Recombinant Pongo abelii Coiled-coil domain-containing protein 47 (CCDC47)

What is CCDC47 and what are its primary functions?

CCDC47 (Coiled-coil domain-containing protein 47) is a protein that serves several critical cellular functions. It is a component of the multi-pass translocon (MPT) complex that mediates insertion of multi-pass membrane proteins into the lipid bilayer of membranes. The MPT complex functions downstream of the SEC61 complex by occluding its lateral gate to promote insertion of subsequent transmembrane regions after the first few transmembrane segments are inserted . Within the MPT complex, CCDC47 is specifically part of the PAT subcomplex, which sequesters highly polar regions in transmembrane domains away from non-polar membrane environments until they can be buried in the fully assembled protein . Additionally, CCDC47 is involved in calcium ion homeostasis regulation in the endoplasmic reticulum and is required for proper protein degradation via the ERAD (ER-associated degradation) pathway . Notably, it plays an essential role in maintaining ER organization during embryogenesis, highlighting its developmental importance .

How does CCDC47 differ between humans and Pongo abelii?

CCDC47 is evolutionarily conserved across primates, but with species-specific variations. When comparing human and Pongo abelii (Sumatran orangutan) CCDC47, the proteins share significant homology in functional domains, especially in regions associated with membrane insertion and calcium homeostasis regulation. The conservation of these domains suggests their critical importance to protein function. While the core functional domains remain highly conserved, there are subtle sequence variations that may influence protein-protein interactions or regulatory mechanisms. These differences potentially reflect species-specific adaptations in endoplasmic reticulum function and protein folding pathways. Notably, variants in human CCDC47 have been associated with specific genetic disorders (Trichohepatoneurodevelopmental Syndrome and Atypical Choroid Plexus Papilloma) , suggesting that even minor variations in this protein can have significant physiological consequences. Comparative studies of human and orangutan CCDC47 can provide valuable insights into the evolution of endoplasmic reticulum organization and protein quality control mechanisms in primates.

What is the role of CCDC47 in the multi-pass translocon complex?

CCDC47 functions as a critical component of the multi-pass translocon (MPT) complex, contributing to the sophisticated mechanism of membrane protein insertion. The protein specifically facilitates the insertion of multi-pass membrane proteins after the SEC61 complex has initiated the process with the first few transmembrane segments . CCDC47's role involves occluding the lateral gate of the SEC61 complex, which prevents premature release of nascent proteins and ensures proper folding and insertion of subsequent transmembrane regions . Within the PAT subcomplex of the MPT, CCDC47 helps to sequester highly polar regions in transmembrane domains, protecting them from the non-polar membrane environment until they can be properly positioned within the interior of the fully assembled protein . This gate-keeping function represents a crucial quality control mechanism for complex membrane protein biogenesis.

Studies using mutagenesis approaches have demonstrated that alterations to CCDC47's interaction domains can significantly impair membrane protein insertion, leading to accumulation of misfolded proteins and activation of ER stress pathways. The temporal and spatial coordination between CCDC47 and other components of the MPT complex remains an active area of research, with implications for understanding various protein misfolding disorders and potential therapeutic interventions targeting this pathway.

How do mutations in CCDC47 contribute to disease pathology?

Mutations in CCDC47 have been associated with serious developmental and neurological disorders. Bi-allelic CCDC47 variants, particularly nonsense or frameshift variants, have been identified in patients with a disorder characterized by various symptoms including developmental delay . These pathogenic variants are predicted to lead to nonsense-mediated mRNA decay or premature truncation of the CCDC47 protein . The primary molecular mechanisms through which CCDC47 mutations cause disease include:

• Disruption of endoplasmic reticulum calcium homeostasis, leading to cellular stress

• Impaired membrane protein insertion, affecting multiple cellular pathways

• Dysfunction of the ERAD pathway, resulting in toxic accumulation of misfolded proteins

• Compromised ER organization during critical developmental periods

These mechanisms collectively contribute to the clinical manifestations observed in patients, including Trichohepatoneurodevelopmental Syndrome and Atypical Choroid Plexus Papilloma . The identification of disease-causing variants has expanded our understanding of CCDC47's essential role in human development and physiology. Research into genotype-phenotype correlations continues to reveal the spectrum of disorders associated with different types of CCDC47 mutations, highlighting the protein's pleiotropic effects on multiple developmental and cellular processes.

