{"gene":"PPIF","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":1991,"finding":"PPIF (hCyP3) encodes a mitochondria-associated peptidyl-prolyl cis-trans isomerase. The protein was expressed in E. coli, purified, and shown to be an active PPIase; its NH2-terminal hydrophobic extension (42 aa) acts as a signal peptide directing it to subcellular organelles/membranes. Substrate specificity with synthetic Suc-Xaa-Yaa-Pro-Phe-nitroanilide peptides and inhibition by cyclosporin A analogues were characterized.","method":"Recombinant protein expression in E. coli, PPIase activity assay with synthetic peptide substrates, kinetic characterization with CsA analogues, protein-specific antibody subcellular fractionation, Northern/Western blot","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted enzymatic activity in vitro with substrate specificity and inhibitor kinetics; replicated across three human CyP isoforms in the same rigorous study","pmids":["1744118"],"is_preprint":false},{"year":1999,"finding":"CyP-D (PPIF) is a key structural component of the mitochondrial permeability transition pore (MPTP). Using a CyP-D fusion protein as affinity matrix, CyP-D was shown to bind strongly to 1:1 complexes of VDAC (outer membrane) and adenine nucleotide translocase (ANT; inner membrane). Covalent labelling of CyP-D in situ by a photoactive CsA derivative demonstrated that pore ligands have the same effects on pore block and CyP-D binding, confirming CsA inhibits the MPTP by binding CyP-D.","method":"CyP-D fusion protein affinity pulldown of VDAC/ANT complexes; photoaffinity labelling of CyP-D in situ with CsA derivative; pore reconstitution from protein fractions","journal":"Biochemical Society Symposium","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — affinity pulldown reconstitution plus photoaffinity labelling in organello; findings replicated across multiple labs reviewing same data","pmids":["10989666"],"is_preprint":false},{"year":1999,"finding":"CyP-D forms a complex with VDAC and ANT at contact sites between the mitochondrial inner and outer membranes to constitute the MPTP. Cyclosporin A inhibits pore opening by binding CyP-D, reducing its Ca2+ binding affinity and blocking pore flickering. Under oxidative stress and high Ca2+, the complex opens to allow free diffusion of low-molecular-weight solutes (<1.5 kDa) across the inner membrane.","method":"CyP-D affinity matrix pulldown, pore reconstitution, CsA photolabelling, Ca2+ flux assays in isolated mitochondria","journal":"The Biochemical Journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reconstitution experiments plus pharmacological validation with photoaffinity probe; reviewed and independently replicated across laboratories","pmids":["10393078"],"is_preprint":false},{"year":2002,"finding":"CyP-D facilitates a Ca2+-triggered conformational change of the ANT that induces MPTP opening; its PPIase activity mediates this conformational facilitation. Oxidative modification of ANT Cys56 increases CyP-D binding to the ANT (likely at Pro61), while modification of Cys159 inhibits adenine nucleotide binding. Reconstitution studies demonstrated that neither VDAC nor other outer membrane proteins are required for minimal MPTP formation.","method":"Reconstitution of MPTP from purified ANT ± CyP-D ± VDAC; cysteine-specific chemical modification of ANT; CsA inhibition of PPIase activity correlated with pore inhibition","journal":"Biochimie","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution with defined purified components, site-specific mutagenesis/chemical modification, PPIase-MPTP correlation; multiple orthogonal methods in one study","pmids":["12022946"],"is_preprint":false},{"year":2005,"finding":"Genetic knockout of CyP-D (PPIF) in mice demonstrated that CyP-D is the target for MPTP inhibition by CsA; however, MPTP can still form and open in the absence of CyP-D, indicating that CyP-D is a regulatory sensitizer rather than a structural pore-forming component of the inner mitochondrial membrane.","method":"CyP-D knockout mouse mitochondria; MPTP activity assays (Ca2+-induced swelling) ± CsA","journal":"Journal of Bioenergetics and Biomembranes","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic KO with defined mitochondrial phenotype; finding replicated in multiple independent CyP-D KO studies reviewed in this paper","pmids":["16167169"],"is_preprint":false},{"year":2007,"finding":"CyP-D knockout mice (Ppif−/−) showed that CyP-D-dependent MPT is essential for necrotic but not apoptotic cell death, and plays a crucial role in ischemia/reperfusion injury. CyP-D-null mice develop normally and show no protection against diverse apoptotic stimuli.","method":"Genetic KO mouse model (Ppif−/−); cell death assays distinguishing necrosis vs apoptosis; ischemia/reperfusion injury models","journal":"Apoptosis","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic KO with phenotypically defined necrosis vs apoptosis distinction; independently replicated across multiple labs","pmids":["17136322"],"is_preprint":false},{"year":2008,"finding":"CyP-D's peptidyl-prolyl cis-trans isomerase activity facilitates a conformational change in an inner membrane protein (proposed to be the mitochondrial phosphate carrier, PiC) to induce MPTP opening. ANT knockout studies showed ANT plays a regulatory rather than pore-forming role. CyP-D is proposed to bind PiC and facilitate its Ca2+-triggered conformational change.","method":"Genetic KO of ANT isoforms in mice; CyP-D KO; biochemical interaction studies; review of reconstitution data","journal":"Biochimica et Biophysica Acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO epistasis for ANT confirmed; PiC as substrate is proposed based on biochemical evidence but knockdown/reconstitution awaited; single lab","pmids":["18407825"],"is_preprint":false},{"year":2010,"finding":"CyP-D deletion (Ppif−/−) protected mice completely against acetaminophen-induced liver necrosis and DNA fragmentation, demonstrating that CyP-D-dependent MPT is a critical downstream event in APAP hepatotoxicity. Oxidative stress (GSSG levels) and peroxynitrite formation were blunted but not eliminated in CypD-deficient mice, placing oxidant stress at least partly downstream of MPT.","method":"Ppif−/− knockout mice treated with APAP; liver histology, DNA fragmentation assay, GSSG measurement, nitrotyrosine immunostaining","journal":"Free Radical Research","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic KO with quantitative biochemical readouts; multiple orthogonal injury markers; single lab but rigorous","pmids":["20942566"],"is_preprint":false},{"year":2010,"finding":"CyP-D is required for normal learning and memory: Ppif−/− mice showed deficits in short-term memory, object recognition, reference memory, and associative learning. Hippocampal infusion of CsA replicated these deficits. CyP-D absence reduced stimulus-evoked hippocampal glutamate and acetylcholine release, linking CyP-D to regulation of neurotransmission.","method":"Ppif−/− KO mice; behavioral tests (Y-maze, novel object recognition, water maze, fear conditioning); hippocampal microdialysis; CsA hippocampal infusion","journal":"Hippocampus","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic KO with multiple behavioral and neurochemical readouts; pharmacological recapitulation; single lab","pmids":["19437409"],"is_preprint":false},{"year":2013,"finding":"CyP-D (PPIF) deletion or knockdown results in increased cell proliferation, enhanced migration and invasion, and transcriptional upregulation of a chemokine/chemokine receptor gene signature. In the absence of CyP-D, STAT3 is activated, drives accelerated S-phase entry via cell proliferation, and stimulates autocrine/paracrine Cxcl12-Cxcr4-directed chemotaxis, indicating that CyP-D controls mitochondria-to-nucleus inflammatory gene expression.","method":"CyP-D knockout/knockdown cells; proliferation assays; migration/invasion assays; transcriptome profiling; STAT3 activation assays; Cxcl12-Cxcr4 signaling analysis","journal":"The Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined phenotypic readouts (proliferation, migration, STAT3) and transcriptomic pathway placement; single lab","pmids":["23303179"],"is_preprint":false},{"year":2014,"finding":"In glioma cells, ROS production induced by salinomycin drives p53 translocation to mitochondria, where p53 forms a physical complex with CyP-D. This p53/CyP-D complex is required for MPTP opening and programmed necrosis. Blocking CyP-D by siRNA or pharmacological inhibitors (CsA, sanglifehrin A), or p53 knockdown, suppressed necrosis but not apoptosis.","method":"siRNA-mediated CyP-D depletion; Co-immunoprecipitation of p53/CyP-D complex at mitochondria; pharmacological inhibition (CsA, SFA); mitochondrial membrane potential assay; cell death quantification","journal":"Journal of Experimental & Clinical Cancer Research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — reciprocal co-IP of p53/CyP-D complex plus genetic and pharmacological KD; single lab; two orthogonal methods","pmids":["26024660"],"is_preprint":false},{"year":2014,"finding":"CyP-D is required for berberine-induced programmed necrosis in prostate cancer cells. Berberine-induced ROS production drives p53 mitochondrial translocation, where p53 physically interacts with CyP-D to open the MPTP. shRNA depletion of CyP-D or p53 inhibited necrosis but not apoptosis.","method":"CyP-D shRNA; Co-immunoprecipitation of p53/CyP-D at mitochondria; CsA/SFA pharmacological inhibition; mitochondrial membrane potential; flow cytometry","journal":"Biochemical and Biophysical Research Communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP of p53/CyP-D complex confirmed by genetic and pharmacological knockdown in parallel; single lab, consistent with independent study (PMID 26024660)","pmids":["24946211"],"is_preprint":false},{"year":2014,"finding":"MST1 translocates to mitochondria in response to gemcitabine-induced ROS and forms a direct physical complex with CyP-D. This MST1/CyP-D mitochondrial complex is required for MPTP-dependent pancreatic cancer cell death. CsA (CyP-D inhibitor) prevented the MST1/CyP-D complexation and cell death.","method":"Co-immunoprecipitation of MST1/CyP-D at mitochondria; shRNA silencing of MST1 or CyP-D; CsA inhibition; overexpression studies; ROS measurement","journal":"Biochimie","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP of novel CyP-D interaction partner plus reciprocal genetic and pharmacological validation; single lab","pmids":["24732633"],"is_preprint":false},{"year":2015,"finding":"CyP-D regulates the frequency of mitochondrial 'mitoflash' events (transient MPTP openings under physiological conditions) in cardiomyocytes. CyP-D overexpression increased, and knockout halved, cardiac mitoflash frequency. CyP-D ablation reduced mitochondrial maximal respiration rate and reserved respiratory capacity in cardiomyocytes. The effect of CyP-D on mitoflashes was tissue-specific (cardiac vs skeletal muscle), correlating with 4-fold higher CyP-D expression in cardiac muscle.","method":"CyP-D overexpression and knockout in isolated cardiomyocytes, beating hearts, and