Cyclophilin G Human

What is Cyclophilin G and what is its basic function in human cells?

Cyclophilin G (PPIG) is a member of the peptidyl-prolyl cis-trans isomerase (PPIase) family of proteins, also known as immunophilins. It catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides, which represents a critical rate-limiting step in protein folding. This enzymatic activity facilitates proper protein folding, transport, and assembly within cells .

Cyclophilin G is distinguished by its specific localization to nuclear speckles, which are nuclear compartments enriched with splicing factors. This localization is functionally significant as Cyclophilin G participates in the regulation of pre-mRNA splicing through interactions with splicing factors such as SC35 and pinin . Unlike other cyclophilins that may be more broadly distributed throughout cells, Cyclophilin G's nuclear speckle localization suggests a specialized role in RNA processing mechanisms.

Beyond its enzymatic function, Cyclophilin G is also known as Clk-associating RS-cyclophilin, CARS-cyclophilin, or SR-cyclophilin, reflecting its interactions with components of the splicing machinery . These alternative names highlight its functional connections to the SR (serine/arginine-rich) protein family, which are key regulators of pre-mRNA splicing.

How does Cyclophilin G relate to other members of the cyclophilin family?

Cyclophilin G is one of at least 18 identified human cyclophilins, each encoded by different genes but sharing a conserved cyclophilin-like domain (CLD). All cyclophilins contain this domain, which typically consists of 145-180 amino acid residues and houses the PPIase activity site . The CLD sequences across different cyclophilins are diverse, but they display high structural conservation, particularly in the regions forming the catalytic pocket .

Among the cyclophilin family, Cyclophilin G stands out for several characteristics:

As the table shows, Cyclophilin G is notably larger (88 kDa, 754 amino acids) than most other family members . This increased size suggests that Cyclophilin G likely contains additional functional domains beyond the conserved CLD that contribute to its specialized functions in pre-mRNA splicing.

What is the structural composition of human Cyclophilin G?

Human Cyclophilin G consists of 754 amino acids with a molecular weight of 88 kDa, making it one of the larger members of the cyclophilin family . Like all cyclophilins, it contains a cyclophilin-like domain (CLD) that houses the PPIase activity. This domain typically consists of 145-180 amino acid residues and is responsible for the protein's enzymatic function .

Based on structural studies of related cyclophilins, the CLD of Cyclophilin G likely adopts the characteristic cyclophilin fold consisting of an eight-stranded anti-parallel β-barrel with two α-helices enclosing the barrel . The catalytic site is located on one face of the molecule, forming a hydrophobic pocket that accommodates the proline residue of substrate proteins .

Commercially available recombinant Cyclophilin G is typically produced as a non-glycosylated polypeptide containing 195 amino acids (representing residues 1-175 of the protein, likely the catalytic domain) with a molecular mass of 21.6 kDa . For research purposes, this recombinant form is often fused to a 20 amino acid His-Tag at the N-terminus to facilitate purification .

The full-length protein likely contains additional domains beyond the CLD that mediate its specific interactions with splicing factors and other nuclear proteins, though the exact domain architecture is not fully characterized in the provided research data.

What cellular processes involve Cyclophilin G?

Cyclophilin G participates in several key cellular processes, with its primary roles centered around nuclear functions:

• Pre-mRNA Splicing Regulation: Cyclophilin G is localized to nuclear speckles, where it interacts with splicing factors SC35 and pinin, indicating a specialized role in the regulation of pre-mRNA splicing . This function distinguishes it from many other cyclophilins and appears to be central to its biological role.

• Protein Folding: Through its PPIase activity, Cyclophilin G catalyzes the cis-trans isomerization of proline peptide bonds, which is often a rate-limiting step in protein folding . This enzymatic function likely impacts the conformational dynamics of nuclear proteins involved in RNA processing.

• Protein Transport and Assembly: Research indicates that Cyclophilin G contributes to the proper transport and assembly of proteins within cells . This function may be particularly relevant for the assembly of splicing complexes in the nucleus.

