dependent manner, enhancing LC3-II interaction and driving excessive mitophagy-ROS-ferroptosis, established a second autophagy-related mechanism by which HPCAL1 promotes ferroptotic cell death.\",\n      \"evidence\": \"Co-immunoprecipitation, ROS and lipid peroxidation fluorescent probes, mitochondrial membrane potential and autophagic flux assays, genetic disruption in mouse I/R and rat IEC-6 H/R models\",\n      \"pmids\": [\"41482082\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the HPCAL1-BNIP3 interaction requires myristoylation or specific EF-hand domains not determined\", \"Relationship between CDH2-targeted autophagy and BNIP3-mediated mitophagy in the same cell not clarified\", \"Single-lab finding in ischemia-reperfusion context, generalizability unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A unified structural and regulatory model explaining how HPCAL1 selectively engages diverse cargo (CDH2, BNIP3, Smad2, PHOX2B) and switches between autophagy receptor, signaling modulator, and nuclear functions remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No crystal or cryo-EM structure of HPCAL1 in complex with any partner\", \"How calcium-myristoyl switching versus calcium-independent nuclear translocation are regulated in the same cell is unknown\", \"Whether HPCAL1 autophagy receptor and signaling functions are coordinated or context-exclusive is unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [5, 6]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CDH2\", \"PHOX2B\", \"BNIP3\", \"SMAD2\", \"PRKCQ\", \"LC3\"],\n    \"other_free_text\": []\n  }\n}\n```"}