Cyclophilin E Human

Basic Research Questions

• What is Cyclophilin E and what are its fundamental cellular functions?

Cyclophilin E (CypE, also known as PPIE or CYP33) is a member of the cyclophilin family of peptidyl-prolyl cis-trans isomerases (PPIases). It is one of 16 cyclophilin isoforms identified in humans and combines RNA-binding capabilities with PPIase enzymatic activity . CypE participates in pre-mRNA splicing as a component of the spliceosome, demonstrating a preference for single-stranded RNA molecules with poly-A and poly-U stretches, suggesting it binds to the poly(A)-region in the 3'-UTR of mRNA molecules . This protein catalyzes the cis-trans isomerization of proline imidic peptide bonds in proteins, which serves as a key regulatory mechanism for protein folding and function . Additionally, CypE inhibits KMT2A (MLL1) activity through its proline isomerase activity, providing a link between proline isomerization and epigenetic regulation . Recent research has also identified CypE as a positive regulator in osteoblast differentiation through its enhancement of Runx2 transcriptional activity .

• What is the molecular structure of Human Cyclophilin E?

Human Cyclophilin E consists of 314 amino acids with distinct functional domains that contribute to its versatile cellular roles. The full protein sequence begins with MATTK RVLYV GGLAE, continuing through to the C-terminal region . The core structure maintains the characteristic cyclophilin fold consisting of an eight-stranded antiparallel β-barrel with associated α-helices, as revealed by X-ray crystallography studies . CypE contains both a peptidyl-prolyl isomerase (PPIase) domain responsible for catalytic activity and an RNA recognition motif (RRM) that enables specific RNA binding. The PPIase domain features a hydrophobic pocket that accommodates proline substrates during the isomerization reaction, while regions outside this canonical active site contribute to isoform-specific substrate recognition . Crystallographic analysis has identified a molecular surface corresponding to the substrate-binding S2 position that represents a site of diversity in the cyclophilin family, potentially explaining the unique functional properties of CypE compared to other cyclophilins . These structural features provide the foundation for understanding CypE's diverse cellular functions.

• How does Cyclophilin E differ from other human cyclophilin family members?

Cyclophilin E exhibits several distinctive features that differentiate it from other members of the human cyclophilin family despite their shared evolutionary conservation:

• What methods are used to measure Cyclophilin E enzymatic activity?

Researchers employ several complementary techniques to measure the peptidyl-prolyl isomerase (PPIase) activity of Cyclophilin E:

The standard tetrapeptide assay utilizes small synthetic peptides containing a proline residue as substrates, allowing spectrophotometric detection of cis-to-trans isomerization rates . This approach can be adapted to test substrate specificity by varying the amino acid sequence surrounding the proline. Thermal stability assays provide an indirect measure of enzymatic activity by detecting changes in protein stability upon binding of known inhibitors like cyclosporin A (CsA) . The temperature at which CypE unfolds shifts in the presence of active site ligands, allowing researchers to screen potential inhibitors.

For more detailed kinetic analysis, protease-coupled assays are valuable, exploiting the fact that certain proteases can only cleave peptides when the target proline is in the trans conformation . The rate of appearance of cleavage products directly correlates with PPIase activity. Nuclear magnetic resonance (NMR) spectroscopy offers perhaps the most direct observation method, allowing researchers to monitor the equilibrium between cis and trans proline conformers in real-time and determine isomerization rates with high precision .

When investigating CypE mutants, comparative activity assays using wild-type protein as a reference point are essential. For example, mutations in the catalytic pocket (R191A, W257A) have been used to demonstrate the requirement of PPIase activity for CypE's function in processes like osteoblast differentiation .

• What experimental models and systems are commonly used to study Cyclophilin E?

Multiple experimental models have been developed to investigate the diverse functions of Cyclophilin E:

For biochemical and structural studies, recombinant human CypE protein expressed in Escherichia coli provides a reliable source of highly purified protein (>95% purity) . This system allows production of both full-length protein and specific domains for focused analyses of structure-function relationships. Cell culture models represent the cornerstone of functional studies, with HEK293T cells commonly used for transfection experiments with wild-type or mutant CypE constructs . For investigating CypE's role in osteoblast differentiation, MC3T3-E1 preosteoblast cells provide an excellent model system, allowing assessment of alkaline phosphatase (ALP) activity and osteogenic gene expression following CypE manipulation .

