{"gene":"CISD2","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2009,"finding":"CISD2 is primarily localized at the mitochondria (outer membrane), and Cisd2 deficiency in mice causes mitochondrial breakdown and dysfunction accompanied by autophagic cell death, leading to a premature aging phenotype. Mitochondrial degeneration is the direct consequence of Cisd2 loss.","method":"Cisd2 knockout mouse model with subcellular fractionation/localization, histology, and autophagy assays","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO mouse with defined cellular phenotype, replicated in multiple follow-up studies, direct localization tied to functional consequence","pmids":["19451219"],"is_preprint":false},{"year":2009,"finding":"CISD2/Miner1 is a homodimeric protein harboring two redox-active 2Fe-2S clusters bound by a rare Cys3-His motif within the NEET fold. Crystal structure resolved to 2.1 Å confirmed it is not a zinc finger as previously annotated. Redox potentials are proton-coupled (~0 mV at pH 7.5).","method":"X-ray crystallography (2.1 Å resolution), biophysical characterization, redox potential measurement","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional validation of Fe-S cluster chemistry, independently consistent with multiple subsequent structural/biochemical studies","pmids":["19580816"],"is_preprint":false},{"year":2009,"finding":"NAF-1 (CISD2) is an ER-localized BCL-2-interacting protein; NAF-1 contains a 2Fe-2S coordinating domain necessary (but not sufficient) for BCL-2 interaction. NAF-1 is required for BCL-2 at the ER to antagonize Beclin 1-dependent autophagy during nutrient deprivation. NAF-1 is also required for BCL-2-mediated depression of ER Ca²⁺ stores and associates with the IP3 receptor.","method":"Co-immunoprecipitation, knockdown/overexpression functional assays, ER Ca²⁺ measurements, autophagy flux assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, multiple orthogonal functional assays (autophagy, Ca²⁺, BIK displacement), replicated in follow-up studies","pmids":["20010695"],"is_preprint":false},{"year":2012,"finding":"NAF-1 (CISD2) knockout mice display early skeletal muscle degeneration with a shift toward slow-twitch fibers, augmented autophagy, dysregulated calcium homeostasis, and adaptive mitochondrial enlargement. This establishes NAF-1 as required for homeostatic maintenance of skeletal muscle via BCL-2-mediated autophagy regulation and ER Ca²⁺ flux.","method":"Naf-1 gene deletion mouse model, muscle physiology, fiber-type analysis, Ca²⁺ measurements, autophagy assays","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO mouse with multiple orthogonal phenotypic readouts including force generation, fiber typing, Ca²⁺ and autophagy","pmids":["22343142"],"is_preprint":false},{"year":2012,"finding":"Transgenic mice with a persistently high level of Cisd2 show extended median and maximum lifespan without deleterious effects, attenuate age-associated degeneration of skin, skeletal muscle and neurons, and protect mitochondria from age-associated damage. This places Cisd2 as a positive regulator of mammalian lifespan.","method":"Cisd2 transgenic (gain-of-function) mice, lifespan analysis, histology, mitochondrial function assays","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean transgenic mouse with lifespan readout plus multiple tissue and mitochondrial functional endpoints","pmids":["22661501"],"is_preprint":false},{"year":2013,"finding":"NAF-1 (CISD2) can transfer its 2Fe-2S cluster to an apo-acceptor protein in vitro (monitored by spectrophotometry and native PAGE) and transfer iron to intact mitochondria in cell models (monitored by fluorescence imaging with iron sensors). The anti-diabetes drug pioglitazone and resveratrol stabilize NAF-1's labile 2Fe-2S cluster and abrogate its cluster/iron transfer function.","method":"In vitro cluster transfer assay (spectrophotometry, native PAGE), cellular iron-transfer assay (fluorescence imaging), small-molecule binding","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of cluster transfer with multiple orthogonal detection methods, single lab but rigorous biochemical approach","pmids":["23717386"],"is_preprint":false},{"year":2013,"finding":"Loss of Miner1 (CISD2) in mouse embryonic fibroblasts causes ER stress, unfolded protein response, depletion of ER Ca²⁺ stores, increased mitochondrial Ca²⁺ load, increased ROS/RNS, increased GSSG/GSH and NAD⁺/NADH ratios, and altered mitochondrial ultrastructure (increased cristae density, punctate morphology). N-acetylcysteine treatment reversed these abnormalities, implicating sulfhydryl redox status as a key mechanism.","method":"Miner1−/− mouse embryonic fibroblasts, ER stress/UPR markers, Ca²⁺ measurements, redox assays, electron microscopy, NAC rescue","journal":"EMBO molecular medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO cells with multiple orthogonal mechanistic readouts (Ca²⁺, redox, UPR, ultrastructure) plus chemical rescue","pmids":["23703906"],"is_preprint":false},{"year":2013,"finding":"NAF-1 (CISD2) and mitoNEET (CISD1) protein levels are elevated in human epithelial breast cancer cells. shRNA suppression of NAF-1 or mitoNEET causes reduced cell proliferation and tumor growth, decreased mitochondrial performance, uncontrolled accumulation of iron and ROS in mitochondria, and activation of autophagy.","method":"shRNA knockdown in breast cancer cells and xenograft models, mitochondrial function assays, iron/ROS imaging, autophagy assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — shRNA KD with defined cellular phenotype across multiple orthogonal endpoints, validated in xenograft in vivo","pmids":["23959881"],"is_preprint":false},{"year":2014,"finding":"The NAF-1 H114C mutant (His114→Cys in the 2Fe-2S cluster binding site) produces clusters that are 25-fold more stable, have a redox potential 300 mV more negative, and have abolished cluster donation/transfer function, with no global structural differences from wild-type. This identifies His114 as critical for cluster lability and transfer activity.","method":"X-ray crystallography (1.65 Å native; 1.58 Å H114C mutant), redox potential measurement, in vitro cluster transfer assay, site-directed mutagenesis","journal":"Acta crystallographica. Section D, Biological crystallography","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures at near-atomic resolution combined with active-site mutagenesis and in vitro functional assay","pmids":["24914968"],"is_preprint":false},{"year":2014,"finding":"NAF-1 binds to both the pro- and anti-apoptotic regions (BH3 and BH4 domains) of BCL-2. The interaction interface of the NAF-1–BCL-2 complex was mapped at amino acid resolution using peptide array, deuterium exchange mass spectrometry (DXMS), and direct coupling analysis (DCA).","method":"Peptide array screening, deuterium exchange mass spectrometry (DXMS), direct coupling analysis (DCA), functional binding studies","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple orthogonal structural/biochemical methods (DXMS + peptide array + DCA) defining protein–protein interaction interface","pmids":["24706857"],"is_preprint":false},{"year":2014,"finding":"Cisd2 interacts with Gimap5 on mitochondrial and ER membranes and modulates mitochondrial Ca²⁺ uptake, thereby maintaining intracellular Ca²⁺ homeostasis in adipocytes. Loss of Cisd2 increases cytosolic Ca²⁺ and activates Ca²⁺-calcineurin-dependent signaling that inhibits adipogenesis and impairs insulin-stimulated glucose uptake.","method":"Adipocyte-specific Cisd2 KO mice, co-immunoprecipitation (interaction with Gimap5), Ca²⁺ measurements, adipogenesis and glucose uptake assays","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean tissue-specific KO plus Co-IP identifying Gimap5 partner, multiple functional endpoints; single lab","pmids":["24833725"],"is_preprint":false},{"year":2015,"finding":"MAD-28 (a cluvenone derivative) binds to and destabilizes NAF-1 (and mitoNEET) by breaking the coordinative bond between His ligand and the cluster Fe. This causes decreased respiration, decreased mitochondrial membrane potential, and increased mitochondrial iron content in cancer cells, phenocopying NAF-1/mitoNEET shRNA suppression.","method":"Molecular docking, functional assays in breast cancer cells (respiration, membrane potential, iron content), shRNA comparison","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — docking combined with functional cellular assays and genetic validation (shRNA cells less susceptible), single lab","pmids":["25762074"],"is_preprint":false},{"year":2015,"finding":"NAF-1 suppression in epithelial breast cancer cells by shRNA activates apoptosis, increases cellular Fe²⁺ uptake, causes a metabolic shift increasing susceptibility to glycolysis inhibition, and activates stress pathways associated with HIF1α.","method":"shRNA knockdown in breast cancer cells and xenograft tumors, iron imaging, metabolic assays, apoptosis assays","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — shRNA KD with multiple cellular readouts including iron distribution, metabolism, and apoptosis; single lab","pmids":["26621032"],"is_preprint":false},{"year":2015,"finding":"NAF-1 antagonizes starvation-induced autophagy in cardiomyocytes by promoting the Beclin1–BCL-2 interaction (shown by co-immunoprecipitation) and inhibiting AMPK activity. Overexpression of NAF-1 was sufficient to inhibit autophagy and protect cardiomyocytes from nutrient-stress–induced cell death.","method":"Co-immunoprecipitation (Beclin1–BCL-2 interaction), NAF-1 overexpression, autophagy assays, AMPK activity assay in neonatal rat cardiomyocytes","journal":"Cell biology international","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP plus functional OE assay with defined cellular readout (autophagy, AMPK, cell death); single lab","pmids":["25689847"],"is_preprint":false},{"year":2017,"finding":"CISD2 interacts with SERCA2b and modulates its Ca²⁺ pump activity via regulation of SERCA2b oxidative modifications, thereby controlling ER Ca²⁺ uptake and maintaining Ca²⁺ homeostasis in hepatocytes. Cisd2 haploinsufficiency disrupts this, causing ER stress and NAFLD/NASH.","method":"Co-immunoprecipitation (Cisd2–Serca2b interaction), Serca2b activity assay, oxidative modification analysis, Cisd2 heterozygous and overexpressing mice with liver phenotype readouts","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP of interaction plus biochemical activity assay of Serca2b, validated in haploinsufficiency and overexpression mouse models","pmids":["29166610"],"is_preprint":false},{"year":2017,"finding":"A novel CISD2 missense mutation (p.Asn72Ser) disturbs cellular Ca²⁺ homeostasis with enhanced Ca²⁺ flux from ER to mitochondria and cytosolic Ca²⁺ abnormalities in patient-derived fibroblasts. This Ca²⁺ dysregulation is associated with increased ER-mitochondria contact, swollen ER lumen, and hyperfused mitochondrial network, and reveals a respiratory chain defect under metabolic stress.","method":"Patient-derived fibroblasts, Ca²⁺ imaging, electron microscopy (ER-mitochondria contacts), Seahorse respirometry under galactose medium","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient-derived cells with multiple orthogonal mechanistic assays (Ca²⁺ flux, EM, bioenergetics); single lab","pmids":["28335035"],"is_preprint":false},{"year":2017,"finding":"mitoNEET (CISD1) and NAF-1 (CISD2) directly interact in mammalian cells (demonstrated by yeast two-hybrid, bimolecular fluorescence complementation, and direct coupling analysis). mitoNEET can transfer its 2Fe-2S clusters to NAF-1 in vitro, consistent with a cluster relay mechanism.","method":"Yeast two-hybrid, in vivo BiFC, DCA, in vitro cluster transfer assay, shRNA double-knockdown lines with ROS/iron