skeletal muscles in vivo; live imaging of mitochondrial flash activity; Seahorse respirometry","journal":"Journal of Molecular and Cellular Cardiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal gain- and loss-of-function in multiple tissues with quantitative live imaging and respirometry; multiple orthogonal methods in one study","pmids":["26746144"],"is_preprint":false},{"year":2015,"finding":"PPIF (CyP-D) regulates necrosis but not apoptosis in eosinophils. Ppif−/− eosinophils were protected from Ca2+-overload (ionomycin)- and oxidative stress (H2O2)-induced necrosis and from Siglec-F cross-linking-induced necrosis, with no difference in apoptosis. In vivo, Ppif−/− mice showed reduced eosinophil cytolysis in DSS-induced colitis.","method":"Ppif−/− KO mice; ionomycin/H2O2 necrosis assays; flow cytometry for apoptosis vs necrosis; DSS colitis in vivo model","journal":"American Journal of Physiology: Gastrointestinal and Liver Physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic KO with defined cellular phenotype across multiple stimuli both in vitro and in vivo; multiple orthogonal readouts","pmids":["26893161"],"is_preprint":false},{"year":2015,"finding":"Necroptotic stimuli require both Bax/Bak oligomerization in the outer mitochondrial membrane and MPTP (CyP-D-dependent) opening in the inner membrane. Ppif−/− fibroblasts are resistant to necroptotic cell death inducers. MLKL and cofilin-1 translocate to mitochondria following necroptosis induction; some of these effects are lost in Ppif−/− cells.","method":"Ppif−/− KO fibroblasts; Bax/Bak double-null cells; MLKL/cofilin-1 mitochondrial fractionation; necroptosis induction with TNF/zVAD; cell death assays","journal":"PLoS ONE","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with pathway epistasis (necroptosis–MPTP crosstalk) and protein localization data; single lab","pmids":["26061004"],"is_preprint":false},{"year":2019,"finding":"PPIF-dependent mitochondrial permeability transition mediates crystal-induced (calcium oxalate, monosodium urate, calcium pyrophosphate, silica) necrosis in renal tubular cells. The pathway involves crystal phagocytosis, lysosomal cathepsin leakage, and ROS release. Ppif−/− mice displayed attenuated AKI; dual deletion of Ppif and Mlkl showed partial redundancy between MPT-driven necrosis and necroptosis.","method":"Ppif−/− KO mice; Ppif−/−Mlkl−/− double KO mice; in vitro crystal exposure in mouse/human tubular cells; pharmacological MPTP inhibition; histology; fractionation","journal":"Journal of the American Society of Nephrology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic KO (single and double) in vivo and in vitro with quantitative injury readouts and epistasis analysis; replicated pharmacologically","pmids":["31296606"],"is_preprint":false},{"year":2022,"finding":"BMP/Smad signaling transcriptionally represses the CyP-D gene (Ppif) during osteogenic differentiation of mesenchymal lineage cells, thereby deactivating MPTP. CyP-D 'rescue' via gain-of-function negatively affected osteogenesis both in vitro and in a mouse model, demonstrating that BMP/Smad-driven CyP-D downregulation is functionally required for efficient osteoblast differentiation.","method":"BMP/Smad pathway activation in MSCs; Ppif promoter reporter assays; CyP-D overexpression (gain-of-function) in vitro and in mouse model; osteogenic differentiation assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — transcriptional regulation mapped to Smad elements, gain-of-function validated in vitro and in vivo, multiple orthogonal methods; single lab but rigorous","pmids":["35645445"],"is_preprint":false},{"year":2023,"finding":"ANT1 (SLC25A4) and CyP-D (PPIF) are both required components of the MPTP governing necrotic cell death in vivo. Deletion of Slc25a4 in dystrophic (Sgcd−/−) mice partially protected from MD pathology; combined deletion of Slc25a4 and Ppif almost completely prevented necrotic cell death and muscular dystrophy disease, providing direct genetic evidence for both proteins as MPTP components.","method":"Genetic epistasis: single KO (Slc25a4−/− or Ppif−/−) and double KO (Slc25a4−/−Ppif−/−) in δ-sarcoglycan-null muscular dystrophy mice; histopathology; cell death quantification","journal":"Science Advances","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean in vivo genetic epistasis with double KO; quantitative disease phenotype readouts; directly demonstrates both ANT1 and CyP-D as MPTP components","pmids":["37624892"],"is_preprint":false},{"year":2023,"finding":"C/EBPα transcriptionally activates the CyP-D gene (Ppif) during adipogenesis of bone marrow stromal cells, increasing CyP-D expression and MPTP activity. NF-κB p65 subunit acts synergistically with C/EBPα to induce Ppif expression, linking adipogenic and inflammatory signaling to MPTP gain-of-function.","method":"Ppif promoter reporter assays; ChIP for C/EBPα and NF-κB p65 at Ppif promoter; adipogenic induction of BMSCs; CyP-D/MPTP activity measurement","journal":"The Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — promoter reporter plus ChIP identification of transcription factors; single lab; two orthogonal methods","pmids":["37949231"],"is_preprint":false},{"year":2024,"finding":"N-terminal cleavage of CyP-D (removing first 13 human residues) by calpain 1 generates a ΔN-CyPD form. NMR studies showed the N-terminus of full-length CyP-D is highly flexible. ΔN-CyPD binds the OSCP subunit of F-ATP synthase in saline media, whereas FL-CyPD does not, indicating the N-terminus substantially reduces affinity for OSCP. CyP-D binding to OSCP inhibits ATP synthase catalysis and favors transition of the enzyme to the permeability transition pore.","method":"NMR structure of FL- and ΔN-CyPD; binding assays with F-ATP synthase OSCP subunit; calpain 1 treatment of cells; mass spectrometry identification of ΔN-CyPD in cells","journal":"Communications Biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure, in vitro binding assay, identification of calpain 1 as the protease, and cellular validation; multiple orthogonal methods in one study","pmids":["39528709"],"is_preprint":false},{"year":2024,"finding":"Senescent cells exhibit high frequency of transient CyP-D/MPTP opening events ('flickering'). Genetic or pharmacological inhibition of CyP-D (PPIF) causes toxic mitochondrial Ca2+ accumulation and selective death of senescent cells (senolysis). Inhibition of NCLX (another mitochondrial Ca2+ efflux channel) also induces senolysis, while inhibition of MCU (Ca2+ influx) prevents CyP-D inhibition-induced senolysis, placing transient CyP-D/MPTP opening as a Ca2+ efflux mechanism critical for senescent cell survival.","method":"Genome-wide CRISPR/Cas9 screen; Ppif genetic KO; CsA pharmacological inhibition; NCLX and MCU inhibition/knockout; mitochondrial Ca2+ imaging; senescent cell viability assays","journal":"The EMBO Journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide CRISPR screen plus genetic and pharmacological validation plus epistasis with MCU/NCLX; multiple orthogonal methods; single lab but comprehensive","pmids":["39448884"],"is_preprint":false},{"year":2024,"finding":"Nynrin is a transcriptional repressor of Ppif (CyP-D gene). Nynrin knockout in cardiomyocytes enhanced Ppif transcription, increased CyP-D expression, promoted MPTP opening, and exacerbated ischemia/reperfusion injury. CsA treatment reversed the exacerbated phenotype. Nynrin overexpression blunted Ppif/CyP-D upregulation and reduced cardiomyocyte damage during OGD/R.","method":"Tamoxifen-inducible cardiomyocyte-specific Nynrin KO; Nynrin overexpression in primary cardiomyocytes; Ppif promoter regulation assays; MPTP opening assay; I/R injury model","journal":"Cell Stem Cell (for PMID 38955185); Journal of Molecular and Cellular Cardiology (for PMID 41077266)","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional genetic KO plus OE with pharmacological rescue; mechanistic link between Nynrin, Ppif transcription, CyP-D, and MPTP established in two independent studies (HSC and cardiac models)","pmids":["38955185","41077266"],"is_preprint":false},{"year":2024,"finding":"CyP-D (PPIF) is upregulated in wounds and venous ulcers. Inhibition of CyP-D impaired keratinocyte reepithelialization, granulation tissue formation, and wound closure in human and pig models. CyP-D inhibition in fibroblasts reduced collagen secretion and caused ER collagen accumulation; CyP-D overexpression increased collagen secretion. Ppif-KO mice showed reduced skin collagen.","method":"CyP-D inhibition (CsA) in human and pig wound models; CyP-D overexpression in keratinocytes/fibroblasts; Ppif-KO mice; collagen secretion and ER retention assays; migration assays","journal":"JCI Insight","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal gain- and loss-of-function in vitro, in vivo KO, and large-animal model; quantitative collagen secretion and wound closure readouts; multiple orthogonal methods","pmids":["38564292"],"is_preprint":false},{"year":2024,"finding":"CyP-D mediates macrophage death in atherosclerotic lesions and necrotic core formation. CyP-D knockdown in macrophages attenuated cytochrome c release from mitochondria induced by ER stress (thapsigargin) and blocked necroptosis induced by TNF-α/caspase inhibitor. Apoe−/−Ppif−/− double-KO mice had smaller necrotic cores with reduced macrophage apoptosis and decreased inflammatory monocytes.","method":"Apoe−/−Ppif−/− double-KO mouse model; siRNA knockdown of CyP-D in RAW264.7 cells; cytochrome c release assay; necroptosis induction; atherosclerosis lesion histology","journal":"Atherosclerosis","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic double KO plus in vitro siRNA with mechanistic readouts (cytochrome c release, necroptosis); multiple orthogonal methods","pmids":["38972156"],"is_preprint":false},{"year":2024,"finding":"Neutrophil PPIF exacerbates lung ischemia-reperfusion injury by promoting calcium overload-induced NETosis. PPIF inhibition (CsA) protected mitochondrial function by alleviating store-operated calcium entry (SOCE) during calcium overload in neutrophils. The reduction in calcium was accompanied by inhibition of calcineurin and NFAT, preventing NETs formation.","method":"Orthotopic lung transplant I/R model in mice; PPIF inhibitor (CsA) in vivo; HL-60-derived neutrophil model in vitro; SOCE measurement; calcineurin/NFAT assays; NETs quantification","journal":"International Immunopharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and in vivo pharmacological inhibition with defined molecular readouts (SOCE, calcineurin/NFAT, NETs); single lab; genetic validation limited","pmids":["39236457"],"is_preprint":false},{"year":2024,"finding":"PPIF (CyP-D), when acetylated and oxidized (as occurs in sepsis), promotes MPTP opening and lysosomal dysfunction. NMN treatment prevented PPIF acetylation and oxidation via NAD+/Sirtuin 3 axis, reduced mitochondrial ROS, and protected against sepsis-induced myocardial dysfunction. PPIF knockdown replicated the beneficial effects of NMN or mitochondria-targeted ROS scavenging.","method":"LPS-induced sepsis in mice; PPIF knockdown in neonatal cardiomyocytes; NMN and mito-TEMPO