• Nuclear Organization: Its specific localization to nuclear speckles suggests a role in maintaining the organization and dynamics of these subnuclear structures, which are hubs for RNA processing factors .

The mechanisms through which Cyclophilin G influences these processes likely involve both its enzymatic PPIase activity, potentially altering the conformation of substrate proteins, and protein-protein interactions mediated by regions outside its catalytic domain.

How can researchers effectively distinguish between direct and indirect effects of Cyclophilin G?

Distinguishing between direct and indirect effects of Cyclophilin G requires carefully designed experimental approaches that isolate its immediate activities from downstream consequences:

• Enzyme Activity Separation Studies: Generate catalytically inactive mutants of Cyclophilin G by introducing point mutations in critical residues of the PPIase domain while preserving protein structure . Compare the effects of wild-type and inactive Cyclophilin G to determine which cellular processes depend specifically on its enzymatic activity versus potential scaffolding functions.

• Time-Course Analysis: Implement acute manipulation systems (such as rapid protein degradation or chemical inhibition) and monitor cellular responses at multiple time points. Direct effects typically manifest shortly after Cyclophilin G manipulation, while indirect effects emerge later in the response cascade .

• Substrate Trapping Approaches: Employ modified versions of Cyclophilin G that can bind but not release substrates, effectively "trapping" direct interaction partners. These approaches can utilize mutations in the catalytic site that permit substrate binding but inhibit catalytic turnover, followed by proteomic identification of trapped proteins .

• In Vitro Reconstitution: Purify Cyclophilin G and candidate substrate proteins, then perform direct enzymatic assays in a defined biochemical system. This approach eliminates cellular complexity and can definitively establish direct enzymatic relationships .

• Domain-Specific Manipulations: Create constructs expressing only specific domains of Cyclophilin G and test which regions are sufficient to produce particular cellular effects. This approach helps map functions to specific protein regions and distinguishes between catalytic and non-catalytic activities .

By systematically applying these approaches, researchers can build a comprehensive picture of Cyclophilin G's direct actions versus secondary consequences, clarifying its precise mechanistic roles in cellular processes.

What experimental approaches are most effective for studying Cyclophilin G's PPIase activity?

Several experimental approaches can be employed to study Cyclophilin G's PPIase activity, each with distinct advantages:

• NMR-Based Assay (1H/1H TOCSY): This protease-free approach provides direct measurement of both substrate binding and catalytic activity . The method uses tetrapeptide substrates (such as AAPF, AFPF, or AGPF) and monitors the cis-trans isomerization through two-dimensional NMR experiments. Key advantages include:

  • Detection of both binding affinity and catalytic turnover

  • No requirement for substrate chemical modifications

  • Ability to measure effects of amino acid substitutions distal to the target proline

  • High sensitivity for substrate binding detection

• Chymotrypsin-Coupled Assay: This traditional approach measures PPIase activity indirectly:

  • Uses peptide substrates containing a chymotrypsin cleavage site

  • The protease cleaves only the trans isomer of the substrate

  • PPIase activity is measured by increased cleavage rate due to acceleration of cis-to-trans conversion

  • Important limitation: Only detects catalysis, not binding

• Thermal Shift Assay: While primarily used for measuring ligand binding (e.g., cyclosporin binding), this approach can indirectly assess functional properties by monitoring changes in protein thermal stability upon ligand binding .

• Computational Approaches: In silico methods based on family-wide structural analysis can characterize molecular features and predict substrate specificity, which is particularly useful for comparative analysis across cyclophilin family members .

For comprehensive characterization of Cyclophilin G's PPIase activity, researchers should consider combining multiple approaches. The NMR-based assay provides the most direct and informative data on both binding and catalytic function, while other methods offer complementary insights into specific aspects of enzyme function.

What are the key considerations when designing inhibitor studies targeting Cyclophilin G?

