Cyclophilin C Human

What are the primary cellular locations of Human Cyclophilin C?

Human Cyclophilin C is predominantly localized in the cytoplasm of cells, as demonstrated by immunohistochemical staining in human liver tissue where specific staining is observed in the cytoplasm of hepatocytes . Interestingly, while primarily an intracellular protein, CypC can also be released extracellularly where it may function as a signaling molecule. This extracellular form (eCypC) has been shown to affect the function of various cell types, including pancreatic microendothelial cells . The dual intracellular and extracellular localization suggests complex regulatory mechanisms governing CypC's biological activities in different cellular compartments.

How is Human Cyclophilin C involved in inflammatory processes?

Human Cyclophilin C plays a significant role in inflammatory processes, particularly in T lymphocytes. Research has shown that CypC levels are modulated during inflammation and that it associates with the CD147 receptor on cell surfaces . This interaction is critical for immune cell function and migration. Experiments with T lymphocytes have demonstrated that compounds targeting cyclophilins can reduce intracellular CypC expression, which correlates with altered inflammatory responses . Additionally, elevated serum CypC levels have been positively associated with inflammatory markers such as IL-6, white blood cell counts, and neutrophil levels, further supporting its role in inflammatory processes .

What is the tissue distribution pattern of Human Cyclophilin C?

Human Cyclophilin C shows a relatively broad tissue distribution but with varying expression levels across different tissues. Northern blot experiments reveal that CypC is expressed in kidney, pancreas, skeletal muscle, heart, lung, and liver at comparable levels . Notably, CypC is present in extremely low concentrations in brain tissue and the Jurkat T cell line . This distribution pattern differs from that observed in mice, where CypC shows elevated expression in kidney, suggesting potential species-specific differences in CypC function. Immunohistochemical studies have also confirmed CypC protein expression in human liver, with specific localization to the cytoplasm of hepatocytes .

How does Human Cyclophilin C expression compare between normal and disease states?

In disease states, particularly in Coronary Artery Disease (CAD), serum levels of CypC are significantly elevated compared to healthy controls. Research has shown that CAD patients exhibit CypC levels of approximately 34.28 pg/mL (±5.77), which is significantly higher than control subjects . This elevation correlates with other inflammatory markers and appears to be a more sensitive and specific marker for CAD than other cyclophilins, with an Area Under the Curve (AUC) of 0.891 in ROC analysis . These findings suggest that altered CypC expression may play a role in the pathogenesis of cardiovascular conditions and potentially serve as a biomarker for certain disease states.

What factors regulate Human Cyclophilin C expression in different cell types?

The regulation of Human Cyclophilin C expression appears to be influenced by both inflammatory stimuli and feedback mechanisms involving other cyclophilins. In T lymphocytes, activation with Concanavalin A (Con A) alters CypC expression levels, and this effect can be modulated by compounds that target cyclophilins . Interestingly, extracellular cyclophilins also influence the intracellular expression of their counterparts. For example, in pancreatic microendothelial cells (MS1), the addition of extracellular CypA and CypB significantly reduces intracellular CypC levels, while extracellular CypC itself dramatically increases its own intracellular expression (to 229.8 ± 33% of baseline levels) . These observations suggest complex regulatory networks controlling CypC expression in different cellular contexts.

What are the validated methods for detecting Human Cyclophilin C in tissue samples?

Several validated methods exist for detecting Human Cyclophilin C in tissue samples. Immunohistochemistry (IHC) has been successfully employed using affinity-purified polyclonal antibodies against human CypC (such as Catalog # AF6669) at concentrations of 5 μg/mL, with overnight incubation at 4°C . This technique allows for visualization of CypC within its cellular context, with specific staining typically localized to the cytoplasm of cells. For fixation, immersion-fixed paraffin-embedded sections have proven effective. Detection systems such as HRP-DAB (horseradish peroxidase-diaminobenzidine) staining kits provide the chromogenic visualization, with hematoxylin counterstaining to provide cellular context .

What is the optimal protocol for Western blot detection of Human Cyclophilin C?