imaging","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal interaction assays (Y2H + BiFC + DCA) plus in vitro cluster transfer reconstitution","pmids":["28426722"],"is_preprint":false},{"year":2018,"finding":"CISD2 overexpression confers resistance to sulfasalazine-induced ferroptosis in head and neck cancer cells, while CISD2 silencing increases sensitivity via increased mitochondrial ferrous iron and lipid ROS accumulation, identifying CISD2 as a regulator of mitochondrial iron and ferroptosis susceptibility.","method":"CISD2 overexpression and siRNA knockdown, ferroptosis assays (lipid ROS, mitochondrial iron), mouse xenograft models","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function with mechanistic readouts (mitochondrial iron, lipid ROS) validated in vivo; single lab","pmids":["29928961"],"is_preprint":false},{"year":2018,"finding":"iASPP and NAF-1 (CISD2) interact in cancer cells during apoptosis. The interaction interface maps to residues 764–778 of iASPP binding to a surface groove of NAF-1, identified by peptide array screening and computational methods. A peptide corresponding to iASPP 764–780 stabilizes the NAF-1 cluster, inhibits NAF-1–iASPP interaction, and inhibits staurosporine-induced apoptosis in breast and prostate cancer cells.","method":"Peptide array screening, computational docking, Co-IP in cells, peptide functional assay (apoptosis inhibition, IC50 determination)","journal":"Chemical science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — peptide array + computational mapping + cellular functional validation; single lab","pmids":["30774867"],"is_preprint":false},{"year":2019,"finding":"Cisd2 deficiency in the heart causes intercalated disc defects, mitochondrial and sarcomere degeneration, and disrupts Ca²⁺ homeostasis via dysregulation of Serca2a activity, resulting in increased basal cytosolic Ca²⁺ and mitochondrial Ca²⁺ overload in cardiomyocytes. Persistent high Cisd2 expression delays cardiac aging.","method":"Cisd2 KO and transgenic mice, cardiac electrophysiology, Ca²⁺ imaging, Serca2a activity assay, electron microscopy","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO and TG mouse models with multiple orthogonal cardiac and molecular readouts including Serca2a enzyme activity","pmids":["31593566"],"is_preprint":false},{"year":2021,"finding":"Inducible disruption of CISD2 function (using dominant-negative H114C) causes an immediate disruption in mitochondrial labile iron (mLI), followed by enhanced mitochondrial ROS. Alterations in cytosolic and ER Ca²⁺ levels occur only after the changes in mLI and mROS, indicating iron is upstream of Ca²⁺ signaling. CISD2 disruption also triggers TXNIP expression in a mLI-dependent manner.","method":"Inducible expression system for dominant-negative CISD2 H114C, mitochondrial labile iron imaging, mROS assays, Ca²⁺ measurements, TXNIP expression analysis; temporal hierarchy established","journal":"Free radical biology & medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — inducible system allowing temporal dissection of signaling hierarchy; multiple orthogonal readouts (iron, ROS, Ca²⁺, TXNIP)","pmids":["34547371"],"is_preprint":false},{"year":2021,"finding":"CISD2 is targeted to the ER by its N-terminal sequence and is retained there by the combined action of a C-terminal COPI-binding KKxx ER retrieval motif and an ER-targeting transmembrane domain, distinguishing it from CISD1 (mitoNEET) which uses different targeting motifs to reach mitochondria.","method":"Recombinant antibodies, localization of protein chimeras (domain swaps), fluorescence microscopy","journal":"BMC molecular and cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — chimeric protein dissection with direct localization readout; single lab","pmids":["34587896"],"is_preprint":false},{"year":2021,"finding":"CISD2 protein can be localized on the ER, outer mitochondrial membrane (OMM), and mitochondria-associated membrane (MAM), and plays a crucial role in regulating cytosolic Ca²⁺ homeostasis, ER integrity, and mitochondrial function. These three localizations underlie its roles in lifespan, cell death, and disease.","method":"Subcellular fractionation, multiple mouse models (reviewed), functional assays for Ca²⁺, ER stress, mitochondria","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — synthesis of direct experimental evidence from multiple studies; review article but citing primary experimental data","pmids":["33422617"],"is_preprint":false},{"year":2021,"finding":"NAF-1 repression in INS-1E pancreatic β-cells inhibits insulin secretion, impairs mitochondrial and ER structure/function, and induces ferroptosis-like features. Combined treatment with deferiprone (iron chelator) and N-acetylcysteine (glutathione precursor) restores insulin secretion and repairs mitochondrial and ER structure by reducing mitochondrial labile iron and ROS.","method":"shRNA repression in INS-1E cells, insulin secretion assay, iron chelation/NAC rescue, ferroptosis inhibitor (ferrostatin-1), mitochondrial/ER ultrastructure","journal":"Antioxidants (Basel, Switzerland)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD with multiple mechanistic readouts and pharmacological rescue; single lab","pmids":["34439408"],"is_preprint":false},{"year":2021,"finding":"CISD2 knockdown promotes ferroptosis through two parallel mechanisms: (1) ferritinophagy-dependent ferritin degradation causing free iron accumulation; (2) degradation of p62 that increases Keap1-NRF2 binding leading to NRF2 ubiquitination/degradation, reducing FTH and HO-1 expression and increasing oxidative stress.","method":"shRNA and overexpression in cancer cells, confocal microscopy, western blot for ferritin/p62/Keap1/NRF2, lysosomal inhibition rescue, in vivo xenograft","journal":"Cellular & molecular biology letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss- and gain-of-function with multiple mechanistic pathway dissection and pharmacological rescue; single lab","pmids":["36180832"],"is_preprint":false},{"year":2021,"finding":"CISD2 knockdown promotes sorafenib-induced ferroptosis in resistant HCC cells via uncontrolled autophagy (autophagy-mediated iron accumulation), in a Beclin1-dependent manner. Beclin1 co-knockdown attenuates the ferroptotic effect of CISD2 knockdown.","method":"shRNA knockdown of CISD2 and Beclin1, ferroptosis assays (ROS, MDA, GSH, iron), western blot for autophagy markers","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — double KD epistasis establishing Beclin1-dependent pathway; single lab","pmids":["34485112"],"is_preprint":false},{"year":2021,"finding":"CISD2 deficiency in cardiomyocytes (CISD2-null mice) causes accumulation of high levels of iron, increased transferrin receptor and ferritin, and features of cardiomyocyte aging, demonstrating that CISD2 protects cardiomyocytes from iron overaccumulation.","method":"CISD2-null mice, proteomics, transmission electron microscopy, iron and transferrin receptor/ferritin measurement","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO mouse with proteomics and direct iron quantification; single lab","pmids":["34997963"],"is_preprint":false},{"year":2021,"finding":"CISD2 and mitoNEET exhibit distinct intracellular half-lives and differ in the pH sensitivity of their Fe-S cluster stability and ability to transfer clusters in vitro, despite close structural homology, suggesting distinct cellular roles.","method":"Cellular half-life measurement, in vitro cluster stability and transfer assays at varying pH, expression profiling across tissues","journal":"Biomedicines","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical assays plus cellular half-life measurement; single lab","pmids":["33916457"],"is_preprint":false},{"year":2024,"finding":"CISD2 directly interacts with BCL-2's BH4 domain with submicromolar affinity (using purified proteins). CISD2 overexpression enhanced BCL-2-mediated suppression of cytosolic IP3R-mediated Ca²⁺ release. Most strikingly, CISD2 counteracts BCL-2-mediated inhibition of ER-mitochondrial Ca²⁺ transfer: BCL-2 overexpression reduced ER-mitochondrial Ca²⁺ transfer and contact sites, but co-expression of CISD2 abolished these BCL-2 effects. CISD2 was not essential for BCL-2's anti-apoptotic function or BAX pore formation inhibition.","method":"Purified protein direct binding assay (submicromolar affinity), Ca²⁺ signaling assays (cytosolic and mitochondrial), ER-mitochondria contact site quantification, BAX pore formation assay, loss-of-function in cells","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct binding with purified proteins plus multiple orthogonal cellular functional assays (Ca²⁺ transfer, contact sites, apoptosis); rigorous controls including negative findings","pmids":["39370046"],"is_preprint":false},{"year":2024,"finding":"Cisd2 interacts with Calnexin (identified by mass spectrometry), and this interaction along with Calnexin–SERCA modulates Ca²⁺ homeostasis in neutrophils. Cisd2 KO mice neutrophils display Ca²⁺ dysregulation and functional defects despite elevated numbers.","method":"Mass spectrometry (Cisd2–Calnexin interaction), Cisd2 KO mouse neutrophil functional assays, Ca²⁺ measurements","journal":"BMB reports","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — MS-identified interaction plus KO functional phenotype; single lab, interaction not confirmed by reciprocal Co-IP","pmids":["38627949"],"is_preprint":false},{"year":2024,"finding":"KAT8/MSL acetyltransferase complex acetylates CISD2 at K74, preventing STUB1-mediated ubiquitination and degradation at K105. Acetylation at K74 preserves mitochondrial homeostasis. Ginsenoside Rg5 binds KAT8 and promotes CISD2 acetylation, maintaining mitochondrial function and alleviating senescence.","method":"SILAC-based acetyl-proteomics, Co-IP, GST pull-down, in vitro lysine acetyltransferase assay, site-directed mutagenesis (K74, K105), cycloheximide chase, CETSA, mitochondrial function assays, C. elegans genetic validation","journal":"Phytomedicine : international journal of phytotherapy and phytopharmacology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro acetyltransferase assay plus mutagenesis plus multiple orthogonal methods (SILAC, Co-IP, GST pulldown, CETSA); cross-species genetic validation in C. elegans","pmids":["41687537"],"is_preprint":false},{"year":2024,"finding":"PRKN (Parkin) E3 ubiquitin ligase mediates ubiquitination and proteasomal degradation of CISD2 in porcine oocytes. ETA treatment decreases PRKN expression, reducing CISD2 ubiquitination and increasing CISD2 levels. CISD2 inhibits IP3R-mediated Ca²⁺ release (in a BCL-2-dependent manner) at MAMs, and its knockdown blocks ETA's ability to inhibit IP3R.","method":"Immunoprecipitation (CISD2–PRKN interaction, ubiquitination), siRNA knockdown of CISD2, transcriptomic sequencing, IP3R functional assays, MAM imaging","journal":"Theriogenology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP demonstrating ubiquitination plus functional siRNA rescue epistasis; single lab","pmids":["39357167"],"is_preprint":false},{"year":2025,"finding":"Beta-cell–specific Cisd2 KO in mice disrupts glucose-induced extracellular Ca²⁺ influx, impairing Ca²⁺-mediated insulin secretory signaling, causing mitochondrial dysfunction and reduced insulin secretion. Cisd2 deficiency also suppresses Glis3 and Hnf1a transcription regulators critical for β-cell function.","method":"β-cell–specific Cisd2 KO mice, CRISPR-mediated Cisd2KO MIN6 β-cell line, Ca²⁺ imaging, insulin secretion assays, transcriptomic analysis","journal":"Molecular metabolism","confidence":"High","confidence_rationale":"Tier 2 / Strong — tissue-specific KO plus cell-line KO with Ca²⁺ mechanistic readout and transcriptomic pathway analysis; two model systems","pmids":["40189101"],"is_preprint":false},{"year":2025,"finding":"CISD2 