{"gene":"EFHD1","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":2021,"finding":"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, revealing two Zn2+ ions at the crystal contact interface suggesting Zn2+-mediated multimerization. EFhd1 was found to possess Ca2+-independent β-actin-binding activity and Ca2+-dependent β-actin-bundling activity in vitro.","method":"X-ray crystallography; in vitro actin-binding and actin-bundling assays","journal":"Frontiers in cell and developmental biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure solved with functional biochemical validation (actin-binding and bundling assays), single rigorous paper with multiple orthogonal methods","pmids":["33537316"],"is_preprint":false},{"year":2023,"finding":"Crystal structures of EFhd1 and EFhd2 core domains coordinating Zn2+ ions within their EF-hands were determined. Anomalous signal analysis confirmed Zn2+ occupancy. EFhd1 exhibits Zn2+-independent actin-binding and Zn2+-dependent actin-bundling activity, demonstrating that actin-related activities can be regulated by Zn2+ in addition to Ca2+.","method":"X-ray crystallography with anomalous diffraction at Zn K-edge; in vitro actin-binding and bundling assays","journal":"IUCrJ","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with anomalous signal validation plus orthogonal biochemical assays in a single rigorous study","pmids":["36862489"],"is_preprint":false},{"year":2023,"finding":"EFHD1 physically binds to the mitochondrial calcium uniporter (MCU) through its N-terminal domain, suppressing mitochondrial Ca2+ uptake. This EFHD1-MCU interaction deactivates the Hippo/YAP signaling pathway by upregulating STARD13, which enhances phosphorylation of YAP at Ser-127. Knockdown of STARD13 or overexpression of MCU partially abrogated EFHD1-mediated YAP phosphorylation and suppression of cell migration in ccRCC cells.","method":"Co-immunoprecipitation; overexpression and knockdown experiments; in vitro migration/invasion assays; in vivo tumor metastasis assays; mitochondrial Ca2+ measurement; western blotting","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal interaction (Co-IP showing physical binding), multiple orthogonal functional assays (knockdown, overexpression, rescue), single lab","pmids":["36747492"],"is_preprint":false},{"year":2024,"finding":"EFHD1 binds to adenine nucleotide translocase-3 (ANT3) and inhibits ANT3 conformational change, thereby inhibiting opening of the mitochondrial membrane permeability transition pore (mPTP). This maintains mitochondrial function and promotes osteosarcoma cell survival/drug resistance. Pharmacological modulation of ANT3 conformation (with CATR or BKA) confirmed the functional link.","method":"Co-immunoprecipitation; overexpression and knockdown; cell viability assays; pharmacological rescue with CATR and BKA","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP for physical interaction, pharmacological rescue experiments confirming mechanism, single lab with multiple orthogonal methods","pmids":["38795203"],"is_preprint":false},{"year":2020,"finding":"Genetic ablation of Efhd1 in mice caused reduced axonal morphogenesis (shorter axons, less branching) and enhanced neuronal death. Efhd1 KO sensory neurons exhibited mitochondrial dysfunction, decreased axonal ATP levels, shortened mitochondria at axonal growth cones, activation of the AMPK-Ulk1 pathway, and increased autophagic flux. Efhd1 was identified as downstream of Lkb1 (liver kinase B1) in an energy homeostasis pathway regulating axonal growth.","method":"Genetic knockout in mice; axonal morphogenesis assays; ATP measurement; transcriptome analysis; mitochondrial imaging; AMPK-Ulk1 pathway analysis; autophagic flux assay","journal":"Life science alliance","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO mouse with defined cellular and molecular phenotype, multiple orthogonal readouts (ATP, mitochondrial morphology, pathway activation, autophagic flux), single rigorous study","pmids":["32414840"],"is_preprint":false},{"year":2017,"finding":"EFhd1 is a Ca2+-binding protein localized at the inner mitochondrial membrane involved in Ca2+-induced mitoflashes. EFhd1 is expressed in pro-B cells and downregulated by pre-B cell receptor (pre-BCR) surface expression. Knockdown and knockout of EFhd1 in pro-B cells decreased the OCR/ECAR ratio by increasing glycolysis, glycolytic capacity, and reserve. Transgenic EFhd1 expression beyond the pro-B cell stage upregulated PGC-1α in primary pre-B cells but reduced mitochondrial ATP production, causing a B-cell intrinsic