Designing effective inhibitor studies for Cyclophilin G requires addressing several critical considerations:

• Selectivity Challenges: The cyclophilin family shares significant structural conservation in the PPIase domain, making selective inhibition challenging. Research indicates that "the currently available small-molecule and peptide-based ligands for this class of enzyme are insufficient for isoform specificity" . To overcome this limitation, inhibitor design should target "regions of the isomerase domain outside the proline-binding surface [that] impart isoform specificity" , particularly the identified S2 binding position that shows diversity among cyclophilins.

• Comprehensive Testing Strategy: A robust evaluation pipeline should include:

  • Thermal shift assays to measure binding affinity

  • NMR-based assays to assess both binding and inhibition of catalytic activity

  • Cell-based assays to evaluate effects on splicing when targeting Cyclophilin G

  • Multiple cyclophilin family members as controls to assess inhibitor selectivity

• Structure-Based Design: Utilize available crystallographic structures of cyclophilin isoforms for comparative analysis and computational simulations to predict binding modes and optimize inhibitor selectivity . Consider fragment-based approaches targeting unique regions of Cyclophilin G rather than the highly conserved active site.

• Biological Validation: Assess inhibitor effects specifically on Cyclophilin G's role in splicing regulation, evaluate nuclear localization of inhibitors, and confirm that observed effects result from specific Cyclophilin G inhibition rather than general PPIase inhibition .

By focusing on unique structural features and implementing a comprehensive testing cascade, researchers can develop and validate selective Cyclophilin G inhibitors despite the challenges posed by the high conservation within the cyclophilin family.

How can researchers effectively isolate and purify Cyclophilin G for experimental studies?

Isolation and purification of Cyclophilin G for experimental studies typically involves recombinant protein expression systems, as extracting native Cyclophilin G from human tissues in sufficient quantities presents significant challenges:

• Expression System Selection: E. coli has been successfully used as an expression system for human Cyclophilin G . This bacterial system allows for high-yield production, though it lacks post-translational modifications that might be present in native Cyclophilin G.

• Construct Design Considerations:

  • Focus on the cyclophilin domain (approximately residues 1-175) rather than the full 754-amino acid protein for higher solubility and stability

  • Add an affinity tag (such as a His-tag) at the N-terminus to facilitate purification

  • Optimize codon usage for the expression system

• Purification Protocol:

  • Begin with affinity chromatography using metal chelation for His-tagged constructs

  • Apply ion exchange chromatography as a secondary purification step

  • Finish with size exclusion chromatography for final polishing and buffer exchange

  • Aim for purity greater than 95% as determined by SDS-PAGE

• Storage Considerations: Based on product information, purified Cyclophilin G should be stored:

  • At 4°C if used within 2-4 weeks

  • At -20°C for longer-term storage

  • With the addition of a carrier protein (0.1% HSA or BSA) for extended stability

  • In buffer containing 20mM Tris-HCl pH-8, 1mM DTT, and 10% glycerol

  • With minimal freeze-thaw cycles to preserve activity

This approach allows researchers to obtain pure, active Cyclophilin G suitable for biochemical, structural, and functional studies. The recombinant protein typically appears as a sterile filtered colorless solution with high purity suitable for experimental applications .

How does Cyclophilin G's enzymatic activity compare with other cyclophilins?

Comparing Cyclophilin G's enzymatic activity with other cyclophilins reveals both shared mechanisms and unique characteristics:

• Substrate Specificity Determinants: While all cyclophilins share the basic PPIase function, structural analysis suggests that "regions of the isomerase domain outside the proline-binding surface impart isoform specificity" . Research identifies "a well-defined molecular surface corresponding to the substrate-binding S2 position that is a site of diversity in the cyclophilin family" . This region likely contributes to Cyclophilin G's unique substrate preferences.

• Tetrapeptide Substrate Utilization: In systematic studies of cyclophilin activity, multiple cyclophilin isoforms including PPIG (Cyclophilin G) showed the ability to bind and catalyze isomerization of standard tetrapeptide substrates such as AAPF, AFPF, and AGPF . This indicates a shared basic catalytic mechanism across family members.