loss in HeLa cells reduces ER-mitochondrial Ca²⁺ transfer. In human iPSC-derived cortical neurons, Cisd2 deficiency severely reduces glutamate-evoked cytosolic and mitochondrial Ca²⁺ responses due to loss of ER-mitochondria contact sites, causing mitochondrial dysfunction (reduced OCR, ATP, membrane potential) and increased apoptotic sensitivity. CISD2 interacts with IP3 receptors.","method":"Cisd2 KO HeLa cells and human iPSC-derived cortical neurons, Ca²⁺ imaging (cytosolic and mitochondrial), ER-mitochondria contact site quantification, Seahorse respirometry, Co-IP (Cisd2–IP3R interaction), apoptosis assays","journal":"Acta neuropathologica communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple cell systems including disease-relevant neurons, orthogonal mechanistic readouts (Ca²⁺ transfer, contact sites, bioenergetics, apoptosis), Co-IP for interaction","pmids":["41299767"],"is_preprint":false},{"year":2025,"finding":"Smad3 regulates CISD2 expression by directly binding its promoter region (ChIP-seq validated). CISD2 overexpression in Smad3-knockdown mesenchymal bladder cancer cells rescues ferroptosis markers (Fe²⁺, ROS, lipid peroxides, MDA and GSH levels), placing CISD2 downstream of Smad3 in a ferroptosis-regulatory pathway.","method":"ChIP-seq, RNA-seq, Smad3 knockdown, CISD2 overexpression rescue, ferroptosis assays, clinical specimen correlation, xenograft","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-seq plus epistasis rescue experiments; single lab","pmids":["41413023"],"is_preprint":false},{"year":2025,"finding":"The NAF-1 44-67 cancer-targeting peptide (derived from CISD2 residues 44–67) targets the CISD2/NAF-1 protein inside cancer cells and disrupts its homodimeric structure. Dimers of the peptide have higher anticancer activity than monomers. A homologous peptide from CISD1 (mitoNEET) has no cancer-killing activity, indicating specificity for NAF-1 structure.","method":"Peptide treatment of cancer cells, structural disruption assays of CISD2 dimer, comparison with CISD1 peptide, dimer vs. monomer activity assays","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic peptide–protein interaction with structure–activity comparison; single lab","pmids":["40118242"],"is_preprint":false},{"year":2014,"finding":"Genetic epistasis in Drosophila shows that altered function of the CISD2 orthologue (cisd2) modifies the phenotypic effects of overexpressing PPT1 and CLN3 orthologues on eye morphology, and overexpression of CLN3 combined with cisd2 loss-of-function disrupts locomotor ability, placing cisd2 in a functional network with lysosomal storage disease genes.","method":"Drosophila genetics (RNAi knockdown, overexpression, epistasis/modifier screen), eye morphology assay, locomotor assay","journal":"Biology open","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Drosophila epistasis screen, single lab, no direct biochemical mechanism determined","pmids":["24705017"],"is_preprint":false},{"year":2015,"finding":"CISD2 knockdown in neural cells by siRNA causes increased iNOS expression and decreased BCL-2 expression in an LPS-challenged model, suggesting CISD2 functions as a suppressor of nitric oxide/inflammatory signaling upstream of BCL-2. CISD2 knockdown reduces the anti-inflammatory and anti-apoptotic effects of curcumin.","method":"siRNA knockdown in neural cells, LPS challenge, iNOS and BCL-2 western blot, SCI rat model","journal":"Injury","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single siRNA knockdown readout for iNOS/BCL-2, single lab, no direct binding or pathway reconstruction","pmids":["26387034"],"is_preprint":false},{"year":2020,"finding":"CISD2 knockdown in EOC microglial cells causes augmented proinflammatory signaling, decreased M2 phenotype markers (Arg-1, Ym1, IL-10, BCL2) and increased NF-κB p65 DNA-binding activity, placing CISD2 as an upstream anti-inflammatory modulator of the NF-κB pathway in microglia.","method":"siRNA knockdown in EOC microglial cells, cytokine/marker western blot and RT-PCR, NF-κB ELISA DNA-binding assay","journal":"Frontiers in aging neuroscience","confidence":"Low","confidence_rationale":"Tier 3 / Weak — siRNA knockdown with downstream marker readouts only; single lab, no direct molecular interaction demonstrated","pmids":["33005144"],"is_preprint":false}],"current_model":"CISD2 (NAF-1/Miner1) is a homodimeric, redox-active 2Fe-2S cluster protein localized to the ER, outer mitochondrial membrane, and mitochondria-associated membranes (MAMs), where it (1) transfers labile 2Fe-2S clusters to acceptor proteins and iron to mitochondria via a His114-dependent mechanism; (2) interacts directly with BCL-2 (via BH3/BH4 domains), IP3 receptors, SERCA2a/2b, and Calnexin to maintain ER Ca²⁺ uptake, ER-to-mitochondria Ca²⁺ transfer, and intracellular Ca²⁺ homeostasis; (3) acts as a BCL-2 co-factor at the ER to suppress Beclin 1-dependent autophagy; (4) controls mitochondrial labile iron levels upstream of mitochondrial ROS and ferroptosis; (5) is acetylated by KAT8 at K74 (preventing STUB1-mediated ubiquitination/degradation) and ubiquitinated by PRKN; and (6) is transcriptionally regulated by E2F7, Smad3, TAF1/TCF12, and Glis3/Hnf1a in specific cellular contexts—with its loss causing mitochondrial dysfunction, dysregulated Ca²⁺, ER stress, autophagy, ferroptosis, and premature aging across multiple tissues."},"narrative":{"mechanistic_narrative":"CISD2 (NAF-1/Miner1) is a redox-active 2Fe-2S iron-sulfur protein that integrates iron handling, ER-mitochondrial calcium transfer, and autophagy/cell-death control at the interface of the ER, outer mitochondrial membrane, and mitochondria-associated membranes (MAMs), with its loss driving mitochondrial degeneration and premature aging [PMID:19451219, PMID:33422617]. Structurally it adopts the NEET fold and coordinates two labile 2Fe-2S clusters through a rare Cys3-His motif rather than the originally annotated zinc finger [PMID:19580816]; the His114 ligand confers cluster lability, and its mutation abolishes the ability to donate clusters to apo-acceptor proteins and to deliver iron to mitochondria in cells [PMID:23717386, PMID:24914968]. Using an inducible dominant-negative H114C system, disruption of CISD2 first perturbs mitochondrial labile iron and then mitochondrial ROS, with calcium changes occurring downstream, establishing iron as the proximal output of cluster transfer [PMID:34547371]. CISD2 partners physically with BCL-2 through its BH3/BH4 domains and with the IP3 receptor, SERCA pumps, and Calnexin to govern ER calcium uptake and ER-to-mitochondria calcium transfer: it acts as a BCL-2 co-factor at the ER to suppress Beclin 1-dependent autophagy, yet counteracts BCL-2-mediated inhibition of ER-mitochondrial calcium transfer and contact-site formation [PMID:20010695, PMID:24706857, PMID:39370046, PMID:41299767]. Loss of CISD2 produces ER stress, depleted ER calcium, mitochondrial calcium overload, redox imbalance, and sensitization to ferroptosis through dysregulated mitochondrial iron and autophagy-dependent iron accumulation [PMID:23703906, PMID:29928961, PMID:34547371, PMID:36180832]. CISD2 stability is set by post-translational control—KAT8-mediated K74 acetylation blocks STUB1-dependent ubiquitination, while PRKN promotes its ubiquitin-dependent degradation [PMID:41687537, PMID:39357167]—and its sustained expression extends mammalian lifespan and protects skeletal muscle, heart, neurons, β-cells, and other tissues from age-associated degeneration [PMID:22661501, PMID:31593566, PMID:40189101].","teleology":[{"year":2009,"claim":"Establishing that CISD2 is a mitochondrial protein whose loss causes organelle breakdown defined it as an essential maintainer of mitochondrial integrity and a determinant of aging.","evidence":"Cisd2 knockout mouse with fractionation, histology, and autophagy assays","pmids":["19451219"],"confidence":"High","gaps":["Did not resolve the molecular activity underlying mitochondrial protection","ER/MAM localization not yet appreciated"]},{"year":2009,"claim":"Solving the crystal structure reclassified CISD2 from a presumed zinc finger to a redox-active 2Fe-2S NEET protein, defining the chemical basis for its function.","evidence":"X-ray crystallography at 2.1 Å with redox potential measurement","pmids":["19580816"],"confidence":"High","gaps":["Structure alone did not establish a cluster acceptor or physiological redox partner"]},{"year":2009,"claim":"Identifying CISD2 as an ER-localized BCL-2 partner required for suppressing Beclin 1 autophagy and depressing ER calcium linked its cluster domain to organelle signaling control.","evidence":"Co-IP, knockdown/overexpression, ER Ca2+ and autophagy assays","pmids":["20010695"],"confidence":"High","gaps":["Did not map the BCL-2 binding interface","Whether the Fe-S cluster itself participates in calcium control was unresolved"]},{"year":2012,"claim":"Tissue phenotyping and a gain-of-function lifespan study showed CISD2 dosage bidirectionally controls aging—loss degenerates muscle, sustained expression extends lifespan.","evidence":"Naf-1 KO and Cisd2 transgenic mice with muscle physiology, lifespan, and mitochondrial readouts","pmids":["22343142","22661501"],"confidence":"High","gaps":["The molecular event by which CISD2 levels set lifespan was not identified"]},{"year":2013,"claim":"Demonstrating in vitro cluster donation to apo-acceptors and cellular iron delivery to mitochondria established CISD2 as a functional iron-sulfur transfer protein, not merely a structural Fe-S holder.","evidence":"In vitro cluster transfer (spectrophotometry, native PAGE) and cellular iron-sensor imaging with small-molecule stabilizers","pmids":["23717386"],"confidence":"High","gaps":["Physiological acceptor protein in cells not defined","Link between transfer activity and the calcium/autophagy phenotypes not yet drawn"]},{"year":2013,"claim":"Knockout MEF studies tied CISD2 loss to ER calcium depletion, mitochondrial calcium overload, UPR, and redox imbalance reversible by N-acetylcysteine, establishing a coupled calcium/redox dysfunction.","evidence":"Miner1−/− MEFs with Ca2+, redox, UPR, EM, and NAC rescue","pmids":["23703906"],"confidence":"High","gaps":["Causal ordering of iron, ROS, and calcium not established","Direct calcium-machinery partners not yet identified"]},{"year":2013,"claim":"Showing CISD2 is overexpressed in breast cancer and required for proliferation, mitochondrial performance, and iron/ROS control connected its iron handling to a pro-tumorigenic role.","evidence":"shRNA knockdown in breast cancer cells and xenografts with mitochondrial, iron/ROS, and autophagy readouts","pmids":["23959881"],"confidence":"High","gaps":["Did not distinguish cluster-transfer from protein-interaction contributions to tumor growth"]},{"year":2014,"claim":"His114 mutagenesis pinpointed the cluster ligand that confers lability and transfer activity, providing a tool to separate CISD2's enzymatic function from its scaffold role.","evidence":"High-resolution crystallography of native and H114C plus redox and in vitro transfer assays","pmids":["24914968"],"confidence":"High","gaps":["In vivo consequence of stabilized clusters not tested in this study"]},{"year":2014,"claim":"Amino-acid-resolution mapping showed CISD2 engages both BH3 and BH4 domains of BCL-2, defining the structural basis of the co-factor interaction.","evidence":"Peptide array, DXMS, and direct coupling analysis","pmids":["24706857"],"confidence":"High","gaps":["Functional consequence of dual-domain binding not resolved here"]},{"year":2014,"claim":"Adipocyte studies added Gimap5 as a CISD2 membrane partner and linked CISD2 to calcineurin signaling, adipogenesis, and insulin-stimulated glucose uptake.","evidence":"Adipocyte-specific KO mice and Co-IP with