{"gene":"EFHD1","run_date":"2026-04-28T17:46:03","timeline":{"discoveries":[{"year":2011,"finding":"EFHD1 (Swiprosin-2) is predicted to contain two EF-hand domains and a coiled-coil domain, and is associated with mitochondria, where it modulates apoptosis and differentiation of neuronal and muscle precursor cells, and is proposed to participate in a cellular response to oxidative stress.","method":"Secondary structure prediction, expression analysis, review of functional data","journal":"Cell communication and signaling : CCS","confidence":"Low","confidence_rationale":"Tier 4 — largely computational/review with limited direct experimental validation of mechanism","pmids":["21244694"],"is_preprint":false},{"year":2017,"finding":"EFHD1 is a Ca2+-binding protein of the inner mitochondrial membrane involved in Ca2+-induced mitoflashes; knockdown or knockout of EFhd1 in pro B cells increases glycolysis and glycolytic capacity, decreasing the OCR/ECAR ratio, while transgenic prolonged expression elevates PGC-1α in pre B cells and reduces mitochondrial ATP production, establishing EFHD1 as a regulator of the metabolic switch from oxidative phosphorylation to glycolysis at the pro-to-pre B cell transition.","method":"shRNA knockdown, CRISPR/Cas9 knockout, transgenic mouse overexpression, Seahorse metabolic flux assay (OCR/ECAR), mitochondrial membrane potential measurement, glucose uptake assay","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 — clean KO and KD with defined metabolic phenotype, corroborated by transgenic overexpression in vivo, multiple orthogonal methods","pmids":["28524857"],"is_preprint":false},{"year":2020,"finding":"Genetic ablation of Efhd1 in mice causes mitochondrial dysfunction, shortened mitochondria at axonal growth cones, decreased axonal ATP levels, activation of the AMPK-Ulk1 pathway, increased autophagic flux, reduced axonal growth and branching, and enhanced neuronal death, placing EFHD1 upstream of AMPK-mediated energy sensing in axonal morphogenesis.","method":"Efhd1 knockout mouse, biochemical ATP measurement, live imaging of mitochondrial morphology, AMPK/Ulk1 phosphorylation assay, autophagic flux measurement, transcriptome analysis","journal":"Life science alliance","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with multiple orthogonal phenotypic and biochemical readouts establishing pathway position","pmids":["32414840"],"is_preprint":false},{"year":2021,"finding":"The crystal structure of the EFhd1 core domain (comprising a proline-rich region C-terminus, two EF-hand domains, and a ligand mimic helix) was solved; structural comparison with EFhd2 and AIF-1 revealed overall similarity; two Zn2+ ions at the crystal contact interface suggest Zn2+-mediated multimerization; EFhd1 exhibits Ca2+-independent β-actin-binding and Ca2+-dependent β-actin-bundling activities in vitro.","method":"X-ray crystallography, in vitro actin-binding and actin-bundling assays, structural comparison","journal":"Frontiers in cell and developmental biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus in vitro biochemical reconstitution of actin-binding and bundling activities","pmids":["33537316"],"is_preprint":false},{"year":2022,"finding":"Efhd1 knockout mice show reduced basal ROS levels and reduced mitoflash events in cardiomyocytes, and are resistant to hypoxic injury, establishing EFHD1 as a required component for cardiac mitoflash activation and a contributor to ischemia-induced cardiomyocyte death.","method":"Efhd1 knockout mouse, ROS measurement, mitoflash imaging, cardiac ischemia-reperfusion model, cardiomyocyte hypoxia assay","journal":"Journal of molecular and cellular cardiology","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with multiple orthogonal readouts (ROS, mitoflash, ischemia resistance)","pmids":["35304170"],"is_preprint":false},{"year":2023,"finding":"EFHD1 physically binds to the mitochondrial calcium uniporter (MCU) through its N-terminal domain; this interaction suppresses mitochondrial Ca2+ uptake and deactivates the Hippo/YAP signaling pathway by upregulating STARD13 to enhance YAP phosphorylation at Ser-127, thereby inhibiting ccRCC cell migration and invasion.","method":"Co-immunoprecipitation, domain-mapping pulldown, overexpression and knockdown in ccRCC cells, mitochondrial Ca2+ measurement, YAP phosphorylation western blot, in vitro migration/invasion assays, in vivo tumor metastasis model","journal":"Cancer science","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP with domain mapping, rescue experiments (MCU OE/STARD13 KD), and in vivo validation","pmids":["36747492"],"is_preprint":false},{"year":2023,"finding":"Crystal structures of EFhd1 and EFhd2 core domains with Zn2+ coordinated within their EF-hands were determined; Zn2+ occupancy at the EF-hands was confirmed by anomalous diffraction at the Zn K-edge; EFhd1 displays Zn2+-independent actin-binding and Zn2+-dependent actin-bundling activity, indicating that actin-regulatory activities of EFhd1 can be regulated by Zn2+ in addition to Ca2+.","method":"X-ray crystallography with anomalous diffraction, in vitro actin-binding and actin-bundling assays","journal":"IUCrJ","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with