Cyclophilin B Human, His

What is Cyclophilin B and what is its primary function in human cells?

Cyclophilin B (CypB) belongs to the cyclophilin family of peptidyl-prolyl isomerases (PPIases) that accelerate protein folding by catalyzing the cis-trans isomerization of proline residues. CypB is primarily localized in the endoplasmic reticulum (ER) where it functions as a molecular chaperone assisting in protein folding and quality control . Unlike other cyclophilin family members, CypB contains an ER retention signal and can be secreted into biological fluids, where it mediates inflammatory responses through interaction with the CD147 receptor on cell surfaces . The PPIase activity of CypB is essential for its protective function against ER stress-induced cell death, as demonstrated by studies showing that isomerase activity-defective mutants (CypB/R62A) increase calcium leakage from the ER, reactive oxygen species (ROS) generation, and decrease mitochondrial membrane potential .

How does His-tagged recombinant Cyclophilin B differ from native human CypB?

His-tagged human Cyclophilin B is a recombinant protein engineered with a polyhistidine tag (typically 6-10 histidine residues) at either the N- or C-terminus to facilitate purification using immobilized metal affinity chromatography (IMAC). While the His-tag provides significant research advantages, researchers should consider potential functional implications:

Structural Considerations:

• The His-tag rarely interferes with the core PPIase domain structure, as it is typically attached to terminal regions

• Crystal structures show that the active site of cyclophilins is formed by residues from the central β-sheet and surrounding loops, which are distant from the termini where tags are attached

Functionality Assessment:
To verify His-tagged CypB functionality, researchers should:

• Perform comparative PPIase activity assays using both tagged and untagged versions

• Assess binding affinity to known partners like Cyclosporin A

• Evaluate chaperone activity through protein folding assays

• Consider tag removal using protease cleavage if interference is detected

How do serum levels of Cyclophilin B vary across the menstrual cycle?

Recent research has revealed significant variations in Cyclophilin B serum concentrations across the menstrual cycle, suggesting hormonal regulation of this protein. A 2023 study monitoring eleven healthy women with normal BMI (21.8 kg/m²) throughout a single menstrual cycle demonstrated a specific pattern of CypB expression :

Methodology for investigating cycle-dependent variations:

• Recruit female subjects with regular menstrual cycles

• Collect blood samples at precisely defined cycle phases (confirmed by hormone measurements)

• Employ ELISA or immunoblotting with anti-CypB antibodies for quantification

• Analyze data using repeated measures ANOVA with post-hoc tests

• Correlate CypB levels with estradiol, progesterone, FSH, and LH measurements

These findings suggest potential roles for CypB in cyclic inflammatory events affecting the female reproductive system and highlight the importance of controlling for menstrual cycle phase when measuring CypB in female subjects .

What is the relationship between Cyclophilin B and other cyclophilins in human disease processes?

The functional distinction between CypB and other cyclophilins, particularly CypA, has significant implications for understanding their roles in disease processes. Recent research demonstrates remarkable specificity in their contributions to pathological conditions:

Non-Alcoholic Steatohepatitis (NASH) Development:
A 2024 study using knockout mouse models revealed that Ppib-/- (CypB KO) mice were protected from developing NASH features in a diet and chemical-induced model, while Ppia-/- (CypA KO) mice developed severe disease comparable to wild-type mice . This demonstrates that:

• CypB plays a necessary role in NASH disease progression

• CypA loss cannot compensate for CypB function in this context

• The specific role of CypB in the ER secretory pathway may be significant to NASH pathogenesis

Research methodology for investigating cyclophilin isoform specificity:

• Generate conditional and tissue-specific knockout models for individual cyclophilins

• Apply isoform-specific inhibitors when available

• Perform rescue experiments by expressing specific cyclophilin isoforms in knockout backgrounds

• Use quantitative proteomics to identify isoform-specific interaction partners

• Evaluate localization patterns through subcellular fractionation and immunofluorescence

This functional specificity extends to other disease contexts and should guide research design when investigating cyclophilin-associated pathologies .

What structural features distinguish Cyclophilin B from other cyclophilin family members?