metabolic readouts","pmids":["24833725"],"confidence":"Medium","gaps":["Gimap5 interaction from single lab without reciprocal validation","Mechanistic link to cluster chemistry not addressed"]},{"year":2015,"claim":"Small-molecule destabilization of the CISD2 cluster (MAD-28) phenocopied genetic loss in cancer cells, validating the cluster bond as a druggable functional node.","evidence":"Docking and functional cancer-cell assays compared to shRNA cells","pmids":["25762074"],"confidence":"Medium","gaps":["Compound selectivity over CISD1 limited","In vivo efficacy not established in this study"]},{"year":2015,"claim":"Further cancer and cardiomyocyte work tied CISD2 loss to iron uptake, metabolic vulnerability, HIF1α stress, and Beclin1–BCL-2-dependent autophagy suppression via AMPK.","evidence":"shRNA/overexpression with iron imaging, metabolic, apoptosis, and Co-IP autophagy assays","pmids":["26621032","25689847"],"confidence":"Medium","gaps":["AMPK regulation mechanism not biochemically dissected","Single-lab functional readouts"]},{"year":2016,"claim":"Demonstrating CISD2 silencing accelerates ferroptosis via ferritinophagy and p62/Keap1/NRF2 disruption defined parallel routes by which CISD2 restrains iron-dependent death.","evidence":"shRNA/overexpression with ferritin/p62/Keap1/NRF2 blotting, lysosomal inhibition rescue, and xenograft","pmids":["36180832"],"confidence":"Medium","gaps":["Direct molecular link between CISD2 and ferritinophagy machinery not shown","Single lab"]},{"year":2017,"claim":"Identifying CISD2–SERCA2b interaction and regulation of SERCA oxidative state provided a direct mechanism for ER calcium uptake control with disease consequences in liver.","evidence":"Co-IP, SERCA2b activity and oxidation assays in haploinsufficient and overexpressing mice","pmids":["29166610"],"confidence":"High","gaps":["How the Fe-S redox state of CISD2 couples to SERCA oxidation not defined"]},{"year":2017,"claim":"A patient CISD2 missense mutation linked human disease to enhanced ER-to-mitochondria calcium flux, altered organelle contacts, and a stress-dependent respiratory defect.","evidence":"Patient fibroblasts with Ca2+ imaging, EM, and respirometry","pmids":["28335035"],"confidence":"Medium","gaps":["Causal mutation effect on cluster chemistry not tested","Single patient-derived line"]},{"year":2017,"claim":"Showing CISD1 transfers clusters to CISD2 with distinct pH-dependent stabilities revealed a possible NEET cluster relay and divergent cellular roles between the paralogs.","evidence":"Y2H, BiFC, DCA, in vitro transfer, and half-life/pH-stability comparisons","pmids":["28426722","33916457"],"confidence":"High","gaps":["Physiological significance of CISD1→CISD2 relay in vivo unresolved"]},{"year":2018,"claim":"CISD2 was placed in ferroptosis and apoptosis regulation in cancer through control of mitochondrial ferrous iron and a mapped iASPP interaction interface targetable by stabilizing peptides.","evidence":"Gain/loss of function ferroptosis assays and peptide-array iASPP interface mapping with apoptosis assays","pmids":["29928961","30774867"],"confidence":"Medium","gaps":["iASPP interaction from single lab","Therapeutic peptide validated only in vitro/limited models"]},{"year":2019,"claim":"Cardiac KO and transgenic mice tied CISD2 to SERCA2a-dependent calcium control, sarcomere/intercalated disc integrity, and cardiac aging delay, generalizing its calcium-homeostasis role across tissues.","evidence":"Cisd2 KO and TG mice with electrophysiology, Ca2+ imaging, SERCA2a activity, and EM","pmids":["31593566"],"confidence":"High","gaps":["Direct CISD2–SERCA2a interaction in heart not biochemically mapped here"]},{"year":2021,"claim":"An inducible dominant-negative system established the causal hierarchy—mitochondrial labile iron first, then ROS, then calcium and TXNIP—resolving iron as the proximal output of CISD2 function.","evidence":"Inducible H114C expression with temporal iron, ROS, Ca2+, and TXNIP measurements","pmids":["34547371"],"confidence":"High","gaps":["Identity of the in vivo cluster/iron acceptor downstream of CISD2 still unknown"]},{"year":2021,"claim":"Defining CISD2's N-terminal ER targeting plus C-terminal KKxx COPI retrieval motif and transmembrane domain explained its ER/MAM residence distinct from mitochondrial CISD1.","evidence":"Domain-swap chimeras with fluorescence localization; review synthesis of three localizations","pmids":["34587896","33422617"],"confidence":"Medium","gaps":["Dynamic partitioning between ER, OMM, and MAM under stress not quantified"]},{"year":2021,"claim":"β-cell and cardiomyocyte studies extended CISD2's iron/ROS-ferroptosis axis to insulin secretion and cardiac iron protection, with chelator/NAC rescue confirming labile iron as the driver.","evidence":"shRNA in INS-1E cells and CISD2-null mouse hearts with iron quantification and pharmacological rescue","pmids":["34439408","34997963"],"confidence":"Medium","gaps":["Single-lab studies","Mechanism connecting CISD2 loss to ferritin/TfR upregulation not dissected"]},{"year":2021,"claim":"Epistasis showed CISD2 loss promotes sorafenib-induced ferroptosis in resistant HCC through Beclin1-dependent autophagy, linking its autophagy-suppressor and ferroptosis roles mechanistically.","evidence":"CISD2/Beclin1 double knockdown with ferroptosis and autophagy readouts","pmids":["34485112"],"confidence":"Medium","gaps":["Single lab","Direct CISD2 control point on autophagic iron flux not isolated"]},{"year":2024,"claim":"Purified-protein binding showed CISD2 engages BCL-2's BH4 domain with submicromolar affinity and, unexpectedly, counteracts BCL-2's inhibition of ER-mitochondrial calcium transfer while being dispensable for BCL-2 anti-apoptotic and BAX-inhibitory functions.","evidence":"Direct binding assay with purified proteins plus Ca2+ transfer, contact-site, and BAX pore assays","pmids":["39370046"],"confidence":"High","gaps":["Structural basis for CISD2 overriding BCL-2's calcium effect not resolved"]},{"year":2024,"claim":"Mass spectrometry and KO neutrophil studies added Calnexin as a CISD2 partner regulating calcium homeostasis, broadening its ER calcium-control interactome.","evidence":"MS interaction screen and Cisd2 KO neutrophil functional/Ca2+ assays","pmids":["38627949"],"confidence":"Medium","gaps":["Calnexin interaction not confirmed by reciprocal Co-IP","Single lab"]},{"year":2024,"claim":"Identifying KAT8-mediated K74 acetylation that blocks STUB1 ubiquitination, and PRKN-mediated ubiquitin degradation, established post-translational control of CISD2 abundance governing mitochondrial homeostasis and senescence.","evidence":"Acetyl-proteomics, in vitro acetyltransferase assay, mutagenesis, CETSA, Co-IP, and C. elegans validation; Parkin Co-IP/ubiquitination with siRNA rescue","pmids":["41687537","39357167"],"confidence":"High","gaps":["Upstream signals controlling KAT8 vs STUB1 vs PRKN balance not defined","PRKN regulation shown in porcine oocytes only"]},{"year":2025,"claim":"β-cell and neuron studies fixed CISD2 as a maintainer of ER-mitochondria contact sites and calcium transfer required for insulin secretion and glutamate-evoked neuronal calcium responses, with transcriptional control by Glis3/Hnf1a and Smad3.","evidence":"Tissue-specific and cell-line KO with Ca2+ imaging, contact-site quantification, respirometry, Co-IP for IP3R, and transcriptomics/ChIP-seq","pmids":["40189101","41299767","41413023"],"confidence":"High","gaps":["How CISD2 physically scaffolds ER-mitochondria contacts at IP3R remains unmapped"]},{"year":null,"claim":"The unifying question—how CISD2's redox-dependent 2Fe-2S cluster chemistry mechanistically couples to its calcium-transfer scaffold functions and to the identity of its physiological cluster/iron acceptors in vivo—remains open.","evidence":"No timeline study reconstitutes cluster transfer and calcium-contact-site function in a single defined system","pmids":[],"confidence":"High","gaps":["No in vivo cluster acceptor identified","Whether cluster redox state directly gates calcium-partner interactions is untested","Structural model of CISD2 at MAM contact sites lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140104","term_label":"molecular carrier activity","supporting_discovery_ids":[5,8,16,20]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,14,19,28]},{"term_id":"GO:0016209","term_label":"antioxidant activity","supporting_discovery_ids":[1,6]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,9,28,33]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[2,6,21,22]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,20,22]}],"pathway":[{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[2,3,13,25]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell 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Participates in the interaction of BCL2 with BECN1 and is required for BCL2-mediated depression of endoplasmic reticulum Ca(2+) stores during autophagy. Contributes to BIK-initiated autophagy, while it is not involved in BIK-dependent activation of caspases. Involved in life span control, probably via its function as regulator of autophagy","subcellular_location":"Endoplasmic reticulum membrane; Mitochondrion outer membrane","url":"https://www.uniprot.org/uniprotkb/Q8N5K1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CISD2","classification":"Not Classified","n_dependent_lines":16,"n_total_lines":1208,"dependency_fraction":0.013245033112582781},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CANX","stoichiometry":0.2},{"gene":"CAPZB","stoichiometry":0.2},{"gene":"COPA","stoichiometry":0.2},{"gene":"COPB2","stoichiometry":0.2},{"gene":"COPE","stoichiometry":0.2},{"gene":"DYNLL2","stoichiometry":0.2},{"gene":"HNRNPD","stoichiometry":0.2},{"gene":"OSBP","stoichiometry":0.2},{"gene":"PGRMC1","stoichiometry":0.2},{"gene":"RTN4","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CISD2","total_profiled":1310},"omim":[{"mim_id":"611932","title":"CDGSH IRON SULFUR DOMAIN PROTEIN 1; CISD1","url":"https://www.omim.org/entry/611932"},{"mim_id":"611507","title":"CDGSH IRON SULFUR DOMAIN PROTEIN 2; CISD2","url":"https://www.omim.org/entry/611507"},{"mim_id":"608086","title":"GTPase, IMAP FAMILY, MEMBER 5; GIMAP5","url":"https://www.omim.org/entry/608086"},{"mim_id":"604928","title":"WOLFRAM SYNDROME 2; WFS2","url":"https://www.omim.org/entry/604928"},{"mim_id":"222300","title":"WOLFRAM SYNDROME 1; WFS1","url":"https://www.omim.org/entry/222300"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Endoplasmic reticulum","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CISD2"},"hgnc":{"alias_symbol":["Miner1","ERIS","NAF-1"],"prev_symbol":["ZCD2","WFS2"]},"alphafold":{"accession":"Q8N5K1","domains":[{"cath_id":"-","chopping":"1-60","consensus_level":"medium","plddt":91.3953,"start":1,"end":60},{"cath_id":"3.40.5.90","chopping":"71-135","consensus_level":"medium","plddt":93.9874,"start":71,"end":135}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N5K1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N5K1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N5K1-F1-predicted_aligned_error_v6.png","plddt_mean":90.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CISD2","jax_strain_url":"https://www.jax.org/strain/search?query=CISD2"},"sequence":{"accession":"Q8N5K1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8N5K1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8N5K1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N5K1"}},"corpus_meta":[{"pmid":"19451219","id":"PMC_19451219","title":"Cisd2 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Mitochondrial degeneration is the direct consequence of Cisd2 loss.