element-specific validation plus in vitro biochemical reconstitution","pmids":["36862489"],"is_preprint":false},{"year":2024,"finding":"EFHD1 binds to adenine nucleotide translocase-3 (ANT3) and inhibits its conformational change, thereby suppressing opening of the mitochondrial permeability transition pore (mPTP); this maintains mitochondrial function and promotes osteosarcoma cell survival and drug resistance.","method":"Co-immunoprecipitation (EFHD1-ANT3 interaction), overexpression and knockdown in OS cell lines, mPTP opening assays, drug sensitivity assays, pharmacological rescue with CATR and BKA","journal":"Cellular and molecular life sciences : CMLS","confidence":"High","confidence_rationale":"Tier 2 — Co-IP identifying binding partner, pharmacological and genetic rescue experiments with defined mechanistic readout","pmids":["38795203"],"is_preprint":false},{"year":2025,"finding":"EFHD1 upregulates SIK3 expression, activating the Hippo signaling pathway to suppress EMT, colorectal cancer cell migration, invasion, and metastasis; SIK3 knockdown partially abrogates the EFHD1-mediated anti-metastatic effects, placing EFHD1 upstream of SIK3 in this pathway.","method":"Overexpression and knockdown in CRC cells, SIK3 knockdown epistasis, western blotting, immunofluorescence, orthotopic xenograft and pulmonary metastasis mouse models","journal":"Journal of Cancer","confidence":"Medium","confidence_rationale":"Tier 2 — epistasis experiment (SIK3 KD rescue), in vivo validation, but single lab study","pmids":["39895792"],"is_preprint":false},{"year":2026,"finding":"EFHD1 acts as a Ca2+-dependent actin crosslinker that stabilizes endoplasmic reticulum-mitochondria contact sites (ERMCS) by detecting spatiotemporal coincidence of inter-organellar proximity and ER Ca2+ release; during MASH, EFHD1 upregulation drives pathological mitochondrial fragmentation via excessive contact persistence, leading to mitochondrial double-stranded RNA escape and activation of a PKR-dependent antiviral stress response that causes hepatocyte damage.","method":"EFHD1 KO and inhibition in human and mouse hepatocyte models, live imaging of ERMCS, mitochondrial morphology analysis, dsRNA detection, PKR pathway assays, Mendelian randomization in humans","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods with in vivo and human genetic validation, but preprint","pmids":["41756893"],"is_preprint":true}],"current_model":"EFHD1 is a mitochondria-localized Ca2+- and Zn2+-binding EF-hand protein that structurally acts as an actin-binding (Ca2+/Zn2+-independent) and actin-bundling (Ca2+/Zn2+-dependent) protein; it regulates mitochondrial Ca2+ handling by binding to MCU (suppressing Ca2+ uptake) and ANT3 (inhibiting mPTP opening), stabilizes ER-mitochondria contact sites in a Ca2+-dependent manner, promotes mitoflash generation and ROS production, and controls cellular energy metabolism and axonal morphogenesis via AMPK-Ulk1 signaling, while also modulating cancer cell behavior through the Hippo/YAP and SIK3 pathways downstream of its mitochondrial Ca2+ regulatory function."},"narrative":{"teleology":[{"year":2011,"claim":"Initial computational and expression analyses established EFHD1 as a predicted two-EF-hand, coiled-coil domain protein associated with mitochondria, setting the stage for mechanistic studies of its role in apoptosis and differentiation.","evidence":"Structure prediction and expression profiling (review/computational)","pmids":["21244694"],"confidence":"Low","gaps":["No direct biochemical or structural validation of domain architecture","Functional roles inferred from expression patterns, not genetic perturbation","Mitochondrial localization not confirmed by suborganellar fractionation"]},{"year":2017,"claim":"Genetic loss-of-function and transgenic gain-of-function experiments revealed that EFHD1 is required at the inner mitochondrial membrane for Ca²⁺-induced mitoflashes and controls the metabolic switch between oxidative phosphorylation and glycolysis during B cell development.","evidence":"shRNA knockdown, CRISPR KO, transgenic mouse overexpression, Seahorse metabolic flux analysis in pro/pre B cells","pmids":["28524857"],"confidence":"High","gaps":["Direct molecular target(s) of EFHD1 at the inner membrane not identified","Mechanism by which EFHD1 promotes mitoflashes not defined","Whether metabolic regulation is Ca²⁺-binding dependent was not tested"]},{"year":2020,"claim":"Efhd1 knockout in mice established EFHD1 as an upstream regulator of mitochondrial ATP supply in axons, linking its loss to AMPK-Ulk1 activation, increased autophagy, and impaired axonal growth and branching.","evidence":"Efhd1 KO mouse, ATP measurement, live mitochondrial imaging, AMPK/Ulk1 phosphorylation, autophagic flux assays in neurons","pmids":["32414840"],"confidence":"High","gaps":["Whether EFHD1 directly maintains ATP production or acts indirectly through Ca²⁺ regulation was unclear","Mitochondrial shortening mechanism at growth cones not resolved","Phenotype not rescued to confirm specificity"]},{"year":2021,"claim":"The crystal structure of the EFHD1 core domain was solved, revealing EF-hand fold similarity to EFhd2 and AIF-1, Zn²⁺-mediated crystal contacts