Genetic manipulation approaches include RNA interference (siRNA) for transient knockdown of CypE expression and CRISPR-Cas9 gene editing for generating stable knockout cell lines . Overexpression systems typically utilize mammalian expression vectors such as pCS4-3HA or pCS4-3Myc vectors for expressing full-length CypE or specific domain constructs . For protein-protein interaction studies, techniques like co-immunoprecipitation and GST pull-down assays are frequently employed to identify and characterize CypE binding partners .

Functional assays specific to CypE's role in RNA processing include RNA binding assays and pre-mRNA splicing assays, while its role in transcriptional regulation can be assessed using luciferase reporter assays with promoters of interest .

Advanced Research Questions

• How can researchers distinguish the specific functions of Cyclophilin E from other cyclophilin family members?

Distinguishing the unique functions of Cyclophilin E from other cyclophilins requires sophisticated experimental strategies to overcome challenges of functional redundancy and structural similarity. A systematic approach begins with expression profiling across cell types and conditions to identify scenarios where CypE expression patterns diverge from other family members . This information guides the selection of appropriate experimental models where CypE-specific functions are most likely to be detectable.

Domain-specific analysis provides critical insights, as CypE's RNA recognition motif (RRM) distinguishes it from cyclophilins that lack this domain . Creating chimeric proteins by swapping domains between cyclophilins can help identify which structural elements are responsible for isoform-specific functions. RNA binding assays focusing on CypE's preference for poly-A and poly-U stretches can further differentiate its activity from other family members with different RNA binding preferences .

For in-depth functional analysis, CRISPR-Cas9 knockout of CypE followed by rescue experiments with different cyclophilin isoforms reveals which functions are uniquely dependent on CypE. Complementary approaches include:

• Substrate identification using proximity labeling or cross-linking techniques

• Proteomic analysis to identify CypE-specific binding partners

• Isoform-specific antibodies for precise localization studies

• Single-cell analyses to account for heterogeneity in expression and function

To enhance specificity of findings, researchers should perform parallel experiments with multiple cyclophilin isoforms and include rescue experiments with catalytically inactive CypE mutants to distinguish structural from enzymatic roles .

• What experimental strategies can identify the RNA targets and binding characteristics of Cyclophilin E?

Characterizing the RNA-binding properties of Cyclophilin E requires a multi-faceted experimental approach focused on both binding specificity and functional consequences. RNA immunoprecipitation (RIP) represents a fundamental technique, where CypE is immunoprecipitated from cellular extracts and associated RNAs are identified through RT-PCR or sequencing . This approach can be enhanced through UV cross-linking (CLIP techniques) to capture direct RNA-protein interactions with nucleotide resolution.

For quantitative binding analysis, electrophoretic mobility shift assays (EMSAs) using purified recombinant CypE and labeled RNA oligonucleotides allow determination of binding affinities for different RNA sequences . Competition assays with unlabeled RNA can further refine understanding of sequence preferences. Surface plasmon resonance (SPR) and fluorescence anisotropy provide complementary biophysical techniques to measure binding kinetics and thermodynamics with various RNA substrates.

Structure-function analyses are essential for understanding the molecular basis of RNA recognition. This includes:

• Generating and testing mutations within the RNA recognition motif (RRM)

• Comparing binding preferences of isolated RRM versus full-length CypE

• Structural studies using X-ray crystallography or NMR to visualize CypE-RNA complexes

Functional validation can be achieved through tethering assays, where CypE is artificially recruited to reporter mRNAs to assess functional consequences on RNA fate . Additionally, transcriptome-wide analyses comparing wild-type and CypE-depleted cells can identify RNA populations affected by CypE activity, revealing its broader impact on RNA processing and gene expression .

