{"gene":"PPIF","run_date":"2026-04-28T19:45:44","timeline":{"discoveries":[{"year":1991,"finding":"PPIF (hCyP3/cyclophilin D) was identified as a distinct human cyclophilin isoform with a 42-amino acid N-terminal hydrophobic extension acting as a mitochondrial targeting sequence, and the purified recombinant protein was shown to possess active peptidyl-prolyl cis-trans isomerase (PPIase) activity that is inhibited by cyclosporin A.","method":"cDNA cloning, recombinant protein expression in E. coli, PPIase enzymatic assay with synthetic peptide substrates, CsA inhibition kinetics, subcellular fractionation/Western blot","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro enzymatic reconstitution with mutagenesis-level substrate specificity analysis; foundational characterization paper","pmids":["1744118"],"is_preprint":false},{"year":1998,"finding":"Cyclophilin D binds strongly to complexes of the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocase (ANT) to form the core structure of the mitochondrial permeability transition pore (mPTP); reconstituted proteoliposomes containing purified VDAC, ANT, and CypD were permeabilized by Ca²⁺ plus phosphate in a cyclosporin A-sensitive manner, establishing the minimal pore composition.","method":"CypD-GST affinity matrix pulldown of mitochondrial membrane proteins, ANT/VDAC co-purification, proteoliposome reconstitution, fluorescein sulphonate permeability assay, CsA inhibition","journal":"European journal of biochemistry","confidence":"High","confidence_rationale":"Tier 1 — direct reconstitution of pore activity in proteoliposomes with purified components, replicated across labs","pmids":["9874241"],"is_preprint":false},{"year":2002,"finding":"CypD overexpression in HEK293 and rat glioma C6 cells desensitizes them to apoptotic stimuli and hyperpolarizes mitochondrial membrane potential; this cytoprotective effect requires intact PPIase activity (shown by site-directed mutagenesis of the catalytic site), yet CypD binding to ANT in GST pulldowns is unaffected by loss of PPIase activity, indicating a PPIase-dependent protective target distinct from ANT.","method":"Live-cell two-photon imaging, site-directed mutagenesis of PPIase active site, mitochondrial membrane potential measurement, GST pulldown (CypD–ANT interaction), apoptosis assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — reconstitution/mutagenesis combined with live imaging and functional apoptosis readouts in a single study","pmids":["12077116"],"is_preprint":false},{"year":2002,"finding":"CypD (cyclophilin D) interacts with the adenine nucleotide translocase (ANT) via ANT Cys160; oxidative cross-linking agents (diamide, phenylarsine oxide) stabilize the ANT 'c' conformation, enhance CypD–ANT binding, and sensitize mPTP opening to Ca²⁺, while alkylation of Cys160 prevents both CypD binding and ADP-mediated mPTP inhibition.","method":"ANT thiol modification with EMA/NEM/PAO/diamide, GST-CypD affinity pulldown, submitochondrial particle cross-linking, mPTP Ca²⁺ sensitivity assays","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal biochemical methods mapping specific cysteine residues; replicated interaction","pmids":["12149099"],"is_preprint":false},{"year":2010,"finding":"Crystal structures of the PPIF (CypD) isomerase domain were determined at high resolution; structural comparison across 14 human cyclophilin isoforms revealed that the substrate-binding S2 surface outside the proline-binding pocket confers isoform specificity for in vivo substrates and drug binding, explaining why CypD activity against short peptides correlates with cyclosporin A ligation.","method":"X-ray crystallography, PPIase enzymatic assay for 15 cyclophilin isoforms, CsA binding assays, computational substrate docking","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 1 — high-resolution crystal structure with enzymatic validation across the family","pmids":["20676357"],"is_preprint":false},{"year":2012,"finding":"In response to oxidative stress, p53 accumulates in the mitochondrial matrix and triggers mPTP opening and necrosis through direct physical interaction with CypD; a robust p53–CypD complex forms during brain ischemia/reperfusion in vivo, and reducing p53 levels or CsA pretreatment prevents complex formation and confers stroke protection.","method":"Co-immunoprecipitation (p53–CypD complex), subcellular fractionation, in vivo mouse stroke model, genetic p53 knockdown, CsA pharmacological inhibition, mPTP opening assay","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP in cells and in vivo, genetic and pharmacological epistasis, multiple orthogonal methods; replicated in later studies","pmids":["22726440"],"is_preprint":false},{"year":2013,"finding":"CypD knockout (Ppif−/−) hearts display altered proteomes with reductions in Krebs cycle enzymes, branched-chain amino acid degradation enzymes, and pyruvate metabolism proteins (including 23% decrease in CPT1), accompanied by decreased acylcarnitine levels, indicating that CypD regulates mitochondrial metabolic pathway composition independently of direct mPTP gating.","method":"Quantitative proteomics (LC-MS/MS) of CypD−/− vs. wild-type mouse hearts, metabolomics (acylcarnitine profiling), enzymatic activity assays (succinate dehydrogenase, electron transfer flavoprotein)","journal":"Journal of molecular and cellular cardiology","confidence":"High","confidence_rationale":"Tier 1–2 — unbiased proteomics plus targeted metabolomics in a clean genetic KO model","pmids":["23262437"],"is_preprint":false},{"year":2013,"finding":"CypD (PPIF) functions as a key physiologic regulator of the mPTP beyond cell death; review synthesizes genetic evidence (Ppif−/− mice) showing CypD regulates transient mPTP openings that modulate mitochondrial Ca²⁺ efflux, bioenergetics, and reactive oxygen species under non-pathological conditions.","method":"Review integrating Ppif−/− genetic mouse model data, mPTP patch-clamp, Ca²⁺ retention assays","journal":"Circulation journal","confidence":"Medium","confidence_rationale":"Tier 3 — review synthesizing prior genetic KO data; no new primary experiments","pmids":["23538482"],"is_preprint":false},{"year":2015,"finding":"RNAi screening identified SPG7 (paraplegin) as a necessary component of the mPTP; biochemical analyses showed the PTP is a heterooligomeric complex of VDAC, SPG7, and CypD; silencing or disruption of SPG7–CypD binding prevented Ca²⁺- and ROS-induced mitochondrial membrane potential depolarization and cell death, positioning CypD as a regulatory subunit whose interaction with SPG7 is required for pore opening.","method":"RNAi screen, Co-immunoprecipitation (SPG7–CypD–VDAC), mitochondrial Ca²⁺ retention assay, ΔΨm measurement, cell death assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 — unbiased RNAi screen plus reciprocal Co-IP and functional epistasis; independent replication reported","pmids":["26387735"],"is_preprint":false},{"year":2016,"finding":"Catalytically active CypD causes aggregation of wild-type p53 into amyloid-type fibrils in vitro; NMR mapping identified CypD active-site residues R55, F60, F113, and W121 as responsible for this activity; Trap1 (mitochondrial Hsp90) normally sequesters CypD in an inhibited complex, and displacement of CypD from Trap1 by influx of unfolded p53 under oxidative stress activates CypD's isomerase activity to trigger mPTP opening.","method":"In vitro CypD–p53 aggregation assay, NMR chemical shift mapping of CypD active site, Gamitrinib (mitochondria-targeted HSP90 inhibitor) treatment, mPTP opening assay in primary MEFs, CypD/p53 genetic epistasis","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution of CypD PPIase activity on p53 substrate, NMR active-site mapping, complemented by genetic epistasis","pmids":["27515399"],"is_preprint":false},{"year":2018,"finding":"CypD phosphorylation is upregulated downstream of RIPK3–PGAM5 signaling during cardiac ischemia-reperfusion; PGAM5 increases CypD phosphorylation to promote mPTP opening and endothelial necroptosis, establishing a Ripk3–PGAM5–CypD–mPTP signaling axis in microvascular injury.","method":"Genetic ablation of Ripk3, melatonin pharmacology, immunoblotting for phospho-CypD and PGAM5, mPTP opening assay, in vivo cardiac IR model, endothelial barrier/permeability assays","journal":"Journal of pineal research","confidence":"Medium","confidence_rationale":"Tier 2–3 — genetic KO plus pharmacological inhibition with defined phosphorylation readout; single lab","pmids":["29770487"],"is_preprint":false},{"year":2022,"finding":"SIRT3 deacetylates CypD at lysine 166 (K166); acetylation of CypD-K166 promotes mPTP opening, increases ROS and mitochondrial membrane potential collapse in spinal cord neurons during neuropathic pain; point mutation CypD-K166R (deacetylation mimetic) abrogates mPTP opening and pain hypersensitivity, identifying K166 acetylation as the functional switch governing CypD-mediated mPTP regulation.","method":"Spared nerve injury mouse model, SIRT3 overexpression/knockout, CypD-K166R point mutation, Co-immunoprecipitation for acetylation, mPTP opening assay, ROS/MMP measurement, behavioral pain assays","journal":"Oxidative medicine and cellular longevity","confidence":"High","confidence_rationale":"Tier 2 — site-specific point mutation combined with Co-IP acetylation and functional in vivo rescue; replicated by multiple subsequent studies","pmids":["36092157"],"is_preprint":false},{"year":2022,"finding":"Endogenous SO₂ sulphenylates CypD specifically at Cys104; C104S mutation in CypD abolishes SO₂-induced sulphenylation and blocks SO₂-mediated inhibition of mPTP opening and cardiomyocyte apoptosis, identifying Cys104 sulphenylation as a novel post-translational modification that suppresses CypD activity.","method":"Biotin-switch sulphenylation assay, site-directed mutagenesis of four CypD cysteine residues (C57S, C104S, C176S, C202S), mPTP opening assay, cytochrome c release, caspase activity, TUNEL assay in neonatal cardiomyocytes","journal":"Frontiers in cell and developmental biology","confidence":"High","confidence_rationale":"Tier 1–2 — direct biochemical detection of modification plus site-specific mutagenesis with functional mPTP and apoptosis readouts","pmids":["35118072"],"is_preprint":false},{"year":2024,"finding":"GCN5L1 (mitochondrial acetyltransferase) promotes CypD acetylation at K166, which is counteracted by SIRT3 (deacetylase); in hypertensive patients, arteriolar CypD acetylation is elevated 280% with reduced Sirt3 and increased GCN5L1; deacetylation-mimetic CypD-K166R mice are protected from vascular oxidative stress, endothelial dysfunction, and Ang II-induced hypertension; endothelial-specific GCN5L1 knockout prevents mitochondrial oxidative stress and metabolic glycolytic switch.","method":"CypD-K166R knock-in mice, endothelial-specific GCN5L1 KO mice, Co-IP for K166 acetylation in patient arterioles, Ang II hypertension model, mitochondrial superoxide measurement, endothelial-dependent relaxation assays, metabolic flux analysis","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 2 — deacetylation-mimetic KI mice plus conditional KO, human patient tissue validation, multiple orthogonal functional endpoints","pmids":["38639088"],"is_preprint":false},{"year":2023,"finding":"CypD directly interacts with ATP5B (ATP synthase beta subunit) to promote mitochondrial ROS release in vascular smooth muscle cells; CypD knockout or CsA inhibition reduces ROS, 8-OHdG production, NLRP3 inflammasome activation, and MMP9 upregulation, defining a CypD–ATP5B–ROS–8-OHdG–NLRP3–MMP9 pathway in intracranial aneurysm pathogenesis.","method":"Co-immunoprecipitation (CypD–ATP5B), CypD−/− mouse model, CsA pharmacological inhibition, ROS measurement, 8-OHdG/NLRP3/MMP9 functional assays, in vivo intracranial aneurysm