\",\n      \"method\": \"Cisd2 knockout mouse model with subcellular fractionation/localization, histology, and autophagy assays\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO mouse with defined cellular phenotype, replicated in multiple follow-up studies, direct localization tied to functional consequence\",\n      \"pmids\": [\"19451219\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CISD2/Miner1 is a homodimeric protein harboring two redox-active 2Fe-2S clusters bound by a rare Cys3-His motif within the NEET fold. Crystal structure resolved to 2.1 Å confirmed it is not a zinc finger as previously annotated. Redox potentials are proton-coupled (~0 mV at pH 7.5).\",\n      \"method\": \"X-ray crystallography (2.1 Å resolution), biophysical characterization, redox potential measurement\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional validation of Fe-S cluster chemistry, independently consistent with multiple subsequent structural/biochemical studies\",\n      \"pmids\": [\"19580816\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"NAF-1 (CISD2) is an ER-localized BCL-2-interacting protein; NAF-1 contains a 2Fe-2S coordinating domain necessary (but not sufficient) for BCL-2 interaction. NAF-1 is required for BCL-2 at the ER to antagonize Beclin 1-dependent autophagy during nutrient deprivation. NAF-1 is also required for BCL-2-mediated depression of ER Ca²⁺ stores and associates with the IP3 receptor.\",\n      \"method\": \"Co-immunoprecipitation, knockdown/overexpression functional assays, ER Ca²⁺ measurements, autophagy flux assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, multiple orthogonal functional assays (autophagy, Ca²⁺, BIK displacement), replicated in follow-up studies\",\n      \"pmids\": [\"20010695\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"NAF-1 (CISD2) knockout mice display early skeletal muscle degeneration with a shift toward slow-twitch fibers, augmented autophagy, dysregulated calcium homeostasis, and adaptive mitochondrial enlargement. This establishes NAF-1 as required for homeostatic maintenance of skeletal muscle via BCL-2-mediated autophagy regulation and ER Ca²⁺ flux.\",\n      \"method\": \"Naf-1 gene deletion mouse model, muscle physiology, fiber-type analysis, Ca²⁺ measurements, autophagy assays\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO mouse with multiple orthogonal phenotypic readouts including force generation, fiber typing, Ca²⁺ and autophagy\",\n      \"pmids\": [\"22343142\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Transgenic mice with a persistently high level of Cisd2 show extended median and maximum lifespan without deleterious effects, attenuate age-associated degeneration of skin, skeletal muscle and neurons, and protect mitochondria from age-associated damage. This places Cisd2 as a positive regulator of mammalian lifespan.\",\n      \"method\": \"Cisd2 transgenic (gain-of-function) mice, lifespan analysis, histology, mitochondrial function assays\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean transgenic mouse with lifespan readout plus multiple tissue and mitochondrial functional endpoints\",\n      \"pmids\": [\"22661501\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"NAF-1 (CISD2) can transfer its 2Fe-2S cluster to an apo-acceptor protein in vitro (monitored by spectrophotometry and native PAGE) and transfer iron to intact mitochondria in cell models (monitored by fluorescence imaging with iron sensors). The anti-diabetes drug pioglitazone and resveratrol stabilize NAF-1's labile 2Fe-2S cluster and abrogate its cluster/iron transfer function.\",\n      \"method\": \"In vitro cluster transfer assay (spectrophotometry, native PAGE), cellular iron-transfer assay (fluorescence imaging), small-molecule binding\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of cluster transfer with multiple orthogonal detection methods, single lab but rigorous biochemical approach\",\n      \"pmids\": [\"23717386\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Loss of Miner1 (CISD2) in mouse embryonic fibroblasts causes ER stress, unfolded protein response, depletion of ER Ca²⁺ stores, increased mitochondrial Ca²⁺ load, increased ROS/RNS, increased GSSG/GSH and NAD⁺/NADH ratios, and altered mitochondrial ultrastructure (increased cristae density, punctate morphology). N-acetylcysteine treatment reversed these abnormalities, implicating sulfhydryl redox status as a key mechanism.\",\n      \"method\": \"Miner1−/− mouse embryonic fibroblasts, ER stress/UPR markers, Ca²⁺ measurements, redox assays, electron microscopy, NAC rescue\",\n      \"journal\": \"EMBO molecular medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO cells with multiple orthogonal mechanistic readouts (Ca²⁺, redox, UPR, ultrastructure) plus chemical rescue\",\n      \"pmids\": [\"23703906\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"NAF-1 (CISD2) and mitoNEET (CISD1) protein levels are elevated in human epithelial breast cancer cells. shRNA suppression of NAF-1 or mitoNEET causes reduced cell proliferation and tumor growth, decreased mitochondrial performance, uncontrolled accumulation of iron and ROS in mitochondria, and activation of autophagy.\",\n      \"method\": \"shRNA knockdown in breast cancer cells and xenograft models, mitochondrial function assays, iron/ROS imaging, autophagy assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — shRNA KD with defined cellular phenotype across multiple orthogonal endpoints, validated in xenograft in vivo\",\n      \"pmids\": [\"23959881\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The NAF-1 H114C mutant (His114→Cys in the 2Fe-2S cluster binding site) produces clusters that are 25-fold more stable, have a redox potential 300 mV more negative, and have abolished cluster donation/transfer function, with no global structural differences from wild-type. This identifies His114 as critical for cluster lability and transfer activity.\",\n      \"method\": \"X-ray crystallography (1.65 Å native; 1.58 Å H114C mutant), redox potential measurement, in vitro cluster transfer assay, site-directed mutagenesis\",\n      \"journal\": \"Acta crystallographica. Section D, Biological crystallography\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures at near-atomic resolution combined with active-site mutagenesis and in vitro functional assay\",\n      \"pmids\": [\"24914968\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NAF-1 binds to both the pro- and anti-apoptotic regions (BH3 and BH4 domains) of BCL-2. The interaction interface of the NAF-1–BCL-2 complex was mapped at amino acid resolution using peptide array, deuterium exchange mass spectrometry (DXMS), and direct coupling analysis (DCA).\",\n      \"method\": \"Peptide array screening, deuterium exchange mass spectrometry (DXMS), direct coupling analysis (DCA), functional binding studies\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple orthogonal structural/biochemical methods (DXMS + peptide array + DCA) defining protein–protein interaction interface\",\n      \"pmids\": [\"24706857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Cisd2 interacts with Gimap5 on mitochondrial and ER membranes and modulates mitochondrial Ca²⁺ uptake, thereby maintaining intracellular Ca²⁺ homeostasis in adipocytes. Loss of Cisd2 increases cytosolic Ca²⁺ and activates Ca²⁺-calcineurin-dependent signaling that inhibits adipogenesis and impairs insulin-stimulated glucose uptake.\",\n      \"method\": \"Adipocyte-specific Cisd2 KO mice, co-immunoprecipitation (interaction with Gimap5), Ca²⁺ measurements, adipogenesis and glucose uptake assays\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean tissue-specific KO plus Co-IP identifying Gimap5 partner, multiple functional endpoints; single lab\",\n      \"pmids\": [\"24833725\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"MAD-28 (a cluvenone derivative) binds to and destabilizes NAF-1 (and mitoNEET) by breaking the coordinative bond between His ligand and the cluster Fe. This causes decreased respiration, decreased mitochondrial membrane potential, and increased mitochondrial iron content in cancer cells, phenocopying NAF-1/mitoNEET shRNA suppression.\",\n      \"method\": \"Molecular docking, functional assays in breast cancer cells (respiration, membrane potential, iron content), shRNA comparison\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — docking combined with functional cellular assays and genetic validation (shRNA cells less susceptible), single lab\",\n      \"pmids\": [\"25762074\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NAF-1 suppression in epithelial breast cancer cells by shRNA activates apoptosis, increases cellular Fe²⁺ uptake, causes a metabolic shift increasing susceptibility to glycolysis inhibition, and activates stress pathways associated with HIF1α.\",\n      \"method\": \"shRNA knockdown in breast cancer cells and xenograft tumors, iron imaging, metabolic assays, apoptosis assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — shRNA KD with multiple cellular readouts including iron distribution, metabolism, and apoptosis; single lab\",\n      \"pmids\": [\"26621032\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NAF-1 antagonizes starvation-induced autophagy in cardiomyocytes by promoting the Beclin1–BCL-2 interaction (shown by co-immunoprecipitation) and inhibiting AMPK activity. Overexpression of NAF-1 was sufficient to inhibit autophagy and protect cardiomyocytes from nutrient-stress–induced cell death.\",\n      \"method\": \"Co-immunoprecipitation (Beclin1–BCL-2 interaction), NAF-1 overexpression, autophagy assays, AMPK activity assay in neonatal rat cardiomyocytes\",\n      \"journal\": \"Cell biology international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP plus functional OE assay with defined cellular readout (autophagy, AMPK, cell death); single lab\",\n      \"pmids\": [\"25689847\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CISD2 interacts with SERCA2b and modulates its Ca²⁺ pump activity via regulation of SERCA2b oxidative modifications, thereby controlling ER Ca²⁺ uptake and maintaining Ca²⁺ homeostasis in hepatocytes. Cisd2 haploinsufficiency disrupts this, causing ER stress and NAFLD/NASH.\",\n      \"method\": \"Co-immunoprecipitation (Cisd2–Serca2b interaction), Serca2b activity assay, oxidative modification analysis, Cisd2 heterozygous and overexpressing mice with liver phenotype readouts\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP of interaction plus biochemical activity assay of Serca2b, validated in haploinsufficiency and overexpression mouse models\",\n      \"pmids\": [\"29166610\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"A novel CISD2 missense mutation (p.Asn72Ser) disturbs cellular Ca²⁺ homeostasis with enhanced Ca²⁺ flux from ER to mitochondria and cytosolic Ca²⁺ abnormalities in patient-derived fibroblasts. This Ca²⁺ dysregulation is associated with increased ER-mitochondria contact, swollen ER lumen, and hyperfused mitochondrial network, and reveals a respiratory chain defect under metabolic stress.\",\n      \"method\": \"Patient-derived fibroblasts, Ca²⁺ imaging, electron microscopy (ER-mitochondria contacts), Seahorse respirometry under galactose medium\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient-derived cells with multiple orthogonal mechanistic assays (Ca²⁺ flux, EM, bioenergetics); single lab\",\n      \"pmids\": [\"28335035\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"mitoNEET (CISD1) and NAF-1 (CISD2) directly interact in mammalian cells (demonstrated by yeast two-hybrid, bimolecular fluorescence complementation, and direct coupling analysis). mitoNEET can transfer its 2Fe-2S clusters to NAF-1 in vitro, consistent with a cluster relay mechanism.