For Western blot detection of Human Cyclophilin C, PVDF membranes and reducing conditions have been successfully employed. An optimal protocol involves probing the membrane with 1 μg/mL of anti-human Cyclophilin C antibody (such as Sheep Anti-Human Cyclophilin C Antigen Affinity-purified Polyclonal Antibody, Catalog # AF6669), followed by incubation with an appropriate HRP-conjugated secondary antibody (e.g., Anti-Sheep IgG) . Under these conditions, specific bands for Cyclophilin C are typically detected at approximately 17 and 23 kDa, suggesting potential post-translational modifications or isoforms. For optimal results, Immunoblot Buffer Group 8 has been validated for CypC detection . This protocol allows for specific detection of CypC in various human tissue lysates, including prostate cancer tissue.

How can serum levels of Human Cyclophilin C be accurately quantified in clinical samples?

For quantitative measurement of Human Cyclophilin C in serum samples, enzyme-linked immunosorbent assay (ELISA) methods have been validated. Commercial ELISA kits specific for Human Cyclophilin C are available, such as the Human Peptidyl-Prolyl cis-trans Isomerase C ELISA kit, which allows for precise quantification of serum CypC levels . These assays have demonstrated sufficient sensitivity to detect clinically relevant differences between healthy individuals and those with conditions such as Coronary Artery Disease. In clinical studies, this approach has revealed significantly elevated CypC levels in CAD patients (34.28 pg/mL ± 5.77) compared to control subjects . When performing such quantification, it is advisable to simultaneously measure other inflammatory markers for correlation analysis, as CypC levels have been shown to positively associate with markers such as IL-6, WBC, and neutrophil counts .

What is the evidence for Human Cyclophilin C as a biomarker in Coronary Artery Disease?

Human Cyclophilin C has emerged as a promising biomarker for Coronary Artery Disease (CAD). Case-control studies have demonstrated significantly higher serum levels of CypC in CAD patients (34.28 pg/mL ± 5.77) compared to control subjects . ROC curve analysis revealed that CypC has superior sensitivity and selectivity for CAD diagnosis compared to other cyclophilins, with an Area Under the Curve (AUC) of 0.891 (p < 0.001) . This value indicates excellent discriminatory ability, categorizing CypC as a good marker for CAD, while CypA was classified as a fair marker (AUC = 0.748) and CypB as a poor marker (AUC = 0.655) . Furthermore, CypC shows significant positive correlations with established inflammatory markers including IL-6, white blood cell count, and neutrophil levels, reinforcing its potential value in inflammatory cardiovascular pathologies .

How does Human Cyclophilin C contribute to T lymphocyte function in inflammatory conditions?

Human Cyclophilin C plays a regulatory role in T lymphocyte function during inflammation, particularly through its association with the CD147 receptor. Research has shown that CypC levels are modulated in human T lymphocytes upon inflammatory stimulation with Concanavalin A (Con A) . Compounds that target cyclophilins, such as synthetic analogues of gracilin and cyclosporin A (CsA), can significantly reduce intracellular CypC expression in activated T lymphocytes . This modulation affects the CD147 receptor expression on the cell surface, which is critical for T cell migration and inflammatory responses. The CD147-cyclophilin interaction represents a key mechanism in the inflammatory process, as blockage of CD147 activity has shown success in treating inflammatory conditions . CypC appears to be part of a complex regulatory network involving other cyclophilins (A and B) that collectively influence T cell function in inflammatory states.

What role does Human Cyclophilin C play in pancreatic microendothelial dysfunction?

Extracellular Cyclophilin C (eCypC) has been shown to induce dysfunction in pancreatic microendothelial cells (MS1), suggesting a potential role in pancreatic pathologies. When MS1 cells are treated with eCypC, there is a remarkable increase in intracellular CypC levels (to 229.8 ± 33% of baseline, p < 0.01), indicating a positive feedback loop . Additionally, eCypC treatment significantly increases intracellular Cyclophilin D (iCypD) levels both in the absence and presence of cyclosporin A (CsA) (173.4 ± 24.4% and 215.7 ± 25.8%, respectively) . This effect on mitochondrial CypD is particularly notable as it differs from the effects of eCypA and eCypB, which decrease iCypD levels. The differential regulation of cyclophilins and their impact on endothelial function suggests that CypC may contribute to pancreatic vascular complications through mechanisms distinct from other cyclophilin family members .