model","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP identifying novel binding partner ATP5B plus genetic KO with defined pathway; single lab","pmids":["37717465"],"is_preprint":false},{"year":2023,"finding":"miR-155-5p in ischemia/reperfusion-derived serum exosomes suppresses NEDD4 expression; NEDD4 normally promotes CypD ubiquitination and degradation; loss of NEDD4 increases CypD protein levels, augmenting mPTP opening and cardiomyocyte apoptosis during myocardial I/R injury.","method":"miRNA inhibitor transfection, luciferase reporter (miR-155-5p targeting NEDD4 3'UTR), shRNA knockdown of NEDD4/CypD, Co-immunoprecipitation of CypD ubiquitination, in vivo I/R mouse model, apoptosis/infarct size assays","journal":"ESC heart failure","confidence":"Medium","confidence_rationale":"Tier 2–3 — epistasis chain (miR-155-5p → NEDD4 → CypD ubiquitination → mPTP) validated with rescue experiments; single lab","pmids":["36631006"],"is_preprint":false},{"year":2024,"finding":"PPIF (CypD) in neutrophils exacerbates lung ischemia-reperfusion injury by promoting store-operated calcium entry (SOCE)-mediated calcium overload, which activates calcineurin/NFAT signaling and drives neutrophil extracellular trap (NET) formation; PPIF inhibition (CsA) or PPIF knockdown alleviates mitochondrial dysfunction, ROS production, and NET formation in a lung transplant model.","method":"Orthotopic lung transplant mouse model, PPIF inhibitor (CsA) and PADI4 inhibitor in vivo, HL-60 neutrophil differentiation model, PPIF siRNA knockdown, SOCE measurement, calcineurin/NFAT pathway assays, NET quantification, mitochondrial function assays","journal":"International immunopharmacology","confidence":"Medium","confidence_rationale":"Tier 2 — direct PPIF mechanistic pathway (SOCE→Ca²⁺ overload→calcineurin/NFAT→NETs) with genetic and pharmacological validation; single lab","pmids":["39236457"],"is_preprint":false},{"year":2025,"finding":"Sirt3-mediated deacetylation of CypD at K167 alleviates P. gingivalis-induced mitochondrial and endothelial dysfunction; CypD-K167 point mutation plasmids and Co-immunoprecipitation confirmed the Sirt3–CypD interaction, and Sirt3 agonist Honokiol rescued endothelial function in vitro and restored vasorelaxation in vivo.","method":"Co-immunoprecipitation (Sirt3–CypD), CypD-K167 point mutation plasmids, Sirt3-specific agonist Honokiol, RNA sequencing of infected endothelial cells, mitochondrial ROS/ΔΨm assays, mouse aortic vasorelaxation ex vivo","journal":"Journal of periodontal research","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP plus site-directed mutation and in vivo validation; single lab, newly published","pmids":["40344434"],"is_preprint":false},{"year":2025,"finding":"CypD (Ppif) ablation prevents cognitive decline, synaptic impairment, and mPTP-mediated mitochondrial damage induced by caspase-3-cleaved tau in the hippocampus; stereotaxic AAV injection of truncated tau in CypD−/− mice showed no mPTP opening or synaptic vesicle protein deregulation, establishing CypD as a necessary downstream effector of pathological tau-driven neurodegeneration.","method":"Stereotaxic hippocampal AAV injection (full-length vs. caspase-3-cleaved tau), CypD−/− and tau−/− KO mice, cognitive behavioral testing (Morris water maze, etc.), synaptic protein analysis, mitochondrial function/mPTP assays","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2 — clean genetic KO epistasis with defined molecular (mPTP) and behavioral endpoints; single lab","pmids":["40023297"],"is_preprint":false},{"year":2025,"finding":"Ppif (CypD) gene knockout in mice protects against sepsis-induced pancreatic injury; Ppif KO reduced serum IL-6 and amylase, improved pancreatic histopathology, decreased apoptosis indices, and ultrastructural analysis revealed that Ppif KO pancreatic acinar cells develop more autophagosomes rather than undergoing mitochondrial swelling and necrotic changes seen in wild-type CLP mice.","method":"Cecal ligation and puncture (CLP) sepsis model in Ppif KO vs. wild-type mice, serum IL-6/amylase ELISA, H&E histology, TUNEL apoptosis assay, transmission electron microscopy of pancreatic ultrastructure","journal":"The Journal of surgical research","confidence":"Medium","confidence_rationale":"Tier 2 — genetic KO with defined molecular and morphological endpoints; single lab, no prior replication","pmids":["41270585"],"is_preprint":false},{"year":2025,"finding":"Female Ppif−/− (CypD-null) mice are more susceptible to hormone-driven (MPA/DMBA) mammary carcinogenesis than wild-type mice, while Ripk3−/− or Mlkl−/− mice are not, indicating that CypD-dependent MPT-driven necrosis acts as an oncosuppressive mechanism specifically restraining HR+ mammary tumor development.","method":"Whole-body Ppif KO, Ripk3 KO, and Mlkl KO female C57BL/6J mice in MPA/DMBA-driven mammary carcinogenesis model, tumor incidence and progression monitoring","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 — clean genetic KO with specific pathway comparison (PPIF vs. necroptosis effectors); single lab","pmids":["40494873"],"is_preprint":false},{"year":2026,"finding":"CypD-dependent mPTP opening is required for mitochondrial swelling and ferroptosis; during ferroptosis, oxidized mitochondrial DNAs (mtDNAs) are released through the CypD-regulated mPTP and activate the cGAS–STING pathway, which promotes ferritinophagy and amplifies ferroptotic signaling; mtDNA repair inhibition synergizes with ferroptosis inducers in suppressing tumor xenografts.","method":"CypD genetic KO and CsA pharmacological inhibition, mPTP opening assay during ferroptosis, oxidized mtDNA detection and release measurement, cGAS-STING pathway activation assay, ferritinophagy assay, xenograft tumor model","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 — genetic and pharmacological CypD manipulation with defined molecular outputs (mtDNA release, cGAS-STING); single lab, newly published","pmids":["41700459"],"is_preprint":false},{"year":2021,"finding":"CypD overexpression in skeletal muscle myofibers increases mitoflash frequency and area with accompanying perinuclear mitochondrial Ca²⁺ efflux; a phospho-resistant CypD-S42A mutant behaves similarly to wild-type CypD overexpression, while expression of only the mitochondrial targeting sequence (CypDN30) does not cause these phenotypes; sodium butyrate feeding reverses CypD-associated mitoflash phenotypes in an ALS mouse model, linking CypD expression level to mitochondrial Ca²⁺ dynamics and mPTP-associated mitoflash activity.","method":"CypD-jRCaMP1b fusion constructs (live Ca²⁺ imaging), CypD-S42A phospho-resistant mutation, mitoflash quantification in isolated myofibers, ALS (SOD1-G93A) mouse model, sodium butyrate dietary supplementation","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 — live imaging with mutation analysis and defined Ca²⁺ efflux readout; single lab","pmids":["34299032"],"is_preprint":false}],"current_model":"PPIF (CypD/cyclophilin D) is a mitochondrial matrix peptidyl-prolyl cis-trans isomerase that acts as the master regulatory subunit of the mitochondrial permeability transition pore (mPTP): it physically binds VDAC, ANT, and SPG7 to form the pore complex and sensitizes pore opening in response to Ca²⁺ and oxidative stress; its activity is regulated by multiple PTMs (acetylation at K166/K167 by GCN5L1, deacetylation by SIRT3; sulphenylation at C104 by SO₂; ubiquitination via NEDD4), by direct protein interactions (p53, Trap1/HSP90, PGAM5, ATP5B, ANT via Cys160), and by phosphorylation at S42, with CypD-dependent mPTP opening driving necrosis, necroptosis, ferroptosis, and ischemia-reperfusion injury across multiple tissues while also mediating physiological Ca²⁺ efflux and oncosuppression in non-stressed contexts."},"narrative":{"teleology":[{"year":1991,"claim":"Establishing that PPIF encodes a distinct mitochondrial cyclophilin with intrinsic PPIase activity resolved the identity of the cyclosporin A-sensitive isomerase in the mitochondrial matrix.","evidence":"cDNA cloning, recombinant expression, PPIase assay with CsA inhibition kinetics, subcellular fractionation","pmids":["1744118"],"confidence":"High","gaps":["Physiological substrates of the PPIase activity were unknown","Relationship to the permeability transition pore had not been tested"]},{"year":1998,"claim":"Reconstitution of a Ca²⁺-sensitive, CsA-inhibitable pore from purified VDAC, ANT, and CypD in proteoliposomes established CypD as a core component of the mPTP, answering how cyclosporin A blocks permeability transition.","evidence":"GST-CypD affinity pulldown, VDAC/ANT co-purification, proteoliposome reconstitution with fluorescein sulphonate permeability assay","pmids":["9874241"],"confidence":"High","gaps":["The stoichiometry and topology of the pore complex were undefined","Whether additional subunits are required in vivo was unknown"]},{"year":2002,"claim":"Mapping the ANT Cys160 thiol as the CypD docking site and showing that PPIase catalytic activity is required for cytoprotection but dispensable for ANT binding separated CypD's pore-regulatory role from its enzymatic function on other substrates.","evidence":"Site-directed mutagenesis of CypD PPIase site and ANT Cys160, GST pulldowns, mPTP Ca²⁺ sensitivity assays, live-cell two-photon imaging, apoptosis readouts","pmids":["12077116","12149099"],"confidence":"High","gaps":["The identity of PPIase-dependent cytoprotective substrate(s) remained unknown","In vivo genetic confirmation (KO mice) was not yet available"]},{"year":2010,"claim":"High-resolution crystal structures of CypD revealed that the S2 surface outside the proline-binding pocket confers isoform specificity, providing a structural framework for selective CypD inhibitor design.","evidence":"X-ray crystallography, PPIase assay across 15 cyclophilin isoforms, CsA binding, computational docking","pmids":["20676357"],"confidence":"High","gaps":["No co-crystal with a physiological substrate or mPTP component was obtained","Structure of CypD within the intact pore complex remained unknown"]},{"year":2012,"claim":"Discovery that mitochondrial p53 triggers mPTP-dependent necrosis through direct complex formation with CypD linked tumor suppressor signaling to the permeability transition pore during ischemia-reperfusion.","evidence":"Reciprocal Co-IP of p53–CypD in cells and in vivo mouse stroke model, genetic p53 knockdown and CsA pharmacological epistasis","pmids":["22726440"],"confidence":"High","gaps":["Binding interface between p53 and CypD was not mapped","Whether p53 is a PPIase substrate or allosteric activator was unclear"]},{"year":2013,"claim":"Unbiased proteomics and metabolomics of CypD-knockout hearts revealed broad remodeling of Krebs cycle and fatty acid oxidation enzymes, establishing that CypD shapes mitochondrial metabolic pathway composition beyond its mPTP gating role.","evidence":"LC-MS/MS quantitative proteomics and acylcarnitine profiling in Ppif⁻/⁻ vs. WT mouse hearts","pmids":["23262437"],"confidence":"High","gaps":["Whether metabolic effects are secondary to chronic mPTP dysregulation or reflect direct PPIase substrates was unresolved","Tissue-specificity of metabolic remodeling was not assessed"]},{"year":2015,"claim":"An unbiased RNAi screen identified SPG7 as a required mPTP component that must interact with CypD for pore opening, expanding the minimal pore model beyond VDAC and ANT.","evidence":"RNAi screen, reciprocal Co-IP of SPG7–CypD–VDAC, mitochondrial Ca²⁺ retention and cell death assays","pmids":["26387735"],"confidence":"High","gaps":["Whether SPG7 is a structural pore subunit or a regulatory cofactor was debated","The exact molecular architecture of the pore remained unresolved"]},{"year":2016,"claim":"NMR mapping showed CypD catalytically drives p53 amyloid-type aggregation via active-site residues R55/F60/F113/W121, and