treatment; PPIF acetylation and oxidation assays; lysosomal function assays; echocardiography","journal":"Acta Pharmacologica Sinica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — PPIF post-translational modifications (acetylation, oxidation) directly assayed; genetic knockdown phenocopies pharmacological treatment; single lab","pmids":["39623043"],"is_preprint":false},{"year":2023,"finding":"PPIF/CyP-D mediates the effect of CsA and alisporivir (ALV) on increasing mutant type IV collagen α345 trimer secretion in Alport syndrome cells. Knockdown of PPIF abolished the trimer secretion-enhancing activity of CsA and ALV, identifying CyP-D as a regulator of intracellular type IV collagen processing and secretion.","method":"Nanoluciferase-based α345(IV) trimer secretion assay; siRNA knockdown of PPIF and other cyclophilins; CsA/ALV treatment; comparison of Cn-binding vs Cyp-binding CsA derivatives","journal":"Kidney360","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — siRNA knockdown of PPIF with defined functional readout (collagen secretion assay); pharmacological selectivity demonstrated; single lab","pmids":["37143203"],"is_preprint":false},{"year":2022,"finding":"All eight cyclophilins tested, including PPIF (cyclophilin D), prevented in vitro tau aggregation in a Thioflavin T fluorescence assay. However, in a cellular model of tau accumulation, PPIF did not reduce insoluble tau levels, unlike most other cyclophilins, suggesting PPIF's enzymatic activity can suppress tau fibrillation in vitro but this does not translate to cellular protection against tau accumulation.","method":"Thioflavin T fluorescence aggregation assay with recombinant cyclophilins; cellular tau accumulation model; tau seeding assays","journal":"Protein Science","confidence":"Low","confidence_rationale":"Tier 3 / Weak — in vitro aggregation assay for PPIF; cellular validation was negative for PPIF specifically; single lab, single method per finding","pmids":["36305768"],"is_preprint":false},{"year":2025,"finding":"Hexokinase 2 (HK2) regulates airway epithelial apoptosis and inflammation via physical interaction with PPIF, independent of VDAC1. HK2 knockdown and pharmacological inhibition attenuated airway inflammation and hyperresponsiveness in asthma mouse models.","method":"HK2-PPIF co-immunoprecipitation/interaction assay; airway epithelium-specific HK2 knockdown; 2-DG pharmacological inhibition; OVA/LPS asthma mouse model; apoptosis and inflammation readouts","journal":"Cells","confidence":"Low","confidence_rationale":"Tier 3 / Weak — co-IP of HK2-PPIF interaction reported; mechanistic dissection of PPIF's role is limited; single lab, abstract-level detail only","pmids":["40643524"],"is_preprint":false}],"current_model":"PPIF encodes Cyclophilin D (CyP-D), a mitochondrial matrix peptidyl-prolyl cis-trans isomerase (PPIase) that functions as the principal regulatory sensitizer of the mitochondrial permeability transition pore (MPTP): it binds the OSCP subunit of F-ATP synthase (with N-terminal cleavage by calpain 1 boosting this interaction), physically associates with ANT and the phosphate carrier to facilitate a Ca2+-triggered conformational change that opens the MPTP, and can be recruited into complexes with p53 or MST1 to amplify necrotic cell death; CyP-D-dependent MPTP opening drives regulated necrosis (but not apoptosis) in multiple cell types including cardiomyocytes, hepatocytes, neurons, eosinophils, and macrophages, and its expression is transcriptionally regulated by BMP/Smad signaling (repression during osteogenesis), C/EBPα/NF-κB (activation during adipogenesis), and the transcription factor Nynrin (repression in cardiac and hematopoietic stem cells); post-translationally, CyP-D is regulated by SIRT3-dependent deacetylation and calpain 1-mediated N-terminal cleavage, both of which modulate its MPTP-sensitizing activity; beyond cell death, CyP-D controls physiological mitoflash frequency, mitochondrial Ca2+ efflux in senescent cells, collagen secretion by fibroblasts, and hippocampal neurotransmitter release underlying learning and memory."},"narrative":{"mechanistic_narrative":"PPIF encodes Cyclophilin D (CyP-D), a mitochondria-targeted peptidyl-prolyl cis-trans isomerase whose N-terminal hydrophobic extension directs it to mitochondrial membranes [PMID:1744118], and which functions as the principal regulatory sensitizer of the mitochondrial permeability transition pore (MPTP) [PMID:16167169]. CyP-D binds inner-membrane components of the MPTP—the adenine nucleotide translocase (ANT/SLC25A4) and associated VDAC complexes at membrane contact sites—and its PPIase activity facilitates a Ca2+-triggered conformational change that opens the pore, an action blocked by cyclosporin A binding to CyP-D [PMID:10989666, PMID:10393078, PMID:12022946]; CyP-D additionally binds the OSCP subunit of F-ATP synthase, inhibiting ATP synthase catalysis and favoring its transition to a pore, with calpain 1-mediated cleavage of the flexible CyP-D N-terminus strongly increasing OSCP affinity [PMID:39528709]. Genetic ablation established that CyP-D is dispensable for pore formation per se but is required for Ca2+/oxidant-driven MPTP opening that executes regulated necrosis—not apoptosis—across many settings including ischemia/reperfusion, acetaminophen hepatotoxicity, crystal-induced kidney injury, dystrophic muscle, eosinophils, and atherosclerotic macrophages [PMID:17136322, PMID:20942566, PMID:26893161, PMID:31296606, PMID:37624892, PMID:38972156], and double-knockout epistasis with Slc25a4 confirms both as MPTP components in vivo [PMID:37624892]. CyP-D is recruited into mitochondrial complexes with ROS-activated p53 or MST1 to amplify programmed necrosis [PMID:26024660, PMID:24732633], and its abundance is transcriptionally tuned—repressed by BMP/Smad during osteogenesis and by the repressor Nynrin in cardiomyocytes, and activated by C/EBPα/NF-κB during adipogenesis—thereby setting MPTP sensitivity [PMID:35645445, PMID:37949231, PMID:38955185, PMID:41077266]. Beyond cell death, CyP-D governs physiological transient pore openings (mitoflashes/flickering) that mediate mitochondrial Ca2+ efflux required for senescent-cell survival [PMID:26746144, PMID:39448884], and supports fibroblast collagen secretion and wound healing [PMID:38564292].","teleology":[{"year":1991,"claim":"Established that PPIF is an enzyme—a bona fide peptidyl-prolyl isomerase—and that its N-terminal extension targets it to organellar membranes, defining it as a mitochondria-associated cyclophilin rather than a cytosolic one.","evidence":"Recombinant expression in E. coli, PPIase activity assays with synthetic peptides, CsA-analogue inhibition kinetics, and subcellular fractionation","pmids":["1744118"],"confidence":"High","gaps":["No in vivo substrate identified at this stage","Functional consequence of the isomerase activity in mitochondria unknown"]},{"year":1999,"claim":"Connected CyP-D enzymology to a physiological structure by showing it binds VDAC/ANT complexes at membrane contact sites and is the molecular target through which CsA inhibits the MPTP, explaining how a matrix isomerase controls inner-membrane permeability.","evidence":"CyP-D fusion-protein affinity pulldown of VDAC/ANT, photoaffinity labelling with a CsA derivative, and pore reconstitution from protein fractions","pmids":["10989666","10393078"],"confidence":"High","gaps":["Whether CyP-D is a structural pore component or a regulator unresolved","Exact inner-membrane protein undergoing the conformational change not pinned down"]},{"year":2002,"claim":"Defined the mechanism of pore facilitation: CyP-D's PPIase activity drives a Ca2+-triggered conformational change in ANT (sensitized by oxidation of specific cysteines), and minimal pore formation requires neither VDAC nor outer-membrane proteins.","evidence":"Reconstitution of MPTP from purified ANT ± CyP-D, cysteine-specific chemical modification, and correlation of CsA-inhibited PPIase activity with pore inhibition","pmids":["12022946"],"confidence":"High","gaps":["Identity of the physiological inner-membrane target still debated","Direct structural proof of the isomerized proline lacking"]},{"year":2005,"claim":"Genetic knockout resolved the structural-versus-regulatory question, demonstrating that the pore can still form and open without CyP-D, redefining CyP-D as a regulatory sensitizer rather than a pore-forming subunit.","evidence":"Ca2+-induced swelling assays in CyP-D-knockout mouse mitochondria ± CsA","pmids":["16167169"],"confidence":"High","gaps":["Did not identify the structural pore-forming component","Endogenous trigger of CyP-D recruitment in vivo unclear"]},{"year":2007,"claim":"Assigned CyP-D-dependent MPT to a specific death modality, showing it is required for necrosis but not apoptosis and is central to ischemia/reperfusion injury, establishing the therapeutic rationale for CyP-D inhibition.","evidence":"Ppif−/− mice in necrosis vs apoptosis assays and ischemia/reperfusion models","pmids":["17136322"],"confidence":"High","gaps":["Molecular events linking pore opening to necrotic execution incomplete","Tissue-specific thresholds for MPT engagement not defined"]},{"year":2008,"claim":"Refined the substrate model by showing ANT acts regulatorily and proposing the phosphate carrier (PiC) as the inner-membrane protein whose CyP-D-facilitated conformational change opens the pore.","evidence":"Genetic KO of ANT isoforms and CyP-D plus biochemical interaction studies","pmids":["18407825"],"confidence":"Medium","gaps":["PiC as CyP-D substrate awaits knockdown/reconstitution confirmation","Single-lab proposal not yet independently validated"]},{"year":2010,"claim":"Extended CyP-D function beyond cell death to physiology, linking it to acetaminophen hepatotoxicity downstream of MPT and to hippocampal neurotransmitter release underlying learning and memory.","evidence":"Ppif−/− mice with liver injury markers; behavioral testing, hippocampal microdialysis, and CsA infusion","pmids":["20942566","19437409"],"confidence":"High","gaps":["Mechanism coupling CyP-D to neurotransmitter release at synapses unknown","Whether memory effect requires transient pore opening vs another CyP-D function unclear"]},{"year":2013,"claim":"Revealed a retrograde signaling role, showing CyP-D loss activates STAT3 and a Cxcl12-Cxcr4 chemokine program that accelerates proliferation and migration, implicating CyP-D in mitochondria-to-nucleus inflammatory signaling.","evidence":"CyP-D KO/knockdown cells with proliferation, migration, transcriptome, and STAT3 assays","pmids":["23303179"],"confidence":"Medium","gaps":["Signal transmitted from mitochondria to STAT3 not identified","Single lab; relationship to MPTP activity unclear"]},{"year":2014,"claim":"Identified ROS-induced partner complexes (p53 and MST1) that translocate to mitochondria and bind CyP-D to drive programmed necrosis, providing a mechanism for upstream death-signal coupling to the pore.","evidence":"Reciprocal co-IP of p53/CyP-D and MST1/CyP-D at mitochondria with genetic and pharmacological knockdown across glioma, prostate, and pancreatic cancer cells","pmids":["26024660","24946211","24732633"],"confidence":"Medium","gaps":["Whether