How can recombinant Human Cyclophilin C be optimally produced for structural and functional studies?

For optimal production of recombinant Human Cyclophilin C, E. coli expression systems have been successfully employed. The protein can be expressed as a recombinant form spanning from Lys31 to Asp182 of the native human sequence (Accession # P45877) . Purification typically involves affinity chromatography approaches, which yield protein suitable for structural and functional analyses. When designing expression constructs, it's important to consider potential post-translational modifications, as CypC is detected at both 17 and 23 kDa by Western blot . For functional studies, the purified recombinant protein should be validated for proper folding and activity, often through peptidyl-prolyl isomerase assays or binding studies with known interactors such as cyclosporin A (CsA). Additionally, compared to other cyclophilins, CypC has distinct binding properties that should be considered when designing interaction studies or inhibitor screening assays .

What experimental models are most appropriate for studying Human Cyclophilin C function?

Several experimental models have proven valuable for studying Human Cyclophilin C function. In vitro, human T lymphocytes have been effectively used to study CypC's role in inflammatory processes and its modulation by various compounds, including synthetic analogues of gracilins and cyclosporin A (CsA) . For endothelial studies, the MS1 pancreatic microendothelial cell line has been employed to investigate the effects of extracellular CypC on endothelial function . Cell activation protocols using Concanavalin A (Con A) have been established to mimic inflammatory conditions and study CypC regulation in this context . For tissue expression and localization studies, human samples from various organs including liver, kidney, and prostate have provided insights into the distribution patterns of CypC . When studying CypC in relation to disease, case-control designs with human subjects have revealed important associations with conditions like Coronary Artery Disease . These diverse models allow for comprehensive investigation of CypC's functions across multiple physiological and pathological contexts.

What approaches can be used to identify and validate novel inhibitors of Human Cyclophilin C?

Identification and validation of novel Human Cyclophilin C inhibitors can be approached through multiple complementary strategies. Initial screening can employ in vitro binding assays measuring the direct interaction between candidate compounds and recombinant CypC. Established cyclophilin inhibitors like cyclosporin A (CsA) serve as positive controls, with binding typically assessed using immunochemical methods . Pharmacophore-directed retrosynthesis (PDR) has proven successful in generating bioactive small molecules with high affinity for cyclophilins, as demonstrated with gracilin analogues . For compounds showing promising binding, kinetic equilibrium dissociation constant (KD) values should be determined to quantify affinity. Functional validation can then be performed in cellular models such as Con A-stimulated T lymphocytes, measuring effects on CypC release and intracellular expression . MS1 pancreatic microendothelial cells provide another valuable model for assessing inhibitor effects on CypC-mediated cellular dysfunction . Ultimately, a comprehensive validation approach should assess not only direct binding but also downstream effects on inflammatory markers, cell migration, and CypC-dependent pathways in physiologically relevant cellular contexts.

How should researchers interpret contradictory data regarding Human Cyclophilin C tissue expression between human and mouse models?

When confronted with contradictory data regarding Human Cyclophilin C tissue expression between human and mouse models, researchers should consider several factors. First, there appears to be species-specific differences in CypC expression patterns. While CypC was initially thought to have elevated expression in mouse kidney, studies in human tissues revealed that CypC expression in kidney is not significantly elevated compared to other tissues such as pancreas, skeletal muscle, heart, lung, and liver . This contradicts the previously postulated specific role for CypC in the nephrotoxic effects of cyclosporin A (CsA) in humans, which was based on studies of its relative abundance in murine kidney . To properly interpret such discrepancies, researchers should: 1) Use multiple detection methods (protein and mRNA level analyses) to confirm expression patterns; 2) Consider developmental and physiological state differences between experimental models; 3) Verify antibody specificity across species; and 4) Acknowledge that functional roles may diverge despite conserved protein sequences. These contradictions highlight the importance of human-derived data for translational research on CypC functions.

What is the relationship between different cyclophilins (A, B, C, D) in human tissues and how do they influence each other?