suggesting multimerization, and demonstrating that EFHD1 binds β-actin independently of Ca²⁺ but bundles actin in a Ca²⁺-dependent manner.","evidence":"X-ray crystallography, in vitro actin cosedimentation and bundling assays","pmids":["33537316"],"confidence":"High","gaps":["Physiological relevance of actin-bundling activity not tested in cells","Zn²⁺-mediated multimerization inferred from crystal contacts only","Which actin pools EFHD1 engages in mitochondria-associated contexts unknown"]},{"year":2022,"claim":"Efhd1 KO in cardiomyocytes demonstrated that EFHD1 is required for basal ROS levels and mitoflash generation, and its absence protects against hypoxia-induced cardiac injury, identifying EFHD1 as a pathophysiologically relevant driver of ischemic cardiomyocyte death.","evidence":"Efhd1 KO mouse, ROS measurement, mitoflash live imaging, cardiac ischemia-reperfusion model","pmids":["35304170"],"confidence":"High","gaps":["Molecular mechanism linking EFHD1 to mitoflash initiation still unknown","Whether protective effect of KO is solely ROS-mediated or involves other pathways not resolved","Heart-specific conditional KO not performed"]},{"year":2023,"claim":"Identification of MCU as a direct binding partner of EFHD1 (via its N-terminal domain) resolved the molecular basis of EFHD1's mitochondrial Ca²⁺ regulatory function, showing that EFHD1 suppresses MCU-mediated Ca²⁺ uptake and downstream Hippo/YAP signaling in renal cancer cells.","evidence":"Reciprocal Co-IP with domain mapping, mitochondrial Ca²⁺ measurement, YAP phosphorylation, rescue with MCU overexpression and STARD13 knockdown, in vivo metastasis model in ccRCC","pmids":["36747492"],"confidence":"High","gaps":["Whether MCU interaction is Ca²⁺-dependent was not tested","Stoichiometry and structural basis of the EFHD1–MCU complex unknown","Generalizability of the STARD13-YAP axis beyond ccRCC not established"]},{"year":2023,"claim":"Zinc coordination within the EF-hands of EFHD1 was confirmed by anomalous diffraction crystallography, establishing that Zn²⁺ — in addition to Ca²⁺ — regulates EFHD1's actin-bundling activity, broadening the signaling inputs controlling this protein.","evidence":"X-ray crystallography with Zn K-edge anomalous diffraction, in vitro actin-binding and bundling assays","pmids":["36862489"],"confidence":"High","gaps":["Whether physiological Zn²⁺ concentrations in mitochondria are sufficient to occupy EF-hands is unknown","No cellular assay of Zn²⁺-dependent actin bundling performed","Relative contributions of Ca²⁺ versus Zn²⁺ in vivo not dissected"]},{"year":2024,"claim":"Discovery that EFHD1 binds ANT3 and inhibits its conformational change to suppress mPTP opening provided a second direct mitochondrial interactor and explained how EFHD1 promotes cell survival and chemoresistance in osteosarcoma.","evidence":"Co-IP of EFHD1–ANT3, mPTP opening assays, pharmacological rescue with CATR and BKA, drug sensitivity assays in osteosarcoma cells","pmids":["38795203"],"confidence":"High","gaps":["Whether EFHD1–MCU and EFHD1–ANT3 interactions are mutually exclusive or simultaneous is unknown","Direct binding interface on ANT3 not mapped","Relevance to non-cancer contexts not assessed"]},{"year":2025,"claim":"Epistasis experiments placed EFHD1 upstream of SIK3 in Hippo pathway activation, extending its tumor-suppressive signaling to colorectal cancer EMT and metastasis.","evidence":"EFHD1 overexpression/knockdown with SIK3 epistasis, western blot, orthotopic xenograft and pulmonary metastasis models in CRC","pmids":["39895792"],"confidence":"Medium","gaps":["Mechanism by which EFHD1 upregulates SIK3 expression is unknown","Single-laboratory study not yet independently confirmed","Relationship between mitochondrial Ca²⁺ regulation and SIK3 induction not tested"]},{"year":null,"claim":"Key unresolved questions include how EFHD1's actin-bundling and Ca²⁺/Zn²⁺-sensing activities coordinate with its MCU and ANT3 interactions in vivo, whether EFHD1 forms a stable complex bridging ER and mitochondria through actin crosslinking under physiological Ca²⁺ dynamics, and how these molecular activities integrate to control tissue-specific metabolic and stress outcomes.","evidence":"","pmids":[],"confidence":"Low","gaps":["No reconstituted system combining MCU binding, ANT3 binding, and actin bundling","In vivo structure-function analysis with EF-hand mutants not performed","Tissue-specific conditional knockouts beyond germline KO are lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[3,6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5,7]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[1,2,4,5,7]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[1,2]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[5,8]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[4,7]}],"complexes":[],"partners":["MCU","ANT3","ACTB","SIK3"],"other_free_text":[]},"mechanistic_narrative":"EFHD1 (Swiprosin-2) is a mitochondrial EF-hand protein that integrates calcium and zinc signaling with mitochondrial function, energy metabolism, and organelle contact site dynamics. Its crystal structure reveals two EF-hand domains capable of coordinating both Ca²⁺ and Zn²⁺, conferring Ca²⁺/Zn²⁺-independent actin-binding and Ca²⁺/Zn²⁺-dependent actin-bundling activities [PMID:33537316, PMID:36862489]. At the inner mitochondrial membrane, EFHD1 physically interacts with MCU to suppress mitochondrial Ca²⁺ uptake and with ANT3 to inhibit mPTP opening, thereby regulating mitoflash generation, ROS production, and cell survival under stress [PMID:36747492, PMID:38795203, PMID:35304170]. Loss of EFHD1 shifts cellular metabolism from oxidative phosphorylation toward glycolysis, activates AMPK-Ulk1 signaling, impairs axonal morphogenesis, and confers resistance to ischemic cardiac injury, while its downstream effects in cancer cells operate through the Hippo/YAP and SIK3 pathways [PMID:28524857, PMID:32414840, PMID:39895792]."