TRAP1 normally sequesters CypD in an inhibited complex, explaining how oxidative stress derepresses CypD's pro-death activity.","evidence":"In vitro CypD–p53 aggregation reconstitution, NMR chemical shift mapping, Gamitrinib (mitochondria-targeted HSP90 inhibitor), genetic epistasis in MEFs","pmids":["27515399"],"confidence":"High","gaps":["In vivo confirmation of TRAP1–CypD stoichiometry and dynamics was lacking","Relevance to non-p53 substrates was not tested"]},{"year":2018,"claim":"Identification of RIPK3–PGAM5-dependent CypD phosphorylation during cardiac ischemia-reperfusion connected necroptotic signaling to mPTP opening in endothelial cells.","evidence":"Ripk3 genetic ablation, phospho-CypD immunoblotting, PGAM5 pathway analysis, in vivo cardiac IR model","pmids":["29770487"],"confidence":"Medium","gaps":["The specific CypD phosphorylation site(s) were not mapped by mass spectrometry","Independent replication in other tissues is needed","Kinase directly phosphorylating CypD was not definitively identified"]},{"year":2021,"claim":"Live imaging of CypD-overexpressing myofibers demonstrated that CypD dosage controls mitoflash frequency and perinuclear Ca²⁺ efflux, with S42 phosphorylation dispensable (S42A behaves as WT), linking CypD expression level to physiological mPTP-dependent Ca²⁺ dynamics in muscle.","evidence":"CypD-jRCaMP1b fusion live Ca²⁺ imaging, S42A phospho-resistant mutant, mitoflash quantification in isolated myofibers, SOD1-G93A ALS model","pmids":["34299032"],"confidence":"Medium","gaps":["S42 phosphorylation role not fully excluded under all stress conditions","Overexpression system limits physiological interpretation","Single lab observation"]},{"year":2022,"claim":"Mapping K166 acetylation (by GCN5L1, reversed by SIRT3) and C104 sulphenylation as opposing regulatory switches resolved how post-translational modifications fine-tune CypD's mPTP-sensitizing activity: K166 acetylation promotes pore opening while C104 sulphenylation suppresses it.","evidence":"CypD-K166R point mutation with in vivo behavioral rescue, biotin-switch sulphenylation assay, C104S mutagenesis, mPTP opening and apoptosis assays in cardiomyocytes and spinal cord neurons","pmids":["36092157","35118072"],"confidence":"High","gaps":["Interplay between acetylation and sulphenylation on the same CypD molecule was not tested","Crystal structure of modified CypD forms was not determined"]},{"year":2023,"claim":"Identification of NEDD4-mediated ubiquitination as a CypD degradation pathway, suppressed by exosomal miR-155-5p during I/R injury, revealed a post-translational mechanism controlling CypD protein levels rather than activity.","evidence":"miR-155-5p inhibitor, luciferase reporter for NEDD4 3′UTR, Co-IP of CypD ubiquitination, in vivo I/R model with infarct size assays","pmids":["36631006"],"confidence":"Medium","gaps":["Ubiquitination site(s) on CypD not mapped","Independent replication needed","Whether NEDD4 directly ubiquitinates CypD or acts through an intermediate was not definitively shown"]},{"year":2024,"claim":"CypD-K166R deacetylation-mimetic knock-in mice demonstrated protection from Ang II-induced hypertension and endothelial dysfunction, and endothelial-specific GCN5L1 knockout prevented mitochondrial oxidative stress, translating the K166 acetylation switch to a defined cardiovascular disease mechanism in vivo.","evidence":"CypD-K166R knock-in mice, endothelial-specific GCN5L1 KO, human patient arteriole Co-IP, Ang II hypertension model, metabolic flux analysis","pmids":["38639088"],"confidence":"High","gaps":["Whether K166 acetylation status is a viable therapeutic biomarker in humans was not tested","Tissue-specificity of GCN5L1-CypD axis beyond endothelium is unknown"]},{"year":2025,"claim":"Genetic epistasis studies in multiple disease models—tau-driven neurodegeneration, sepsis-induced pancreatic injury, and hormone-driven mammary carcinogenesis—collectively established CypD as a convergent downstream effector: its ablation protects against pathological mPTP opening in neuronal and acinar contexts while paradoxically increasing tumor susceptibility, revealing an oncosuppressive function of MPT-driven necrosis.","evidence":"Ppif⁻/⁻ mice with hippocampal AAV-tau injection, CLP sepsis model, MPA/DMBA mammary carcinogenesis model with comparison to Ripk3⁻/⁻ and Mlkl⁻/⁻ mice","pmids":["40023297","41270585","40494873"],"confidence":"Medium","gaps":["Mechanism by which CypD-dependent necrosis specifically suppresses HR+ tumors is unknown","Whether CypD's oncosuppressive role involves its PPIase activity or mPTP gating is not distinguished","Independent replication of carcinogenesis finding is needed"]},{"year":2025,"claim":"CypD-dependent mPTP opening was shown to mediate oxidized mtDNA release during ferroptosis, activating the cGAS–STING pathway to promote ferritinophagy and amplify ferroptotic cell death, extending CypD's role to a new form of regulated cell death.","evidence":"CypD KO and CsA inhibition during ferroptosis, oxidized mtDNA release measurement, cGAS–STING activation assay, ferritinophagy assay, tumor xenograft model","pmids":["41700459"],"confidence":"Medium","gaps":["Whether CypD directly senses lipid peroxidation signals or is activated indirectly is unknown","The ferroptosis findings have not been independently replicated","Structural basis for CypD-dependent mtDNA release channel is undefined"]},{"year":null,"claim":"The high-resolution structure of CypD within the assembled mPTP complex, the full catalog of PPIase substrates in the mitochondrial matrix, and the integration of multiple PTMs (acetylation, sulphenylation, phosphorylation, ubiquitination) into a unified regulatory model remain major open questions.","evidence":"","pmids":[],"confidence":"Low","gaps":["No cryo-EM or structural model of the intact mPTP with CypD bound","Comprehensive identification of mitochondrial PPIase substrates has not been performed","Quantitative model integrating combinatorial PTM effects on CypD activity is lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016853","term_label":"isomerase activity","supporting_discovery_ids":[0,4,9]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,2,8,11,12]},{"term_id":"GO:0044183","term_label":"protein folding chaperone","supporting_discovery_ids":[9]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,1,6,7,22]}],"pathway":[{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[1,5,8,10,11,21]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[3,5,12,13]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[6]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[16,19]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[19]}],"complexes":["mitochondrial permeability transition pore (mPTP)"],"partners":["VDAC1","SLC25A4","SPG7","TP53","TRAP1","ATP5F1B","SIRT3","PGAM5"],"other_free_text":[]},"mechanistic_narrative":"PPIF (cyclophilin D) is a mitochondrial matrix peptidyl-prolyl cis-trans isomerase that functions as the principal regulatory subunit of the mitochondrial permeability transition pore (mPTP), gating Ca²⁺- and oxidative stress-induced mitochondrial permeability transition to control necrosis, ferroptosis, and physiological Ca²⁺ efflux. CypD assembles with VDAC and ANT (binding ANT via Cys160) to form the minimal pore complex, and additionally interacts with SPG7, ATP5B, p53, and TRAP1/HSP90 to integrate diverse stress signals into mPTP opening [PMID:9874241, PMID:12149099, PMID:26387735, PMID:22726440, PMID:27515399, PMID:37717465]. Its activity is tuned by acetylation at K166/K167 (promoted by GCN5L1, reversed by SIRT3), sulphenylation at C104, NEDD4-mediated ubiquitination, and RIPK3–PGAM5-dependent phosphorylation, with K166 deacetylation-mimetic knock-in mice showing protection from vascular oxidative stress and hypertension [PMID:36092157, PMID:38639088, PMID:35118072, PMID:36631006, PMID:29770487]. CypD-dependent mPTP opening also drives oxidized mtDNA release to activate cGAS–STING signaling during ferroptosis and acts as an oncosuppressive mechanism in hormone receptor-positive mammary tumorigenesis [PMID:41700459, PMID:40494873]."},"prefetch_data":{"uniprot":{"accession":"P30405","full_name":"Peptidyl-prolyl cis-trans isomerase F, mitochondrial","aliases":["Cyclophilin D","CyP-D","CypD","Cyclophilin F","Mitochondrial cyclophilin","CyP-M","Rotamase F"],"length_aa":207,"mass_kda":22.0,"function":"PPIase that catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides and may therefore assist protein folding (PubMed:20676357). Involved in regulation of the mitochondrial permeability transition pore (mPTP) (PubMed:26387735). 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GPX4 protein stability via OTUB1.","date":"2022","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/36369321","citation_count":259,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"34800366","id":"PMC_34800366","title":"Quantitative high-confidence human mitochondrial proteome and its dynamics in cellular context.","date":"2021","source":"Cell metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/34800366","citation_count":239,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"20676357","id":"PMC_20676357","title":"Structural and biochemical characterization of the human cyclophilin family of peptidyl-prolyl isomerases.","date":"2010","source":"PLoS biology","url":"https://pubmed.ncbi.nlm.nih.gov/20676357","citation_count":238,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"17207965","id":"PMC_17207965","title":"hORFeome v3.1: a resource of human open reading frames representing over 10,000 human genes.","date":"2007","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/17207965","citation_count":222,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"23538482","id":"PMC_23538482","title":"Physiologic functions of cyclophilin D and the mitochondrial permeability transition pore.","date":"2013","source":"Circulation journal : official journal of the Japanese Circulation Society","url":"https://pubmed.ncbi.nlm.nih.gov/23538482","citation_count":219,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"29568061","id":"PMC_29568061","title":"An AP-MS- and BioID-compatible MAC-tag enables comprehensive mapping of protein interactions and subcellular localizations.","date":"2018","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/29568061","citation_count":201,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"21516116","id":"PMC_21516116","title":"Next-generation sequencing to generate interactome datasets.","date":"2011","source":"Nature methods","url":"https://pubmed.ncbi.nlm.nih.gov/21516116","citation_count":200,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"32203420","id":"PMC_32203420","title":"Systems analysis of RhoGEF and RhoGAP regulatory proteins reveals spatially organized RAC1 signalling from integrin adhesions.","date":"2020","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/32203420","citation_count":194,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"1744118","id":"PMC_1744118","title":"The cyclophilin multigene family of peptidyl-prolyl isomerases. Characterization of three separate human isoforms.","date":"1991","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/1744118","citation_count":185,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"26387735","id":"PMC_26387735","title":"SPG7 Is an Essential and Conserved Component of the Mitochondrial Permeability Transition Pore.","date":"2015","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/26387735","citation_count":169,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"12077116","id":"PMC_12077116","title":"Mitochondrial targeted cyclophilin D protects cells from cell death by peptidyl prolyl isomerization.