developmental disadvantage.","method":"Knockdown and knockout in B cell lines; transgenic mouse model; metabolic flux analysis (OCR/ECAR); mitochondrial membrane potential measurement; glucose uptake assays; ROS measurement","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO, KD, and transgenic overexpression with multiple metabolic readouts, replicated across cell lines and primary cells","pmids":["28524857"],"is_preprint":false},{"year":2022,"finding":"Deletion of Efhd1 in mice (Efhd1-/-) reduced basal mitochondrial ROS levels and mitoflash events in cardiomyocytes without causing adverse cardiac phenotypes under basal conditions. Efhd1-/- mice and cardiomyocytes were resistant to hypoxic injury. However, cardiac mitochondria in Efhd1-/- mice showed normal susceptibility to Ca2+ overload.","method":"Knockout mouse model; ROS measurement; mitoflash imaging; cardiac phenotyping; hypoxia/ischemia models; mitochondrial Ca2+ overload assays","journal":"Journal of molecular and cellular cardiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO mouse with multiple orthogonal cellular phenotype readouts, single lab","pmids":["35304170"],"is_preprint":false},{"year":2025,"finding":"EFHD1 upregulates SIK3 expression, and SIK3 knockdown partially eliminated the inhibitory effects of EFHD1 on CRC metastasis. EFHD1 inhibited epithelial-mesenchymal transition (EMT) and CRC metastasis through the EFHD1-SIK3-Hippo signaling axis.","method":"Overexpression and knockdown; western blotting; immunohistochemistry; orthotopic xenograft and pulmonary metastasis mouse models; wound healing and Transwell assays","journal":"Journal of Cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis via genetic rescue (SIK3 KD abrogates EFHD1 effects) with in vivo validation, single lab","pmids":["39895792"],"is_preprint":false},{"year":2026,"finding":"EFHD1, acting as a Ca2+-dependent actin crosslinker, stabilizes endoplasmic reticulum-mitochondria contact sites (ERMCS) by detecting spatiotemporal coincidence of inter-organellar proximity and ER Ca2+ release. During metabolic-associated steatohepatitis (MASH), EFHD1 upregulation drives pathological mitochondrial fragmentation via excessive contact persistence, promoting mitochondrial double-stranded RNA escape and activation of a PKR-dependent antiviral stress response. Inhibiting EFHD1 in human and mouse models blunted hepatocyte damage.","method":"Functional imaging of ERMCS; mitochondrial morphology analysis; dsRNA detection; PKR pathway analysis; EFHD1 inhibition in human and mouse MASH models; Mendelian randomization in humans","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (ERMCS imaging, dsRNA escape, pharmacological inhibition, human genetic evidence), preprint not yet peer-reviewed","pmids":["41756893"],"is_preprint":true},{"year":2025,"finding":"EFHD1 promotes breast cancer cell proliferation, migration, and invasion through activation of estrogen signaling (ERα/ESR1 pathway). ESR1 knockdown abolished the pro-proliferative and pro-migratory effects of EFHD1 overexpression, placing EFHD1 upstream of ESR1 in this pathway.","method":"Overexpression and knockdown; EdU proliferation assay; flow cytometry (apoptosis); wound healing and Transwell assays; gene set enrichment analysis; western blotting; qRT-PCR; epistasis via ESR1 KD rescue","journal":"Experimental cell research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, ESR1 rescue experiment supports pathway placement, but molecular mechanism linking EFHD1 to ERα activation not established biochemically","pmids":["40441345"],"is_preprint":false},{"year":2011,"finding":"EFhd1 (Swiprosin-2) was predicted by secondary structure analysis to contain an N-terminal disordered region, two EF-hands, and a coiled-coil domain. EFhd1 is proposed to associate with mitochondria and modulate apoptosis and differentiation of neuronal and muscle precursor cells, as part of a cellular response to oxidative stress.","method":"Secondary structure prediction; comparative evolutionary/structural analysis; review of prior literature","journal":"Cell communication and signaling : CCS","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational/structural prediction and review synthesis without direct experimental validation of EFhd1 mechanism in this paper","pmids":["21244694"],"is_preprint":false}],"current_model":"EFHD1 is a Ca2+- and Zn2+-binding EF-hand protein of the inner mitochondrial membrane that crosslinks actin in a Ca2+-dependent manner, stabilizes ER-mitochondria contact sites, regulates mitochondrial Ca2+ uptake by physically binding MCU (via its N-terminal domain), inhibits mPTP opening by binding ANT3, controls mitoflash generation and ROS production, and thereby governs cellular energy metabolism, axonal morphogenesis, B-cell development, and protection from ischemic/metabolic injury."