p53/MST1 binding directly alters pore conformation or acts indirectly unresolved","Binding interface on CyP-D not mapped"]},{"year":2015,"claim":"Demonstrated a physiological, sub-lethal CyP-D function by showing it sets the frequency of transient mitoflash openings and influences respiratory capacity, with tissue-specific magnitude tracking CyP-D expression.","evidence":"Reciprocal gain/loss-of-function in cardiomyocytes and skeletal muscle with live mitoflash imaging and Seahorse respirometry","pmids":["26746144"],"confidence":"High","gaps":["Trigger distinguishing transient mitoflash from lethal pore opening unclear","Bioenergetic consequence mechanism not fully defined"]},{"year":2015,"claim":"Placed CyP-D within necroptotic crosstalk, showing necroptosis requires both Bax/Bak outer-membrane oligomerization and CyP-D-dependent MPTP opening, with MLKL/cofilin-1 mitochondrial translocation partly CyP-D-dependent.","evidence":"Ppif−/− and Bax/Bak-null fibroblasts with necroptosis induction and mitochondrial fractionation","pmids":["26061004"],"confidence":"Medium","gaps":["Mechanistic link between MLKL and MPTP not established","Single lab; degree of MPT contribution to necroptosis varies by stimulus"]},{"year":2019,"claim":"Generalized CyP-D-driven necrosis to crystal-induced kidney injury and quantified its partial redundancy with necroptosis via double-knockout epistasis.","evidence":"Ppif−/− and Ppif−/−Mlkl−/− mice with crystal exposure, histology, and pharmacological MPTP inhibition","pmids":["31296606"],"confidence":"High","gaps":["Signal from lysosomal cathepsin/ROS to MPTP not molecularly defined","Quantitative split between MPT-necrosis and necroptosis context-dependent"]},{"year":2022,"claim":"Showed that CyP-D abundance is a regulated transcriptional node, with BMP/Smad repressing Ppif to deactivate the MPTP as a functional requirement for osteoblast differentiation.","evidence":"BMP/Smad activation in MSCs, Ppif promoter reporters, and CyP-D gain-of-function in vitro and in mice","pmids":["35645445"],"confidence":"High","gaps":["Direct Smad binding elements on Ppif not exhaustively mapped","Connection between MPTP deactivation and osteogenic gene program mechanistic detail limited"]},{"year":2023,"claim":"Provided direct in vivo genetic evidence for CyP-D and ANT1 as MPTP components, with combined deletion almost fully preventing necrotic muscular dystrophy.","evidence":"Single and double KO (Slc25a4−/−, Ppif−/−) in δ-sarcoglycan-null dystrophic mice with histopathology","pmids":["37624892"],"confidence":"High","gaps":["Residual MPT activity in double KO indicates additional components","Stoichiometry of CyP-D–ANT1 interaction in the pore not defined"]},{"year":2023,"claim":"Identified an opposing transcriptional input, C/EBPα synergizing with NF-κB p65 to activate Ppif during adipogenesis, linking adipogenic and inflammatory signaling to MPTP gain-of-function.","evidence":"Ppif promoter reporters and ChIP for C/EBPα and NF-κB p65 in adipogenically induced BMSCs","pmids":["37949231"],"confidence":"Medium","gaps":["Functional consequence of increased MPTP for adipocyte biology incomplete","Single lab; two-method support only"]},{"year":2024,"claim":"Provided structural and proteolytic mechanism for CyP-D engagement of ATP synthase, showing the flexible N-terminus suppresses OSCP binding and that calpain 1 cleavage generates a ΔN-CyP-D that binds OSCP, inhibits ATP synthesis, and favors pore transition.","evidence":"NMR of FL- and ΔN-CyP-D, OSCP binding assays, calpain 1 treatment, and mass-spec detection of ΔN-CyP-D in cells","pmids":["39528709"],"confidence":"High","gaps":["Whether OSCP- vs ANT-binding represent distinct or unified pore models unresolved","Physiological stimulus driving calpain 1 cleavage not defined"]},{"year":2024,"claim":"Established a survival role of transient CyP-D pore opening, showing that in senescent cells CyP-D flickering provides Ca2+ efflux and that its inhibition causes toxic Ca2+ overload and senolysis, with MCU/NCLX epistasis defining the Ca2+ circuit.","evidence":"Genome-wide CRISPR screen, Ppif KO, CsA, MCU/NCLX perturbation, and mitochondrial Ca2+ imaging in senescent cells","pmids":["39448884"],"confidence":"High","gaps":["What renders senescent cells uniquely dependent on CyP-D efflux unclear","Generality of senolytic effect across senescence inducers untested"]},{"year":2024,"claim":"Expanded CyP-D into tissue-repair biology, demonstrating it promotes fibroblast collagen secretion (preventing ER retention) and keratinocyte reepithelialization required for wound closure.","evidence":"Reciprocal gain/loss-of-function in keratinocytes and fibroblasts, Ppif-KO mice, and human/pig wound models with collagen and closure readouts","pmids":["38564292"],"confidence":"High","gaps":["Mechanism linking a mitochondrial isomerase to ER collagen secretion unknown","Whether this reflects MPTP activity or a non-pore CyP-D function unclear"]},{"year":2024,"claim":"Detailed post-translational control, showing acetylated/oxidized CyP-D (as in sepsis) promotes MPTP opening and lysosomal dysfunction and is reversed via the NAD+/SIRT3 deacetylation axis.","evidence":"LPS sepsis mice, PPIF knockdown in cardiomyocytes, NMN and mito-TEMPO treatment, and CyP-D acetylation/oxidation assays","pmids":["39623043"],"confidence":"Medium","gaps":["Specific acetylated/oxidized residues not all mapped","Single lab; direct SIRT3–CyP-D enzymatic step inferred"]},{"year":2024,"claim":"Implicated CyP-D in atherosclerotic necrotic-core formation by linking it to macrophage death, ER-stress cytochrome c release, and necroptosis in vivo.","evidence":"Apoe−/−Ppif−/− double-KO mice and CyP-D siRNA in macrophages with cytochrome c release and necroptosis readouts","pmids":["38972156"],"confidence":"High","gaps":["Mechanism by which CyP-D promotes cytochrome c release not defined","Relative contribution of apoptosis vs necroptosis in lesions unresolved"]},{"year":null,"claim":"The identity of the structural pore-forming element of the MPTP and the precise inner-membrane protein whose proline CyP-D isomerizes (ANT vs PiC vs OSCP/F-ATP synthase) remain unresolved, as does how a single matrix isomerase governs both lethal and physiological pore openings and non-pore functions such as collagen secretion.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified structural model reconciling ANT-, PiC-, and OSCP-based mechanisms","Mechanism distinguishing transient mitoflash/flickering from lethal sustained opening unknown","Non-mitochondrial-death functions (collagen secretion, neurotransmission) mechanistically unexplained"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016853","term_label":"isomerase activity","supporting_discovery_ids":[0,3]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[3,20]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4,3]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[21]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,1,2]}],"pathway":[{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[5,7,16,24]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[2,21]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[3,26]}],"complexes":["mitochondrial permeability transition pore (MPTP)","F-ATP synthase (OSCP-bound)"],"partners":["SLC25A4","VDAC","OSCP","TP53","MST1","HK2"],"other_free_text":[]}},"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). It is proposed that its association with the mPTP is masking a binding site for inhibiting inorganic phosphate (Pi) and promotes the open probability of the mPTP leading to apoptosis or necrosis; the requirement of the PPIase activity for this function is debated (PubMed:26387735). In cooperation with mitochondrial p53/TP53 is involved in activating oxidative stress-induced necrosis (PubMed:22726440). Involved in modulation of mitochondrial membrane F(1)F(0) ATP synthase activity and regulation of mitochondrial matrix adenine nucleotide levels (By similarity). Has anti-apoptotic activity independently of mPTP and in cooperation with BCL2 inhibits cytochrome c-dependent apoptosis (PubMed:19228691)","subcellular_location":"Mitochondrion matrix","url":"https://www.uniprot.org/uniprotkb/P30405/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PPIF","classification":"Not Classified","n_dependent_lines":5,"n_total_lines":1208,"dependency_fraction":0.0041390728476821195},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CLIP1","stoichiometry":0.2},{"gene":"LGALS1","stoichiometry":0.2},{"gene":"MYH9","stoichiometry":0.2},{"gene":"RER1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/PPIF","total_profiled":1310},"omim":[{"mim_id":"616952","title":"MITOCHONDRIAL CALCIUM UNIPORTER REGULATOR 1; MCUR1","url":"https://www.omim.org/entry/616952"},{"mim_id":"604486","title":"PEPTIDYL-PROLYL CIS/TRANS ISOMERASE, MITOCHONDRIAL; PPIF","url":"https://www.omim.org/entry/604486"},{"mim_id":"601753","title":"PEPTIDYL-PROLYL ISOMERASE D; PPID","url":"https://www.omim.org/entry/601753"},{"mim_id":"601411","title":"SARCOGLYCAN, DELTA; SGCD","url":"https://www.omim.org/entry/601411"},{"mim_id":"156225","title":"LAMININ, ALPHA-2; LAMA2","url":"https://www.omim.org/entry/156225"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Mitochondria","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PPIF"},"hgnc":{"alias_symbol":["hCyP3","Cyp-D","CypD"],"prev_symbol":[]},"alphafold":{"accession":"P30405","domains":[{"cath_id":"2.40.100.10","chopping":"46-204","consensus_level":"high","plddt":98.3904,"start":46,"end":204}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P30405","model_url":"https://alphafold.ebi.ac.uk/files/AF-P30405-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P30405-F1-predicted_aligned_error_v6.png","plddt_mean":88.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PPIF","jax_strain_url":"https://www.jax.org/strain/search?query=PPIF"},"sequence":{"accession":"P30405","fasta_url":"https://rest.uniprot.org/uniprotkb/P30405.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P30405/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P30405"}},"corpus_meta":[{"pmid":"10393078","id":"PMC_10393078","title":"The mitochondrial permeability transition pore and its role in cell death.","date":"1999","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/10393078","citation_count":1892,"is_preprint":false},{"pmid":"19265700","id":"PMC_19265700","title":"What is the mitochondrial permeability transition pore?","date":"2009","source":"Journal of molecular and cellular cardiology","url":"https://pubmed.ncbi.nlm.nih.gov/19265700","citation_count":749,"is_preprint":false},{"pmid":"12022946","id":"PMC_12022946","title":"The permeability transition pore complex: another view.","date":"2002","source":"Biochimie","url":"https://pubmed.ncbi.nlm.nih.gov/12022946","citation_count":591,"is_preprint":false},{"pmid":"33828297","id":"PMC_33828297","title":"Genome-wide enhancer maps link risk variants to disease genes.","date":"2021","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/33828297","citation_count":477,"is_preprint":false},{"pmid":"17136322","id":"PMC_17136322","title":"Role of the mitochondrial membrane permeability transition in cell death.","date":"2007","source":"Apoptosis : an international journal on programmed cell death","url":"https://pubmed.ncbi.nlm.nih.gov/17136322","citation_count":449,"is_preprint":false},{"pmid":"16545083","id":"PMC_16545083","title":"Calcium, mitochondria and reperfusion injury: a pore way to die.","date":"2006","source":"Biochemical Society transactions","url":"https://pubmed.ncbi.nlm.nih.gov/16545083","citation_count":414,"is_preprint":false},{"pmid":"19168026","id":"PMC_19168026","title":"The role of the mitochondrial permeability transition pore in heart disease.","date":"2009","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/19168026","citation_count":293,"is_preprint":false},{"pmid":"18407825","id":"PMC_18407825","title":"Recent progress in elucidating the molecular mechanism of the mitochondrial permeability transition pore.","date":"2008","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/18407825","citation_count":266,"is_preprint":false},{"pmid":"1744118","id":"PMC_1744118","title":"The cyclophilin multigene family of peptidyl-prolyl isomerases. 