• How does the peptidyl-prolyl isomerase activity of Cyclophilin E contribute to pre-mRNA splicing?

Cyclophilin E's peptidyl-prolyl isomerase (PPIase) activity plays a critical role in pre-mRNA splicing through multiple mechanisms that depend on its ability to catalyze conformational changes in spliceosomal proteins. As a component of the spliceosome, CypE facilitates the dynamic rearrangements required during the splicing cycle by isomerizing specific proline residues in spliceosomal proteins . This enzymatic activity enables precise conformational transitions necessary for spliceosome assembly, catalysis, and disassembly.

The dual functionality of CypE is particularly important for splicing regulation - its RRM domain recognizes specific RNA sequences (especially those with poly-A and poly-U stretches), while its PPIase domain simultaneously modifies protein conformations . This combination allows CypE to coordinate RNA binding with protein conformational changes at specific sites within the spliceosome.

Experimental evidence for the importance of CypE's PPIase activity in splicing comes from multiple approaches:

• Use of PPIase-deficient mutants demonstrates splicing defects that cannot be rescued without catalytic activity

• Cyclosporin A inhibition of CypE's PPIase activity disrupts normal splicing patterns

• Mass spectrometry studies identify isomerized prolines in spliceosomal proteins that serve as CypE substrates

• In vitro splicing assays with recombinant wild-type and mutant CypE reveal stage-specific requirements for PPIase activity

The spliceosome undergoes extensive remodeling during the splicing cycle, requiring numerous proteins to adopt different conformations . CypE's ability to catalyze these conformational switches through proline isomerization represents a fundamental regulatory mechanism in RNA processing that impacts gene expression patterns across the transcriptome .

• How does Cyclophilin E regulate KMT2A activity and what are the implications for epigenetic regulation?

Cyclophilin E exerts a significant regulatory effect on KMT2A (also known as MLL1, Mixed-Lineage Leukemia 1), a histone methyltransferase that catalyzes H3K4 methylation associated with active transcription. This regulation represents a direct link between proline isomerization and epigenetic control mechanisms. CypE inhibits KMT2A activity through a mechanism that critically depends on its peptidyl-prolyl isomerase function . The interaction involves direct binding between CypE and KMT2A, followed by isomerization of specific proline residues in KMT2A, which alters its conformation and reduces its histone methyltransferase activity .

The molecular mechanism has been extensively characterized through:

• Co-immunoprecipitation studies demonstrating physical interaction between CypE and KMT2A

• Histone methyltransferase assays showing decreased H3K4 methylation in the presence of active CypE

• Experiments with PPIase-deficient CypE mutants that fail to inhibit KMT2A activity

• Domain mapping identifying critical regions required for the CypE-KMT2A interaction

This regulatory relationship has profound implications for epigenetic control of gene expression. By inhibiting KMT2A, CypE can repress the expression of KMT2A target genes, which are often associated with developmental processes and cell fate decisions . This creates a mechanism for dynamic regulation of gene expression programs, where the catalytic activity of CypE serves as a reversible switch affecting chromatin state.

The CypE-KMT2A regulatory axis likely plays important roles in both normal development and disease contexts, particularly in systems where precise control of gene expression timing is critical . This mechanism represents a novel layer of epigenetic regulation where post-translational modifications (proline isomerization) directly impact histone modifications.

• What is the role of Cyclophilin E in osteoblast differentiation and bone formation?

Recent research has established Cyclophilin E as a positive regulator of osteoblast differentiation, revealing a previously unrecognized role in skeletal biology . Through both gain and loss of function experiments, CypE has been shown to enhance osteoblast differentiation as measured by increased alkaline phosphatase (ALP) staining and expression . The molecular mechanism involves CypE's enhancement of Runx2 transcriptional activity, with Runx2 being the master transcription factor controlling osteoblast differentiation and bone formation .