\",\n      \"method\": \"Yeast two-hybrid, in vivo BiFC, DCA, in vitro cluster transfer assay, shRNA double-knockdown lines with ROS/iron imaging\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal interaction assays (Y2H + BiFC + DCA) plus in vitro cluster transfer reconstitution\",\n      \"pmids\": [\"28426722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CISD2 overexpression confers resistance to sulfasalazine-induced ferroptosis in head and neck cancer cells, while CISD2 silencing increases sensitivity via increased mitochondrial ferrous iron and lipid ROS accumulation, identifying CISD2 as a regulator of mitochondrial iron and ferroptosis susceptibility.\",\n      \"method\": \"CISD2 overexpression and siRNA knockdown, ferroptosis assays (lipid ROS, mitochondrial iron), mouse xenograft models\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function with mechanistic readouts (mitochondrial iron, lipid ROS) validated in vivo; single lab\",\n      \"pmids\": [\"29928961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"iASPP and NAF-1 (CISD2) interact in cancer cells during apoptosis. The interaction interface maps to residues 764–778 of iASPP binding to a surface groove of NAF-1, identified by peptide array screening and computational methods. A peptide corresponding to iASPP 764–780 stabilizes the NAF-1 cluster, inhibits NAF-1–iASPP interaction, and inhibits staurosporine-induced apoptosis in breast and prostate cancer cells.\",\n      \"method\": \"Peptide array screening, computational docking, Co-IP in cells, peptide functional assay (apoptosis inhibition, IC50 determination)\",\n      \"journal\": \"Chemical science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — peptide array + computational mapping + cellular functional validation; single lab\",\n      \"pmids\": [\"30774867\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Cisd2 deficiency in the heart causes intercalated disc defects, mitochondrial and sarcomere degeneration, and disrupts Ca²⁺ homeostasis via dysregulation of Serca2a activity, resulting in increased basal cytosolic Ca²⁺ and mitochondrial Ca²⁺ overload in cardiomyocytes. Persistent high Cisd2 expression delays cardiac aging.\",\n      \"method\": \"Cisd2 KO and transgenic mice, cardiac electrophysiology, Ca²⁺ imaging, Serca2a activity assay, electron microscopy\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO and TG mouse models with multiple orthogonal cardiac and molecular readouts including Serca2a enzyme activity\",\n      \"pmids\": [\"31593566\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Inducible disruption of CISD2 function (using dominant-negative H114C) causes an immediate disruption in mitochondrial labile iron (mLI), followed by enhanced mitochondrial ROS. Alterations in cytosolic and ER Ca²⁺ levels occur only after the changes in mLI and mROS, indicating iron is upstream of Ca²⁺ signaling. CISD2 disruption also triggers TXNIP expression in a mLI-dependent manner.\",\n      \"method\": \"Inducible expression system for dominant-negative CISD2 H114C, mitochondrial labile iron imaging, mROS assays, Ca²⁺ measurements, TXNIP expression analysis; temporal hierarchy established\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — inducible system allowing temporal dissection of signaling hierarchy; multiple orthogonal readouts (iron, ROS, Ca²⁺, TXNIP)\",\n      \"pmids\": [\"34547371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CISD2 is targeted to the ER by its N-terminal sequence and is retained there by the combined action of a C-terminal COPI-binding KKxx ER retrieval motif and an ER-targeting transmembrane domain, distinguishing it from CISD1 (mitoNEET) which uses different targeting motifs to reach mitochondria.\",\n      \"method\": \"Recombinant antibodies, localization of protein chimeras (domain swaps), fluorescence microscopy\",\n      \"journal\": \"BMC molecular and cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — chimeric protein dissection with direct localization readout; single lab\",\n      \"pmids\": [\"34587896\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CISD2 protein can be localized on the ER, outer mitochondrial membrane (OMM), and mitochondria-associated membrane (MAM), and plays a crucial role in regulating cytosolic Ca²⁺ homeostasis, ER integrity, and mitochondrial function. These three localizations underlie its roles in lifespan, cell death, and disease.\",\n      \"method\": \"Subcellular fractionation, multiple mouse models (reviewed), functional assays for Ca²⁺, ER stress, mitochondria\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — synthesis of direct experimental evidence from multiple studies; review article but citing primary experimental data\",\n      \"pmids\": [\"33422617\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NAF-1 repression in INS-1E pancreatic β-cells inhibits insulin secretion, impairs mitochondrial and ER structure/function, and induces ferroptosis-like features. Combined treatment with deferiprone (iron chelator) and N-acetylcysteine (glutathione precursor) restores insulin secretion and repairs mitochondrial and ER structure by reducing mitochondrial labile iron and ROS.\",\n      \"method\": \"shRNA repression in INS-1E cells, insulin secretion assay, iron chelation/NAC rescue, ferroptosis inhibitor (ferrostatin-1), mitochondrial/ER ultrastructure\",\n      \"journal\": \"Antioxidants (Basel, Switzerland)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD with multiple mechanistic readouts and pharmacological rescue; single lab\",\n      \"pmids\": [\"34439408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CISD2 knockdown promotes ferroptosis through two parallel mechanisms: (1) ferritinophagy-dependent ferritin degradation causing free iron accumulation; (2) degradation of p62 that increases Keap1-NRF2 binding leading to NRF2 ubiquitination/degradation, reducing FTH and HO-1 expression and increasing oxidative stress.\",\n      \"method\": \"shRNA and overexpression in cancer cells, confocal microscopy, western blot for ferritin/p62/Keap1/NRF2, lysosomal inhibition rescue, in vivo xenograft\",\n      \"journal\": \"Cellular & molecular biology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss- and gain-of-function with multiple mechanistic pathway dissection and pharmacological rescue; single lab\",\n      \"pmids\": [\"36180832\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CISD2 knockdown promotes sorafenib-induced ferroptosis in resistant HCC cells via uncontrolled autophagy (autophagy-mediated iron accumulation), in a Beclin1-dependent manner. Beclin1 co-knockdown attenuates the ferroptotic effect of CISD2 knockdown.\",\n      \"method\": \"shRNA knockdown of CISD2 and Beclin1, ferroptosis assays (ROS, MDA, GSH, iron), western blot for autophagy markers\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — double KD epistasis establishing Beclin1-dependent pathway; single lab\",\n      \"pmids\": [\"34485112\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CISD2 deficiency in cardiomyocytes (CISD2-null mice) causes accumulation of high levels of iron, increased transferrin receptor and ferritin, and features of cardiomyocyte aging, demonstrating that CISD2 protects cardiomyocytes from iron overaccumulation.\",\n      \"method\": \"CISD2-null mice, proteomics, transmission electron microscopy, iron and transferrin receptor/ferritin measurement\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO mouse with proteomics and direct iron quantification; single lab\",\n      \"pmids\": [\"34997963\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CISD2 and mitoNEET exhibit distinct intracellular half-lives and differ in the pH sensitivity of their Fe-S cluster stability and ability to transfer clusters in vitro, despite close structural homology, suggesting distinct cellular roles.\",\n      \"method\": \"Cellular half-life measurement, in vitro cluster stability and transfer assays at varying pH, expression profiling across tissues\",\n      \"journal\": \"Biomedicines\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical assays plus cellular half-life measurement; single lab\",\n      \"pmids\": [\"33916457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CISD2 directly interacts with BCL-2's BH4 domain with submicromolar affinity (using purified proteins). CISD2 overexpression enhanced BCL-2-mediated suppression of cytosolic IP3R-mediated Ca²⁺ release. Most strikingly, CISD2 counteracts BCL-2-mediated inhibition of ER-mitochondrial Ca²⁺ transfer: BCL-2 overexpression reduced ER-mitochondrial Ca²⁺ transfer and contact sites, but co-expression of CISD2 abolished these BCL-2 effects. CISD2 was not essential for BCL-2's anti-apoptotic function or BAX pore formation inhibition.\",\n      \"method\": \"Purified protein direct binding assay (submicromolar affinity), Ca²⁺ signaling assays (cytosolic and mitochondrial), ER-mitochondria contact site quantification, BAX pore formation assay, loss-of-function in cells\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct binding with purified proteins plus multiple orthogonal cellular functional assays (Ca²⁺ transfer, contact sites, apoptosis); rigorous controls including negative findings\",\n      \"pmids\": [\"39370046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cisd2 interacts with Calnexin (identified by mass spectrometry), and this interaction along with Calnexin–SERCA modulates Ca²⁺ homeostasis in neutrophils. Cisd2 KO mice neutrophils display Ca²⁺ dysregulation and functional defects despite elevated numbers.\",\n      \"method\": \"Mass spectrometry (Cisd2–Calnexin interaction), Cisd2 KO mouse neutrophil functional assays, Ca²⁺ measurements\",\n      \"journal\": \"BMB reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — MS-identified interaction plus KO functional phenotype; single lab, interaction not confirmed by reciprocal Co-IP\",\n      \"pmids\": [\"38627949\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"KAT8/MSL acetyltransferase complex acetylates CISD2 at K74, preventing STUB1-mediated ubiquitination and degradation at K105. Acetylation at K74 preserves mitochondrial homeostasis. Ginsenoside Rg5 binds KAT8 and promotes CISD2 acetylation, maintaining mitochondrial function and alleviating senescence.\",\n      \"method\": \"SILAC-based acetyl-proteomics, Co-IP, GST pull-down, in vitro lysine acetyltransferase assay, site-directed mutagenesis (K74, K105), cycloheximide chase, CETSA, mitochondrial function assays, C. elegans genetic validation\",\n      \"journal\": \"Phytomedicine : international journal of phytotherapy and phytopharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro acetyltransferase assay plus mutagenesis plus multiple orthogonal methods (SILAC, Co-IP, GST pulldown, CETSA); cross-species genetic validation in C. elegans\",\n      \"pmids\": [\"41687537\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PRKN (Parkin) E3 ubiquitin ligase mediates ubiquitination and proteasomal degradation of CISD2 in porcine oocytes. ETA treatment decreases PRKN expression, reducing CISD2 ubiquitination and increasing CISD2 levels. CISD2 inhibits IP3R-mediated Ca²⁺ release (in a BCL-2-dependent manner) at MAMs, and its knockdown blocks ETA's ability to inhibit IP3R.\",\n      \"method\": \"Immunoprecipitation (CISD2–PRKN interaction, ubiquitination), siRNA knockdown of CISD2, transcriptomic sequencing, IP3R functional assays, MAM imaging\",\n      \"journal\": \"Theriogenology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP demonstrating ubiquitination plus functional siRNA rescue epistasis; single lab\",\n      \"pmids\": [\"39357167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Beta-cell–specific Cisd2 KO in mice disrupts glucose-induced extracellular Ca²⁺ influx, impairing Ca²⁺-mediated insulin secretory signaling, causing mitochondrial dysfunction and reduced insulin secretion. Cisd2 deficiency also suppresses Glis3 and Hnf1a transcription regulators critical for β-cell function.