• Correlation Between Activities: Research has demonstrated "a strict correlation between the ability to bind cyclosporin and activity against the tetrapeptide substrates" . This relationship appears consistent across the cyclophilin family, suggesting that the basic catalytic mechanism is conserved despite variations in substrate preferences.

• Structural Conservation vs. Functional Diversity: Despite having "very similar secondary structural elements" across the cyclophilin family, functional studies suggest that Cyclophilin G has evolved specialized roles, particularly in nuclear processes and pre-mRNA splicing, distinguishing it functionally from other family members.

• Size and Domain Contributions: With 754 amino acids and 88 kDa molecular weight, Cyclophilin G is substantially larger than many other cyclophilins (e.g., CypA at 18 kDa) , suggesting it may have additional functional domains that influence its enzymatic behavior and substrate selection.

This combination of shared catalytic mechanisms with distinctive structural features likely underlies Cyclophilin G's specialized roles in nuclear processes, particularly in pre-mRNA splicing regulation.

How is Cyclophilin G involved in pre-mRNA splicing?

Cyclophilin G plays a specialized role in pre-mRNA splicing through several key mechanisms:

• Nuclear Speckle Localization: Cyclophilin G is distinctly localized to nuclear speckles, which are nuclear domains enriched in pre-mRNA splicing factors . This specific localization pattern suggests a dedicated function in splicing processes rather than broader cellular roles.

• Direct Interaction with Splicing Factors: Research demonstrates that Cyclophilin G interacts with essential splicing components, specifically the splicing factors SC35 (SRSF2) and pinin . These interactions suggest that Cyclophilin G may affect the function, conformation, or localization of these critical splicing regulators.

• Conformational Modulation of Splicing Proteins: Through its PPIase activity, Cyclophilin G likely catalyzes proline isomerization in splicing-related proteins . This enzymatic activity can serve as a regulatory mechanism by altering protein conformations, which in turn affects protein-protein interactions or functional activities within the splicing machinery.

• Pre-mRNA Processing Regulation: The alternative name "SR-cyclophilin" indicates a functional relationship with SR (serine/arginine-rich) proteins, which are major regulators of constitutive and alternative splicing . This suggests Cyclophilin G may influence splice site selection and exon inclusion/exclusion decisions.

The combination of Cyclophilin G's enzymatic activity and its specific interactions with splicing machinery components positions it as a potential regulatory factor in pre-mRNA processing, possibly affecting both constitutive splicing efficiency and alternative splicing outcomes.

What experimental models are most suitable for studying Cyclophilin G's physiological functions?

Selecting appropriate experimental models is crucial for investigating Cyclophilin G's physiological functions, particularly in relation to its role in splicing:

• Cell Line Selection:

  • Human cell lines with robust splicing activity (HeLa, HEK293) maintain the native context for Cyclophilin G function

  • Neuronal cell lines (SH-SY5Y) or stem cell models with complex splicing patterns allow investigation of Cyclophilin G's role in regulating tissue-specific alternative splicing

  • Inducible expression systems (Tet-On/Tet-Off) provide temporal control for analyzing Cyclophilin G's effects

• Genetic Manipulation Approaches:

  • CRISPR/Cas9 genome editing to generate knockout cell lines or introduce tagged versions of Cyclophilin G at endogenous loci

  • Creation of point mutations in the PPIase domain to distinguish enzymatic from scaffolding functions

  • RNA interference (siRNA/shRNA) for temporal studies of Cyclophilin G depletion

  • Comparison of full-length vs. domain-specific constructs to map functional regions

• Functional Assays:

  • In vitro splicing assays using nuclear extracts to directly assess Cyclophilin G's effect on splicing efficiency

  • RNA-Seq analysis to evaluate global changes in splicing patterns following Cyclophilin G manipulation

  • Minigene reporter assays to study effects on specific splicing events

  • Live cell imaging with fluorescently tagged Cyclophilin G to study dynamics within nuclear speckles

• Biochemical Approaches:

  • Co-immunoprecipitation to identify interaction partners in splicing complexes

  • Nuclear speckle isolation followed by proteomic analysis

  • In vitro reconstitution of splicing reactions with purified components

The most productive research strategy would combine multiple model systems, allowing complementary insights into Cyclophilin G's physiological functions while controlling for potential artifacts or limitations inherent to any single experimental approach.