Experimental evidence demonstrates that CypE overexpression significantly increases BMP4-induced ALP staining and the mRNA expression of osteogenic markers . This effect is dependent on CypE's peptidyl-prolyl isomerase activity, as PPIase-deficient mutants (CypE R191A and CypE W257A) fail to enhance osteoblast differentiation . The mechanistic details were elucidated through a series of experiments showing that:

• CypE enhances Runx2 transcriptional activity through its PPIase function

• The Akt signaling pathway is involved in mediating CypE's effects on osteoblast differentiation

• CypE increases the transcriptional activity of both ALP and osteocalcin (OC) genes, key markers of osteoblast differentiation

These findings have significant implications for understanding bone development and potential therapeutic approaches for bone disorders. The identification of CypE as a positive regulator in this process suggests that targeting its activity could provide novel strategies for enhancing bone formation in conditions such as osteoporosis . Additionally, this research highlights how the peptidyl-prolyl isomerase activity of cyclophilins can influence tissue-specific differentiation programs through regulation of key transcription factors.

• What approaches can identify physiological substrates of Cyclophilin E?

Identifying the physiological substrates of Cyclophilin E represents a major challenge in understanding its diverse cellular functions. A comprehensive strategy combines multiple complementary approaches focused on capturing the transient enzyme-substrate interactions characteristic of PPIases.

Substrate trapping methods utilize catalytically inactive CypE mutants that can bind but not release substrates, allowing for stable complex formation that can be analyzed by mass spectrometry . This approach can be enhanced through chemical cross-linking to stabilize transient interactions before analysis. Proximity-based labeling techniques, such as BioID or APEX2 fusions with CypE, enable identification of proteins in close spatial proximity to CypE in living cells, providing a more physiological context for potential substrate discovery .

Biochemical screening approaches include:

• Peptide library screening to identify sequence motifs preferentially isomerized by CypE

• Proline-directed conformational antibodies that specifically recognize cis or trans conformations

• Proteomic analysis of proline isomerization changes upon CypE manipulation

For validation of candidate substrates, researchers employ multiple complementary techniques:

Computational approaches can supplement experimental methods by predicting potential substrates based on structural compatibility and sequence features . Integration of transcriptomic, proteomic, and interactomic data provides a systems-level view to prioritize candidate substrates for experimental validation. This multi-faceted approach is essential for building a comprehensive understanding of CypE's substrate network and cellular functions .

• How can researchers develop isoform-specific inhibitors for Cyclophilin E?

Developing isoform-specific inhibitors for Cyclophilin E presents significant challenges due to the high conservation of active sites across the cyclophilin family, but several strategic approaches can overcome these obstacles . Structure-based design strategies form the foundation of this effort, utilizing high-resolution crystal structures of CypE to identify unique structural features that distinguish it from other cyclophilins . Computational analysis has revealed that regions outside the canonical active site, particularly the substrate-binding S2 position, represent sites of diversity that can be exploited for selective inhibitor design .

The development pathway should include:

• Comparative structural analysis of all human cyclophilin isoforms to identify CypE-specific features

• Virtual screening of chemical libraries against the CypE active site and adjacent regions

• Fragment-based approaches to build selectivity by targeting multiple binding pockets

• Structure-activity relationship studies to optimize selectivity while maintaining potency

Validation of inhibitor candidates requires rigorous testing across multiple cyclophilin isoforms using:

A promising strategy involves developing dual-targeting inhibitors that simultaneously engage both the PPIase active site and the RNA-binding domain unique to CypE . This approach could achieve dramatically increased selectivity over cyclophilins that lack the RNA-binding domain. Additionally, allosteric inhibitors that bind outside the highly conserved active site represent a valuable alternative strategy for achieving isoform selectivity .

While challenging, the development of CypE-specific inhibitors would provide valuable tools for dissecting its functions and potentially lead to therapeutic applications in contexts where selective modulation of CypE activity is beneficial .

• What evidence links Cyclophilin E to human disease pathogenesis?

While direct evidence linking Cyclophilin E specifically to human diseases remains limited, several lines of investigation suggest potential roles in pathological processes. Cyclophilins as a family have been implicated in various diseases, with reports of upregulated expression in many human cancers and strong correlations between cyclophilin overexpression and malignant transformation . CypE's role in regulating KMT2A activity is particularly relevant to disease contexts, as KMT2A is a histone methyltransferase frequently involved in leukemic translocations . By inhibiting KMT2A activity through its PPIase function, CypE may influence gene expression patterns relevant to cancer development and progression.