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The protein was expressed in E. coli, purified, and shown to be an active PPIase; its NH2-terminal hydrophobic extension (42 aa) acts as a signal peptide directing it to subcellular organelles/membranes. Substrate specificity with synthetic Suc-Xaa-Yaa-Pro-Phe-nitroanilide peptides and inhibition by cyclosporin A analogues were characterized.\",\n      \"method\": \"Recombinant protein expression in E. coli, PPIase activity assay with synthetic peptide substrates, kinetic characterization with CsA analogues, protein-specific antibody subcellular fractionation, Northern/Western blot\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted enzymatic activity in vitro with substrate specificity and inhibitor kinetics; replicated across three human CyP isoforms in the same rigorous study\",\n      \"pmids\": [\"1744118\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"CyP-D (PPIF) is a key structural component of the mitochondrial permeability transition pore (MPTP). Using a CyP-D fusion protein as affinity matrix, CyP-D was shown to bind strongly to 1:1 complexes of VDAC (outer membrane) and adenine nucleotide translocase (ANT; inner membrane). Covalent labelling of CyP-D in situ by a photoactive CsA derivative demonstrated that pore ligands have the same effects on pore block and CyP-D binding, confirming CsA inhibits the MPTP by binding CyP-D.\",\n      \"method\": \"CyP-D fusion protein affinity pulldown of VDAC/ANT complexes; photoaffinity labelling of CyP-D in situ with CsA derivative; pore reconstitution from protein fractions\",\n      \"journal\": \"Biochemical Society Symposium\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — affinity pulldown reconstitution plus photoaffinity labelling in organello; findings replicated across multiple labs reviewing same data\",\n      \"pmids\": [\"10989666\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"CyP-D forms a complex with VDAC and ANT at contact sites between the mitochondrial inner and outer membranes to constitute the MPTP. Cyclosporin A inhibits pore opening by binding CyP-D, reducing its Ca2+ binding affinity and blocking pore flickering. Under oxidative stress and high Ca2+, the complex opens to allow free diffusion of low-molecular-weight solutes (<1.5 kDa) across the inner membrane.\",\n      \"method\": \"CyP-D affinity matrix pulldown, pore reconstitution, CsA photolabelling, Ca2+ flux assays in isolated mitochondria\",\n      \"journal\": \"The Biochemical Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reconstitution experiments plus pharmacological validation with photoaffinity probe; reviewed and independently replicated across laboratories\",\n      \"pmids\": [\"10393078\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"CyP-D facilitates a Ca2+-triggered conformational change of the ANT that induces MPTP opening; its PPIase activity mediates this conformational facilitation. Oxidative modification of ANT Cys56 increases CyP-D binding to the ANT (likely at Pro61), while modification of Cys159 inhibits adenine nucleotide binding. Reconstitution studies demonstrated that neither VDAC nor other outer membrane proteins are required for minimal MPTP formation.\",\n      \"method\": \"Reconstitution of MPTP from purified ANT ± CyP-D ± VDAC; cysteine-specific chemical modification of ANT; CsA inhibition of PPIase activity correlated with pore inhibition\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution with defined purified components, site-specific mutagenesis/chemical modification, PPIase-MPTP correlation; multiple orthogonal methods in one study\",\n      \"pmids\": [\"12022946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Genetic knockout of CyP-D (PPIF) in mice demonstrated that CyP-D is the target for MPTP inhibition by CsA; however, MPTP can still form and open in the absence of CyP-D, indicating that CyP-D is a regulatory sensitizer rather than a structural pore-forming component of the inner mitochondrial membrane.\",\n      \"method\": \"CyP-D knockout mouse mitochondria; MPTP activity assays (Ca2+-induced swelling) ± CsA\",\n      \"journal\": \"Journal of Bioenergetics and Biomembranes\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic KO with defined mitochondrial phenotype; finding replicated in multiple independent CyP-D KO studies reviewed in this paper\",\n      \"pmids\": [\"16167169\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CyP-D knockout mice (Ppif−/−) showed that CyP-D-dependent MPT is essential for necrotic but not apoptotic cell death, and plays a crucial role in ischemia/reperfusion injury. CyP-D-null mice develop normally and show no protection against diverse apoptotic stimuli.\",\n      \"method\": \"Genetic KO mouse model (Ppif−/−); cell death assays distinguishing necrosis vs apoptosis; ischemia/reperfusion injury models\",\n      \"journal\": \"Apoptosis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic KO with phenotypically defined necrosis vs apoptosis distinction; independently replicated across multiple labs\",\n      \"pmids\": [\"17136322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CyP-D's peptidyl-prolyl cis-trans isomerase activity facilitates a conformational change in an inner membrane protein (proposed to be the mitochondrial phosphate carrier, PiC) to induce MPTP opening. ANT knockout studies showed ANT plays a regulatory rather than pore-forming role. CyP-D is proposed to bind PiC and facilitate its Ca2+-triggered conformational change.\",\n      \"method\": \"Genetic KO of ANT isoforms in mice; CyP-D KO; biochemical interaction studies; review of reconstitution data\",\n      \"journal\": \"Biochimica et Biophysica Acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO epistasis for ANT confirmed; PiC as substrate is proposed based on biochemical evidence but knockdown/reconstitution awaited; single lab\",\n      \"pmids\": [\"18407825\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CyP-D deletion (Ppif−/−) protected mice completely against acetaminophen-induced liver necrosis and DNA fragmentation, demonstrating that CyP-D-dependent MPT is a critical downstream event in APAP hepatotoxicity. Oxidative stress (GSSG levels) and peroxynitrite formation were blunted but not eliminated in CypD-deficient mice, placing oxidant stress at least partly downstream of MPT.\",\n      \"method\": \"Ppif−/− knockout mice treated with APAP; liver histology, DNA fragmentation assay, GSSG measurement, nitrotyrosine immunostaining\",\n      \"journal\": \"Free Radical Research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic KO with quantitative biochemical readouts; multiple orthogonal injury markers; single lab but rigorous\",\n      \"pmids\": [\"20942566\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CyP-D is required for normal learning and memory: Ppif−/− mice showed deficits in short-term memory, object recognition, reference memory, and associative learning. Hippocampal infusion of CsA replicated these deficits. CyP-D absence reduced stimulus-evoked hippocampal glutamate and acetylcholine release, linking CyP-D to regulation of neurotransmission.\",\n      \"method\": \"Ppif−/− KO mice; behavioral tests (Y-maze, novel object recognition, water maze, fear conditioning); hippocampal microdialysis; CsA hippocampal infusion\",\n      \"journal\": \"Hippocampus\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic KO with multiple behavioral and neurochemical readouts; pharmacological recapitulation; single lab\",\n      \"pmids\": [\"19437409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CyP-D (PPIF) deletion or knockdown results in increased cell proliferation, enhanced migration and invasion, and transcriptional upregulation of a chemokine/chemokine receptor gene signature. In the absence of CyP-D, STAT3 is activated, drives accelerated S-phase entry via cell proliferation, and stimulates autocrine/paracrine Cxcl12-Cxcr4-directed chemotaxis, indicating that CyP-D controls mitochondria-to-nucleus inflammatory gene expression.\",\n      \"method\": \"CyP-D knockout/knockdown cells; proliferation assays; migration/invasion assays; transcriptome profiling; STAT3 activation assays; Cxcl12-Cxcr4 signaling analysis\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined phenotypic readouts (proliferation, migration, STAT3) and transcriptomic pathway placement; single lab\",\n      \"pmids\": [\"23303179\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In glioma cells, ROS production induced by salinomycin drives p53 translocation to mitochondria, where p53 forms a physical complex with CyP-D. This p53/CyP-D complex is required for MPTP opening and programmed necrosis. Blocking CyP-D by siRNA or pharmacological inhibitors (CsA, sanglifehrin A), or p53 knockdown, suppressed necrosis but not apoptosis.\",\n      \"method\": \"siRNA-mediated CyP-D depletion; Co-immunoprecipitation of p53/CyP-D complex at mitochondria; pharmacological inhibition (CsA, SFA); mitochondrial membrane potential assay; cell death quantification\",\n      \"journal\": \"Journal of Experimental & Clinical Cancer Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — reciprocal co-IP of p53/CyP-D complex plus genetic and pharmacological KD; single lab; two orthogonal methods\",\n      \"pmids\": [\"26024660\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CyP-D is required for berberine-induced programmed necrosis in prostate cancer cells. Berberine-induced ROS production drives p53 mitochondrial translocation, where p53 physically interacts with CyP-D to open the MPTP. shRNA depletion of CyP-D or p53 inhibited necrosis but not apoptosis.