Cyclophilin F Rat
Description
Mitochondrial Permeability Transition Pore (mPTP) Regulation
Cyclophilin F directly binds to the mPTP complex, increasing its open probability during oxidative stress . This interaction is modulated by:
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Calcium Sensitivity: CypD knockout models show enhanced mitochondrial calcium retention, altering NADH/NAD+ ratios and metabolic enzyme acetylation .
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Cooperative Partners: Synergizes with mitochondrial p53 to activate necrosis under oxidative stress .
ATP Synthase Modulation
Cyclophilin F regulates FF ATP synthase activity through direct interaction with the enzyme’s lateral stalk, influencing mitochondrial matrix adenine nucleotide levels .
Disease Models
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Cardiac Ischemia/Reperfusion Injury: CypD inhibition reduces infarct size by 40% in rat models via mPTP closure .
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Neurodegeneration: Aberrant CypD-mPTP interactions exacerbate oxidative damage in Alzheimer’s disease models .
Therapeutic Targeting
Recent studies identified isoform-selective CypD inhibitors (e.g., macrocycle A14) that exploit non-conserved S2 pocket residues, achieving >50-fold selectivity over other cyclophilins .
Product Specs
Q&A
What are the key functional roles of Cyclophilin F in mitochondrial biology?
Cyclophilin F (CypF) is a mitochondrial peptidyl-prolyl cis-trans isomerase that regulates protein folding and mitochondrial permeability transition pore (mPTP) activity. It modulates the F1F0 ATP synthase complex, influencing adenine nucleotide levels and oxidative stress responses . CypF also exhibits anti-apoptotic activity independent of mPTP, interacting with BCL2 to inhibit cytochrome c-dependent apoptosis .
Experimental Design Considerations
To study CypF’s mitochondrial roles:
How is recombinant Cyclophilin F Rat produced and purified?
CypF is expressed in E. coli as a non-glycosylated, His-tagged polypeptide (200 amino acids, 21.2 kDa). Purification involves proprietary chromatographic techniques, yielding a sterile, phosphate-buffered solution with 10% glycerol and 1 mM DTT .
Optimizing Protein Stability
| Condition | Recommendation | Rationale |
|---|---|---|
| Short-term storage | 4°C for 2–4 weeks | Preserve enzymatic activity |
| Long-term storage | -20°C with 0.1% HSA/BSA | Prevent aggregation and degradation |
What experimental approaches are used to study Cyclophilin F's role in apoptosis?
CypF’s anti-apoptotic function is investigated via:
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In vitro assays: TUNEL staining or caspase activity measurements in cell lines overexpressing CypF.
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Mitochondrial membrane potential: JC-1 or rhodamine-123 staining to monitor cytochrome c release.
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BCL2 interaction studies: Co-IP or proximity ligation assays to confirm binding .
Data Contradiction Analysis
Conflicting results in apoptosis studies may arise from:
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Model variability: Use of immortalized vs primary cells.
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Stimuli intensity: Oxidative stress vs hypoxia-induced damage.
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CypF isoform specificity: Cross-reactivity with other cyclophilins (e.g., CyP-D) .
How does Cyclophilin F's interaction with F1F0 ATP synthase affect mitochondrial function?
CypF modulates ATP synthase activity, altering mitochondrial matrix adenine nucleotide levels. This interaction may influence ATP production under stress conditions, such as ischemia-reperfusion injury .
Methodological Challenges
| Challenge | Solution |
|---|---|
| Direct binding detection | Native PAGE or cryo-EM structural analysis |
| Functional redundancy | CRISPR-Cas9 knockout in mitochondrial models |
What are the challenges in studying Cyclophilin F in vivo vs in vitro?
| In Vitro Limitations | In Vivo Solutions |
|---|---|
| Lack of mitochondrial dynamics | Use of Langendorff-perfused hearts |
| Recombinant protein artifacts | Adeno-associated virus (AAV)-mediated gene delivery |
| Simplified stress models | Ischemia-reperfusion protocols in rodents |
How to optimize storage and handling of Cyclophilin F Rat recombinant protein?
Critical Parameters
| Factor | Optimal Condition | Impact |
|---|---|---|
| Freezing medium | 10% glycerol + 1 mM DTT | Prevents oxidation and aggregation |
| Thawing method | Slow thawing at 4°C | Maintains structural integrity |
What methods are used to assess Cyclophilin F's peptidyl-prolyl cis-trans isomerase activity?
Enzymatic Assays
How does Cyclophilin F's His-tag affect its biochemical studies?
The N-terminal His-tag may:
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Interfere with substrate binding by steric hindrance.
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Enhance solubility in E. coli but alter mitochondrial localization signals.
Mitigation Strategies
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Tag removal: Use thrombin or enterokinase cleavage sites.
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Competition assays: Compare tagged vs untagged CypF activity.
What are the limitations of using E. coli-expressed Cyclophilin F in mammalian studies?
| Limitation | Impact | Solution |
|---|---|---|
| Absence of PTMs | Altered mitochondrial targeting signals | Co-expression with mammalian chaperones |
| Host strain variability | Inconsistent yields | Optimize induction conditions (e.g., IPTG concentration) |
Product Science Overview
Cyclophilin F, also known as Peptidyl-prolyl cis-trans isomerase F (PPIase F), is a mitochondrial protein that plays a crucial role in protein folding and mitochondrial function. The recombinant form of Cyclophilin F from rats is often used in research to study its functions and interactions.
Recombinant Rat Cyclophilin F is typically produced in Escherichia coli expression systems. The protein is a single, non-glycosylated polypeptide chain containing 200 amino acids, with a molecular mass of approximately 21.2 kDa . It is often fused to a His-tag at the N-terminus to facilitate purification .
Cyclophilin F accelerates the folding of proteins by catalyzing the cis-trans isomerization of proline imidic peptide bonds in oligopeptides . It is involved in the regulation of the mitochondrial permeability transition pore (mPTP), which is crucial for maintaining mitochondrial function and integrity . Cyclophilin F, in cooperation with mitochondrial TP53, plays a role in activating oxidative stress-induced necrosis .
Additionally, Cyclophilin F modulates the activity of mitochondrial membrane F1F0 ATP synthase and regulates mitochondrial matrix adenine nucleotide levels . It also exhibits anti-apoptotic activity independently of mPTP and, in cooperation with BCL2, inhibits cytochrome c-dependent apoptosis .