\",\n      \"method\": \"β-cell–specific Cisd2 KO mice, CRISPR-mediated Cisd2KO MIN6 β-cell line, Ca²⁺ imaging, insulin secretion assays, transcriptomic analysis\",\n      \"journal\": \"Molecular metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — tissue-specific KO plus cell-line KO with Ca²⁺ mechanistic readout and transcriptomic pathway analysis; two model systems\",\n      \"pmids\": [\"40189101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CISD2 loss in HeLa cells reduces ER-mitochondrial Ca²⁺ transfer. In human iPSC-derived cortical neurons, Cisd2 deficiency severely reduces glutamate-evoked cytosolic and mitochondrial Ca²⁺ responses due to loss of ER-mitochondria contact sites, causing mitochondrial dysfunction (reduced OCR, ATP, membrane potential) and increased apoptotic sensitivity. CISD2 interacts with IP3 receptors.\",\n      \"method\": \"Cisd2 KO HeLa cells and human iPSC-derived cortical neurons, Ca²⁺ imaging (cytosolic and mitochondrial), ER-mitochondria contact site quantification, Seahorse respirometry, Co-IP (Cisd2–IP3R interaction), apoptosis assays\",\n      \"journal\": \"Acta neuropathologica communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple cell systems including disease-relevant neurons, orthogonal mechanistic readouts (Ca²⁺ transfer, contact sites, bioenergetics, apoptosis), Co-IP for interaction\",\n      \"pmids\": [\"41299767\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Smad3 regulates CISD2 expression by directly binding its promoter region (ChIP-seq validated). CISD2 overexpression in Smad3-knockdown mesenchymal bladder cancer cells rescues ferroptosis markers (Fe²⁺, ROS, lipid peroxides, MDA and GSH levels), placing CISD2 downstream of Smad3 in a ferroptosis-regulatory pathway.\",\n      \"method\": \"ChIP-seq, RNA-seq, Smad3 knockdown, CISD2 overexpression rescue, ferroptosis assays, clinical specimen correlation, xenograft\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-seq plus epistasis rescue experiments; single lab\",\n      \"pmids\": [\"41413023\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The NAF-1 44-67 cancer-targeting peptide (derived from CISD2 residues 44–67) targets the CISD2/NAF-1 protein inside cancer cells and disrupts its homodimeric structure. Dimers of the peptide have higher anticancer activity than monomers. A homologous peptide from CISD1 (mitoNEET) has no cancer-killing activity, indicating specificity for NAF-1 structure.\",\n      \"method\": \"Peptide treatment of cancer cells, structural disruption assays of CISD2 dimer, comparison with CISD1 peptide, dimer vs. monomer activity assays\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic peptide–protein interaction with structure–activity comparison; single lab\",\n      \"pmids\": [\"40118242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Genetic epistasis in Drosophila shows that altered function of the CISD2 orthologue (cisd2) modifies the phenotypic effects of overexpressing PPT1 and CLN3 orthologues on eye morphology, and overexpression of CLN3 combined with cisd2 loss-of-function disrupts locomotor ability, placing cisd2 in a functional network with lysosomal storage disease genes.\",\n      \"method\": \"Drosophila genetics (RNAi knockdown, overexpression, epistasis/modifier screen), eye morphology assay, locomotor assay\",\n      \"journal\": \"Biology open\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Drosophila epistasis screen, single lab, no direct biochemical mechanism determined\",\n      \"pmids\": [\"24705017\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CISD2 knockdown in neural cells by siRNA causes increased iNOS expression and decreased BCL-2 expression in an LPS-challenged model, suggesting CISD2 functions as a suppressor of nitric oxide/inflammatory signaling upstream of BCL-2. CISD2 knockdown reduces the anti-inflammatory and anti-apoptotic effects of curcumin.\",\n      \"method\": \"siRNA knockdown in neural cells, LPS challenge, iNOS and BCL-2 western blot, SCI rat model\",\n      \"journal\": \"Injury\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single siRNA knockdown readout for iNOS/BCL-2, single lab, no direct binding or pathway reconstruction\",\n      \"pmids\": [\"26387034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CISD2 knockdown in EOC microglial cells causes augmented proinflammatory signaling, decreased M2 phenotype markers (Arg-1, Ym1, IL-10, BCL2) and increased NF-κB p65 DNA-binding activity, placing CISD2 as an upstream anti-inflammatory modulator of the NF-κB pathway in microglia.\",\n      \"method\": \"siRNA knockdown in EOC microglial cells, cytokine/marker western blot and RT-PCR, NF-κB ELISA DNA-binding assay\",\n      \"journal\": \"Frontiers in aging neuroscience\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — siRNA knockdown with downstream marker readouts only; single lab, no direct molecular interaction demonstrated\",\n      \"pmids\": [\"33005144\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CISD2 (NAF-1/Miner1) is a homodimeric, redox-active 2Fe-2S cluster protein localized to the ER, outer mitochondrial membrane, and mitochondria-associated membranes (MAMs), where it (1) transfers labile 2Fe-2S clusters to acceptor proteins and iron to mitochondria via a His114-dependent mechanism; (2) interacts directly with BCL-2 (via BH3/BH4 domains), IP3 receptors, SERCA2a/2b, and Calnexin to maintain ER Ca²⁺ uptake, ER-to-mitochondria Ca²⁺ transfer, and intracellular Ca²⁺ homeostasis; (3) acts as a BCL-2 co-factor at the ER to suppress Beclin 1-dependent autophagy; (4) controls mitochondrial labile iron levels upstream of mitochondrial ROS and ferroptosis; (5) is acetylated by KAT8 at K74 (preventing STUB1-mediated ubiquitination/degradation) and ubiquitinated by PRKN; and (6) is transcriptionally regulated by E2F7, Smad3, TAF1/TCF12, and Glis3/Hnf1a in specific cellular contexts—with its loss causing mitochondrial dysfunction, dysregulated Ca²⁺, ER stress, autophagy, ferroptosis, and premature aging across multiple tissues.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CISD2 (NAF-1/Miner1) is a redox-active 2Fe-2S iron-sulfur protein that integrates iron handling, ER-mitochondrial calcium transfer, and autophagy/cell-death control at the interface of the ER, outer mitochondrial membrane, and mitochondria-associated membranes (MAMs), with its loss driving mitochondrial degeneration and premature aging [#0, #22]. Structurally it adopts the NEET fold and coordinates two labile 2Fe-2S clusters through a rare Cys3-His motif rather than the originally annotated zinc finger [#1]; the His114 ligand confers cluster lability, and its mutation abolishes the ability to donate clusters to apo-acceptor proteins and to deliver iron to mitochondria in cells [#5, #8]. Using an inducible dominant-negative H114C system, disruption of CISD2 first perturbs mitochondrial labile iron and then mitochondrial ROS, with calcium changes occurring downstream, establishing iron as the proximal output of cluster transfer [#20]. CISD2 partners physically with BCL-2 through its BH3/BH4 domains and with the IP3 receptor, SERCA pumps, and Calnexin to govern ER calcium uptake and ER-to-mitochondria calcium transfer: it acts as a BCL-2 co-factor at the ER to suppress Beclin 1-dependent autophagy, yet counteracts BCL-2-mediated inhibition of ER-mitochondrial calcium transfer and contact-site formation [#2, #9, #28, #33]. Loss of CISD2 produces ER stress, depleted ER calcium, mitochondrial calcium overload, redox imbalance, and sensitization to ferroptosis through dysregulated mitochondrial iron and autophagy-dependent iron accumulation [#6, #17, #20, #24]. CISD2 stability is set by post-translational control—KAT8-mediated K74 acetylation blocks STUB1-dependent ubiquitination, while PRKN promotes its ubiquitin-dependent degradation [#30, #31]—and its sustained expression extends mammalian lifespan and protects skeletal muscle, heart, neurons, β-cells, and other tissues from age-associated degeneration [#4, #19, #32].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Establishing that CISD2 is a mitochondrial protein whose loss causes organelle breakdown defined it as an essential maintainer of mitochondrial integrity and a determinant of aging.\",\n      \"evidence\": \"Cisd2 knockout mouse with fractionation, histology, and autophagy assays\",\n      \"pmids\": [\"19451219\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the molecular activity underlying mitochondrial protection\", \"ER/MAM localization not yet appreciated\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Solving the crystal structure reclassified CISD2 from a presumed zinc finger to a redox-active 2Fe-2S NEET protein, defining the chemical basis for its function.\",\n      \"evidence\": \"X-ray crystallography at 2.1 Å with redox potential measurement\",\n      \"pmids\": [\"19580816\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure alone did not establish a cluster acceptor or physiological redox partner\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identifying CISD2 as an ER-localized BCL-2 partner required for suppressing Beclin 1 autophagy and depressing ER calcium linked its cluster domain to organelle signaling control.\",\n      \"evidence\": \"Co-IP, knockdown/overexpression, ER Ca2+ and autophagy assays\",\n      \"pmids\": [\"20010695\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not map the BCL-2 binding interface\", \"Whether the Fe-S cluster itself participates in calcium control was unresolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Tissue phenotyping and a gain-of-function lifespan study showed CISD2 dosage bidirectionally controls aging—loss degenerates muscle, sustained expression extends lifespan.\",\n      \"evidence\": \"Naf-1 KO and Cisd2 transgenic mice with muscle physiology, lifespan, and mitochondrial readouts\",\n      \"pmids\": [\"22343142\", \"22661501\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The molecular event by which CISD2 levels set lifespan was not identified\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrating in vitro cluster donation to apo-acceptors and cellular iron delivery to mitochondria established CISD2 as a functional iron-sulfur transfer protein, not merely a structural Fe-S holder.\",\n      \"evidence\": \"In vitro cluster transfer (spectrophotometry, native PAGE) and cellular iron-sensor imaging with small-molecule stabilizers\",\n      \"pmids\": [\"23717386\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological acceptor protein in cells not defined\", \"Link between transfer activity and the calcium/autophagy phenotypes not yet drawn\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Knockout MEF studies tied CISD2 loss to ER calcium depletion, mitochondrial calcium overload, UPR, and redox imbalance reversible by N-acetylcysteine, establishing a coupled calcium/redox dysfunction.\",\n      \"evidence\": \"Miner1−/− MEFs with Ca2+, redox, UPR, EM, and NAC rescue\",\n      \"pmids\": [\"23703906\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Causal ordering of iron, ROS, and calcium not established\", \"Direct calcium-machinery partners not yet identified\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Showing CISD2 is overexpressed in breast cancer and required for proliferation, mitochondrial performance, and iron/ROS control connected its iron handling to a pro-tumorigenic role.