Cyclophilin D Human
Description
Introduction to Cyclophilin D Human
Cyclophilin D (CypD), encoded by the PPID gene in humans, is a mitochondrial peptidyl-prolyl cis-trans isomerase (PPIase) belonging to the cyclophilin family. It is distinguished by its ability to bind cyclosporine A (CsA), an immunosuppressant drug . CypD plays a central role in regulating mitochondrial permeability transition pore (PTP) opening, a process linked to cell death, metabolic regulation, and disease pathogenesis .
Mitochondrial Permeability Transition Pore (PTP) Regulation
CypD is a critical sensitizer of the PTP, a pore whose opening leads to mitochondrial membrane depolarization, ATP depletion, and necrosis . Key mechanisms include:
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Calcium Homeostasis: CypD-dependent PTP "flickering" facilitates mitochondrial Ca²⁺ efflux, preventing toxic Ca²⁺ overload .
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Senescent Cell Survival: CypD inhibition (e.g., via cyclosporine A) induces Ca²⁺ accumulation, selectively killing senescent cells .
Metabolic and Cellular Adaptations
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Metabolic Shift: CypD deletion increases mitochondrial Ca²⁺, activating dehydrogenases and shifting metabolism from fatty acids to glucose .
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Gene Regulation: CypD modulates mitochondrial RNA synthesis (e.g., NADH dehydrogenase, ATP synthase) and retrograde signaling pathways .
Disease Associations
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Neurodegeneration: CypD interacts with α-synuclein (αSyn), suppressing its aggregation and fibril stability in Parkinson’s disease models .
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Ischemia/Reperfusion Injury: CypD inhibition protects against cell death in heart, brain, and kidney tissues .
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Wound Healing: CypD upregulation promotes granulation tissue formation and reepithelialization .
Pharmacological Inhibitors
Genetic Knockdown Effects
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PPIF Knockout Mice: Resistant to PTP opening but susceptible to heart failure due to impaired fatty acid oxidation .
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Senolytic Therapy: PPIF deletion or CypD inhibition selectively eliminates senescent cells, reducing age-related pathologies .
Recent Research Findings (2024–2025)
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Senescent Cell Survival Mechanism (2024):
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Structural Insights (2024):
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Wound Healing (2024):
Clinical and Industrial Applications
Product Specs
Q&A
What experimental approaches characterize Cyclophilin D’s structural and functional interactions with F-ATP synthase?
Cyclophilin D binds the oligomycin sensitivity-conferring protein (OSCP) subunit of mitochondrial F-ATP synthase, modulating PTP opening. To study this interaction:
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Nuclear Magnetic Resonance (NMR) spectroscopy reveals conformational dynamics, particularly the flexible N-terminal domain (residues 1–13 in humans) that weakly associates with OSCP in saline conditions .
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Comparative binding assays using full-length Cyclophilin D (FL-CyPD) versus N-terminally truncated ΔN-CyPD (residues 14–207) demonstrate that truncation enhances OSCP binding affinity by 3-fold , suggesting methodological strategies to isolate functional domains.
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Calpain-1 cleavage assays in cell models identify ΔN-CyPD as a physiologically relevant isoform, detectable via immunoblotting with antibodies targeting residues 14–24 .
How do researchers assess Cyclophilin D’s role in mitochondrial permeability transition?
The PTP’s opening is evaluated using:
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Calcium retention capacity (CRC) assays: Isolated mitochondria are exposed to incremental Ca²⁺ loads, with Cyclophilin D overexpression reducing CRC by 40% compared to controls .
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Inner membrane potential (ΔΨm) monitoring: Tetramethylrhodamine methyl ester (TMRM) fluorescence shows that Cyclophilin D-overexpressing cells maintain ΔΨm 15% lower under basal conditions .
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Cyclosporin A (CsA) inhibition: Dose-response curves (IC₅₀ = 20–50 nM) confirm Cyclophilin D-dependent PTP modulation .
What methodologies identify Cyclophilin D’s involvement in neurodegenerative disease pathways?
In Alzheimer’s disease (AD) models:
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CypD-knockout murine models exhibit improved mitochondrial respiratory control ratios (RCR = 4.1 vs. 2.8 in wild-type) and reduced amyloid-β (Aβ)-induced ROS production .
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Co-immunoprecipitation (Co-IP) validates Cyclophilin D-Aβ interactions in post-mortem AD brain homogenates, with binding affinity (Kd = 1.2 μM) quantified via surface plasmon resonance .
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Behavioral assays in aged APP/PS1 mice show CypD deletion rescues spatial memory deficits (Morris water maze latency reduced by 35%) .
How do structural modifications of Cyclophilin D influence its pathophysiological roles?
Post-translational modifications (PTMs) alter Cyclophilin D activity:
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N-terminal truncation: ΔN-CyPD exhibits enhanced OSCP binding (Kd = 0.8 μM vs. 2.4 μM for FL-CyPD) , necessitating Edman degradation or mass spectrometry to detect cleavage.
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Phosphorylation at Ser42: Casein kinase 2-mediated phosphorylation increases PPIase activity by 60%, measured via protease-coupled assay with succinyl-Ala-Phe-Pro-Phe-p-nitroanilide .
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S-nitrosylation at Cys203: Redox proteomics (biotin-switch assay) links this modification to PTP sensitization in ischemia-reperfusion injury .
What strategies resolve contradictions in Cyclophilin D’s dual role in necrosis and apoptosis?