},"prefetch_data":{"uniprot":{"accession":"Q9BUP0","full_name":"EF-hand domain-containing protein D1","aliases":["EF-hand domain-containing protein 1","Swiprosin-2"],"length_aa":239,"mass_kda":26.9,"function":"Acts as a calcium sensor for mitochondrial flash (mitoflash) activation, an event characterized by stochastic bursts of superoxide production (PubMed:26975899). May play a role in neuronal differentiation (By similarity)","subcellular_location":"Mitochondrion inner membrane","url":"https://www.uniprot.org/uniprotkb/Q9BUP0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EFHD1","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/EFHD1","total_profiled":1310},"omim":[{"mim_id":"611617","title":"EF-HAND DOMAIN FAMILY, MEMBER D1; EFHD1","url":"https://www.omim.org/entry/611617"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Golgi apparatus","reliability":"Supported"},{"location":"Nucleoli fibrillar center","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"blood vessel","ntpm":313.4},{"tissue":"brain","ntpm":324.6}],"url":"https://www.proteinatlas.org/search/EFHD1"},"hgnc":{"alias_symbol":["FLJ13612"],"prev_symbol":[]},"alphafold":{"accession":"Q9BUP0","domains":[{"cath_id":"1.10.238.10","chopping":"79-174","consensus_level":"high","plddt":89.2779,"start":79,"end":174},{"cath_id":"1.20.5","chopping":"184-239","consensus_level":"medium","plddt":81.8459,"start":184,"end":239}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BUP0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BUP0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BUP0-F1-predicted_aligned_error_v6.png","plddt_mean":77.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EFHD1","jax_strain_url":"https://www.jax.org/strain/search?query=EFHD1"},"sequence":{"accession":"Q9BUP0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BUP0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BUP0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BUP0"}},"corpus_meta":[{"pmid":"21244694","id":"PMC_21244694","title":"Fraternal twins: Swiprosin-1/EFhd2 and Swiprosin-2/EFhd1, two homologous EF-hand containing calcium binding adaptor proteins with distinct functions.","date":"2011","source":"Cell communication and signaling : CCS","url":"https://pubmed.ncbi.nlm.nih.gov/21244694","citation_count":68,"is_preprint":false},{"pmid":"28524857","id":"PMC_28524857","title":"A defined metabolic state in pre B cells governs B-cell development and is counterbalanced by Swiprosin-2/EFhd1.","date":"2017","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/28524857","citation_count":58,"is_preprint":false},{"pmid":"24861485","id":"PMC_24861485","title":"Aberrant promoter methylation of PPP1R3C and EFHD1 in plasma of colorectal cancer patients.","date":"2014","source":"Cancer medicine","url":"https://pubmed.ncbi.nlm.nih.gov/24861485","citation_count":37,"is_preprint":false},{"pmid":"36747492","id":"PMC_36747492","title":"EFHD1, a novel mitochondrial regulator of tumor metastasis in clear cell renal cell carcinoma.","date":"2023","source":"Cancer science","url":"https://pubmed.ncbi.nlm.nih.gov/36747492","citation_count":25,"is_preprint":false},{"pmid":"32414840","id":"PMC_32414840","title":"Regulation of axonal morphogenesis by the mitochondrial protein Efhd1.","date":"2020","source":"Life science alliance","url":"https://pubmed.ncbi.nlm.nih.gov/32414840","citation_count":23,"is_preprint":false},{"pmid":"35304170","id":"PMC_35304170","title":"EFHD1 ablation inhibits cardiac mitoflash activation and protects cardiomyocytes from ischemia.","date":"2022","source":"Journal of molecular and cellular cardiology","url":"https://pubmed.ncbi.nlm.nih.gov/35304170","citation_count":19,"is_preprint":false},{"pmid":"33537316","id":"PMC_33537316","title":"Structural and Biochemical Characterization of EFhd1/Swiprosin-2, an Actin-Binding Protein in Mitochondria.","date":"2021","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/33537316","citation_count":18,"is_preprint":false},{"pmid":"38795203","id":"PMC_38795203","title":"EFHD1 promotes osteosarcoma proliferation and drug resistance by inhibiting the opening of the mitochondrial membrane permeability transition pore (mPTP) by binding to ANT3.","date":"2024","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/38795203","citation_count":8,"is_preprint":false},{"pmid":"23909416","id":"PMC_23909416","title":"Monoclonal antibodies to discriminate the EF hand containing calcium binding adaptor proteins EFhd1 and