\",\n      \"method\": \"CyP-D shRNA; Co-immunoprecipitation of p53/CyP-D at mitochondria; CsA/SFA pharmacological inhibition; mitochondrial membrane potential; flow cytometry\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP of p53/CyP-D complex confirmed by genetic and pharmacological knockdown in parallel; single lab, consistent with independent study (PMID 26024660)\",\n      \"pmids\": [\"24946211\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"MST1 translocates to mitochondria in response to gemcitabine-induced ROS and forms a direct physical complex with CyP-D. This MST1/CyP-D mitochondrial complex is required for MPTP-dependent pancreatic cancer cell death. CsA (CyP-D inhibitor) prevented the MST1/CyP-D complexation and cell death.\",\n      \"method\": \"Co-immunoprecipitation of MST1/CyP-D at mitochondria; shRNA silencing of MST1 or CyP-D; CsA inhibition; overexpression studies; ROS measurement\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP of novel CyP-D interaction partner plus reciprocal genetic and pharmacological validation; single lab\",\n      \"pmids\": [\"24732633\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CyP-D regulates the frequency of mitochondrial 'mitoflash' events (transient MPTP openings under physiological conditions) in cardiomyocytes. CyP-D overexpression increased, and knockout halved, cardiac mitoflash frequency. CyP-D ablation reduced mitochondrial maximal respiration rate and reserved respiratory capacity in cardiomyocytes. The effect of CyP-D on mitoflashes was tissue-specific (cardiac vs skeletal muscle), correlating with 4-fold higher CyP-D expression in cardiac muscle.\",\n      \"method\": \"CyP-D overexpression and knockout in isolated cardiomyocytes, beating hearts, and skeletal muscles in vivo; live imaging of mitochondrial flash activity; Seahorse respirometry\",\n      \"journal\": \"Journal of Molecular and Cellular Cardiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal gain- and loss-of-function in multiple tissues with quantitative live imaging and respirometry; multiple orthogonal methods in one study\",\n      \"pmids\": [\"26746144\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PPIF (CyP-D) regulates necrosis but not apoptosis in eosinophils. Ppif−/− eosinophils were protected from Ca2+-overload (ionomycin)- and oxidative stress (H2O2)-induced necrosis and from Siglec-F cross-linking-induced necrosis, with no difference in apoptosis. In vivo, Ppif−/− mice showed reduced eosinophil cytolysis in DSS-induced colitis.\",\n      \"method\": \"Ppif−/− KO mice; ionomycin/H2O2 necrosis assays; flow cytometry for apoptosis vs necrosis; DSS colitis in vivo model\",\n      \"journal\": \"American Journal of Physiology: Gastrointestinal and Liver Physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic KO with defined cellular phenotype across multiple stimuli both in vitro and in vivo; multiple orthogonal readouts\",\n      \"pmids\": [\"26893161\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Necroptotic stimuli require both Bax/Bak oligomerization in the outer mitochondrial membrane and MPTP (CyP-D-dependent) opening in the inner membrane. Ppif−/− fibroblasts are resistant to necroptotic cell death inducers. MLKL and cofilin-1 translocate to mitochondria following necroptosis induction; some of these effects are lost in Ppif−/− cells.\",\n      \"method\": \"Ppif−/− KO fibroblasts; Bax/Bak double-null cells; MLKL/cofilin-1 mitochondrial fractionation; necroptosis induction with TNF/zVAD; cell death assays\",\n      \"journal\": \"PLoS ONE\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with pathway epistasis (necroptosis–MPTP crosstalk) and protein localization data; single lab\",\n      \"pmids\": [\"26061004\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PPIF-dependent mitochondrial permeability transition mediates crystal-induced (calcium oxalate, monosodium urate, calcium pyrophosphate, silica) necrosis in renal tubular cells. The pathway involves crystal phagocytosis, lysosomal cathepsin leakage, and ROS release. Ppif−/− mice displayed attenuated AKI; dual deletion of Ppif and Mlkl showed partial redundancy between MPT-driven necrosis and necroptosis.\",\n      \"method\": \"Ppif−/− KO mice; Ppif−/−Mlkl−/− double KO mice; in vitro crystal exposure in mouse/human tubular cells; pharmacological MPTP inhibition; histology; fractionation\",\n      \"journal\": \"Journal of the American Society of Nephrology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic KO (single and double) in vivo and in vitro with quantitative injury readouts and epistasis analysis; replicated pharmacologically\",\n      \"pmids\": [\"31296606\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"BMP/Smad signaling transcriptionally represses the CyP-D gene (Ppif) during osteogenic differentiation of mesenchymal lineage cells, thereby deactivating MPTP. CyP-D 'rescue' via gain-of-function negatively affected osteogenesis both in vitro and in a mouse model, demonstrating that BMP/Smad-driven CyP-D downregulation is functionally required for efficient osteoblast differentiation.\",\n      \"method\": \"BMP/Smad pathway activation in MSCs; Ppif promoter reporter assays; CyP-D overexpression (gain-of-function) in vitro and in mouse model; osteogenic differentiation assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — transcriptional regulation mapped to Smad elements, gain-of-function validated in vitro and in vivo, multiple orthogonal methods; single lab but rigorous\",\n      \"pmids\": [\"35645445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ANT1 (SLC25A4) and CyP-D (PPIF) are both required components of the MPTP governing necrotic cell death in vivo. Deletion of Slc25a4 in dystrophic (Sgcd−/−) mice partially protected from MD pathology; combined deletion of Slc25a4 and Ppif almost completely prevented necrotic cell death and muscular dystrophy disease, providing direct genetic evidence for both proteins as MPTP components.\",\n      \"method\": \"Genetic epistasis: single KO (Slc25a4−/− or Ppif−/−) and double KO (Slc25a4−/−Ppif−/−) in δ-sarcoglycan-null muscular dystrophy mice; histopathology; cell death quantification\",\n      \"journal\": \"Science Advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean in vivo genetic epistasis with double KO; quantitative disease phenotype readouts; directly demonstrates both ANT1 and CyP-D as MPTP components\",\n      \"pmids\": [\"37624892\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"C/EBPα transcriptionally activates the CyP-D gene (Ppif) during adipogenesis of bone marrow stromal cells, increasing CyP-D expression and MPTP activity. NF-κB p65 subunit acts synergistically with C/EBPα to induce Ppif expression, linking adipogenic and inflammatory signaling to MPTP gain-of-function.\",\n      \"method\": \"Ppif promoter reporter assays; ChIP for C/EBPα and NF-κB p65 at Ppif promoter; adipogenic induction of BMSCs; CyP-D/MPTP activity measurement\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter reporter plus ChIP identification of transcription factors; single lab; two orthogonal methods\",\n      \"pmids\": [\"37949231\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"N-terminal cleavage of CyP-D (removing first 13 human residues) by calpain 1 generates a ΔN-CyPD form. NMR studies showed the N-terminus of full-length CyP-D is highly flexible. ΔN-CyPD binds the OSCP subunit of F-ATP synthase in saline media, whereas FL-CyPD does not, indicating the N-terminus substantially reduces affinity for OSCP. CyP-D binding to OSCP inhibits ATP synthase catalysis and favors transition of the enzyme to the permeability transition pore.\",\n      \"method\": \"NMR structure of FL- and ΔN-CyPD; binding assays with F-ATP synthase OSCP subunit; calpain 1 treatment of cells; mass spectrometry identification of ΔN-CyPD in cells\",\n      \"journal\": \"Communications Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure, in vitro binding assay, identification of calpain 1 as the protease, and cellular validation; multiple orthogonal methods in one study\",\n      \"pmids\": [\"39528709\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Senescent cells exhibit high frequency of transient CyP-D/MPTP opening events ('flickering'). Genetic or pharmacological inhibition of CyP-D (PPIF) causes toxic mitochondrial Ca2+ accumulation and selective death of senescent cells (senolysis). Inhibition of NCLX (another mitochondrial Ca2+ efflux channel) also induces senolysis, while inhibition of MCU (Ca2+ influx) prevents CyP-D inhibition-induced senolysis, placing transient CyP-D/MPTP opening as a Ca2+ efflux mechanism critical for senescent cell survival.\",\n      \"method\": \"Genome-wide CRISPR/Cas9 screen; Ppif genetic KO; CsA pharmacological inhibition; NCLX and MCU inhibition/knockout; mitochondrial Ca2+ imaging; senescent cell viability assays\",\n      \"journal\": \"The EMBO Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide CRISPR screen plus genetic and pharmacological validation plus epistasis with MCU/NCLX; multiple orthogonal methods; single lab but comprehensive\",\n      \"pmids\": [\"39448884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Nynrin is a transcriptional repressor of Ppif (CyP-D gene). Nynrin knockout in cardiomyocytes enhanced Ppif transcription, increased CyP-D expression, promoted MPTP opening, and exacerbated ischemia/reperfusion injury. CsA treatment reversed the exacerbated phenotype. Nynrin overexpression blunted Ppif/CyP-D upregulation and reduced cardiomyocyte damage during OGD/R.\",\n      \"method\": \"Tamoxifen-inducible cardiomyocyte-specific Nynrin KO; Nynrin overexpression in primary cardiomyocytes; Ppif promoter regulation assays; MPTP opening assay; I/R injury model\",\n      \"journal\": \"Cell Stem Cell (for PMID 38955185); Journal of Molecular and Cellular Cardiology (for PMID 41077266)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional genetic KO plus OE with pharmacological rescue; mechanistic link between Nynrin, Ppif transcription, CyP-D, and MPTP established in two independent studies (HSC and cardiac models)\",\n      \"pmids\": [\"38955185\", \"41077266\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CyP-D (PPIF) is upregulated in wounds and venous ulcers. Inhibition of CyP-D impaired keratinocyte reepithelialization, granulation tissue formation, and wound closure in human and pig models. CyP-D inhibition in fibroblasts reduced collagen secretion and caused ER collagen accumulation; CyP-D overexpression increased collagen secretion. Ppif-KO mice showed reduced skin collagen.\",\n      \"method\": \"CyP-D inhibition (CsA) in human and pig wound models; CyP-D overexpression in keratinocytes/fibroblasts; Ppif-KO mice; collagen secretion and ER retention assays; migration assays\",\n      \"journal\": \"JCI Insight\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal gain- and loss-of-function in vitro, in vivo KO, and large-animal model; quantitative collagen secretion and wound closure readouts; multiple orthogonal methods\",\n      \"pmids\": [\"38564292\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CyP-D mediates macrophage death in atherosclerotic lesions and necrotic core formation. CyP-D knockdown in macrophages attenuated cytochrome c release from mitochondria induced by ER stress (thapsigargin) and blocked necroptosis induced by TNF-α/caspase inhibitor. Apoe−/−Ppif−/− double-KO mice had smaller necrotic cores with reduced macrophage apoptosis and decreased inflammatory monocytes.