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12077116","citation_count":149,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"18461144","id":"PMC_18461144","title":"Systematic identification of mRNAs recruited to argonaute 2 by specific microRNAs and corresponding changes in transcript abundance.","date":"2008","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/18461144","citation_count":148,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":25686,"output_tokens":4631,"usd":0.073261},"stage2":{"model":"claude-opus-4-6","input_tokens":8103,"output_tokens":3068,"usd":0.175822},"total_usd":0.59477,"stage1_batch_id":"msgbatch_01SUR8NDzVrNmyszWyJeCct7","stage2_batch_id":"msgbatch_01G7Yfutko3cDWzejrwH5AMx","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":38088,"output_tokens":6976,"usd":0.109452},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":10708,"output_tokens":4158,"usd":0.236235}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2016,\n      \"finding\": \"CypD (PPIF) causes strong aggregation of wild-type p53 protein into amyloid-type fibrils in vitro through its peptidyl-prolyl isomerase activity; the responsible active-site residues were mapped by NMR to R55, F60, F113, and W121. CypD is normally held in check by Trap1 (mitochondrial HSP90), and oxidative stress displaces CypD from Trap1, liberating its isomerase activity to isomerize mPTP structural components and p53, inducing pore opening.\",\n      \"method\": \"In vitro reconstitution of p53 aggregation assay, active-site mutagenesis, NMR chemical shift mapping, pharmacological inhibition of Trap1 with Gamitrinib in primary MEFs\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution in vitro with mutagenesis and NMR structural mapping, plus functional cell-based validation\",\n      \"pmids\": [\"27515399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SIRT3 deacetylates CypD at lysine 166 (K166); acetylation of CypD-K166 promotes mPTP opening, whereas the deacetylation-mimetic K166R point mutation abolishes SNI-induced mitochondrial dysfunction and neuropathic pain in mice.\",\n      \"method\": \"Western blot for acetylation levels, K166R point mutation knock-in mice, in vivo spared nerve injury model, mPTP opening assays, ROS and MMP measurements\",\n      \"journal\": \"Oxidative medicine and cellular longevity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — site-specific mutation with clear phenotypic rescue replicated across multiple functional readouts\",\n      \"pmids\": [\"36092157\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"GCN5L1 (mitochondrial acetyltransferase) acetylates CypD at K166, promoting endothelial dysfunction and hypertension; SIRT3 counteracts this by deacetylating CypD-K166. Deacetylation-mimetic CypD-K166R mice are protected from vascular oxidative stress and angiotensin II-induced hypertension.\",\n      \"method\": \"CypD-K166R deacetylation-mimetic mice, endothelial-specific GCN5L1 knockout mice, Ang II hypertension model, measurement of mitochondrial superoxide, endothelial-dependent relaxation, and blood pressure\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal genetic models (K166R knock-in, cell-type-specific KO) with rigorous functional phenotyping\",\n      \"pmids\": [\"38639088\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Endogenous SO2 sulphenylates CypD specifically at Cysteine 104 (C104), inhibiting mPTP opening and protecting cardiomyocytes from apoptosis; C104S mutation in CypD abolishes SO2-induced sulphenylation and blocks the inhibitory effect on mPTP opening.\",\n      \"method\": \"Biotin switch analysis for sulphenylation detection, CypD cysteine-to-serine mutant plasmids (C104S and others), mPTP opening assay, cytochrome c release, caspase activity in H9c2 cells\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — site-specific mutagenesis identifying the modification site combined with functional mPTP readout\",\n      \"pmids\": [\"35118072\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Activated RIPK3 upregulates PGAM5, which increases CypD phosphorylation, obligating endothelial cells to undergo necroptosis via augmenting mPTP opening; melatonin interrupts endothelial necroptosis by blocking the RIPK3–PGAM5–CypD signal cascade.\",\n      \"method\": \"Genetic ablation of Ripk3, pharmacological inhibition, measurement of mPTP opening, endothelial permeability and necroptosis markers in cardiac microvascular IR injury model\",\n      \"journal\": \"Journal of pineal research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO plus pharmacological inhibition with functional phenotypic readouts, single lab\",\n      \"pmids\": [\"29770487\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RIP3/MLKL-dependent necroptosis in prolactinoma cells involves the PGAM5/CypD pathway; bromocriptine activates this pathway to induce mPTP-mediated necroptosis.\",\n      \"method\": \"Immunohistochemistry in patient tissue, siRNA/pharmacological inhibition in MMQ cells, Necrostatin-1 rescue, ultrastructural analysis by electron microscopy\",\n      \"journal\": \"Biomedicine & pharmacotherapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological and genetic perturbation with ultrastructural validation, single lab\",\n      \"pmids\": [\"30611988\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CypD directly interacts with ATP5B to promote ROS release in vascular smooth muscle cells, contributing to intracranial aneurysm formation; CypD-/- mice showed reduced ROS, 8-OHdG, NLRP3, and MMP9 levels and decreased aneurysm incidence.\",\n      \"method\": \"Co-immunoprecipitation (CypD–ATP5B interaction), CypD knockout mice, cyclosporin A pharmacological inhibition, in vivo aneurysm model, ROS and MMP9 activity assays\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP identifying binding partner, genetic KO with in vivo phenotype, pharmacological validation\",\n      \"pmids\": [\"37717465\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NEDD4 E3 ubiquitin ligase promotes CypD ubiquitination; miR-155-5p targets NEDD4, reducing CypD ubiquitination and thereby increasing CypD levels to promote mPTP-dependent cardiomyocyte apoptosis in ischemia-reperfusion injury.\",\n      \"method\": \"Luciferase reporter assay (miR-155-5p targeting NEDD4), co-immunoprecipitation for CypD ubiquitination, shRNA knockdowns, exosome isolation and injection in I/R mouse model\",\n      \"journal\": \"ESC heart failure\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP for ubiquitination, genetic knockdown rescue experiments in vitro and in vivo\",\n      \"pmids\": [\"36631006\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Knockdown of CypD (and ANT) attenuates mortalin-depletion-induced mitochondrial permeability and cell death in vemurafenib-resistant B-RafV600E melanoma cells; MEK/ERK inhibition also suppresses this mitochondrial death mechanism, placing CypD downstream of MEK/ERK in this pathway.\",\n      \"method\": \"siRNA knockdown of CypD and ANT, pharmacological MEK/ERK inhibition, mitochondrial permeability assays, xenograft tumor model\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic knockdown with pathway epistasis and in vivo validation, single lab\",\n      \"pmids\": [\"33440231\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Neutrophil PPIF (CypD) promotes calcium overload-induced neutrophil extracellular trap (NET) formation during lung ischemia-reperfusion injury; PPIF inhibition with cyclosporin A alleviated store-operated calcium entry (SOCE), ROS production, and NET formation via calcineurin/NFAT pathway suppression.\",\n      \"method\": \"Orthotopic lung transplant mouse model, PPIF inhibitor (cyclosporin A) treatment, HL-60 cell neutrophil model in vitro, PPIF siRNA interference, calcineurin/NFAT pathway analysis\",\n      \"journal\": \"International immunopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic and pharmacological inhibition with in vivo and in vitro mechanistic pathway characterization, single lab\",\n      \"pmids\": [\"39236457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Sirt3-mediated deacetylation of CypD at K167 (equivalent to K166) alleviates Porphyromonas gingivalis-induced mitochondrial dysfunction and endothelial dysfunction; CypD K167 point mutation plasmids and Co-immunoprecipitation confirmed the Sirt3–CypD interaction.\",\n      \"method\": \"Co-immunoprecipitation, CypD K167 point mutation plasmids, Honokiol (Sirt3 agonist) treatment, RNA sequencing, in vivo mouse oral inoculation model\",\n      \"journal\": \"Journal of periodontal research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with site-specific mutation and in vivo validation, single lab\",\n      \"pmids\": [\"40344434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CypD ablation in parvalbumin (PV) interneurons prevents neonatal sevoflurane-induced mitochondrial calcium dysregulation, oxidative stress, loss of mitochondrial membrane potential, synaptic deficits, and cognitive impairment; conditional CypD knockout (PpifF/F-PVCre mice) confirmed a cell-type-specific role of CypD in these neurons.\",\n      \"method\": \"Conditional CypD knockout (PpifF/F-PVCre), behavioral tests (NOR, MWM, social interaction), calcium imaging, Golgi staining, whole-cell patch-clamp, Western blot, immunofluorescence\",\n      \"journal\": \"Chemico-biological interactions\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — conditional cell-type-specific genetic KO with multiple orthogonal readouts, single lab\",\n      \"pmids\": [\"40784489\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CypD ablation (CypD-/-) prevents cognitive decline, synaptic impairment, and mPTP-mediated mitochondrial damage induced by caspase-3-cleaved tau expressed in mouse hippocampus, establishing CypD as a required mediator of truncated tau-induced neurodegeneration.\",\n      \"method\": \"CypD(-/-) and tau(-/-) knockout mice, stereotaxic AAV hippocampal injection of GFP-tagged tau constructs, cognitive behavioral tests, synaptic protein analysis, mitochondrial function assays\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with in vivo epistasis and multiple mechanistic readouts, single lab\",\n      \"pmids\": [\"40023297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CypD dependent mPTP opening mediates mitochondrial swelling during ferroptosis and is required for the release of oxidized mitochondrial DNA (mtDNA), which then activates the cGAS-STING pathway to promote ferritinophagy and ferroptosis.\",\n      \"method\": \"Pharmacological and genetic inhibition of CypD/mPTP, measurement of oxidized mtDNA release, cGAS-STING pathway activation assays, mouse xenograft tumor models\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic and pharmacological CypD inhibition with mechanistic pathway characterization in vitro and in vivo\",\n      \"pmids\": [\"41700459\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PDZD8 knockdown reduces CypD expression and alleviates Ca2+ flow into mitochondria, decreasing pancreatic β-cell apoptosis; CypD overexpression reversed the protective effect of PDZD8 knockdown, placing CypD downstream of PDZD8-mediated ER-mitochondria contact (MAM) and mitochondrial Ca2+ overload.\",\n      \"method\": \"PDZD8 knockdown, CypD overexpression rescue, proximity ligation assay (VDAC1-IP3R1 interaction), JC-1 mitochondrial membrane potential, flow cytometry, transmission electron microscopy\",\n      \"journal\": \"Diabetes & metabolism journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis by rescue overexpression, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"39069376\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Whole-body deletion of Ppif (encoding CypD) increases susceptibility to hormone receptor-positive mammary carcinogenesis in mice, while deletion of Ripk3 or Mlkl does not, indicating that CypD-regulated MPT-driven necrosis specifically restrains HR+ mammary tumorigenesis.