EFhd2.","date":"2013","source":"Monoclonal antibodies in immunodiagnosis and immunotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/23909416","citation_count":8,"is_preprint":false},{"pmid":"36862489","id":"PMC_36862489","title":"Structural and biochemical insights into Zn2+-bound EF-hand proteins, EFhd1 and EFhd2.","date":"2023","source":"IUCrJ","url":"https://pubmed.ncbi.nlm.nih.gov/36862489","citation_count":3,"is_preprint":false},{"pmid":"39895792","id":"PMC_39895792","title":"EFHD1 Activates SIK3 to Limit Colorectal Cancer Initiation and Progression via the Hippo Pathway.","date":"2025","source":"Journal of Cancer","url":"https://pubmed.ncbi.nlm.nih.gov/39895792","citation_count":1,"is_preprint":false},{"pmid":"41279427","id":"PMC_41279427","title":"The Function of Efhd1 + Telocytes in the Synovial Lymphatic System and Inflammatory-Erosive Arthritis.","date":"2026","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/41279427","citation_count":1,"is_preprint":false},{"pmid":"40441345","id":"PMC_40441345","title":"EFHD1 promotes breast cancer progression through estrogen signaling.","date":"2025","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/40441345","citation_count":1,"is_preprint":false},{"pmid":"40689352","id":"PMC_40689352","title":"Sevoflurane induces Ca2+ overload and EFHD1 upregulation, driving pyroptosis in SCLC cells.","date":"2025","source":"Cytotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/40689352","citation_count":0,"is_preprint":false},{"pmid":"41756893","id":"PMC_41756893","title":"Excessive Ca2+-dependent ER-mitochondrial contact stabilization by EFHD1 drives liver injury.","date":"2026","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/41756893","citation_count":0,"is_preprint":false},{"pmid":"41377507","id":"PMC_41377507","title":"Assessment of the Role of Telocyte Zbtb16 in Lymphatic Drainage using Efhd1-CreERT2 x Zbtb16flox/flox Mice.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/41377507","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.11.28.691161","title":"Assessment of the Role of Telocyte Zbtb16 in Lymphatic Drainage using Efhd1-CreERT2 x Zbtb16  <sup>flox/flox</sup>  Mice","date":"2025-12-01","source":"bioRxiv","url":"https://doi.org/10.1101/2025.11.28.691161","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.11.03.685859","title":"The Function of  <i>Efhd1</i>  <sup>+</sup>  Telocytes in the Synovial Lymphatic System and Inflammatory-Erosive Arthritis","date":"2025-11-05","source":"bioRxiv","url":"https://doi.org/10.1101/2025.11.03.685859","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.09.20.614203","title":"Identification of genes conferring individual-level variation responsible for metabolic dysfunction-associated steatohepatitis using single-cell eQTL analysis","date":"2024-09-24","source":"bioRxiv","url":"https://doi.org/10.1101/2024.09.20.614203","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.08.11.24311581","title":"Integrative Multi-omics Approach Reveals the Molecular Characterization and Differences of ECM-PI3K-Akt Pathway among Coronary Artery Bypass Grafting Conduits with Clinical Implications","date":"2024-08-14","source":"bioRxiv","url":"https://doi.org/10.1101/2024.08.11.24311581","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12588,"output_tokens":2695,"usd":0.039094},"stage2":{"model":"claude-opus-4-6","input_tokens":6016,"output_tokens":2888,"usd":0.15342},"total_usd":0.192514,"stage1_batch_id":"msgbatch_0118RScg3WGaGkaZRtWZbtce","stage2_batch_id":"msgbatch_01Cpfzvtbj42aqp5oYZ5Te2s","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2011,\n      \"finding\": \"EFHD1 (Swiprosin-2) is predicted to contain two EF-hand domains and a coiled-coil domain, and is associated with mitochondria, where it modulates apoptosis and differentiation of neuronal and muscle precursor cells, and is proposed to participate in a cellular response to oxidative stress.\",\n      \"method\": \"Secondary structure prediction, expression analysis, review of functional data\",\n      \"journal\": \"Cell communication and signaling : CCS\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 — largely computational/review with limited direct experimental validation of mechanism\",\n      \"pmids\": [\"21244694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"EFHD1 is a Ca2+-binding protein of the inner mitochondrial membrane involved in Ca2+-induced mitoflashes; knockdown or knockout of EFhd1 in pro B cells increases glycolysis and glycolytic capacity, decreasing the OCR/ECAR ratio, while transgenic prolonged expression elevates PGC-1α in pre B cells and reduces mitochondrial ATP production, establishing EFHD1 as a regulator of the metabolic switch from oxidative phosphorylation to glycolysis at the pro-to-pre B cell transition.\",\n      \"method\": \"shRNA knockdown, CRISPR/Cas9 knockout, transgenic mouse overexpression, Seahorse metabolic flux assay (OCR/ECAR), mitochondrial membrane potential measurement, glucose uptake assay\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO and KD with defined metabolic phenotype, corroborated by transgenic overexpression in vivo, multiple orthogonal methods\",\n      \"pmids\": [\"28524857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Genetic ablation of Efhd1 in mice causes mitochondrial dysfunction, shortened mitochondria at axonal growth cones, decreased axonal ATP levels, activation of the AMPK-Ulk1 pathway, increased autophagic flux, reduced axonal growth and branching, and enhanced neuronal death, placing EFHD1 upstream of AMPK-mediated energy sensing in axonal morphogenesis.