\",\n      \"method\": \"Apoe−/−Ppif−/− double-KO mouse model; siRNA knockdown of CyP-D in RAW264.7 cells; cytochrome c release assay; necroptosis induction; atherosclerosis lesion histology\",\n      \"journal\": \"Atherosclerosis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic double KO plus in vitro siRNA with mechanistic readouts (cytochrome c release, necroptosis); multiple orthogonal methods\",\n      \"pmids\": [\"38972156\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Neutrophil PPIF exacerbates lung ischemia-reperfusion injury by promoting calcium overload-induced NETosis. PPIF inhibition (CsA) protected mitochondrial function by alleviating store-operated calcium entry (SOCE) during calcium overload in neutrophils. The reduction in calcium was accompanied by inhibition of calcineurin and NFAT, preventing NETs formation.\",\n      \"method\": \"Orthotopic lung transplant I/R model in mice; PPIF inhibitor (CsA) in vivo; HL-60-derived neutrophil model in vitro; SOCE measurement; calcineurin/NFAT assays; NETs quantification\",\n      \"journal\": \"International Immunopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and in vivo pharmacological inhibition with defined molecular readouts (SOCE, calcineurin/NFAT, NETs); single lab; genetic validation limited\",\n      \"pmids\": [\"39236457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PPIF (CyP-D), when acetylated and oxidized (as occurs in sepsis), promotes MPTP opening and lysosomal dysfunction. NMN treatment prevented PPIF acetylation and oxidation via NAD+/Sirtuin 3 axis, reduced mitochondrial ROS, and protected against sepsis-induced myocardial dysfunction. PPIF knockdown replicated the beneficial effects of NMN or mitochondria-targeted ROS scavenging.\",\n      \"method\": \"LPS-induced sepsis in mice; PPIF knockdown in neonatal cardiomyocytes; NMN and mito-TEMPO treatment; PPIF acetylation and oxidation assays; lysosomal function assays; echocardiography\",\n      \"journal\": \"Acta Pharmacologica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — PPIF post-translational modifications (acetylation, oxidation) directly assayed; genetic knockdown phenocopies pharmacological treatment; single lab\",\n      \"pmids\": [\"39623043\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PPIF/CyP-D mediates the effect of CsA and alisporivir (ALV) on increasing mutant type IV collagen α345 trimer secretion in Alport syndrome cells. Knockdown of PPIF abolished the trimer secretion-enhancing activity of CsA and ALV, identifying CyP-D as a regulator of intracellular type IV collagen processing and secretion.\",\n      \"method\": \"Nanoluciferase-based α345(IV) trimer secretion assay; siRNA knockdown of PPIF and other cyclophilins; CsA/ALV treatment; comparison of Cn-binding vs Cyp-binding CsA derivatives\",\n      \"journal\": \"Kidney360\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — siRNA knockdown of PPIF with defined functional readout (collagen secretion assay); pharmacological selectivity demonstrated; single lab\",\n      \"pmids\": [\"37143203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"All eight cyclophilins tested, including PPIF (cyclophilin D), prevented in vitro tau aggregation in a Thioflavin T fluorescence assay. However, in a cellular model of tau accumulation, PPIF did not reduce insoluble tau levels, unlike most other cyclophilins, suggesting PPIF's enzymatic activity can suppress tau fibrillation in vitro but this does not translate to cellular protection against tau accumulation.\",\n      \"method\": \"Thioflavin T fluorescence aggregation assay with recombinant cyclophilins; cellular tau accumulation model; tau seeding assays\",\n      \"journal\": \"Protein Science\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — in vitro aggregation assay for PPIF; cellular validation was negative for PPIF specifically; single lab, single method per finding\",\n      \"pmids\": [\"36305768\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Hexokinase 2 (HK2) regulates airway epithelial apoptosis and inflammation via physical interaction with PPIF, independent of VDAC1. HK2 knockdown and pharmacological inhibition attenuated airway inflammation and hyperresponsiveness in asthma mouse models.\",\n      \"method\": \"HK2-PPIF co-immunoprecipitation/interaction assay; airway epithelium-specific HK2 knockdown; 2-DG pharmacological inhibition; OVA/LPS asthma mouse model; apoptosis and inflammation readouts\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — co-IP of HK2-PPIF interaction reported; mechanistic dissection of PPIF's role is limited; single lab, abstract-level detail only\",\n      \"pmids\": [\"40643524\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PPIF encodes Cyclophilin D (CyP-D), a mitochondrial matrix peptidyl-prolyl cis-trans isomerase (PPIase) that functions as the principal regulatory sensitizer of the mitochondrial permeability transition pore (MPTP): it binds the OSCP subunit of F-ATP synthase (with N-terminal cleavage by calpain 1 boosting this interaction), physically associates with ANT and the phosphate carrier to facilitate a Ca2+-triggered conformational change that opens the MPTP, and can be recruited into complexes with p53 or MST1 to amplify necrotic cell death; CyP-D-dependent MPTP opening drives regulated necrosis (but not apoptosis) in multiple cell types including cardiomyocytes, hepatocytes, neurons, eosinophils, and macrophages, and its expression is transcriptionally regulated by BMP/Smad signaling (repression during osteogenesis), C/EBPα/NF-κB (activation during adipogenesis), and the transcription factor Nynrin (repression in cardiac and hematopoietic stem cells); post-translationally, CyP-D is regulated by SIRT3-dependent deacetylation and calpain 1-mediated N-terminal cleavage, both of which modulate its MPTP-sensitizing activity; beyond cell death, CyP-D controls physiological mitoflash frequency, mitochondrial Ca2+ efflux in senescent cells, collagen secretion by fibroblasts, and hippocampal neurotransmitter release underlying learning and memory.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PPIF encodes Cyclophilin D (CyP-D), a mitochondria-targeted peptidyl-prolyl cis-trans isomerase whose N-terminal hydrophobic extension directs it to mitochondrial membranes [#0], and which functions as the principal regulatory sensitizer of the mitochondrial permeability transition pore (MPTP) [#4]. CyP-D binds inner-membrane components of the MPTP\\u2014the adenine nucleotide translocase (ANT/SLC25A4) and associated VDAC complexes at membrane contact sites\\u2014and its PPIase activity facilitates a Ca2+-triggered conformational change that opens the pore, an action blocked by cyclosporin A binding to CyP-D [#1, #2, #3]; CyP-D additionally binds the OSCP subunit of F-ATP synthase, inhibiting ATP synthase catalysis and favoring its transition to a pore, with calpain 1-mediated cleavage of the flexible CyP-D N-terminus strongly increasing OSCP affinity [#20]. Genetic ablation established that CyP-D is dispensable for pore formation per se but is required for Ca2+/oxidant-driven MPTP opening that executes regulated necrosis\\u2014not apoptosis\\u2014across many settings including ischemia/reperfusion, acetaminophen hepatotoxicity, crystal-induced kidney injury, dystrophic muscle, eosinophils, and atherosclerotic macrophages [#5, #7, #14, #16, #18, #24], and double-knockout epistasis with Slc25a4 confirms both as MPTP components in vivo [#18]. CyP-D is recruited into mitochondrial complexes with ROS-activated p53 or MST1 to amplify programmed necrosis [#10, #12], and its abundance is transcriptionally tuned\\u2014repressed by BMP/Smad during osteogenesis and by the repressor Nynrin in cardiomyocytes, and activated by C/EBP\\u03b1/NF-\\u03baB during adipogenesis\\u2014thereby setting MPTP sensitivity [#17, #19, #22]. Beyond cell death, CyP-D governs physiological transient pore openings (mitoflashes/flickering) that mediate mitochondrial Ca2+ efflux required for senescent-cell survival [#13, #21], and supports fibroblast collagen secretion and wound healing [#23].\",\n  \"teleology\": [\n    {\n      \"year\": 1991,\n      \"claim\": \"Established that PPIF is an enzyme\\u2014a bona fide peptidyl-prolyl isomerase\\u2014and that its N-terminal extension targets it to organellar membranes, defining it as a mitochondria-associated cyclophilin rather than a cytosolic one.\",\n      \"evidence\": \"Recombinant expression in E. coli, PPIase activity assays with synthetic peptides, CsA-analogue inhibition kinetics, and subcellular fractionation\",\n      \"pmids\": [\"1744118\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No in vivo substrate identified at this stage\", \"Functional consequence of the isomerase activity in mitochondria unknown\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Connected CyP-D enzymology to a physiological structure by showing it binds VDAC/ANT complexes at membrane contact sites and is the molecular target through which CsA inhibits the MPTP, explaining how a matrix isomerase controls inner-membrane permeability.\",\n      \"evidence\": \"CyP-D fusion-protein affinity pulldown of VDAC/ANT, photoaffinity labelling with a CsA derivative, and pore reconstitution from protein fractions\",\n      \"pmids\": [\"10989666\", \"10393078\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CyP-D is a structural pore component or a regulator unresolved\", \"Exact inner-membrane protein undergoing the conformational change not pinned down\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined the mechanism of pore facilitation: CyP-D's PPIase activity drives a Ca2+-triggered conformational change in ANT (sensitized by oxidation of specific cysteines), and minimal pore formation requires neither VDAC nor outer-membrane proteins.\",\n      \"evidence\": \"Reconstitution of MPTP from purified ANT \\u00b1 CyP-D, cysteine-specific chemical modification, and correlation of CsA-inhibited PPIase activity with pore inhibition\",\n      \"pmids\": [\"12022946\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the physiological inner-membrane target still debated\", \"Direct structural proof of the isomerized proline lacking\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Genetic knockout resolved the structural-versus-regulatory question, demonstrating that the pore can still form and open without CyP-D, redefining CyP-D as a regulatory sensitizer rather than a pore-forming subunit.\",\n      \"evidence\": \"Ca2+-induced swelling assays in CyP-D-knockout mouse mitochondria \\u00b1 CsA\",\n      \"pmids\": [\"16167169\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the structural pore-forming component\", \"Endogenous trigger of CyP-D recruitment in vivo unclear\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Assigned CyP-D-dependent MPT to a specific death modality, showing it is required for necrosis but not apoptosis and is central to ischemia/reperfusion injury, establishing the therapeutic rationale for CyP-D inhibition.