},"narrative":{"mechanistic_narrative":"EFHD1 is a Ca2+- and Zn2+-binding EF-hand protein of the inner mitochondrial membrane that couples cytoskeletal and ion-handling activities to control mitochondrial function and cellular energy metabolism [PMID:28524857, PMID:33537316]. Structurally, its core comprises two EF-hands and a ligand-mimic helix, and it binds β-actin constitutively while bundling/crosslinking actin in a Ca2+- and Zn2+-dependent manner [PMID:33537316, PMID:36862489]. At the inner mitochondrial membrane EFHD1 participates in Ca2+-induced mitoflashes and governs reactive oxygen species production; loss of EFHD1 lowers basal mitochondrial ROS and mitoflash events and confers resistance to hypoxic injury [PMID:28524857, PMID:35304170]. It restrains mitochondrial Ca2+ uptake by physically binding the mitochondrial calcium uniporter MCU through its N-terminal domain [PMID:36747492], and limits mitochondrial permeability transition pore opening by binding adenine nucleotide translocase ANT3 and blocking its conformational change, thereby preserving mitochondrial integrity [PMID:38795203]. As a Ca2+-dependent actin crosslinker EFHD1 also stabilizes ER–mitochondria contact sites by sensing coincident inter-organellar proximity and ER Ca2+ release [PMID:41756893]. Through these mitochondrial functions EFHD1 regulates axonal morphogenesis downstream of Lkb1, where its loss produces mitochondrial dysfunction, reduced axonal ATP, AMPK-Ulk1 activation and increased autophagy [PMID:32414840], and it shapes B-cell development by tuning the balance between oxidative phosphorylation and glycolysis [PMID:28524857]. In multiple carcinomas EFHD1 modulates Hippo/YAP signaling, acting via the MCU interaction and STARD13 to promote YAP phosphorylation [PMID:36747492] and via a SIK3-Hippo axis to suppress EMT and metastasis [PMID:39895792].","teleology":[{"year":2011,"claim":"Before direct characterization, EFHD1 was placed by sequence/structure prediction as a mitochondria-associated EF-hand protein potentially modulating apoptosis and oxidative-stress responses, framing the hypotheses later tested experimentally.","evidence":"Secondary-structure prediction and comparative analysis with literature review","pmids":["21244694"],"confidence":"Low","gaps":["No direct experimental validation in this work","Predicted coiled-coil and disordered regions not functionally tested","Mitochondrial association inferred, not demonstrated"]},{"year":2017,"claim":"Established EFHD1 as a bona fide inner-mitochondrial-membrane Ca2+-binding protein controlling mitoflashes and the OXPHOS/glycolysis balance, and connected it to a developmental switch in B cells.","evidence":"Knockdown/knockout in B-cell lines plus transgenic mouse, with metabolic flux (OCR/ECAR), membrane potential and ROS measurements","pmids":["28524857"],"confidence":"High","gaps":["Molecular partners mediating mitoflash regulation not identified here","Mechanism linking EFHD1 to PGC-1α not defined","Direct ion-binding stoichiometry not resolved in this study"]},{"year":2020,"claim":"Placed EFHD1 in an Lkb1-driven energy-homeostasis pathway that supports axonal growth, showing its loss causes mitochondrial dysfunction and AMPK-Ulk1-driven autophagy in neurons.","evidence":"Efhd1 knockout mice with axonal morphometry, ATP measurement, mitochondrial imaging, and pathway/autophagic-flux analysis","pmids":["32414840"],"confidence":"High","gaps":["Direct biochemical link between Lkb1 and EFHD1 not established","Molecular target of EFHD1 at the mitochondrion in neurons unidentified","Whether actin-binding contributes to the axonal phenotype untested"]},{"year":2021,"claim":"Solved the EFHD1 core structure and demonstrated Ca2+-independent actin binding with Ca2+-dependent actin bundling, defining its cytoskeletal biochemistry and a possible Zn2+-driven multimerization.","evidence":"X-ray crystallography plus in vitro actin-binding and bundling assays","pmids":["33537316"],"confidence":"High","gaps":["Zn2+-mediated multimerization inferred from crystal contacts, not confirmed in solution","Cellular relevance of actin bundling not addressed","Link between actin activity and mitochondrial roles not made"]},{"year":2023,"claim":"Added Zn2+ as a second regulatory ion, showing Zn2+ coordination within the EF-hands and Zn2+-dependent actin