\",\n      \"method\": \"Efhd1 knockout mouse, biochemical ATP measurement, live imaging of mitochondrial morphology, AMPK/Ulk1 phosphorylation assay, autophagic flux measurement, transcriptome analysis\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with multiple orthogonal phenotypic and biochemical readouts establishing pathway position\",\n      \"pmids\": [\"32414840\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The crystal structure of the EFhd1 core domain (comprising a proline-rich region C-terminus, two EF-hand domains, and a ligand mimic helix) was solved; structural comparison with EFhd2 and AIF-1 revealed overall similarity; two Zn2+ ions at the crystal contact interface suggest Zn2+-mediated multimerization; EFhd1 exhibits Ca2+-independent β-actin-binding and Ca2+-dependent β-actin-bundling activities in vitro.\",\n      \"method\": \"X-ray crystallography, in vitro actin-binding and actin-bundling assays, structural comparison\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus in vitro biochemical reconstitution of actin-binding and bundling activities\",\n      \"pmids\": [\"33537316\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Efhd1 knockout mice show reduced basal ROS levels and reduced mitoflash events in cardiomyocytes, and are resistant to hypoxic injury, establishing EFHD1 as a required component for cardiac mitoflash activation and a contributor to ischemia-induced cardiomyocyte death.\",\n      \"method\": \"Efhd1 knockout mouse, ROS measurement, mitoflash imaging, cardiac ischemia-reperfusion model, cardiomyocyte hypoxia assay\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with multiple orthogonal readouts (ROS, mitoflash, ischemia resistance)\",\n      \"pmids\": [\"35304170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EFHD1 physically binds to the mitochondrial calcium uniporter (MCU) through its N-terminal domain; this interaction suppresses mitochondrial Ca2+ uptake and deactivates the Hippo/YAP signaling pathway by upregulating STARD13 to enhance YAP phosphorylation at Ser-127, thereby inhibiting ccRCC cell migration and invasion.\",\n      \"method\": \"Co-immunoprecipitation, domain-mapping pulldown, overexpression and knockdown in ccRCC cells, mitochondrial Ca2+ measurement, YAP phosphorylation western blot, in vitro migration/invasion assays, in vivo tumor metastasis model\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP with domain mapping, rescue experiments (MCU OE/STARD13 KD), and in vivo validation\",\n      \"pmids\": [\"36747492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Crystal structures of EFhd1 and EFhd2 core domains with Zn2+ coordinated within their EF-hands were determined; Zn2+ occupancy at the EF-hands was confirmed by anomalous diffraction at the Zn K-edge; EFhd1 displays Zn2+-independent actin-binding and Zn2+-dependent actin-bundling activity, indicating that actin-regulatory activities of EFhd1 can be regulated by Zn2+ in addition to Ca2+.\",\n      \"method\": \"X-ray crystallography with anomalous diffraction, in vitro actin-binding and actin-bundling assays\",\n      \"journal\": \"IUCrJ\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with element-specific validation plus in vitro biochemical reconstitution\",\n      \"pmids\": [\"36862489\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EFHD1 binds to adenine nucleotide translocase-3 (ANT3) and inhibits its conformational change, thereby suppressing opening of the mitochondrial permeability transition pore (mPTP); this maintains mitochondrial function and promotes osteosarcoma cell survival and drug resistance.\",\n      \"method\": \"Co-immunoprecipitation (EFHD1-ANT3 interaction), overexpression and knockdown in OS cell lines, mPTP opening assays, drug sensitivity assays, pharmacological rescue with CATR and BKA\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP identifying binding partner, pharmacological and genetic rescue experiments with defined mechanistic readout\",\n      \"pmids\": [\"38795203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"EFHD1 upregulates SIK3 expression, activating the Hippo signaling pathway to suppress EMT, colorectal cancer cell migration, invasion, and metastasis; SIK3 knockdown partially abrogates the EFHD1-mediated anti-metastatic effects, placing EFHD1 upstream of SIK3 in this pathway.\",\n      \"method\": \"Overexpression and knockdown in CRC cells, SIK3 knockdown epistasis, western blotting, immunofluorescence, orthotopic xenograft and pulmonary metastasis mouse models\",\n      \"journal\": \"Journal of Cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — epistasis experiment (SIK3 KD rescue), in vivo validation, but single lab study\",\n      \"pmids\": [\"39895792\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"EFHD1 acts as a Ca2+-dependent actin crosslinker that stabilizes endoplasmic reticulum-mitochondria contact sites (ERMCS) by detecting spatiotemporal coincidence of inter-organellar proximity and ER Ca2+ release; during MASH, EFHD1 upregulation drives pathological mitochondrial fragmentation via excessive contact persistence, leading to mitochondrial double-stranded RNA escape and activation of a PKR-dependent antiviral stress response that causes hepatocyte damage.