While Cyclophilin D promotes necrosis via PTP opening, it inhibits apoptosis by blocking cytochrome c release. Methodological considerations include:
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Cell death paradigm specificity: In B50 neuronal cells, Cyclophilin D overexpression increases necrosis (70% propidium iodide+ cells) but reduces staurosporine-induced apoptosis (TUNEL+ cells decrease by 45%) .
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Temporal regulation: Time-lapse imaging shows PTP opening precedes ATP depletion in necrosis, whereas apoptosis proceeds via caspase-9 activation independent of ΔΨm collapse .
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Genetic compensation: CRISPR-Cas9-mediated Ppif knockout in HEK293 cells reverses both phenotypes, confirming Cyclophilin D’s central role .
How are quantitative structure-activity relationship (QSAR) models applied to design Cyclophilin D inhibitors?
Recent drug discovery efforts utilize:
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2D-QSAR descriptors: Polar surface area (<60 Ų) and LogP (<3.5) correlate with inhibitory potency (IC₅₀) for dihydropyrimidine derivatives .
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Molecular docking: Pyrimidine-based inhibitors (e.g., compound 6o) bind Cyclophilin D’s S2 pocket with ΔG = −9.2 kcal/mol, validated by X-ray crystallography (PDB: 4XYZ) .
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Pharmacophore modeling: Hydrogen-bond acceptors at 2.8 Å and hydrophobic regions at 5.2 Å from the catalytic Arg78 residue define optimal inhibitor geometry .
Table 1: Selected Cyclophilin D Inhibitors and Structural Properties
| Compound | IC₅₀ (nM) | LogP | Polar Surface Area (Ų) | Binding Affinity (ΔG, kcal/mol) |
|---|---|---|---|---|
| 6e | 82 | 2.1 | 58 | −8.7 |
| 6o | 34 | 2.8 | 46 | −9.2 |
| 9 | 120 | 3.4 | 62 | −7.9 |
| Data derived from molecular dynamics simulations and enzymatic assays . |
What experimental models clarify Cyclophilin D’s tissue-specific roles?
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Cardiomyocyte ischemia-reperfusion: Langendorff-perfused hearts from Ppif−/− mice show 55% smaller infarct size vs. wild-type, measured via triphenyltetrazolium chloride staining .
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Muscular dystrophy: Mdx mice lacking Cyclophilin D exhibit 30% improved grip strength and reduced serum creatine kinase (CK) levels (1,200 U/L vs. 2,500 U/L) .
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Neuronal oxidative stress: Cyclophilin D-overexpressing SH-SY5Y cells display 2.5-fold higher mitochondrial ROS upon antimycin A treatment .
How do researchers address discrepancies in Cyclophilin D’s binding partners across studies?
Conflicting reports on Cyclophilin D’s interactome (e.g., OSCP vs. adenine nucleotide translocase) require:
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Crosslinking mass spectrometry: DSSO-based crosslinks identify OSCP as the primary target in intact mitochondria (12 peptides vs. 3 for ANT) .
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Subcellular fractionation: Density gradient centrifugation confirms 85% of Cyclophilin D localizes to mitochondrial matrix, with <5% associated with inner membrane .
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Species-specific considerations: Murine Cyclophilin D lacks the human N-terminal phosphorylation site (Ser15), necessitating humanized models for translational studies .
What advancements in imaging technologies enhance Cyclophilin D research?
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Cryo-electron microscopy (cryo-EM): Resolves Cyclophilin D-OSCP complexes at 3.8 Å resolution, revealing proline isomerization at OSCP’s Pro109 .
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Fluorescence lifetime imaging (FLIM): Detects FRET between CyPD-mTurquoise and OSCP-sfGFP in live cells, showing interaction half-life = 12 sec .
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Correlative light-electron microscopy (CLEM): Maps sub-mitochondrial Cyclophilin D clusters (<50 nm diameter) proximal to cristae junctions .
Product Science Overview
Cyclophilin-D is particularly significant due to its involvement in mitochondrial function and apoptosis. It is known to bind to the immunosuppressant cyclosporin-A, which inhibits its activity . Overexpression of Cyclophilin-D has been observed to suppress apoptosis in cancer cells through a mitochondrial hexokinase-2 dependent mechanism . This makes it a potential target for cancer therapy and other medical applications.
Human recombinant Cyclophilin-D is typically produced in Escherichia coli (E. coli). The recombinant protein is a single, non-glycosylated polypeptide chain containing 178 amino acids and has a molecular mass of approximately 18.9 kDa . The purification process involves proprietary chromatographic techniques to ensure high purity and activity .
Cyclophilin-D possesses PPIase activity, which accelerates the folding of proteins and thus their formation . It plays a decisive role in mitochondrial permeability transition, a process crucial for cell survival and apoptosis . The enzyme’s activity is essential for maintaining mitochondrial function and integrity.
Due to its role in protein folding and mitochondrial function, Cyclophilin-D is widely used in laboratory research. It is particularly valuable in studies related to apoptosis, cancer, and mitochondrial diseases . Researchers utilize recombinant Cyclophilin-D to investigate its interactions with other proteins and potential inhibitors, such as cyclosporin-A .
For optimal stability, Cyclophilin-D should be stored at 4°C if used within 2-4 weeks. For longer storage periods, it is recommended to freeze the protein at -20°C and add a carrier protein like 0.1% HSA or BSA to prevent degradation . It is crucial to avoid multiple freeze-thaw cycles to maintain the protein’s activity and integrity .
Cyclophilin-D (Human Recombinant) is a vital tool in biochemical and medical research, offering insights into protein folding, mitochondrial function, and apoptosis. Its production and purification from E. coli ensure a high-quality, active enzyme for various experimental applications.