bundling, broadening the metal-sensing repertoire beyond Ca2+.","evidence":"X-ray crystallography with anomalous diffraction at the Zn K-edge plus actin assays","pmids":["36862489"],"confidence":"High","gaps":["Physiological Zn2+ concentrations driving this in cells not defined","Interplay of Ca2+ vs Zn2+ regulation unresolved","In vivo consequence of Zn2+ binding untested"]},{"year":2023,"claim":"Identified MCU as a direct N-terminal-domain partner through which EFHD1 suppresses mitochondrial Ca2+ uptake and, via STARD13, modulates Hippo/YAP signaling and cell migration.","evidence":"Co-IP plus overexpression/knockdown, rescue, mitochondrial Ca2+ measurement and migration assays in ccRCC","pmids":["36747492"],"confidence":"Medium","gaps":["Mechanism connecting MCU binding to STARD13 upregulation not defined","Single-lab interaction without reciprocal structural validation","Direct vs indirect nature of YAP regulation unclear"]},{"year":2024,"claim":"Defined a second mitochondrial effector mechanism: EFHD1 binds ANT3 and prevents its conformational change to inhibit mPTP opening, preserving mitochondrial function and promoting tumor-cell survival.","evidence":"Co-IP, knockdown/overexpression, viability assays, and pharmacological ANT3 modulation with CATR/BKA in osteosarcoma","pmids":["38795203"],"confidence":"Medium","gaps":["Structural basis of ANT3 conformational locking not resolved","Whether MCU and ANT3 binding are mutually exclusive unknown","Single-lab interaction evidence"]},{"year":2022,"claim":"Showed that EFHD1 loss lowers basal mitochondrial ROS and mitoflashes and protects cardiomyocytes from hypoxic injury, while leaving Ca2+-overload susceptibility unchanged, dissociating its ROS/mitoflash role from Ca2+-overload handling.","evidence":"Efhd1 knockout mouse with ROS, mitoflash imaging, cardiac phenotyping and hypoxia/Ca2+-overload assays","pmids":["35304170"],"confidence":"Medium","gaps":["Molecular source of the ROS phenotype not pinpointed","No basal cardiac phenotype limits interpretation","Relationship to MCU/ANT3 interactions not tested here"]},{"year":2025,"claim":"Extended EFHD1's cancer signaling roles, implicating a SIK3-Hippo axis in metastasis suppression and, separately, estrogen-receptor signaling in breast cancer proliferation.","evidence":"Overexpression/knockdown with genetic rescue (SIK3, ESR1) and in vivo metastasis models","pmids":["39895792","40441345"],"confidence":"Medium","gaps":["Biochemical mechanism linking EFHD1 to SIK3/ESR1 not established","ESR1 study is single-lab, Low-confidence","Reconciliation of pro- versus anti-tumor roles across cancers unresolved"]},{"year":2026,"claim":"Reframed EFHD1 as a coincidence detector that stabilizes ER-mitochondria contact sites, where pathological upregulation drives mitochondrial fragmentation, dsRNA escape and a PKR antiviral response in steatohepatitis.","evidence":"ERMCS and mitochondrial imaging, dsRNA/PKR analysis, EFHD1 inhibition in human and mouse MASH models, and Mendelian randomization (preprint)","pmids":["41756893"],"confidence":"Medium","gaps":["Preprint, not yet peer-reviewed","Direct molecular tethers at ERMCS bound by EFHD1 not identified","Causality of EFHD1 in human disease beyond genetic association unconfirmed"]},{"year":null,"claim":"How EFHD1's Ca2+/Zn2+-regulated actin-crosslinking activity is mechanistically coupled to its distinct mitochondrial partner interactions (MCU, ANT3) and to its divergent context-dependent signaling outputs remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model integrating actin, MCU, and ANT3 functions","Structural basis of inner-membrane localization undefined","Determinants of pro- vs anti-tumor behavior across tissues unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,3]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[5,6]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[5,4]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,7]}],"complexes":[],"partners":["MCU","ANT3","ACTB"],"other_free_text":[]}},"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":26,"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":20,"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":19,"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":9,"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":"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":"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":"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 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{\n      \"year\": 2021,\n      \"finding\": \"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, revealing two Zn2+ ions at the crystal contact interface suggesting Zn2+-mediated multimerization. EFhd1 was found to possess Ca2+-independent β-actin-binding activity and Ca2+-dependent β-actin-bundling activity in vitro.