\",\n      \"method\": \"EFHD1 KO and inhibition in human and mouse hepatocyte models, live imaging of ERMCS, mitochondrial morphology analysis, dsRNA detection, PKR pathway assays, Mendelian randomization in humans\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods with in vivo and human genetic validation, but preprint\",\n      \"pmids\": [\"41756893\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"EFHD1 is a mitochondria-localized Ca2+- and Zn2+-binding EF-hand protein that structurally acts as an actin-binding (Ca2+/Zn2+-independent) and actin-bundling (Ca2+/Zn2+-dependent) protein; it regulates mitochondrial Ca2+ handling by binding to MCU (suppressing Ca2+ uptake) and ANT3 (inhibiting mPTP opening), stabilizes ER-mitochondria contact sites in a Ca2+-dependent manner, promotes mitoflash generation and ROS production, and controls cellular energy metabolism and axonal morphogenesis via AMPK-Ulk1 signaling, while also modulating cancer cell behavior through the Hippo/YAP and SIK3 pathways downstream of its mitochondrial Ca2+ regulatory function.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"EFHD1 (Swiprosin-2) is a mitochondrial EF-hand protein that integrates calcium and zinc signaling with mitochondrial function, energy metabolism, and organelle contact site dynamics. Its crystal structure reveals two EF-hand domains capable of coordinating both Ca²⁺ and Zn²⁺, conferring Ca²⁺/Zn²⁺-independent actin-binding and Ca²⁺/Zn²⁺-dependent actin-bundling activities [PMID:33537316, PMID:36862489]. At the inner mitochondrial membrane, EFHD1 physically interacts with MCU to suppress mitochondrial Ca²⁺ uptake and with ANT3 to inhibit mPTP opening, thereby regulating mitoflash generation, ROS production, and cell survival under stress [PMID:36747492, PMID:38795203, PMID:35304170]. Loss of EFHD1 shifts cellular metabolism from oxidative phosphorylation toward glycolysis, activates AMPK-Ulk1 signaling, impairs axonal morphogenesis, and confers resistance to ischemic cardiac injury, while its downstream effects in cancer cells operate through the Hippo/YAP and SIK3 pathways [PMID:28524857, PMID:32414840, PMID:39895792].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Initial computational and expression analyses established EFHD1 as a predicted two-EF-hand, coiled-coil domain protein associated with mitochondria, setting the stage for mechanistic studies of its role in apoptosis and differentiation.\",\n      \"evidence\": \"Structure prediction and expression profiling (review/computational)\",\n      \"pmids\": [\"21244694\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No direct biochemical or structural validation of domain architecture\",\n        \"Functional roles inferred from expression patterns, not genetic perturbation\",\n        \"Mitochondrial localization not confirmed by suborganellar fractionation\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Genetic loss-of-function and transgenic gain-of-function experiments revealed that EFHD1 is required at the inner mitochondrial membrane for Ca²⁺-induced mitoflashes and controls the metabolic switch between oxidative phosphorylation and glycolysis during B cell development.\",\n      \"evidence\": \"shRNA knockdown, CRISPR KO, transgenic mouse overexpression, Seahorse metabolic flux analysis in pro/pre B cells\",\n      \"pmids\": [\"28524857\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct molecular target(s) of EFHD1 at the inner membrane not identified\",\n        \"Mechanism by which EFHD1 promotes mitoflashes not defined\",\n        \"Whether metabolic regulation is Ca²⁺-binding dependent was not tested\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Efhd1 knockout in mice established EFHD1 as an upstream regulator of mitochondrial ATP supply in axons, linking its loss to AMPK-Ulk1 activation, increased autophagy, and impaired axonal growth and branching.\",\n      \"evidence\": \"Efhd1 KO mouse, ATP measurement, live mitochondrial imaging, AMPK/Ulk1 phosphorylation, autophagic flux assays in neurons\",\n      \"pmids\": [\"32414840\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether EFHD1 directly maintains ATP production or acts indirectly through Ca²⁺ regulation was unclear\",\n        \"Mitochondrial shortening mechanism at growth cones not resolved\",\n        \"Phenotype not rescued to confirm specificity\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"The crystal structure of the EFHD1 core domain was solved, revealing EF-hand fold similarity to EFhd2 and AIF-1, Zn²⁺-mediated crystal contacts suggesting multimerization, and demonstrating that EFHD1 binds β-actin independently of Ca²⁺ but bundles actin in a Ca²⁺-dependent manner.