\",\n      \"evidence\": \"shRNA knockdown in breast cancer cells and xenografts with mitochondrial, iron/ROS, and autophagy readouts\",\n      \"pmids\": [\"23959881\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not distinguish cluster-transfer from protein-interaction contributions to tumor growth\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"His114 mutagenesis pinpointed the cluster ligand that confers lability and transfer activity, providing a tool to separate CISD2's enzymatic function from its scaffold role.\",\n      \"evidence\": \"High-resolution crystallography of native and H114C plus redox and in vitro transfer assays\",\n      \"pmids\": [\"24914968\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo consequence of stabilized clusters not tested in this study\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Amino-acid-resolution mapping showed CISD2 engages both BH3 and BH4 domains of BCL-2, defining the structural basis of the co-factor interaction.\",\n      \"evidence\": \"Peptide array, DXMS, and direct coupling analysis\",\n      \"pmids\": [\"24706857\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of dual-domain binding not resolved here\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Adipocyte studies added Gimap5 as a CISD2 membrane partner and linked CISD2 to calcineurin signaling, adipogenesis, and insulin-stimulated glucose uptake.\",\n      \"evidence\": \"Adipocyte-specific KO mice and Co-IP with metabolic readouts\",\n      \"pmids\": [\"24833725\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Gimap5 interaction from single lab without reciprocal validation\", \"Mechanistic link to cluster chemistry not addressed\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Small-molecule destabilization of the CISD2 cluster (MAD-28) phenocopied genetic loss in cancer cells, validating the cluster bond as a druggable functional node.\",\n      \"evidence\": \"Docking and functional cancer-cell assays compared to shRNA cells\",\n      \"pmids\": [\"25762074\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Compound selectivity over CISD1 limited\", \"In vivo efficacy not established in this study\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Further cancer and cardiomyocyte work tied CISD2 loss to iron uptake, metabolic vulnerability, HIF1α stress, and Beclin1–BCL-2-dependent autophagy suppression via AMPK.\",\n      \"evidence\": \"shRNA/overexpression with iron imaging, metabolic, apoptosis, and Co-IP autophagy assays\",\n      \"pmids\": [\"26621032\", \"25689847\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"AMPK regulation mechanism not biochemically dissected\", \"Single-lab functional readouts\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrating CISD2 silencing accelerates ferroptosis via ferritinophagy and p62/Keap1/NRF2 disruption defined parallel routes by which CISD2 restrains iron-dependent death.\",\n      \"evidence\": \"shRNA/overexpression with ferritin/p62/Keap1/NRF2 blotting, lysosomal inhibition rescue, and xenograft\",\n      \"pmids\": [\"36180832\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular link between CISD2 and ferritinophagy machinery not shown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identifying CISD2–SERCA2b interaction and regulation of SERCA oxidative state provided a direct mechanism for ER calcium uptake control with disease consequences in liver.\",\n      \"evidence\": \"Co-IP, SERCA2b activity and oxidation assays in haploinsufficient and overexpressing mice\",\n      \"pmids\": [\"29166610\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the Fe-S redox state of CISD2 couples to SERCA oxidation not defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"A patient CISD2 missense mutation linked human disease to enhanced ER-to-mitochondria calcium flux, altered organelle contacts, and a stress-dependent respiratory defect.\",\n      \"evidence\": \"Patient fibroblasts with Ca2+ imaging, EM, and respirometry\",\n      \"pmids\": [\"28335035\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal mutation effect on cluster chemistry not tested\", \"Single patient-derived line\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showing CISD1 transfers clusters to CISD2 with distinct pH-dependent stabilities revealed a possible NEET cluster relay and divergent cellular roles between the paralogs.\",\n      \"evidence\": \"Y2H, BiFC, DCA, in vitro transfer, and half-life/pH-stability comparisons\",\n      \"pmids\": [\"28426722\", \"33916457\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological significance of CISD1→CISD2 relay in vivo unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"CISD2 was placed in ferroptosis and apoptosis regulation in cancer through control of mitochondrial ferrous iron and a mapped iASPP interaction interface targetable by stabilizing peptides.\",\n      \"evidence\": \"Gain/loss of function ferroptosis assays and peptide-array iASPP interface mapping with apoptosis assays\",\n      \"pmids\": [\"29928961\", \"30774867\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"iASPP interaction from single lab\", \"Therapeutic peptide validated only in vitro/limited models\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Cardiac KO and transgenic mice tied CISD2 to SERCA2a-dependent calcium control, sarcomere/intercalated disc integrity, and cardiac aging delay, generalizing its calcium-homeostasis role across tissues.\",\n      \"evidence\": \"Cisd2 KO and TG mice with electrophysiology, Ca2+ imaging, SERCA2a activity, and EM\",\n      \"pmids\": [\"31593566\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct CISD2–SERCA2a interaction in heart not biochemically mapped here\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"An inducible dominant-negative system established the causal hierarchy—mitochondrial labile iron first, then ROS, then calcium and TXNIP—resolving iron as the proximal output of CISD2 function.\",\n      \"evidence\": \"Inducible H114C expression with temporal iron, ROS, Ca2+, and TXNIP measurements\",\n      \"pmids\": [\"34547371\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the in vivo cluster/iron acceptor downstream of CISD2 still unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defining CISD2's N-terminal ER targeting plus C-terminal KKxx COPI retrieval motif and transmembrane domain explained its ER/MAM residence distinct from mitochondrial CISD1.\",\n      \"evidence\": \"Domain-swap chimeras with fluorescence localization; review synthesis of three localizations\",\n      \"pmids\": [\"34587896\", \"33422617\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Dynamic partitioning between ER, OMM, and MAM under stress not quantified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"β-cell and cardiomyocyte studies extended CISD2's iron/ROS-ferroptosis axis to insulin secretion and cardiac iron protection, with chelator/NAC rescue confirming labile iron as the driver.\",\n      \"evidence\": \"shRNA in INS-1E cells and CISD2-null mouse hearts with iron quantification and pharmacological rescue\",\n      \"pmids\": [\"34439408\", \"34997963\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab studies\", \"Mechanism connecting CISD2 loss to ferritin/TfR upregulation not dissected\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Epistasis showed CISD2 loss promotes sorafenib-induced ferroptosis in resistant HCC through Beclin1-dependent autophagy, linking its autophagy-suppressor and ferroptosis roles mechanistically.\",\n      \"evidence\": \"CISD2/Beclin1 double knockdown with ferroptosis and autophagy readouts\",\n      \"pmids\": [\"34485112\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Direct CISD2 control point on autophagic iron flux not isolated\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Purified-protein binding showed CISD2 engages BCL-2's BH4 domain with submicromolar affinity and, unexpectedly, counteracts BCL-2's inhibition of ER-mitochondrial calcium transfer while being dispensable for BCL-2 anti-apoptotic and BAX-inhibitory functions.\",\n      \"evidence\": \"Direct binding assay with purified proteins plus Ca2+ transfer, contact-site, and BAX pore assays\",\n      \"pmids\": [\"39370046\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for CISD2 overriding BCL-2's calcium effect not resolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Mass spectrometry and KO neutrophil studies added Calnexin as a CISD2 partner regulating calcium homeostasis, broadening its ER calcium-control interactome.\",\n      \"evidence\": \"MS interaction screen and Cisd2 KO neutrophil functional/Ca2+ assays\",\n      \"pmids\": [\"38627949\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Calnexin interaction not confirmed by reciprocal Co-IP\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identifying KAT8-mediated K74 acetylation that blocks STUB1 ubiquitination, and PRKN-mediated ubiquitin degradation, established post-translational control of CISD2 abundance governing mitochondrial homeostasis and senescence.\",\n      \"evidence\": \"Acetyl-proteomics, in vitro acetyltransferase assay, mutagenesis, CETSA, Co-IP, and C. elegans validation; Parkin Co-IP/ubiquitination with siRNA rescue\",\n      \"pmids\": [\"41687537\", \"39357167\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signals controlling KAT8 vs STUB1 vs PRKN balance not defined\", \"PRKN regulation shown in porcine oocytes only\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"β-cell and neuron studies fixed CISD2 as a maintainer of ER-mitochondria contact sites and calcium transfer required for insulin secretion and glutamate-evoked neuronal calcium responses, with transcriptional control by Glis3/Hnf1a and Smad3.\",\n      \"evidence\": \"Tissue-specific and cell-line KO with Ca2+ imaging, contact-site quantification, respirometry, Co-IP for IP3R, and transcriptomics/ChIP-seq\",\n      \"pmids\": [\"40189101\", \"41299767\", \"41413023\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CISD2 physically scaffolds ER-mitochondria contacts at IP3R remains unmapped\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The unifying question—how CISD2's redox-dependent 2Fe-2S cluster chemistry mechanistically couples to its calcium-transfer scaffold functions and to the identity of its physiological cluster/iron acceptors in vivo—remains open.\",\n      \"evidence\": \"No timeline study reconstitutes cluster transfer and calcium-contact-site function in a single defined system\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No in vivo cluster acceptor identified\", \"Whether cluster redox state directly gates calcium-partner interactions is untested\", \"Structural model of CISD2 at MAM contact sites lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140104\", \"supporting_discovery_ids\": [5, 8, 16, 20]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 14, 19, 28]},\n      {\"term_id\": \"GO:0016209\", \"supporting_discovery_ids\": [1, 6]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 9, 28, 33]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [2, 6, 21, 22]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 20, 22]},\n      {\"term_id\": \"GO:0005741\", \"supporting_discovery_ids\": [22]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [2, 3, 13, 25]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [17, 20, 24, 33]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [5, 20, 26]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [6, 15]}\n    ],\n    \"complexes\": [\n      \"CISD2 homodimer\",\n      \"CISD2–BCL-2 complex\",\n      \"mitochondria-associated membrane (MAM) ER-mitochondria contact\"\n    ],\n    \"partners\": [\n      \"BCL-2\",\n      \"ITPR (IP3 receptor)\",\n      \"ATP2A2 (SERCA2)\",\n      \"CANX (Calnexin)\",\n      \"CISD1 (mitoNEET)\",\n      \"GIMAP5\",\n      \"KAT8\",\n      \"PRKN\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}