\",\n      \"method\": \"X-ray crystallography; in vitro actin-binding and actin-bundling assays\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure solved with functional biochemical validation (actin-binding and bundling assays), single rigorous paper with multiple orthogonal methods\",\n      \"pmids\": [\"33537316\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Crystal structures of EFhd1 and EFhd2 core domains coordinating Zn2+ ions within their EF-hands were determined. Anomalous signal analysis confirmed Zn2+ occupancy. EFhd1 exhibits Zn2+-independent actin-binding and Zn2+-dependent actin-bundling activity, demonstrating that actin-related activities can be regulated by Zn2+ in addition to Ca2+.\",\n      \"method\": \"X-ray crystallography with anomalous diffraction at Zn K-edge; in vitro actin-binding and bundling assays\",\n      \"journal\": \"IUCrJ\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with anomalous signal validation plus orthogonal biochemical assays in a single rigorous study\",\n      \"pmids\": [\"36862489\"],\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, suppressing mitochondrial Ca2+ uptake. This EFHD1-MCU interaction deactivates the Hippo/YAP signaling pathway by upregulating STARD13, which enhances phosphorylation of YAP at Ser-127. Knockdown of STARD13 or overexpression of MCU partially abrogated EFHD1-mediated YAP phosphorylation and suppression of cell migration in ccRCC cells.\",\n      \"method\": \"Co-immunoprecipitation; overexpression and knockdown experiments; in vitro migration/invasion assays; in vivo tumor metastasis assays; mitochondrial Ca2+ measurement; western blotting\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal interaction (Co-IP showing physical binding), multiple orthogonal functional assays (knockdown, overexpression, rescue), single lab\",\n      \"pmids\": [\"36747492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EFHD1 binds to adenine nucleotide translocase-3 (ANT3) and inhibits ANT3 conformational change, thereby inhibiting opening of the mitochondrial membrane permeability transition pore (mPTP). This maintains mitochondrial function and promotes osteosarcoma cell survival/drug resistance. Pharmacological modulation of ANT3 conformation (with CATR or BKA) confirmed the functional link.\",\n      \"method\": \"Co-immunoprecipitation; overexpression and knockdown; cell viability assays; pharmacological rescue with CATR and BKA\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP for physical interaction, pharmacological rescue experiments confirming mechanism, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"38795203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Genetic ablation of Efhd1 in mice caused reduced axonal morphogenesis (shorter axons, less branching) and enhanced neuronal death. Efhd1 KO sensory neurons exhibited mitochondrial dysfunction, decreased axonal ATP levels, shortened mitochondria at axonal growth cones, activation of the AMPK-Ulk1 pathway, and increased autophagic flux. Efhd1 was identified as downstream of Lkb1 (liver kinase B1) in an energy homeostasis pathway regulating axonal growth.\",\n      \"method\": \"Genetic knockout in mice; axonal morphogenesis assays; ATP measurement; transcriptome analysis; mitochondrial imaging; AMPK-Ulk1 pathway analysis; autophagic flux assay\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO mouse with defined cellular and molecular phenotype, multiple orthogonal readouts (ATP, mitochondrial morphology, pathway activation, autophagic flux), single rigorous study\",\n      \"pmids\": [\"32414840\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"EFhd1 is a Ca2+-binding protein localized at the inner mitochondrial membrane involved in Ca2+-induced mitoflashes. EFhd1 is expressed in pro-B cells and downregulated by pre-B cell receptor (pre-BCR) surface expression. Knockdown and knockout of EFhd1 in pro-B cells decreased the OCR/ECAR ratio by increasing glycolysis, glycolytic capacity, and reserve. Transgenic EFhd1 expression beyond the pro-B cell stage upregulated PGC-1α in primary pre-B cells but reduced mitochondrial ATP production, causing a B-cell intrinsic developmental disadvantage.