Human cyclophilins share a conserved PPIase domain but exhibit distinct functional characteristics driven by specific structural differences:

Key structural distinctions of CypB:

• Contains an N-terminal signal sequence directing it to the ER

• Features an ER retention signal (HEEL) at its C-terminus

• Possesses a core PPIase domain with specific substrate specificity

Comparative structural analysis:
The cyclophilin family shows greatest conformational divergence in the S2 pocket region, which influences substrate specificity . Structural studies reveal that cyclophilins can be categorized by:

• β1-β2 loop configuration:

  • "Full-length" loops (PPIB/CypB, PPIC, PPID, PPIE, PPIF, PPIG, PPIH, PPIL6, NKTR, RanBP2)

  • "Deleted" loops (PPIL1, PPIL2, PPIL3, PPIL4, SDCCAG-10, PPWD1)

• Active site residue variations:

  • CypB contains a critical tryptophan residue (equivalent to Trp121 in CypA) that forms a hydrogen bond with substrates

  • Some cyclophilins have alternative residues (histidine or tyrosine) at this position, affecting substrate binding

• α1-β3 loop region:

  • This region shows substantial structural diversity among cyclophilin family members

  • Influences interaction with binding partners and subcellular localization

Experimental approaches to study these structural distinctions include X-ray crystallography, NMR spectroscopy, and hydrogen-deuterium exchange mass spectrometry to map interaction surfaces and conformational dynamics .

How does the PPIase activity of Cyclophilin B contribute to its protective role against ER stress?

The peptidyl-prolyl isomerase (PPIase) activity of CypB plays a critical role in protecting cells against ER stress-induced apoptosis through several mechanisms:

PPIase-dependent cytoprotection:
Research demonstrates that wild-type CypB attenuates ER stress-induced cell death, while PPIase-defective mutants (CypB/R62A) exacerbate it . The specific mechanisms include:

• Protein folding acceleration:

  • CypB catalyzes the rate-limiting cis-trans isomerization of proline residues

  • This activity directly accelerates protein folding, reducing misfolded protein burden

• ER calcium homeostasis maintenance:

  • PPIase-defective CypB increases Ca²⁺ leakage from the ER

  • Wild-type CypB helps maintain proper calcium storage in the ER lumen

• Mitochondrial integrity preservation:

  • CypB PPIase activity helps maintain mitochondrial membrane potential during ER stress

  • This prevents activation of the intrinsic apoptotic pathway

• ROS generation suppression:

  • Functional CypB limits reactive oxygen species production during ER stress

  • This protective effect requires intact PPIase activity

• Chaperone network integration:

  • CypB interacts with other ER stress-related chaperones including BiP and Grp94

  • These interactions require functional PPIase domains to maintain the ER protein folding network

To investigate these mechanisms, researchers can employ experimental approaches including PPIase activity assays with purified proteins, calcium imaging in ER stress conditions, ROS detection assays, and co-immunoprecipitation studies to map interactions with other chaperones .

What are the optimal methods for purifying His-tagged human Cyclophilin B for structural and functional studies?

Purification of His-tagged human Cyclophilin B requires careful optimization to maintain structural integrity and enzymatic activity. The following protocol integrates best practices for high-yield, high-purity preparations:

Expression system selection:

• E. coli BL21(DE3) for high yield of soluble protein

• Mammalian expression systems (HEK293 or CHO cells) when post-translational modifications are required

Optimized purification workflow:

• Immobilized Metal Affinity Chromatography (IMAC):

  • Use Ni-NTA or Co-TALON resins (Co²⁺ typically provides higher specificity)

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol

  • Include 5-10 mM imidazole in binding buffer to reduce non-specific binding

  • Elute with 250-300 mM imidazole gradient

• Size Exclusion Chromatography:

  • Further purify using Superdex 75 column

  • Buffer: 20 mM HEPES pH 7.5, 150 mM NaCl

  • Monitor for monomeric state (~21 kDa for His-tagged CypB)

• Quality control assessments:

  • SDS-PAGE (>95% purity)

  • Western blot with anti-His and anti-CypB antibodies

  • Circular dichroism to verify secondary structure

  • PPIase activity assay using standard peptide substrates

Considerations for specific applications:

• For crystallography: Include final polishing step with ion exchange chromatography

• For functional assays: Verify activity with PPIase assay using tetrapeptide substrate

• For binding studies: Consider tag removal using TEV or PreScission protease

Proper storage in 20 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol at -80°C maintains activity for >6 months. Avoid repeated freeze-thaw cycles.

How can researchers effectively study Cyclophilin B-mediated cyclosporin A internalization in human T-lymphocytes?