The involvement of CypE in pre-mRNA splicing as a component of the spliceosome suggests potential roles in splicing-related disorders . Mutations in spliceosomal components are associated with various human diseases, and alterations in CypE function could potentially contribute to splicing dysregulation observed in certain pathological conditions. Additionally, CypE's recently discovered role as a positive regulator in osteoblast differentiation indicates possible involvement in bone disorders . Dysregulation of CypE expression or activity could potentially impact bone formation and homeostasis, with implications for conditions such as osteoporosis or other metabolic bone diseases.

Research approaches to further investigate disease connections include:

• Expression analysis in patient samples compared to healthy controls

• Genetic association studies examining CypE variants in disease populations

• Functional studies of disease-associated variants or expression changes

• Development of animal models with altered CypE expression or activity

These investigations may reveal new therapeutic opportunities targeting CypE in specific disease contexts, particularly in cancers with aberrant KMT2A activity or bone disorders characterized by impaired osteoblast function .

• How do post-translational modifications affect Cyclophilin E function?

Post-translational modifications (PTMs) likely play crucial roles in regulating Cyclophilin E activity, localization, and protein interactions, though this area remains underexplored in the current literature. A systematic investigation of CypE PTMs would provide valuable insights into additional regulatory mechanisms controlling its diverse cellular functions.

Potential PTMs affecting CypE include phosphorylation, which may modulate enzymatic activity or protein-protein interactions; ubiquitination, affecting protein stability and turnover; SUMOylation, potentially influencing nuclear localization or transcriptional regulation; and acetylation, which could impact DNA/RNA binding properties or catalytic activity . The dynamic interplay between these modifications likely creates a complex regulatory network that fine-tunes CypE function in different cellular contexts.

A comprehensive methodological approach to characterize CypE PTMs includes:

Physiologically, PTMs may serve as molecular switches that regulate CypE in response to cellular signaling events, stress conditions, or developmental cues. For example, phosphorylation could alter CypE's ability to interact with splicing factors or transcriptional regulators, providing a mechanism to coordinate its activity with cell state . Similarly, modifications affecting subcellular localization could partition CypE between different cellular compartments to control access to substrates or binding partners.

Understanding the PTM landscape of CypE would not only reveal fundamental regulatory mechanisms but could also identify new therapeutic targets, as enzymes responsible for these modifications might provide alternative approaches to modulate CypE function in disease contexts .

• What emerging technologies and methodologies are advancing Cyclophilin E research?

The field of Cyclophilin E research stands to benefit significantly from several emerging technologies and methodological advances that address current limitations in understanding this multifunctional protein. Cryo-electron microscopy represents a transformative approach for visualizing CypE within larger macromolecular complexes like the spliceosome, potentially revealing how it interacts with RNA and protein partners in native contexts . This technique overcomes limitations of traditional structural biology methods by capturing dynamic assemblies in near-native states.

Chemical biology innovations are providing unprecedented tools for studying CypE:

• Activity-based probes that selectively label active CypE in complex cellular environments

• Photo-crosslinking amino acid analogs incorporated into CypE to capture transient interactions

• Bifunctional molecules that target both the PPIase domain and RNA-binding region for enhanced specificity

Advanced genomic engineering approaches include:

Computational advances are equally important, with machine learning algorithms now capable of predicting protein-protein interactions, potential substrates, and effects of mutations with increasing accuracy . Molecular dynamics simulations provide insights into conformational changes and substrate binding mechanisms that are difficult to capture experimentally.

Single-cell and spatial technologies offer new perspectives on CypE function in heterogeneous cell populations and tissues, potentially revealing cell type-specific roles that would be masked in bulk analyses. These technological advances collectively address the "scarcity of experimental data for cyclophilin isoforms" noted in the literature and promise to significantly accelerate progress in understanding CypE's diverse cellular functions .