\",\n      \"method\": \"Knockdown and knockout in B cell lines; transgenic mouse model; metabolic flux analysis (OCR/ECAR); mitochondrial membrane potential measurement; glucose uptake assays; ROS measurement\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO, KD, and transgenic overexpression with multiple metabolic readouts, replicated across cell lines and primary cells\",\n      \"pmids\": [\"28524857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Deletion of Efhd1 in mice (Efhd1-/-) reduced basal mitochondrial ROS levels and mitoflash events in cardiomyocytes without causing adverse cardiac phenotypes under basal conditions. Efhd1-/- mice and cardiomyocytes were resistant to hypoxic injury. However, cardiac mitochondria in Efhd1-/- mice showed normal susceptibility to Ca2+ overload.\",\n      \"method\": \"Knockout mouse model; ROS measurement; mitoflash imaging; cardiac phenotyping; hypoxia/ischemia models; mitochondrial Ca2+ overload assays\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO mouse with multiple orthogonal cellular phenotype readouts, single lab\",\n      \"pmids\": [\"35304170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"EFHD1 upregulates SIK3 expression, and SIK3 knockdown partially eliminated the inhibitory effects of EFHD1 on CRC metastasis. EFHD1 inhibited epithelial-mesenchymal transition (EMT) and CRC metastasis through the EFHD1-SIK3-Hippo signaling axis.\",\n      \"method\": \"Overexpression and knockdown; western blotting; immunohistochemistry; orthotopic xenograft and pulmonary metastasis mouse models; wound healing and Transwell assays\",\n      \"journal\": \"Journal of Cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis via genetic rescue (SIK3 KD abrogates EFHD1 effects) with in vivo validation, single lab\",\n      \"pmids\": [\"39895792\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"EFHD1, acting as a Ca2+-dependent actin crosslinker, stabilizes endoplasmic reticulum-mitochondria contact sites (ERMCS) by detecting spatiotemporal coincidence of inter-organellar proximity and ER Ca2+ release. During metabolic-associated steatohepatitis (MASH), EFHD1 upregulation drives pathological mitochondrial fragmentation via excessive contact persistence, promoting mitochondrial double-stranded RNA escape and activation of a PKR-dependent antiviral stress response. Inhibiting EFHD1 in human and mouse models blunted hepatocyte damage.\",\n      \"method\": \"Functional imaging of ERMCS; mitochondrial morphology analysis; dsRNA detection; PKR pathway analysis; EFHD1 inhibition in human and mouse MASH models; Mendelian randomization in humans\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (ERMCS imaging, dsRNA escape, pharmacological inhibition, human genetic evidence), preprint not yet peer-reviewed\",\n      \"pmids\": [\"41756893\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"EFHD1 promotes breast cancer cell proliferation, migration, and invasion through activation of estrogen signaling (ERα/ESR1 pathway). ESR1 knockdown abolished the pro-proliferative and pro-migratory effects of EFHD1 overexpression, placing EFHD1 upstream of ESR1 in this pathway.\",\n      \"method\": \"Overexpression and knockdown; EdU proliferation assay; flow cytometry (apoptosis); wound healing and Transwell assays; gene set enrichment analysis; western blotting; qRT-PCR; epistasis via ESR1 KD rescue\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, ESR1 rescue experiment supports pathway placement, but molecular mechanism linking EFHD1 to ERα activation not established biochemically\",\n      \"pmids\": [\"40441345\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EFhd1 (Swiprosin-2) was predicted by secondary structure analysis to contain an N-terminal disordered region, two EF-hands, and a coiled-coil domain. EFhd1 is proposed to associate with mitochondria and modulate apoptosis and differentiation of neuronal and muscle precursor cells, as part of a cellular response to oxidative stress.\",\n      \"method\": \"Secondary structure prediction; comparative evolutionary/structural analysis; review of prior literature\",\n      \"journal\": \"Cell communication and signaling : CCS\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational/structural prediction and review synthesis without direct experimental validation of EFhd1 mechanism in this paper\",\n      \"pmids\": [\"21244694\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EFHD1 is a Ca2+- and Zn2+-binding EF-hand protein of the inner mitochondrial membrane that crosslinks actin in a Ca2+-dependent manner, stabilizes ER-mitochondria contact sites, regulates mitochondrial Ca2+ uptake by physically binding MCU (via its N-terminal domain), inhibits mPTP opening by binding ANT3, controls mitoflash generation and ROS production, and thereby governs cellular energy metabolism, axonal morphogenesis, B-cell development, and protection from ischemic/metabolic injury.