\",\n      \"method\": \"Ppif-/-, Ripk3-/-, and Mlkl-/- C57BL/6J mice in MPA/DMBA mammary carcinogenesis model; tumor incidence and histology\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with pathway-specific epistasis (comparison with Ripk3-/- and Mlkl-/-) in vivo, single lab\",\n      \"pmids\": [\"40494873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CypD overexpression in skeletal muscle myofibers increases mitoflash frequency and area and is associated with perinuclear mitochondrial Ca2+ efflux during mitoflashes; the phospho-resistant S42A mutation in CypD does not abolish this phenotype. Sodium butyrate feeding reverses CypD-associated mitoflash changes and reduces ectopic CypD upregulation.\",\n      \"method\": \"CypD and CypD-S42A fusion constructs with mitochondrial Ca2+ sensor jRCaMP1b, live imaging in mouse myofibers, mitoflash quantification, dietary butyrate feeding in ALS mouse model\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — association between CypD expression level and mitoflash phenotype; S42A mutation result is correlative without full mechanistic follow-up\",\n      \"pmids\": [\"34299032\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"SIRT3 in central amygdala GABAergic neurons deacetylates CypD at K166, inhibiting mPTP opening; CypD-K166R deacetylation-mimetic mutation prevents mitochondrial dysfunction and depressive-like behaviors comorbid with chronic pain in SNI mice.\",\n      \"method\": \"Stereotaxic AAV-mediated SIRT3 overexpression in CeA GABAergic neurons, CypD-K166R knock-in mice, behavioral tests, ROS/MMP/MnSOD measurements, cyclosporin A pharmacological inhibition\",\n      \"journal\": \"European journal of pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — site-specific deacetylation-mimetic mutation with in vivo behavioral and mitochondrial phenotype rescue, single lab\",\n      \"pmids\": [\"41643830\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PPIF encodes Cyclophilin D (CypD), a mitochondrial matrix peptidyl-prolyl isomerase that acts as the primary regulator of the mitochondrial permeability transition pore (mPTP): its isomerase activity directly promotes mPTP opening by isomerizing pore structural components (and substrates such as p53), it is activated by stress-induced displacement from its inhibitory Trap1 complex, its activity is modulated post-translationally by SIRT3-mediated deacetylation and GCN5L1-mediated acetylation at K166, by SO2-mediated sulphenylation at C104, and by NEDD4-dependent ubiquitination, and it interacts with binding partners including ATP5B and ANT; collectively these mechanisms link upstream signals (Ca2+ overload, oxidative stress, RIPK3–PGAM5 necroptotic signaling, ER–mitochondria Ca2+ transfer via PDZD8/IP3R1/VDAC1) to mPTP opening and consequent cell death (necrosis, necroptosis, apoptosis, or ferroptosis via oxidized mtDNA/cGAS-STING release) across cardiac, neuronal, vascular, skeletal, and immune cell contexts.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries from KEPT papers only.\n\n**Classification:**\n\nPapers 18, 29 (corpus) — EXCLUDE: These describe CypD from the cypemycin biosynthesis pathway in bacteria (a flavin-dependent decarboxylase/RiPP enzyme), completely unrelated to mammalian PPIF/CypD.\n\nPaper 19 (corpus) — EXCLUDE: Describes CYP2D (cytochrome P450 2D) family, alias collision.\n\nPaper 42 (corpus) — EXCLUDE: Retraction notice.\n\nPaper 46 (corpus) — EXCLUDE: PPIF = \"personal and professional identity formation\" in medical education, alias collision.\n\nGene2pubmed papers 1-13, 15, 18, 19, 21, 22, 24-26, 30 — EXCLUDE: Genome/proteomics resources, GWAS, interactome maps with no specific PPIF mechanism, miRNA studies without PPIF-specific findings. Exception: papers 14, 16, 17, 21, 23, 27, 28, 29 contain PPIF/CypD-specific mechanistic findings.\n\nAll remaining papers about mammalian/vertebrate CypD (PPIF) — KEEP.\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1991,\n      \"finding\": \"PPIF (hCyP3/cyclophilin D) was identified as a distinct human cyclophilin isoform with a 42-amino acid N-terminal hydrophobic extension acting as a mitochondrial targeting sequence, and the purified recombinant protein was shown to possess active peptidyl-prolyl cis-trans isomerase (PPIase) activity that is inhibited by cyclosporin A.\",\n      \"method\": \"cDNA cloning, recombinant protein expression in E. coli, PPIase enzymatic assay with synthetic peptide substrates, CsA inhibition kinetics, subcellular fractionation/Western blot\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic reconstitution with mutagenesis-level substrate specificity analysis; foundational characterization paper\",\n      \"pmids\": [\"1744118\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Cyclophilin D binds strongly to complexes of the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocase (ANT) to form the core structure of the mitochondrial permeability transition pore (mPTP); reconstituted proteoliposomes containing purified VDAC, ANT, and CypD were permeabilized by Ca²⁺ plus phosphate in a cyclosporin A-sensitive manner, establishing the minimal pore composition.\",\n      \"method\": \"CypD-GST affinity matrix pulldown of mitochondrial membrane proteins, ANT/VDAC co-purification, proteoliposome reconstitution, fluorescein sulphonate permeability assay, CsA inhibition\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct reconstitution of pore activity in proteoliposomes with purified components, replicated across labs\",\n      \"pmids\": [\"9874241\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"CypD overexpression in HEK293 and rat glioma C6 cells desensitizes them to apoptotic stimuli and hyperpolarizes mitochondrial membrane potential; this cytoprotective effect requires intact PPIase activity (shown by site-directed mutagenesis of the catalytic site), yet CypD binding to ANT in GST pulldowns is unaffected by loss of PPIase activity, indicating a PPIase-dependent protective target distinct from ANT.\",\n      \"method\": \"Live-cell two-photon imaging, site-directed mutagenesis of PPIase active site, mitochondrial membrane potential measurement, GST pulldown (CypD–ANT interaction), apoptosis assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — reconstitution/mutagenesis combined with live imaging and functional apoptosis readouts in a single study\",\n      \"pmids\": [\"12077116\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"CypD (cyclophilin D) interacts with the adenine nucleotide translocase (ANT) via ANT Cys160; oxidative cross-linking agents (diamide, phenylarsine oxide) stabilize the ANT 'c' conformation, enhance CypD–ANT binding, and sensitize mPTP opening to Ca²⁺, while alkylation of Cys160 prevents both CypD binding and ADP-mediated mPTP inhibition.\",\n      \"method\": \"ANT thiol modification with EMA/NEM/PAO/diamide, GST-CypD affinity pulldown, submitochondrial particle cross-linking, mPTP Ca²⁺ sensitivity assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal biochemical methods mapping specific cysteine residues; replicated interaction\",\n      \"pmids\": [\"12149099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Crystal structures of the PPIF (CypD) isomerase domain were determined at high resolution; structural comparison across 14 human cyclophilin isoforms revealed that the substrate-binding S2 surface outside the proline-binding pocket confers isoform specificity for in vivo substrates and drug binding, explaining why CypD activity against short peptides correlates with cyclosporin A ligation.\",\n      \"method\": \"X-ray crystallography, PPIase enzymatic assay for 15 cyclophilin isoforms, CsA binding assays, computational substrate docking\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution crystal structure with enzymatic validation across the family\",\n      \"pmids\": [\"20676357\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In response to oxidative stress, p53 accumulates in the mitochondrial matrix and triggers mPTP opening and necrosis through direct physical interaction with CypD; a robust p53–CypD complex forms during brain ischemia/reperfusion in vivo, and reducing p53 levels or CsA pretreatment prevents complex formation and confers stroke protection.\",\n      \"method\": \"Co-immunoprecipitation (p53–CypD complex), subcellular fractionation, in vivo mouse stroke model, genetic p53 knockdown, CsA pharmacological inhibition, mPTP opening assay\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP in cells and in vivo, genetic and pharmacological epistasis, multiple orthogonal methods; replicated in later studies\",\n      \"pmids\": [\"22726440\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CypD knockout (Ppif−/−) hearts display altered proteomes with reductions in Krebs cycle enzymes, branched-chain amino acid degradation enzymes, and pyruvate metabolism proteins (including 23% decrease in CPT1), accompanied by decreased acylcarnitine levels, indicating that CypD regulates mitochondrial metabolic pathway composition independently of direct mPTP gating.\",\n      \"method\": \"Quantitative proteomics (LC-MS/MS) of CypD−/− vs. wild-type mouse hearts, metabolomics (acylcarnitine profiling), enzymatic activity assays (succinate dehydrogenase, electron transfer flavoprotein)\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — unbiased proteomics plus targeted metabolomics in a clean genetic KO model\",\n      \"pmids\": [\"23262437\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CypD (PPIF) functions as a key physiologic regulator of the mPTP beyond cell death; review synthesizes genetic evidence (Ppif−/− mice) showing CypD regulates transient mPTP openings that modulate mitochondrial Ca²⁺ efflux, bioenergetics, and reactive oxygen species under non-pathological conditions.\",\n      \"method\": \"Review integrating Ppif−/− genetic mouse model data, mPTP patch-clamp, Ca²⁺ retention assays\",\n      \"journal\": \"Circulation journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — review synthesizing prior genetic KO data; no new primary experiments\",\n      \"pmids\": [\"23538482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RNAi screening identified SPG7 (paraplegin) as a necessary component of the mPTP; biochemical analyses showed the PTP is a heterooligomeric complex of VDAC, SPG7, and CypD; silencing or disruption of SPG7–CypD binding prevented Ca²⁺- and ROS-induced mitochondrial membrane potential depolarization and cell death, positioning CypD as a regulatory subunit whose interaction with SPG7 is required for pore opening.\",\n      \"method\": \"RNAi screen, Co-immunoprecipitation (SPG7–CypD–VDAC), mitochondrial Ca²⁺ retention assay, ΔΨm measurement, cell death assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — unbiased RNAi screen plus reciprocal Co-IP and functional epistasis; independent replication reported\",\n      \"pmids\": [\"26387735\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Catalytically active CypD causes aggregation of wild-type p53 into amyloid-type fibrils in vitro; NMR mapping identified CypD active-site residues R55, F60, F113, and W121 as responsible for this activity; Trap1 (mitochondrial Hsp90) normally sequesters CypD in an inhibited complex, and displacement of CypD from Trap1 by influx of unfolded p53 under oxidative stress activates CypD's isomerase activity to trigger mPTP opening.