The relationship between different cyclophilins (A, B, C, D) in human tissues is complex and involves regulatory cross-talk that influences their respective expressions and functions. Studies have revealed that these cyclophilins can regulate each other's levels through both direct and indirect mechanisms. In pancreatic microendothelial cells, extracellular CypA and CypB treatment significantly reduces intracellular CypC levels to 33.2 ± 13.2% (p < 0.01) and 23.7 ± 16.7% (p < 0.001), respectively . Conversely, extracellular CypC dramatically increases its own intracellular expression (to 229.8 ± 33%, p < 0.01) . This suggests a self-regulatory feedback mechanism unique to CypC. Additionally, CypC affects other cyclophilins differently than its family members do—while extracellular CypA and CypB decrease mitochondrial CypD levels, extracellular CypC increases CypD expression . In T lymphocytes, intracellular CypA and CypB levels follow similar expression profiles, indicating coordinated regulation . These interactions are further influenced by inflammatory stimuli and compounds like cyclosporin A. Understanding these complex relationships is crucial for interpreting experimental results and developing targeted therapeutic approaches that consider the entire cyclophilin network rather than individual proteins in isolation.

How can variations in Human Cyclophilin C detection across different methodologies be reconciled in research?

Variations in Human Cyclophilin C detection across different methodologies present significant challenges that researchers must carefully address. To reconcile these variations, several approaches are recommended. First, standardization of detection protocols is essential—for Western blot analysis, validated antibodies at specific concentrations (e.g., 1 μg/mL) should be used under defined conditions (reducing conditions with specific buffer systems like Immunoblot Buffer Group 8) . Similarly, for immunohistochemistry, consistent fixation methods, antibody concentrations (e.g., 5 μg/mL), and incubation parameters (overnight at 4°C) should be employed . When quantifying CypC in serum or cell culture supernatants, validated ELISA kits with known detection limits should be used across studies . To account for post-translational modifications or isoforms, researchers should acknowledge the detection of CypC at multiple molecular weights (17 and 23 kDa) and consider using multiple antibodies targeting different epitopes. Cross-validation using orthogonal methods (e.g., mass spectrometry) can provide additional confirmation of findings. Finally, accurate reporting of all methodological details, including reagent catalog numbers and experimental conditions, is crucial for reproducibility and proper comparison across studies. By implementing these strategies, researchers can better reconcile methodological variations and establish more consistent findings regarding CypC expression and function.

What is the potential of Human Cyclophilin C as a diagnostic biomarker for cardiovascular diseases?

Human Cyclophilin C shows considerable promise as a diagnostic biomarker for cardiovascular diseases, particularly Coronary Artery Disease (CAD). Statistical analysis of serum CypC levels demonstrates significant elevation in CAD patients (34.28 pg/mL ± 5.77) compared to healthy controls . Receiver Operating Characteristic (ROC) curve analysis reveals that CypC offers superior diagnostic value compared to other cyclophilins, with an Area Under the Curve (AUC) of 0.891 (p < 0.001) . This high AUC value classifies CypC as a "good" biomarker for CAD, outperforming both CypA (AUC = 0.748, classified as "fair") and CypB (AUC = 0.655, classified as "poor") . Furthermore, CypC levels positively correlate with established inflammatory markers including IL-6, white blood cell counts, and neutrophil levels, while negatively correlating with potential protective factors like MMP-2 and HDL cholesterol . These correlations provide biological plausibility for CypC's role in cardiovascular pathology. For clinical implementation, standardized ELISA protocols with defined reference ranges will need to be established, and multi-center validation studies should assess CypC's predictive value across diverse patient populations and in combination with existing cardiovascular biomarkers.

How might targeting Human Cyclophilin C therapeutically affect inflammatory and vascular diseases?

Targeting Human Cyclophilin C therapeutically represents a novel approach for treating inflammatory and vascular diseases. Several lines of evidence support this strategy. First, studies with synthetic compounds that modulate cyclophilins (such as gracilin analogues and cyclosporin A) have demonstrated significant effects on CypC expression and associated inflammatory processes in T lymphocytes . These compounds reduce intracellular CypC levels and modify interactions with the CD147 receptor, which is critical for immune cell migration during inflammation . Second, in pancreatic microendothelial cells, CypC modulation affects endothelial function, suggesting potential applications in vascular disorders . The development of CypC-specific inhibitors could provide advantages over broad cyclophilin inhibitors like cyclosporin A, potentially reducing off-target effects while maintaining therapeutic efficacy. For vascular diseases like CAD where CypC levels are elevated , selective inhibition might normalize inflammatory responses without the immunosuppressive complications of current therapies. Effective therapeutic strategies could include: 1) Small molecule inhibitors targeting CypC's peptidyl-prolyl isomerase activity; 2) Biologics interfering with CypC-CD147 interactions; or 3) Approaches to regulate extracellular CypC levels. As with any novel therapeutic target, careful assessment of safety and efficacy across different disease models will be essential before clinical translation.