\",\n      \"evidence\": \"X-ray crystallography, in vitro actin cosedimentation and bundling assays\",\n      \"pmids\": [\"33537316\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Physiological relevance of actin-bundling activity not tested in cells\",\n        \"Zn²⁺-mediated multimerization inferred from crystal contacts only\",\n        \"Which actin pools EFHD1 engages in mitochondria-associated contexts unknown\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Efhd1 KO in cardiomyocytes demonstrated that EFHD1 is required for basal ROS levels and mitoflash generation, and its absence protects against hypoxia-induced cardiac injury, identifying EFHD1 as a pathophysiologically relevant driver of ischemic cardiomyocyte death.\",\n      \"evidence\": \"Efhd1 KO mouse, ROS measurement, mitoflash live imaging, cardiac ischemia-reperfusion model\",\n      \"pmids\": [\"35304170\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Molecular mechanism linking EFHD1 to mitoflash initiation still unknown\",\n        \"Whether protective effect of KO is solely ROS-mediated or involves other pathways not resolved\",\n        \"Heart-specific conditional KO not performed\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of MCU as a direct binding partner of EFHD1 (via its N-terminal domain) resolved the molecular basis of EFHD1's mitochondrial Ca²⁺ regulatory function, showing that EFHD1 suppresses MCU-mediated Ca²⁺ uptake and downstream Hippo/YAP signaling in renal cancer cells.\",\n      \"evidence\": \"Reciprocal Co-IP with domain mapping, mitochondrial Ca²⁺ measurement, YAP phosphorylation, rescue with MCU overexpression and STARD13 knockdown, in vivo metastasis model in ccRCC\",\n      \"pmids\": [\"36747492\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether MCU interaction is Ca²⁺-dependent was not tested\",\n        \"Stoichiometry and structural basis of the EFHD1–MCU complex unknown\",\n        \"Generalizability of the STARD13-YAP axis beyond ccRCC not established\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Zinc coordination within the EF-hands of EFHD1 was confirmed by anomalous diffraction crystallography, establishing that Zn²⁺ — in addition to Ca²⁺ — regulates EFHD1's actin-bundling activity, broadening the signaling inputs controlling this protein.\",\n      \"evidence\": \"X-ray crystallography with Zn K-edge anomalous diffraction, in vitro actin-binding and bundling assays\",\n      \"pmids\": [\"36862489\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether physiological Zn²⁺ concentrations in mitochondria are sufficient to occupy EF-hands is unknown\",\n        \"No cellular assay of Zn²⁺-dependent actin bundling performed\",\n        \"Relative contributions of Ca²⁺ versus Zn²⁺ in vivo not dissected\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Discovery that EFHD1 binds ANT3 and inhibits its conformational change to suppress mPTP opening provided a second direct mitochondrial interactor and explained how EFHD1 promotes cell survival and chemoresistance in osteosarcoma.\",\n      \"evidence\": \"Co-IP of EFHD1–ANT3, mPTP opening assays, pharmacological rescue with CATR and BKA, drug sensitivity assays in osteosarcoma cells\",\n      \"pmids\": [\"38795203\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether EFHD1–MCU and EFHD1–ANT3 interactions are mutually exclusive or simultaneous is unknown\",\n        \"Direct binding interface on ANT3 not mapped\",\n        \"Relevance to non-cancer contexts not assessed\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Epistasis experiments placed EFHD1 upstream of SIK3 in Hippo pathway activation, extending its tumor-suppressive signaling to colorectal cancer EMT and metastasis.\",\n      \"evidence\": \"EFHD1 overexpression/knockdown with SIK3 epistasis, western blot, orthotopic xenograft and pulmonary metastasis models in CRC\",\n      \"pmids\": [\"39895792\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism by which EFHD1 upregulates SIK3 expression is unknown\",\n        \"Single-laboratory study not yet independently confirmed\",\n        \"Relationship between mitochondrial Ca²⁺ regulation and SIK3 induction not tested\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include how EFHD1's actin-bundling and Ca²⁺/Zn²⁺-sensing activities coordinate with its MCU and ANT3 interactions in vivo, whether EFHD1 forms a stable complex bridging ER and mitochondria through actin crosslinking under physiological Ca²⁺ dynamics, and how these molecular activities integrate to control tissue-specific metabolic and stress outcomes.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No reconstituted system combining MCU binding, ANT3 binding, and actin bundling\",\n        \"In vivo structure-function analysis with EF-hand mutants not performed\",\n        \"Tissue-specific conditional knockouts beyond germline KO are lacking\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [3, 6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [1, 2, 4, 5, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [5, 8]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [4, 7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"MCU\",\n      \"ANT3\",\n      \"ACTB\",\n      \"SIK3\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}