\",\n      \"method\": \"In vitro CypD–p53 aggregation assay, NMR chemical shift mapping of CypD active site, Gamitrinib (mitochondria-targeted HSP90 inhibitor) treatment, mPTP opening assay in primary MEFs, CypD/p53 genetic epistasis\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution of CypD PPIase activity on p53 substrate, NMR active-site mapping, complemented by genetic epistasis\",\n      \"pmids\": [\"27515399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CypD phosphorylation is upregulated downstream of RIPK3–PGAM5 signaling during cardiac ischemia-reperfusion; PGAM5 increases CypD phosphorylation to promote mPTP opening and endothelial necroptosis, establishing a Ripk3–PGAM5–CypD–mPTP signaling axis in microvascular injury.\",\n      \"method\": \"Genetic ablation of Ripk3, melatonin pharmacology, immunoblotting for phospho-CypD and PGAM5, mPTP opening assay, in vivo cardiac IR model, endothelial barrier/permeability assays\",\n      \"journal\": \"Journal of pineal research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — genetic KO plus pharmacological inhibition with defined phosphorylation readout; single lab\",\n      \"pmids\": [\"29770487\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SIRT3 deacetylates CypD at lysine 166 (K166); acetylation of CypD-K166 promotes mPTP opening, increases ROS and mitochondrial membrane potential collapse in spinal cord neurons during neuropathic pain; point mutation CypD-K166R (deacetylation mimetic) abrogates mPTP opening and pain hypersensitivity, identifying K166 acetylation as the functional switch governing CypD-mediated mPTP regulation.\",\n      \"method\": \"Spared nerve injury mouse model, SIRT3 overexpression/knockout, CypD-K166R point mutation, Co-immunoprecipitation for acetylation, mPTP opening assay, ROS/MMP measurement, behavioral pain assays\",\n      \"journal\": \"Oxidative medicine and cellular longevity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — site-specific point mutation combined with Co-IP acetylation and functional in vivo rescue; replicated by multiple subsequent studies\",\n      \"pmids\": [\"36092157\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Endogenous SO₂ sulphenylates CypD specifically at Cys104; C104S mutation in CypD abolishes SO₂-induced sulphenylation and blocks SO₂-mediated inhibition of mPTP opening and cardiomyocyte apoptosis, identifying Cys104 sulphenylation as a novel post-translational modification that suppresses CypD activity.\",\n      \"method\": \"Biotin-switch sulphenylation assay, site-directed mutagenesis of four CypD cysteine residues (C57S, C104S, C176S, C202S), mPTP opening assay, cytochrome c release, caspase activity, TUNEL assay in neonatal cardiomyocytes\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct biochemical detection of modification plus site-specific mutagenesis with functional mPTP and apoptosis readouts\",\n      \"pmids\": [\"35118072\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"GCN5L1 (mitochondrial acetyltransferase) promotes CypD acetylation at K166, which is counteracted by SIRT3 (deacetylase); in hypertensive patients, arteriolar CypD acetylation is elevated 280% with reduced Sirt3 and increased GCN5L1; deacetylation-mimetic CypD-K166R mice are protected from vascular oxidative stress, endothelial dysfunction, and Ang II-induced hypertension; endothelial-specific GCN5L1 knockout prevents mitochondrial oxidative stress and metabolic glycolytic switch.\",\n      \"method\": \"CypD-K166R knock-in mice, endothelial-specific GCN5L1 KO mice, Co-IP for K166 acetylation in patient arterioles, Ang II hypertension model, mitochondrial superoxide measurement, endothelial-dependent relaxation assays, metabolic flux analysis\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — deacetylation-mimetic KI mice plus conditional KO, human patient tissue validation, multiple orthogonal functional endpoints\",\n      \"pmids\": [\"38639088\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CypD directly interacts with ATP5B (ATP synthase beta subunit) to promote mitochondrial ROS release in vascular smooth muscle cells; CypD knockout or CsA inhibition reduces ROS, 8-OHdG production, NLRP3 inflammasome activation, and MMP9 upregulation, defining a CypD–ATP5B–ROS–8-OHdG–NLRP3–MMP9 pathway in intracranial aneurysm pathogenesis.\",\n      \"method\": \"Co-immunoprecipitation (CypD–ATP5B), CypD−/− mouse model, CsA pharmacological inhibition, ROS measurement, 8-OHdG/NLRP3/MMP9 functional assays, in vivo intracranial aneurysm model\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP identifying novel binding partner ATP5B plus genetic KO with defined pathway; single lab\",\n      \"pmids\": [\"37717465\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"miR-155-5p in ischemia/reperfusion-derived serum exosomes suppresses NEDD4 expression; NEDD4 normally promotes CypD ubiquitination and degradation; loss of NEDD4 increases CypD protein levels, augmenting mPTP opening and cardiomyocyte apoptosis during myocardial I/R injury.\",\n      \"method\": \"miRNA inhibitor transfection, luciferase reporter (miR-155-5p targeting NEDD4 3'UTR), shRNA knockdown of NEDD4/CypD, Co-immunoprecipitation of CypD ubiquitination, in vivo I/R mouse model, apoptosis/infarct size assays\",\n      \"journal\": \"ESC heart failure\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — epistasis chain (miR-155-5p → NEDD4 → CypD ubiquitination → mPTP) validated with rescue experiments; single lab\",\n      \"pmids\": [\"36631006\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PPIF (CypD) in neutrophils exacerbates lung ischemia-reperfusion injury by promoting store-operated calcium entry (SOCE)-mediated calcium overload, which activates calcineurin/NFAT signaling and drives neutrophil extracellular trap (NET) formation; PPIF inhibition (CsA) or PPIF knockdown alleviates mitochondrial dysfunction, ROS production, and NET formation in a lung transplant model.\",\n      \"method\": \"Orthotopic lung transplant mouse model, PPIF inhibitor (CsA) and PADI4 inhibitor in vivo, HL-60 neutrophil differentiation model, PPIF siRNA knockdown, SOCE measurement, calcineurin/NFAT pathway assays, NET quantification, mitochondrial function assays\",\n      \"journal\": \"International immunopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct PPIF mechanistic pathway (SOCE→Ca²⁺ overload→calcineurin/NFAT→NETs) with genetic and pharmacological validation; single lab\",\n      \"pmids\": [\"39236457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Sirt3-mediated deacetylation of CypD at K167 alleviates P. gingivalis-induced mitochondrial and endothelial dysfunction; CypD-K167 point mutation plasmids and Co-immunoprecipitation confirmed the Sirt3–CypD interaction, and Sirt3 agonist Honokiol rescued endothelial function in vitro and restored vasorelaxation in vivo.\",\n      \"method\": \"Co-immunoprecipitation (Sirt3–CypD), CypD-K167 point mutation plasmids, Sirt3-specific agonist Honokiol, RNA sequencing of infected endothelial cells, mitochondrial ROS/ΔΨm assays, mouse aortic vasorelaxation ex vivo\",\n      \"journal\": \"Journal of periodontal research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP plus site-directed mutation and in vivo validation; single lab, newly published\",\n      \"pmids\": [\"40344434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CypD (Ppif) ablation prevents cognitive decline, synaptic impairment, and mPTP-mediated mitochondrial damage induced by caspase-3-cleaved tau in the hippocampus; stereotaxic AAV injection of truncated tau in CypD−/− mice showed no mPTP opening or synaptic vesicle protein deregulation, establishing CypD as a necessary downstream effector of pathological tau-driven neurodegeneration.\",\n      \"method\": \"Stereotaxic hippocampal AAV injection (full-length vs. caspase-3-cleaved tau), CypD−/− and tau−/− KO mice, cognitive behavioral testing (Morris water maze, etc.), synaptic protein analysis, mitochondrial function/mPTP assays\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic KO epistasis with defined molecular (mPTP) and behavioral endpoints; single lab\",\n      \"pmids\": [\"40023297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Ppif (CypD) gene knockout in mice protects against sepsis-induced pancreatic injury; Ppif KO reduced serum IL-6 and amylase, improved pancreatic histopathology, decreased apoptosis indices, and ultrastructural analysis revealed that Ppif KO pancreatic acinar cells develop more autophagosomes rather than undergoing mitochondrial swelling and necrotic changes seen in wild-type CLP mice.\",\n      \"method\": \"Cecal ligation and puncture (CLP) sepsis model in Ppif KO vs. wild-type mice, serum IL-6/amylase ELISA, H&E histology, TUNEL apoptosis assay, transmission electron microscopy of pancreatic ultrastructure\",\n      \"journal\": \"The Journal of surgical research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with defined molecular and morphological endpoints; single lab, no prior replication\",\n      \"pmids\": [\"41270585\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Female Ppif−/− (CypD-null) mice are more susceptible to hormone-driven (MPA/DMBA) mammary carcinogenesis than wild-type mice, while Ripk3−/− or Mlkl−/− mice are not, indicating that CypD-dependent MPT-driven necrosis acts as an oncosuppressive mechanism specifically restraining HR+ mammary tumor development.\",\n      \"method\": \"Whole-body Ppif KO, Ripk3 KO, and Mlkl KO female C57BL/6J mice in MPA/DMBA-driven mammary carcinogenesis model, tumor incidence and progression monitoring\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic KO with specific pathway comparison (PPIF vs. necroptosis effectors); single lab\",\n      \"pmids\": [\"40494873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"CypD-dependent mPTP opening is required for mitochondrial swelling and ferroptosis; during ferroptosis, oxidized mitochondrial DNAs (mtDNAs) are released through the CypD-regulated mPTP and activate the cGAS–STING pathway, which promotes ferritinophagy and amplifies ferroptotic signaling; mtDNA repair inhibition synergizes with ferroptosis inducers in suppressing tumor xenografts.\",\n      \"method\": \"CypD genetic KO and CsA pharmacological inhibition, mPTP opening assay during ferroptosis, oxidized mtDNA detection and release measurement, cGAS-STING pathway activation assay, ferritinophagy assay, xenograft tumor model\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic and pharmacological CypD manipulation with defined molecular outputs (mtDNA release, cGAS-STING); single lab, newly published\",\n      \"pmids\": [\"41700459\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CypD overexpression in skeletal muscle myofibers increases mitoflash frequency and area with accompanying perinuclear mitochondrial Ca²⁺ efflux; a phospho-resistant CypD-S42A mutant behaves similarly to wild-type CypD overexpression, while expression of only the mitochondrial targeting sequence (CypDN30) does not cause these phenotypes; sodium butyrate feeding reverses CypD-associated mitoflash phenotypes in an ALS mouse model, linking CypD expression level to mitochondrial Ca²⁺ dynamics and mPTP-associated mitoflash activity.\",\n      \"method\": \"CypD-jRCaMP1b fusion constructs (live Ca²⁺ imaging), CypD-S42A phospho-resistant mutation, mitoflash quantification in isolated myofibers, ALS (SOD1-G93A) mouse model, sodium butyrate dietary supplementation\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — live imaging with mutation analysis and defined Ca²⁺ efflux readout; single lab\",\n      \"pmids\": [\"34299032\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PPIF (CypD/cyclophilin D) is a mitochondrial matrix peptidyl-prolyl cis-trans isomerase that acts as the master regulatory subunit of the mitochondrial permeability transition pore (mPTP): it physically binds VDAC, ANT, and SPG7 to form the pore complex and sensitizes pore opening in response to Ca²⁺ and oxidative stress; its activity is regulated by multiple PTMs (acetylation at K166/K167 by GCN5L1, deacetylation by SIRT3; sulphenylation at C104 by SO₂; ubiquitination via NEDD4), by direct protein interactions (p53, Trap1/HSP90, PGAM5, ATP5B, ANT via Cys160), and by phosphorylation at S42, with CypD-dependent mPTP opening driving necrosis, necroptosis, ferroptosis, and ischemia-reperfusion injury across multiple tissues while also mediating physiological Ca²⁺ efflux and oncosuppression in non-stressed contexts.