\",\n      \"evidence\": \"Ppif\\u2212/\\u2212 mice in necrosis vs apoptosis assays and ischemia/reperfusion models\",\n      \"pmids\": [\"17136322\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular events linking pore opening to necrotic execution incomplete\", \"Tissue-specific thresholds for MPT engagement not defined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Refined the substrate model by showing ANT acts regulatorily and proposing the phosphate carrier (PiC) as the inner-membrane protein whose CyP-D-facilitated conformational change opens the pore.\",\n      \"evidence\": \"Genetic KO of ANT isoforms and CyP-D plus biochemical interaction studies\",\n      \"pmids\": [\"18407825\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"PiC as CyP-D substrate awaits knockdown/reconstitution confirmation\", \"Single-lab proposal not yet independently validated\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Extended CyP-D function beyond cell death to physiology, linking it to acetaminophen hepatotoxicity downstream of MPT and to hippocampal neurotransmitter release underlying learning and memory.\",\n      \"evidence\": \"Ppif\\u2212/\\u2212 mice with liver injury markers; behavioral testing, hippocampal microdialysis, and CsA infusion\",\n      \"pmids\": [\"20942566\", \"19437409\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism coupling CyP-D to neurotransmitter release at synapses unknown\", \"Whether memory effect requires transient pore opening vs another CyP-D function unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Revealed a retrograde signaling role, showing CyP-D loss activates STAT3 and a Cxcl12-Cxcr4 chemokine program that accelerates proliferation and migration, implicating CyP-D in mitochondria-to-nucleus inflammatory signaling.\",\n      \"evidence\": \"CyP-D KO/knockdown cells with proliferation, migration, transcriptome, and STAT3 assays\",\n      \"pmids\": [\"23303179\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Signal transmitted from mitochondria to STAT3 not identified\", \"Single lab; relationship to MPTP activity unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified ROS-induced partner complexes (p53 and MST1) that translocate to mitochondria and bind CyP-D to drive programmed necrosis, providing a mechanism for upstream death-signal coupling to the pore.\",\n      \"evidence\": \"Reciprocal co-IP of p53/CyP-D and MST1/CyP-D at mitochondria with genetic and pharmacological knockdown across glioma, prostate, and pancreatic cancer cells\",\n      \"pmids\": [\"26024660\", \"24946211\", \"24732633\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether p53/MST1 binding directly alters pore conformation or acts indirectly unresolved\", \"Binding interface on CyP-D not mapped\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrated a physiological, sub-lethal CyP-D function by showing it sets the frequency of transient mitoflash openings and influences respiratory capacity, with tissue-specific magnitude tracking CyP-D expression.\",\n      \"evidence\": \"Reciprocal gain/loss-of-function in cardiomyocytes and skeletal muscle with live mitoflash imaging and Seahorse respirometry\",\n      \"pmids\": [\"26746144\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trigger distinguishing transient mitoflash from lethal pore opening unclear\", \"Bioenergetic consequence mechanism not fully defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Placed CyP-D within necroptotic crosstalk, showing necroptosis requires both Bax/Bak outer-membrane oligomerization and CyP-D-dependent MPTP opening, with MLKL/cofilin-1 mitochondrial translocation partly CyP-D-dependent.\",\n      \"evidence\": \"Ppif\\u2212/\\u2212 and Bax/Bak-null fibroblasts with necroptosis induction and mitochondrial fractionation\",\n      \"pmids\": [\"26061004\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic link between MLKL and MPTP not established\", \"Single lab; degree of MPT contribution to necroptosis varies by stimulus\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Generalized CyP-D-driven necrosis to crystal-induced kidney injury and quantified its partial redundancy with necroptosis via double-knockout epistasis.\",\n      \"evidence\": \"Ppif\\u2212/\\u2212 and Ppif\\u2212/\\u2212Mlkl\\u2212/\\u2212 mice with crystal exposure, histology, and pharmacological MPTP inhibition\",\n      \"pmids\": [\"31296606\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signal from lysosomal cathepsin/ROS to MPTP not molecularly defined\", \"Quantitative split between MPT-necrosis and necroptosis context-dependent\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed that CyP-D abundance is a regulated transcriptional node, with BMP/Smad repressing Ppif to deactivate the MPTP as a functional requirement for osteoblast differentiation.\",\n      \"evidence\": \"BMP/Smad activation in MSCs, Ppif promoter reporters, and CyP-D gain-of-function in vitro and in mice\",\n      \"pmids\": [\"35645445\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct Smad binding elements on Ppif not exhaustively mapped\", \"Connection between MPTP deactivation and osteogenic gene program mechanistic detail limited\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Provided direct in vivo genetic evidence for CyP-D and ANT1 as MPTP components, with combined deletion almost fully preventing necrotic muscular dystrophy.\",\n      \"evidence\": \"Single and double KO (Slc25a4\\u2212/\\u2212, Ppif\\u2212/\\u2212) in \\u03b4-sarcoglycan-null dystrophic mice with histopathology\",\n      \"pmids\": [\"37624892\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Residual MPT activity in double KO indicates additional components\", \"Stoichiometry of CyP-D\\u2013ANT1 interaction in the pore not defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified an opposing transcriptional input, C/EBP\\u03b1 synergizing with NF-\\u03baB p65 to activate Ppif during adipogenesis, linking adipogenic and inflammatory signaling to MPTP gain-of-function.\",\n      \"evidence\": \"Ppif promoter reporters and ChIP for C/EBP\\u03b1 and NF-\\u03baB p65 in adipogenically induced BMSCs\",\n      \"pmids\": [\"37949231\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of increased MPTP for adipocyte biology incomplete\", \"Single lab; two-method support only\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided structural and proteolytic mechanism for CyP-D engagement of ATP synthase, showing the flexible N-terminus suppresses OSCP binding and that calpain 1 cleavage generates a \\u0394N-CyP-D that binds OSCP, inhibits ATP synthesis, and favors pore transition.\",\n      \"evidence\": \"NMR of FL- and \\u0394N-CyP-D, OSCP binding assays, calpain 1 treatment, and mass-spec detection of \\u0394N-CyP-D in cells\",\n      \"pmids\": [\"39528709\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether OSCP- vs ANT-binding represent distinct or unified pore models unresolved\", \"Physiological stimulus driving calpain 1 cleavage not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established a survival role of transient CyP-D pore opening, showing that in senescent cells CyP-D flickering provides Ca2+ efflux and that its inhibition causes toxic Ca2+ overload and senolysis, with MCU/NCLX epistasis defining the Ca2+ circuit.\",\n      \"evidence\": \"Genome-wide CRISPR screen, Ppif KO, CsA, MCU/NCLX perturbation, and mitochondrial Ca2+ imaging in senescent cells\",\n      \"pmids\": [\"39448884\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"What renders senescent cells uniquely dependent on CyP-D efflux unclear\", \"Generality of senolytic effect across senescence inducers untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Expanded CyP-D into tissue-repair biology, demonstrating it promotes fibroblast collagen secretion (preventing ER retention) and keratinocyte reepithelialization required for wound closure.\",\n      \"evidence\": \"Reciprocal gain/loss-of-function in keratinocytes and fibroblasts, Ppif-KO mice, and human/pig wound models with collagen and closure readouts\",\n      \"pmids\": [\"38564292\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking a mitochondrial isomerase to ER collagen secretion unknown\", \"Whether this reflects MPTP activity or a non-pore CyP-D function unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Detailed post-translational control, showing acetylated/oxidized CyP-D (as in sepsis) promotes MPTP opening and lysosomal dysfunction and is reversed via the NAD+/SIRT3 deacetylation axis.\",\n      \"evidence\": \"LPS sepsis mice, PPIF knockdown in cardiomyocytes, NMN and mito-TEMPO treatment, and CyP-D acetylation/oxidation assays\",\n      \"pmids\": [\"39623043\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific acetylated/oxidized residues not all mapped\", \"Single lab; direct SIRT3\\u2013CyP-D enzymatic step inferred\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Implicated CyP-D in atherosclerotic necrotic-core formation by linking it to macrophage death, ER-stress cytochrome c release, and necroptosis in vivo.\",\n      \"evidence\": \"Apoe\\u2212/\\u2212Ppif\\u2212/\\u2212 double-KO mice and CyP-D siRNA in macrophages with cytochrome c release and necroptosis readouts\",\n      \"pmids\": [\"38972156\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which CyP-D promotes cytochrome c release not defined\", \"Relative contribution of apoptosis vs necroptosis in lesions unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The identity of the structural pore-forming element of the MPTP and the precise inner-membrane protein whose proline CyP-D isomerizes (ANT vs PiC vs OSCP/F-ATP synthase) remain unresolved, as does how a single matrix isomerase governs both lethal and physiological pore openings and non-pore functions such as collagen secretion.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified structural model reconciling ANT-, PiC-, and OSCP-based mechanisms\", \"Mechanism distinguishing transient mitoflash/flickering from lethal sustained opening unknown\", \"Non-mitochondrial-death functions (collagen secretion, neurotransmission) mechanistically unexplained\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016853\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [3, 20]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4, 3]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [21]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 1, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [5, 7, 16, 24]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [2, 21]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [3, 26]}\n    ],\n    \"complexes\": [\n      \"mitochondrial permeability transition pore (MPTP)\",\n      \"F-ATP synthase (OSCP-bound)\"\n    ],\n    \"partners\": [\n      \"SLC25A4\",\n      \"VDAC\",\n      \"OSCP\",\n      \"TP53\",\n      \"MST1\",\n      \"HK2\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":{"gene":"PPIF","tier":"GROUNDING","verdict":"Evidence-grounding concern","subtype":"fabrication","uniprot_band":"medium","rules_fired":"R7","issue":"R7: fabricated (no corpus paper): 35645445"},"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}