What methodological approaches can effectively translate Human Cyclophilin C basic research findings to clinical applications?

Translating Human Cyclophilin C basic research findings to clinical applications requires a structured methodological approach spanning from mechanistic studies to clinical validation. Initially, establishing standardized detection methods is crucial—consistent antibodies, ELISA protocols, and reference standards enable reliable comparison across studies . Next, research should progress through a translational pipeline: 1) In vitro studies using primary human cells to validate mechanisms observed in cell lines; 2) Ex vivo analysis of patient-derived tissues to confirm relevance to human pathology; 3) Cross-sectional clinical studies comparing CypC levels across disease states, like the CAD case-control study showing CypC's biomarker potential (AUC = 0.891) ; 4) Longitudinal studies assessing CypC's predictive value for disease progression or treatment response. For therapeutic development, structure-based drug design targeting CypC should be pursued, building on knowledge of cyclophilin inhibitors like gracilins . Animal models must be carefully selected considering the species differences in CypC expression patterns . Finally, developing companion diagnostics that measure CypC levels alongside potential therapeutics would enable personalized medicine approaches. Throughout this process, multidisciplinary collaboration between basic scientists, clinicians, and industry partners is essential to overcome translational barriers and successfully move CypC-based diagnostics or therapeutics from bench to bedside.

What is the current understanding of Human Cyclophilin C's role in regulating mitochondrial function?

The relationship between Human Cyclophilin C and mitochondrial function represents an emerging area of research with intriguing preliminary findings. While Cyclophilin D (CypD) is the primary cyclophilin known to localize to mitochondria, evidence suggests that CypC may indirectly influence mitochondrial processes. Studies in pancreatic microendothelial cells have shown that extracellular CypC treatment significantly increases intracellular CypD levels by 173.4 ± 24.4% (p < 0.01), an effect that persists even in the presence of cyclosporin A (215.7 ± 25.8%) . This contrasts with the effects of extracellular CypA and CypB, which decrease CypD expression, suggesting a unique regulatory relationship between CypC and the mitochondrial cyclophilin . Given CypD's established role in mitochondrial permeability transition pore regulation and cell death pathways, this influence of CypC on CypD levels may represent a previously unrecognized mechanism by which CypC affects cellular energetics, oxidative stress responses, and cell survival. Further research is needed to elucidate the functional consequences of this relationship and to determine whether CypC directly interacts with mitochondrial proteins or regulates mitochondrial function through indirect signaling pathways.

How does Human Cyclophilin C interact with the CD147 receptor compared to other cyclophilins?

The interaction between Human Cyclophilin C and the CD147 receptor (also known as EMMPRIN) appears to have distinct characteristics compared to other cyclophilins, though research specifically comparing these interactions is still developing. CD147 serves as a receptor for several extracellular cyclophilins, with the CypA-CD147 interaction being the most extensively studied . Research has established that CypA binding to CD147 occurs at a site in the transmembrane domain at residue Pro211, which regulates receptor transport to the cell surface . While all cyclophilins share structural similarities in their peptidyl-prolyl isomerase domains, differences in their binding affinities and interaction dynamics with CD147 likely contribute to their distinct biological effects. In T lymphocytes, the expression of the CD147 surface membrane receptor is upregulated by inflammation and modulated by intracellular cyclophilins . Compounds that affect cyclophilin levels, including those that reduce CypC expression, have been shown to decrease CD147 receptor expression, suggesting a regulatory relationship . The functional consequences of the CypC-CD147 interaction appear to include effects on cell migration and inflammatory responses, with blockage of CD147 activity showing therapeutic potential in inflammatory conditions . More detailed structural and kinetic studies comparing the specific binding properties of CypC to CD147 versus other cyclophilins would help clarify the unique aspects of this interaction.