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PPIF encodes Cyclophilin D (CypD), a mitochondrial matrix peptidyl-prolyl isomerase that serves as the principal regulator of mitochondrial permeability transition pore (mPTP) opening, linking diverse upstream stress signals to mitochondrial-dependent cell death across cardiac, vascular, neuronal, immune, and tumor cell contexts. CypD's isomerase activity — mapped to active-site residues R55, F60, F113, and W121 — directly promotes mPTP opening and can isomerize substrates such as p53 into amyloid-type aggregates; this activity is normally restrained by sequestration with the mitochondrial chaperone Trap1, from which CypD is released upon oxidative stress [PMID:27515399]. CypD activity is bidirectionally controlled by acetylation at K166: GCN5L1-mediated acetylation promotes mPTP opening and endothelial dysfunction, whereas SIRT3-mediated deacetylation is protective, as demonstrated by K166R deacetylation-mimetic knock-in mice that resist neuropathic pain, hypertension, and depression-like behaviors [PMID:36092157, PMID:38639088, PMID:41643830]. CypD-dependent mPTP opening also mediates ferroptotic release of oxidized mitochondrial DNA to activate cGAS-STING signaling [PMID:41700459], drives necroptosis downstream of the RIPK3–PGAM5 axis [PMID:29770487], and is required for truncated tau-induced neurodegeneration [PMID:40023297], while whole-body CypD loss increases susceptibility to HR-positive mammary carcinogenesis, indicating CypD-regulated necrosis exerts tumor-suppressive functions [PMID:40494873].\",\n  \"teleology\": [\n    {\n      \"year\": 2016,\n      \"claim\": \"The molecular basis of CypD's mPTP-promoting activity was resolved: its peptidyl-prolyl isomerase active site (R55, F60, F113, W121) directly isomerizes p53 into amyloid fibrils, and this activity is held in check by Trap1 until oxidative stress displaces the complex, establishing CypD as a conditionally activated enzymatic trigger of pore opening.\",\n      \"evidence\": \"In vitro reconstitution of p53 aggregation, NMR chemical shift mapping, active-site mutagenesis, and Gamitrinib-mediated Trap1 inhibition in primary MEFs\",\n      \"pmids\": [\"27515399\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Trap1 displacement is the sole mechanism of CypD activation in vivo\", \"Identity of endogenous mPTP structural substrates isomerized by CypD beyond p53\", \"No structural model of the CypD–Trap1 inhibitory complex\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"CypD was positioned downstream of necroptotic signaling: RIPK3 upregulates PGAM5, which increases CypD phosphorylation and consequent mPTP opening, establishing a defined signal transduction path from necroptosis kinases to mitochondrial permeability transition.\",\n      \"evidence\": \"Ripk3 genetic ablation and pharmacological inhibition in cardiac microvascular ischemia-reperfusion model with mPTP and necroptosis marker readouts\",\n      \"pmids\": [\"29770487\", \"30611988\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The specific phosphorylation site(s) on CypD modified by PGAM5 are not mapped\", \"Whether PGAM5 phosphorylates CypD directly or through an intermediary kinase\", \"Phenotype not yet confirmed in CypD phospho-site mutant knock-in mice\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"CypD was shown to participate in mPTP-dependent cell death in drug-resistant melanoma cells downstream of MEK/ERK signaling, and to interact functionally with ANT, broadening the pathological contexts and identifying epistatic partners of CypD at the pore.\",\n      \"evidence\": \"siRNA knockdown of CypD and ANT with MEK/ERK pharmacological inhibition in vemurafenib-resistant melanoma cells and xenograft models\",\n      \"pmids\": [\"33440231\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CypD and ANT interact directly in these cells was not shown by co-IP\", \"The mechanism by which MEK/ERK regulates CypD expression or activity is undefined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"The critical post-translational regulatory switch on CypD was identified: SIRT3-mediated deacetylation of K166 inhibits mPTP opening, and SO2-mediated sulphenylation of C104 independently inhibits mPTP, establishing two distinct inhibitory modifications on CypD.\",\n      \"evidence\": \"CypD-K166R knock-in mice rescued from neuropathic pain after spared nerve injury; CypD-C104S mutagenesis abolished SO2-induced mPTP inhibition in H9c2 cardiomyocytes\",\n      \"pmids\": [\"36092157\", \"35118072\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether K166 acetylation and C104 sulphenylation interact or are independent regulatory axes\", \"The acetyltransferase responsible for K166 acetylation was not yet identified in 2022\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"CypD was found to directly interact with ATP5B (ATP synthase β-subunit) to promote ROS release in vascular smooth muscle cells, and NEDD4 E3 ligase was identified as a ubiquitin ligase controlling CypD protein levels, adding a binding partner and a degradation pathway to the regulatory network.\",\n      \"evidence\": \"Co-IP of CypD–ATP5B and CypD-KO mice in intracranial aneurysm model; Co-IP for CypD ubiquitination by NEDD4 with miR-155-5p epistasis in ischemia-reperfusion model\",\n      \"pmids\": [\"37717465\", \"36631006\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reciprocal Co-IP for CypD–ATP5B not reported\", \"Whether NEDD4-dependent ubiquitination targets CypD for proteasomal or lysosomal degradation is unresolved\", \"The specific ubiquitination sites on CypD are unmapped\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"The acetylation-promoting enzyme was identified as GCN5L1, completing the K166 acetylation/deacetylation circuit; simultaneously, CypD-dependent mPTP opening was shown to mediate oxidized mtDNA release that activates cGAS-STING to drive ferroptosis, expanding CypD's role beyond necrosis/necroptosis to ferroptotic cell death.\",\n      \"evidence\": \"Endothelial-specific GCN5L1 KO and CypD-K166R mice in angiotensin II hypertension model; genetic and pharmacological CypD inhibition with oxidized mtDNA and cGAS-STING pathway measurements in tumor xenografts\",\n      \"pmids\": [\"38639088\", \"41700459\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct in vitro acetylation of CypD by GCN5L1 has not been reconstituted with purified proteins\", \"Whether cGAS-STING activation requires CypD isomerase activity specifically or just pore opening\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"CypD was placed downstream of ER–mitochondria calcium transfer: PDZD8 at mitochondria-associated membranes drives Ca2+ overload that upregulates CypD to induce β-cell apoptosis, and CypD in neutrophils promotes store-operated Ca2+ entry and NET formation, revealing CypD as a calcium-responsive effector in non-canonical immune and metabolic cell death contexts.\",\n      \"evidence\": \"PDZD8 knockdown with CypD overexpression rescue in pancreatic β-cells; PPIF siRNA and cyclosporin A in neutrophil/HL-60 cells and lung transplant model\",\n      \"pmids\": [\"39069376\", \"39236457\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CypD expression is transcriptionally upregulated by Ca2+ or stabilized post-translationally in these settings\", \"The mechanism linking CypD to SOCE/calcineurin/NFAT in neutrophils is indirect\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Cell-type-specific and disease-context roles of CypD were delineated: conditional CypD ablation in parvalbumin interneurons prevents anesthetic neurotoxicity, CypD loss is required for truncated tau-induced neurodegeneration, and whole-body CypD deletion paradoxically increases HR+ mammary tumor susceptibility, demonstrating CypD-regulated necrosis has both pathogenic and tumor-suppressive functions.\",\n      \"evidence\": \"PpifF/F-PVCre conditional KO with behavioral and electrophysiology; CypD-/- mice with AAV-tau hippocampal injection; Ppif-/- vs Ripk3-/- vs Mlkl-/- in MPA/DMBA mammary carcinogenesis\",\n      \"pmids\": [\"40784489\", \"40023297\", \"40494873\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The specific mPTP composition in parvalbumin interneurons is unknown\", \"How CypD-regulated necrosis suppresses HR+ but not other tumor types is unexplained\", \"Whether CypD's tumor-suppressive role depends on its isomerase activity or solely on pore opening\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular identity and stoichiometry of the mPTP pore complex that CypD regulates remain unresolved; it is unknown whether CypD's isomerase activity or a non-enzymatic scaffolding function is the primary driver of pore opening in vivo, and no high-resolution structure of CypD engaged with its pore target exists.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No reconstituted mPTP with defined subunit composition\", \"No cryo-EM or crystal structure of CypD bound to a pore component\", \"The relative contributions of isomerase activity vs. protein–protein interaction to mPTP regulation are unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016853\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 2, 3, 6, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 1, 2, 3, 6, 11, 12, 13, 14]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [3, 4, 5, 7, 8, 13, 14, 15]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [0, 1, 2, 3, 6, 11]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [9, 13]}\n    ],\n    \"complexes\": [\n      \"mitochondrial permeability transition pore (mPTP)\",\n      \"CypD–Trap1 inhibitory complex\"\n    ],\n    \"partners\": [\n      \"TRAP1\",\n      \"ATP5B\",\n      \"SIRT3\",\n      \"GCN5L1\",\n      \"NEDD4\",\n      \"PGAM5\",\n      \"ANT\",\n      \"PDZD8\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"PPIF (cyclophilin D) is a mitochondrial matrix peptidyl-prolyl cis-trans isomerase that functions as the principal regulatory subunit of the mitochondrial permeability transition pore (mPTP), gating Ca²⁺- and oxidative stress-induced mitochondrial permeability transition to control necrosis, ferroptosis, and physiological Ca²⁺ efflux. CypD assembles with VDAC and ANT (binding ANT via Cys160) to form the minimal pore complex, and additionally interacts with SPG7, ATP5B, p53, and TRAP1/HSP90 to integrate diverse stress signals into mPTP opening [PMID:9874241, PMID:12149099, PMID:26387735, PMID:22726440, PMID:27515399, PMID:37717465]. Its activity is tuned by acetylation at K166/K167 (promoted by GCN5L1, reversed by SIRT3), sulphenylation at C104, NEDD4-mediated ubiquitination, and RIPK3–PGAM5-dependent phosphorylation, with K166 deacetylation-mimetic knock-in mice showing protection from vascular oxidative stress and hypertension [PMID:36092157, PMID:38639088, PMID:35118072, PMID:36631006, PMID:29770487]. CypD-dependent mPTP opening also drives oxidized mtDNA release to activate cGAS–STING signaling during ferroptosis and acts as an oncosuppressive mechanism in hormone receptor-positive mammary tumorigenesis [PMID:41700459, PMID:40494873].\",\n  \"teleology\": [\n    {\n      \"year\": 1991,\n      \"claim\": \"Establishing that PPIF encodes a distinct mitochondrial cyclophilin with intrinsic PPIase activity resolved the identity of the cyclosporin A-sensitive isomerase in the mitochondrial matrix.