Studying CypB-mediated cyclosporin A (CsA) internalization requires specialized techniques to track both the protein and drug. Based on established research methodologies , the following approach is recommended:

Preparation of CsA-CypB complex:

• Incubate purified His-tagged CypB with CsA at 1:1 molar ratio

• Confirm complex formation using isothermal titration calorimetry or fluorescence polarization

Binding and internalization assay:

• Isolate peripheral blood T-lymphocytes using Ficoll-Hypaque gradient and negative selection

• Label CypB with fluorescent tag (e.g., Alexa Fluor 488) or radiolabel (³H or ¹²⁵I)

• Label CsA with distinct fluorophore (e.g., Alexa Fluor 647) or tritium (³H-CsA)

• Incubate cells with labeled CypB-CsA complex at 4°C (binding) or 37°C (internalization)

• Analyze using:

  • Flow cytometry for quantitative assessment

  • Confocal microscopy for localization studies

  • Scintillation counting for radiolabeled components

Key parameters to measure:

• Binding kinetics (Kd and number of binding sites)

• Internalization rate of the complex

• Differential accumulation of CypB versus CsA

• Receptor recycling dynamics

• Effect on T-cell activation using CD3-stimulated proliferation assays

Research has demonstrated that both free CypB and the CsA-CypB complex bind to T-lymphocyte surfaces with similar affinity (Kd values), but the complex shows different internalization dynamics with preferential accumulation of CsA within cells . This methodology allows detailed investigation of how CypB may enhance the immunosuppressive effects of CsA through targeted drug delivery.

What experimental approaches can best elucidate the role of Cyclophilin B in NASH progression?

Recent evidence demonstrating that CypB knockout mice are protected from NASH development opens significant research opportunities . To further elucidate CypB's role in NASH pathogenesis, researchers should consider these methodological approaches:

In vivo models and tissue-specific manipulations:

• Conditional knockout strategies:

  • Generate hepatocyte-specific Ppib knockout using Alb-Cre

  • Create stellate cell-specific deletion using GFAP-Cre

  • Develop macrophage-specific deletion using LysM-Cre to assess inflammatory contributions

• CypB inhibitor studies:

  • Test pan-cyclophilin inhibitors (e.g., CRV431/reconfilstat) versus selective CypB inhibitors

  • Establish dose-response relationships and treatment windows

  • Measure histological outcomes, inflammatory markers, and fibrosis progression

Mechanistic investigations:

• ER stress pathway analysis:

  • Evaluate UPR markers (PERK, IRE1α, ATF6) in CypB-deficient versus wild-type hepatocytes

  • Measure ER calcium homeostasis using Fura-2 or genetically encoded calcium indicators

  • Assess mitochondrial function and ROS production

• Multi-omics approaches:

  • Perform RNA-seq to identify transcriptional networks affected by CypB deletion

  • Use proteomics to identify CypB client proteins in hepatocytes

  • Apply lipidomics to characterize lipid profile changes in CypB-deficient livers

• Cell-specific contributions:

  • Analyze hepatocyte lipid accumulation (Oil Red O staining)

  • Measure stellate cell activation markers (α-SMA, collagen)

  • Assess inflammatory cell recruitment and polarization

The experimental design should compare wild-type, CypA knockout, and CypB knockout mice under standard diet, western diet, CCl₄ treatment, or combined challenges to fully understand the specificity of CypB's contribution to NASH pathogenesis .

How can the relationship between Cyclophilin B and hormonal regulation be thoroughly investigated?

The discovery that CypB serum levels fluctuate across the menstrual cycle suggests important connections between CypB and hormonal regulation that warrant further investigation . A comprehensive research approach should include:

In vitro hormone response studies:

• Cell culture models:

  • Treat primary human cells or relevant cell lines with physiological concentrations of estradiol, progesterone, FSH, and LH

  • Measure CypB gene expression, protein levels, and secretion

  • Analyze promoter activity using luciferase reporter assays to identify hormone-responsive elements

• Receptor antagonist experiments:

  • Apply specific estrogen and progesterone receptor antagonists

  • Use siRNA knockdown of specific hormone receptors

  • Identify receptor subtypes mediating CypB regulation

In vivo models for hormonal manipulation:

• Ovariectomized rodent models with hormone replacement:

  • Surgically remove ovaries to eliminate endogenous hormones

  • Administer controlled hormone replacement regimens

  • Measure serum and tissue CypB levels under different hormonal conditions

• Transgenic approaches:

  • Generate CypB reporter mice to visualize expression patterns

  • Create conditional CypB knockout in hormone-responsive tissues

  • Assess reproductive phenotypes in CypB-deficient animals

Clinical studies with enhanced controls:

• Design studies accounting for:

  • Precise cycle phase determination using hormone measurements

  • Oral contraceptive use and other medications

  • Age, BMI, and other potential confounders

• Longitudinal sampling across multiple cycles to assess:

  • Consistency of CypB fluctuations

  • Correlation with specific hormone peaks

  • Relationship to cycle-dependent inflammatory markers

The demonstrated correlation between CypB, LH (r=0.743, p=0.009 at periovulatory phase), and FSH (r=0.633, p=0.036 at mid-luteal phase) provides a foundation for investigating potential regulatory relationships and functional significance in reproductive physiology .