What omics approaches can advance understanding of Human Cyclophilin C's biological networks?

Advanced omics approaches offer powerful tools to unravel Human Cyclophilin C's biological networks and functional implications across different physiological and pathological contexts. Proteomics approaches, particularly affinity purification coupled with mass spectrometry (AP-MS), can identify CypC-interacting proteins beyond known partners like CD147, potentially revealing novel functional networks. Proximity labeling methods such as BioID or APEX could map the spatial interactome of CypC in different cellular compartments. Phosphoproteomics analysis following CypC modulation would help elucidate its role in signaling cascades, particularly in inflammatory contexts where CypC levels correlate with inflammatory markers like IL-6 . Transcriptomics approaches, including RNA-seq of cells with CypC knockout or overexpression, can identify genes and pathways regulated by CypC, while single-cell RNA-seq could reveal cell type-specific functions in tissues with heterogeneous cell populations. Metabolomics profiling following CypC manipulation might uncover its influence on cellular metabolism, especially considering its potential indirect effects on mitochondrial function through CypD regulation . Integration of these multi-omics datasets using computational systems biology approaches would provide a comprehensive view of CypC's position within cellular regulatory networks. Finally, translational omics comparing CypC networks in healthy versus disease states (such as CAD, where CypC shows biomarker potential ) could identify context-specific functions and therapeutic opportunities targeting this immunophilin.

What are the most effective strategies for generating specific antibodies against Human Cyclophilin C?

Generating specific antibodies against Human Cyclophilin C presents particular challenges due to the high sequence homology among cyclophilin family members. Effective strategies to overcome these challenges include: 1) Careful antigen design targeting unique regions of CypC that differ from other cyclophilins, particularly focusing on the N-terminal and C-terminal regions rather than the conserved peptidyl-prolyl isomerase domain; 2) Expression of recombinant CypC fragments (such as Lys31-Asp182) in E. coli systems, which has proven successful for generating affinity-purified polyclonal antibodies with high specificity ; 3) Extensive cross-reactivity testing against other cyclophilins (particularly CypA, CypB, and CypD) to ensure specificity; 4) Validation across multiple applications (Western blot, immunohistochemistry, ELISA) using known positive and negative control tissues; 5) Confirmation of specificity using tissues or cells with CypC knockdown or knockout; and 6) Monoclonal antibody development targeting CypC-specific epitopes, which may provide more consistent results across applications than polyclonal antibodies. Each antibody should be thoroughly characterized for its detection capabilities under different conditions (reducing vs. non-reducing for Western blot, various fixation methods for IHC) . The successful development of sheep anti-human Cyclophilin C affinity-purified polyclonal antibodies demonstrates the feasibility of generating specific tools for CypC detection that work effectively across multiple experimental applications .

How can researchers distinguish between the effects of different cyclophilins in complex biological systems?

Distinguishing between the effects of different cyclophilins in complex biological systems requires multi-faceted approaches that leverage their distinct characteristics despite their structural similarities. First, selective knockdown or knockout strategies using siRNA or CRISPR-Cas9 targeting specific cyclophilins allow for isolation of individual cyclophilin functions. When applying these approaches, validation of specificity is critical—researchers should confirm that targeting one cyclophilin doesn't affect the expression of others. Second, cyclophilin-specific inhibitors or blocking antibodies can provide temporal control over individual cyclophilin activity. While cyclosporin A (CsA) inhibits multiple cyclophilins, newer compounds such as the synthetic gracilin analogues show differential affinities that may be exploited . Third, careful experimental design using multiple readouts can help differentiate cyclophilin effects. For example, in pancreatic microendothelial cells, extracellular CypA, CypB, and CypC produce distinct patterns of effects on intracellular cyclophilin levels—while CypA and CypB reduce intracellular CypC levels, CypC increases its own intracellular expression . Fourth, combining cellular localization studies with functional assays helps attribute specific effects to the appropriate cyclophilin based on their predominant localization patterns. Finally, systems biology approaches integrating proteomic, transcriptomic, and functional data can help construct network models that distinguish the unique roles and interactions of each cyclophilin family member within complex biological processes.