\",\n      \"evidence\": \"cDNA cloning, recombinant expression, PPIase assay with CsA inhibition kinetics, subcellular fractionation\",\n      \"pmids\": [\"1744118\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological substrates of the PPIase activity were unknown\", \"Relationship to the permeability transition pore had not been tested\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Reconstitution of a Ca²⁺-sensitive, CsA-inhibitable pore from purified VDAC, ANT, and CypD in proteoliposomes established CypD as a core component of the mPTP, answering how cyclosporin A blocks permeability transition.\",\n      \"evidence\": \"GST-CypD affinity pulldown, VDAC/ANT co-purification, proteoliposome reconstitution with fluorescein sulphonate permeability assay\",\n      \"pmids\": [\"9874241\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The stoichiometry and topology of the pore complex were undefined\", \"Whether additional subunits are required in vivo was unknown\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Mapping the ANT Cys160 thiol as the CypD docking site and showing that PPIase catalytic activity is required for cytoprotection but dispensable for ANT binding separated CypD's pore-regulatory role from its enzymatic function on other substrates.\",\n      \"evidence\": \"Site-directed mutagenesis of CypD PPIase site and ANT Cys160, GST pulldowns, mPTP Ca²⁺ sensitivity assays, live-cell two-photon imaging, apoptosis readouts\",\n      \"pmids\": [\"12077116\", \"12149099\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The identity of PPIase-dependent cytoprotective substrate(s) remained unknown\", \"In vivo genetic confirmation (KO mice) was not yet available\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"High-resolution crystal structures of CypD revealed that the S2 surface outside the proline-binding pocket confers isoform specificity, providing a structural framework for selective CypD inhibitor design.\",\n      \"evidence\": \"X-ray crystallography, PPIase assay across 15 cyclophilin isoforms, CsA binding, computational docking\",\n      \"pmids\": [\"20676357\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No co-crystal with a physiological substrate or mPTP component was obtained\", \"Structure of CypD within the intact pore complex remained unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Discovery that mitochondrial p53 triggers mPTP-dependent necrosis through direct complex formation with CypD linked tumor suppressor signaling to the permeability transition pore during ischemia-reperfusion.\",\n      \"evidence\": \"Reciprocal Co-IP of p53–CypD in cells and in vivo mouse stroke model, genetic p53 knockdown and CsA pharmacological epistasis\",\n      \"pmids\": [\"22726440\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding interface between p53 and CypD was not mapped\", \"Whether p53 is a PPIase substrate or allosteric activator was unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Unbiased proteomics and metabolomics of CypD-knockout hearts revealed broad remodeling of Krebs cycle and fatty acid oxidation enzymes, establishing that CypD shapes mitochondrial metabolic pathway composition beyond its mPTP gating role.\",\n      \"evidence\": \"LC-MS/MS quantitative proteomics and acylcarnitine profiling in Ppif⁻/⁻ vs. WT mouse hearts\",\n      \"pmids\": [\"23262437\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether metabolic effects are secondary to chronic mPTP dysregulation or reflect direct PPIase substrates was unresolved\", \"Tissue-specificity of metabolic remodeling was not assessed\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"An unbiased RNAi screen identified SPG7 as a required mPTP component that must interact with CypD for pore opening, expanding the minimal pore model beyond VDAC and ANT.\",\n      \"evidence\": \"RNAi screen, reciprocal Co-IP of SPG7–CypD–VDAC, mitochondrial Ca²⁺ retention and cell death assays\",\n      \"pmids\": [\"26387735\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SPG7 is a structural pore subunit or a regulatory cofactor was debated\", \"The exact molecular architecture of the pore remained unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"NMR mapping showed CypD catalytically drives p53 amyloid-type aggregation via active-site residues R55/F60/F113/W121, and TRAP1 normally sequesters CypD in an inhibited complex, explaining how oxidative stress derepresses CypD's pro-death activity.\",\n      \"evidence\": \"In vitro CypD–p53 aggregation reconstitution, NMR chemical shift mapping, Gamitrinib (mitochondria-targeted HSP90 inhibitor), genetic epistasis in MEFs\",\n      \"pmids\": [\"27515399\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo confirmation of TRAP1–CypD stoichiometry and dynamics was lacking\", \"Relevance to non-p53 substrates was not tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identification of RIPK3–PGAM5-dependent CypD phosphorylation during cardiac ischemia-reperfusion connected necroptotic signaling to mPTP opening in endothelial cells.\",\n      \"evidence\": \"Ripk3 genetic ablation, phospho-CypD immunoblotting, PGAM5 pathway analysis, in vivo cardiac IR model\",\n      \"pmids\": [\"29770487\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The specific CypD phosphorylation site(s) were not mapped by mass spectrometry\", \"Independent replication in other tissues is needed\", \"Kinase directly phosphorylating CypD was not definitively identified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Live imaging of CypD-overexpressing myofibers demonstrated that CypD dosage controls mitoflash frequency and perinuclear Ca²⁺ efflux, with S42 phosphorylation dispensable (S42A behaves as WT), linking CypD expression level to physiological mPTP-dependent Ca²⁺ dynamics in muscle.\",\n      \"evidence\": \"CypD-jRCaMP1b fusion live Ca²⁺ imaging, S42A phospho-resistant mutant, mitoflash quantification in isolated myofibers, SOD1-G93A ALS model\",\n      \"pmids\": [\"34299032\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"S42 phosphorylation role not fully excluded under all stress conditions\", \"Overexpression system limits physiological interpretation\", \"Single lab observation\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Mapping K166 acetylation (by GCN5L1, reversed by SIRT3) and C104 sulphenylation as opposing regulatory switches resolved how post-translational modifications fine-tune CypD's mPTP-sensitizing activity: K166 acetylation promotes pore opening while C104 sulphenylation suppresses it.\",\n      \"evidence\": \"CypD-K166R point mutation with in vivo behavioral rescue, biotin-switch sulphenylation assay, C104S mutagenesis, mPTP opening and apoptosis assays in cardiomyocytes and spinal cord neurons\",\n      \"pmids\": [\"36092157\", \"35118072\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Interplay between acetylation and sulphenylation on the same CypD molecule was not tested\", \"Crystal structure of modified CypD forms was not determined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of NEDD4-mediated ubiquitination as a CypD degradation pathway, suppressed by exosomal miR-155-5p during I/R injury, revealed a post-translational mechanism controlling CypD protein levels rather than activity.\",\n      \"evidence\": \"miR-155-5p inhibitor, luciferase reporter for NEDD4 3′UTR, Co-IP of CypD ubiquitination, in vivo I/R model with infarct size assays\",\n      \"pmids\": [\"36631006\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitination site(s) on CypD not mapped\", \"Independent replication needed\", \"Whether NEDD4 directly ubiquitinates CypD or acts through an intermediate was not definitively shown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"CypD-K166R deacetylation-mimetic knock-in mice demonstrated protection from Ang II-induced hypertension and endothelial dysfunction, and endothelial-specific GCN5L1 knockout prevented mitochondrial oxidative stress, translating the K166 acetylation switch to a defined cardiovascular disease mechanism in vivo.\",\n      \"evidence\": \"CypD-K166R knock-in mice, endothelial-specific GCN5L1 KO, human patient arteriole Co-IP, Ang II hypertension model, metabolic flux analysis\",\n      \"pmids\": [\"38639088\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether K166 acetylation status is a viable therapeutic biomarker in humans was not tested\", \"Tissue-specificity of GCN5L1-CypD axis beyond endothelium is unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Genetic epistasis studies in multiple disease models—tau-driven neurodegeneration, sepsis-induced pancreatic injury, and hormone-driven mammary carcinogenesis—collectively established CypD as a convergent downstream effector: its ablation protects against pathological mPTP opening in neuronal and acinar contexts while paradoxically increasing tumor susceptibility, revealing an oncosuppressive function of MPT-driven necrosis.\",\n      \"evidence\": \"Ppif⁻/⁻ mice with hippocampal AAV-tau injection, CLP sepsis model, MPA/DMBA mammary carcinogenesis model with comparison to Ripk3⁻/⁻ and Mlkl⁻/⁻ mice\",\n      \"pmids\": [\"40023297\", \"41270585\", \"40494873\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which CypD-dependent necrosis specifically suppresses HR+ tumors is unknown\", \"Whether CypD's oncosuppressive role involves its PPIase activity or mPTP gating is not distinguished\", \"Independent replication of carcinogenesis finding is needed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"CypD-dependent mPTP opening was shown to mediate oxidized mtDNA release during ferroptosis, activating the cGAS–STING pathway to promote ferritinophagy and amplify ferroptotic cell death, extending CypD's role to a new form of regulated cell death.\",\n      \"evidence\": \"CypD KO and CsA inhibition during ferroptosis, oxidized mtDNA release measurement, cGAS–STING activation assay, ferritinophagy assay, tumor xenograft model\",\n      \"pmids\": [\"41700459\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CypD directly senses lipid peroxidation signals or is activated indirectly is unknown\", \"The ferroptosis findings have not been independently replicated\", \"Structural basis for CypD-dependent mtDNA release channel is undefined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The high-resolution structure of CypD within the assembled mPTP complex, the full catalog of PPIase substrates in the mitochondrial matrix, and the integration of multiple PTMs (acetylation, sulphenylation, phosphorylation, ubiquitination) into a unified regulatory model remain major open questions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No cryo-EM or structural model of the intact mPTP with CypD bound\", \"Comprehensive identification of mitochondrial PPIase substrates has not been performed\", \"Quantitative model integrating combinatorial PTM effects on CypD activity is lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016853\", \"supporting_discovery_ids\": [0, 4, 9]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 2, 8, 11, 12]},\n      {\"term_id\": \"GO:0044183\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 1, 6, 7, 22]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [1, 5, 8, 10, 11, 21]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [3, 5, 12, 13]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [16, 19]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [19]}\n    ],\n    \"complexes\": [\n      \"mitochondrial permeability transition pore (mPTP)\"\n    ],\n    \"partners\": [\n      \"VDAC1\",\n      \"SLC25A4\",\n      \"SPG7\",\n      \"TP53\",\n      \"TRAP1\",\n      \"ATP5F1B\",\n      \"SIRT3\",\n      \"PGAM5\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}