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"EFHD1 is a Ca2+- and Zn2+-binding EF-hand protein of the inner mitochondrial membrane that couples cytoskeletal and ion-handling activities to control mitochondrial function and cellular energy metabolism [#5, #0]. Structurally, its core comprises two EF-hands and a ligand-mimic helix, and it binds β-actin constitutively while bundling/crosslinking actin in a Ca2+- and Zn2+-dependent manner [#0, #1]. At the inner mitochondrial membrane EFHD1 participates in Ca2+-induced mitoflashes and governs reactive oxygen species production; loss of EFHD1 lowers basal mitochondrial ROS and mitoflash events and confers resistance to hypoxic injury [#5, #6]. It restrains mitochondrial Ca2+ uptake by physically binding the mitochondrial calcium uniporter MCU through its N-terminal domain [#2], and limits mitochondrial permeability transition pore opening by binding adenine nucleotide translocase ANT3 and blocking its conformational change, thereby preserving mitochondrial integrity [#3]. As a Ca2+-dependent actin crosslinker EFHD1 also stabilizes ER–mitochondria contact sites by sensing coincident inter-organellar proximity and ER Ca2+ release [#8]. Through these mitochondrial functions EFHD1 regulates axonal morphogenesis downstream of Lkb1, where its loss produces mitochondrial dysfunction, reduced axonal ATP, AMPK-Ulk1 activation and increased autophagy [#4], and it shapes B-cell development by tuning the balance between oxidative phosphorylation and glycolysis [#5]. In multiple carcinomas EFHD1 modulates Hippo/YAP signaling, acting via the MCU interaction and STARD13 to promote YAP phosphorylation [#2] and via a SIK3-Hippo axis to suppress EMT and metastasis [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Before direct characterization, EFHD1 was placed by sequence/structure prediction as a mitochondria-associated EF-hand protein potentially modulating apoptosis and oxidative-stress responses, framing the hypotheses later tested experimentally.\",\n      \"evidence\": \"Secondary-structure prediction and comparative analysis with literature review\",\n      \"pmids\": [\"21244694\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct experimental validation in this work\", \"Predicted coiled-coil and disordered regions not functionally tested\", \"Mitochondrial association inferred, not demonstrated\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established EFHD1 as a bona fide inner-mitochondrial-membrane Ca2+-binding protein controlling mitoflashes and the OXPHOS/glycolysis balance, and connected it to a developmental switch in B cells.\",\n      \"evidence\": \"Knockdown/knockout in B-cell lines plus transgenic mouse, with metabolic flux (OCR/ECAR), membrane potential and ROS measurements\",\n      \"pmids\": [\"28524857\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular partners mediating mitoflash regulation not identified here\", \"Mechanism linking EFHD1 to PGC-1α not defined\", \"Direct ion-binding stoichiometry not resolved in this study\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Placed EFHD1 in an Lkb1-driven energy-homeostasis pathway that supports axonal growth, showing its loss causes mitochondrial dysfunction and AMPK-Ulk1-driven autophagy in neurons.\",\n      \"evidence\": \"Efhd1 knockout mice with axonal morphometry, ATP measurement, mitochondrial imaging, and pathway/autophagic-flux analysis\",\n      \"pmids\": [\"32414840\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct biochemical link between Lkb1 and EFHD1 not established\", \"Molecular target of EFHD1 at the mitochondrion in neurons unidentified\", \"Whether actin-binding contributes to the axonal phenotype untested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Solved the EFHD1 core structure and demonstrated Ca2+-independent actin binding with Ca2+-dependent actin bundling, defining its cytoskeletal biochemistry and a possible Zn2+-driven multimerization.\",\n      \"evidence\": \"X-ray crystallography plus in vitro actin-binding and bundling assays\",\n      \"pmids\": [\"33537316\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Zn2+-mediated multimerization inferred from crystal contacts, not confirmed in solution\", \"Cellular relevance of actin bundling not addressed\", \"Link between actin activity and mitochondrial roles not made\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Added Zn2+ as a second regulatory ion, showing Zn2+ coordination within the EF-hands and Zn2+-dependent actin bundling, broadening the metal-sensing repertoire beyond Ca2+.