What are the challenges in crystallizing Cyclophilin G for structural studies?

Crystallizing Cyclophilin G for structural studies presents several technical challenges that researchers must address:

• Size and Complexity Barriers: With 754 amino acids and 88 kDa molecular weight, full-length Cyclophilin G is significantly larger than other successfully crystallized cyclophilins (e.g., CypA at 18 kDa) . This large size likely incorporates multiple domains with potential flexible linkers that can impede crystal formation. The most practical approach involves focusing on crystallizing the catalytic domain (approximately 20 kDa) rather than the full-length protein .

• Expression and Purification Hurdles: Larger proteins often face solubility challenges when overexpressed. Researchers should:

  • Screen multiple expression systems (bacterial, insect, mammalian)

  • Optimize buffer conditions to enhance solubility

  • Employ multi-step chromatography including affinity, ion exchange, and size exclusion steps to achieve the homogeneity required for crystallization

• Crystallization Optimization Requirements: Extensive screening of crystallization conditions is necessary, including:

  • High-throughput screening of thousands of conditions varying precipitants, pH, salt concentration, and temperature

  • Co-crystallization with binding partners (cyclosporin derivatives or interaction partners like SC35 fragments) to stabilize the protein

  • Surface engineering to reduce entropy and promote crystal contacts

• Alternative Structural Approaches: If crystallization proves exceptionally difficult, researchers can consider:

  • Cryo-electron microscopy, which doesn't require crystals and can handle conformational heterogeneity

  • NMR spectroscopy for the isolated catalytic domain (~20 kDa)

  • Integrative structural biology combining low-resolution techniques with computational modeling

Based on research showing successful crystallization of seven human cyclophilin domains , the catalytic domain of Cyclophilin G should be amenable to crystallization with proper optimization, even if the full-length protein proves challenging.

How does Cyclophilin G differ functionally from other cyclophilins in terms of substrate specificity?

Understanding Cyclophilin G's substrate specificity compared to other cyclophilins requires examining both structural features and biochemical properties:

• Structural Determinants of Specificity: While all cyclophilins share a conserved cyclophilin-like domain (CLD), research identifies important variations that influence substrate recognition:

  • "Regions of the isomerase domain outside the proline-binding surface impart isoform specificity"

  • There exists "a well-defined molecular surface corresponding to the substrate-binding S2 position that is a site of diversity in the cyclophilin family"

  • The extended active site that interacts with residues flanking the target proline varies among cyclophilins, likely contributing to Cyclophilin G's unique substrate preferences

• Cellular Context Specialization: Cyclophilin G's specific localization to nuclear speckles (unlike other cyclophilins) suggests it has evolved to target substrates relevant to pre-mRNA processing and splicing . This spatial restriction likely narrows its physiological substrate range compared to more broadly distributed cyclophilins.

• Size and Domain Architecture Influence: At 88 kDa, Cyclophilin G is substantially larger than many other cyclophilins, suggesting additional domains that may influence substrate selection through extended binding surfaces or allosteric mechanisms .

• Specific Protein Interactions: Cyclophilin G's documented interactions with splicing factors SC35 and pinin indicate it may preferentially target substrates involved in the splicing machinery , distinguishing it from other cyclophilins with different interaction profiles.

To experimentally determine Cyclophilin G's substrate specificity compared to other cyclophilins, researchers should employ:

• Peptide library screening with varied amino acid compositions flanking the proline residue

• Direct comparative binding and activity measurements across cyclophilin isoforms

• Structural analysis of substrate complexes

• Computational modeling of substrate interactions

These combined approaches can elucidate the molecular basis for Cyclophilin G's unique substrate preferences and functional specialization.