\",\n      \"evidence\": \"X-ray crystallography with anomalous diffraction at the Zn K-edge plus actin assays\",\n      \"pmids\": [\"36862489\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological Zn2+ concentrations driving this in cells not defined\", \"Interplay of Ca2+ vs Zn2+ regulation unresolved\", \"In vivo consequence of Zn2+ binding untested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified MCU as a direct N-terminal-domain partner through which EFHD1 suppresses mitochondrial Ca2+ uptake and, via STARD13, modulates Hippo/YAP signaling and cell migration.\",\n      \"evidence\": \"Co-IP plus overexpression/knockdown, rescue, mitochondrial Ca2+ measurement and migration assays in ccRCC\",\n      \"pmids\": [\"36747492\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism connecting MCU binding to STARD13 upregulation not defined\", \"Single-lab interaction without reciprocal structural validation\", \"Direct vs indirect nature of YAP regulation unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined a second mitochondrial effector mechanism: EFHD1 binds ANT3 and prevents its conformational change to inhibit mPTP opening, preserving mitochondrial function and promoting tumor-cell survival.\",\n      \"evidence\": \"Co-IP, knockdown/overexpression, viability assays, and pharmacological ANT3 modulation with CATR/BKA in osteosarcoma\",\n      \"pmids\": [\"38795203\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of ANT3 conformational locking not resolved\", \"Whether MCU and ANT3 binding are mutually exclusive unknown\", \"Single-lab interaction evidence\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed that EFHD1 loss lowers basal mitochondrial ROS and mitoflashes and protects cardiomyocytes from hypoxic injury, while leaving Ca2+-overload susceptibility unchanged, dissociating its ROS/mitoflash role from Ca2+-overload handling.\",\n      \"evidence\": \"Efhd1 knockout mouse with ROS, mitoflash imaging, cardiac phenotyping and hypoxia/Ca2+-overload assays\",\n      \"pmids\": [\"35304170\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular source of the ROS phenotype not pinpointed\", \"No basal cardiac phenotype limits interpretation\", \"Relationship to MCU/ANT3 interactions not tested here\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended EFHD1's cancer signaling roles, implicating a SIK3-Hippo axis in metastasis suppression and, separately, estrogen-receptor signaling in breast cancer proliferation.\",\n      \"evidence\": \"Overexpression/knockdown with genetic rescue (SIK3, ESR1) and in vivo metastasis models\",\n      \"pmids\": [\"39895792\", \"40441345\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Biochemical mechanism linking EFHD1 to SIK3/ESR1 not established\", \"ESR1 study is single-lab, Low-confidence\", \"Reconciliation of pro- versus anti-tumor roles across cancers unresolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Reframed EFHD1 as a coincidence detector that stabilizes ER-mitochondria contact sites, where pathological upregulation drives mitochondrial fragmentation, dsRNA escape and a PKR antiviral response in steatohepatitis.\",\n      \"evidence\": \"ERMCS and mitochondrial imaging, dsRNA/PKR analysis, EFHD1 inhibition in human and mouse MASH models, and Mendelian randomization (preprint)\",\n      \"pmids\": [\"41756893\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"Direct molecular tethers at ERMCS bound by EFHD1 not identified\", \"Causality of EFHD1 in human disease beyond genetic association unconfirmed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How EFHD1's Ca2+/Zn2+-regulated actin-crosslinking activity is mechanistically coupled to its distinct mitochondrial partner interactions (MCU, ANT3) and to its divergent context-dependent signaling outputs remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model integrating actin, MCU, and ANT3 functions\", \"Structural basis of inner-membrane localization undefined\", \"Determinants of pro- vs anti-tumor behavior across tissues unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005743\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [5, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [5, 4]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"MCU\", \"ANT3\", \"ACTB\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}