What methodological approaches can be used to study Cyclophilin G in disease contexts?

Studying Cyclophilin G in disease contexts requires methodological approaches that connect its molecular functions to pathological processes:

• Expression Analysis in Disease:

  • Tissue microarray analysis to examine Cyclophilin G expression across normal and disease tissues

  • Transcriptomic profiling to analyze PPIG mRNA expression in disease datasets (TCGA, GEO)

  • Single-cell analysis to examine cell type-specific expression in heterogeneous disease tissues

• Genetic Association Studies:

  • Analysis of PPIG genetic variants through next-generation sequencing of patient cohorts

  • Examination of genome-wide association studies for disease associations at the PPIG locus (2q31.1)

  • Focus on splicing-related diseases given Cyclophilin G's role in pre-mRNA processing

• Functional Disease Models:

  • Patient-derived cell models (primary cells or iPSCs) from individuals with relevant disorders

  • Disease-specific cellular stress models to determine if Cyclophilin G modifies responses to pathological conditions

  • Comparison of Cyclophilin G function and splicing patterns between patient and control cells

• Splicing Analysis in Disease Contexts:

  • RNA-seq with specialized analysis for splicing events to identify disease-specific patterns potentially regulated by Cyclophilin G

  • Minigene assays with disease-associated variants to test how Cyclophilin G manipulation affects their splicing

  • Direct measurement of Cyclophilin G's impact on disease-associated splicing defects

• Therapeutic Targeting Approaches:

  • Small molecule inhibitor screening with a focus on isoform selectivity

  • Development of methods to modulate Cyclophilin G expression or activity in disease contexts

  • Targeting of Cyclophilin G-dependent splicing events relevant to disease pathology

By implementing these methodological approaches, researchers can establish connections between Cyclophilin G's molecular functions and disease mechanisms, potentially identifying new therapeutic opportunities.

How does Cyclophilin G interact with other splicing factors at the molecular level?

Understanding the molecular interactions between Cyclophilin G and splicing factors reveals several key mechanisms:

• Characterized Interaction Partners: Research specifically identifies two splicing factors that interact with Cyclophilin G:

  • SC35 (SRSF2): A serine/arginine-rich splicing factor essential for spliceosome assembly

  • Pinin: A nuclear speckle-associated protein involved in mRNA splicing and export

• Interaction Mechanisms:

  • PPIase-Mediated Regulation: Cyclophilin G likely recognizes and isomerizes specific proline residues in splicing factors, causing conformational changes that alter their activity, interactions, or localization . This enzymatic activity represents a primary mode of action consistent with the fundamental function of cyclophilins.

  • Domain-Specific Interactions: Alternative names like "Clk-associating RS-cyclophilin" and "SR-cyclophilin" suggest specialized interactions with RS domain-containing proteins . RS domains are characteristic of many splicing factors and mediate protein-protein interactions within the spliceosome.

  • Nuclear Speckle Co-localization: Cyclophilin G's specific localization to nuclear speckles places it in close proximity to the splicing machinery, facilitating interactions with resident splicing factors .

• Functional Consequences:

  • Regulation of Splicing Factor Activity: Isomerization of proline residues in splicing factors could serve as a molecular switch that alters RNA binding capacity, protein-protein interactions, or catalytic activities .

  • Spliceosome Assembly Modulation: Interactions with SC35 and pinin suggest Cyclophilin G may influence the dynamic assembly and disassembly of spliceosomes .

  • Integration of Signaling and Splicing: The association with Clk (CDC-like kinase) indicated by one of its alternative names suggests Cyclophilin G might function at the interface between cellular signaling pathways and the splicing machinery .

To fully elucidate these interactions at the molecular level, additional structural studies, detailed domain mapping, and functional assays investigating how Cyclophilin G's enzymatic activity affects splicing factor function would be necessary. The current data provides a foundation for hypothesizing interaction mechanisms, but further research is needed to determine precise molecular details.