What is the relationship between CCDC47 and calcium homeostasis in the endoplasmic reticulum?

CCDC47 plays a crucial role in maintaining calcium ion homeostasis in the endoplasmic reticulum (ER), a function that impacts numerous cellular processes . The protein contains calcium-binding domains that facilitate its interaction with calcium ions and other calcium-regulatory proteins within the ER. Through these interactions, CCDC47 helps regulate calcium ion flux, storage, and release, contributing to the precise control of ER calcium levels necessary for optimal protein folding, processing, and secretion.

Disturbances in CCDC47 function can lead to dysregulation of ER calcium homeostasis, triggering ER stress responses and potentially contributing to calcium-dependent pathologies. Research has shown that:

• Depletion of CCDC47 increases cytosolic calcium levels

• CCDC47 interacts with key calcium-handling proteins including STIM1 and SERCA

• Calcium binding to CCDC47 modulates its interaction with the translocon complex

• Mutations in calcium-binding domains of CCDC47 disrupt protein folding quality control

These findings establish CCDC47 as an important regulator of calcium-dependent processes in the ER, with implications for understanding cellular stress responses and developing therapeutic strategies targeting calcium homeostasis in diseases characterized by ER dysfunction.

What are the optimal conditions for expressing and purifying recombinant Pongo abelii CCDC47?

The optimal expression and purification of recombinant Pongo abelii CCDC47 requires careful consideration of expression systems, tags, and purification strategies. Based on current protocols, the following approach yields high-quality protein:

Expression System:
E. coli has been successfully used for expressing recombinant CCDC47 , though mammalian expression systems like HEK293 cells may provide better post-translational modifications for functional studies .

Construct Design:

• Full-length mature protein (typically amino acids 21-483)

• N-terminal His-tag for purification

• Codon optimization for the expression host

Expression Conditions:

• For E. coli: Induction with 0.5-1 mM IPTG at OD600 of 0.6-0.8

• Temperature reduction to 18-25°C after induction

• Extended expression time (16-20 hours) at reduced temperature

Purification Protocol:

• Cell lysis in Tris/PBS-based buffer with protease inhibitors

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

• Size exclusion chromatography to remove aggregates

• Final buffer containing Tris/PBS, 6% Trehalose, pH 8.0

Storage Considerations:
The purified protein should be stored as a lyophilized powder or in solution with 50% glycerol at -20°C/-80°C . For working aliquots, storage at 4°C is suitable for up to one week, but repeated freeze-thaw cycles should be avoided .

Researchers should verify protein quality through SDS-PAGE (>90% purity) and functional assays appropriate to the specific research questions being addressed.

How can I validate the functional activity of recombinant CCDC47 in vitro?

Validating the functional activity of recombinant CCDC47 in vitro requires multiple complementary approaches focusing on its diverse cellular functions. Recommended validation methods include:

1. Membrane Insertion Assays:

• Reconstituted in vitro translation systems with radiolabeled substrate proteins

• Microsomes or liposomes to monitor insertion efficiency

• Crosslinking assays to detect interactions with SEC61 complex components

2. Calcium Binding Studies:

• Isothermal titration calorimetry (ITC) to determine calcium binding affinity

• Circular dichroism (CD) spectroscopy to assess calcium-induced conformational changes

• Calcium flux assays using fluorescent indicators in reconstituted systems

3. Protein-Protein Interaction Analyses:

• Pull-down assays with known interaction partners (SEC61 complex components)

• Surface plasmon resonance (SPR) to measure binding kinetics

• Proximity ligation assays in cellular systems

4. ER Stress Response Monitoring:

• Complementation assays in CCDC47-depleted cells

• Analysis of unfolded protein response (UPR) markers

• Measurement of ERAD pathway activity using model substrates

5. Structural Integrity Assessment:

A comprehensive validation approach should combine multiple methods, with careful attention to buffer conditions that preserve native protein conformation, particularly the inclusion of calcium ions at physiologically relevant concentrations where appropriate.

What analytical techniques are most effective for studying CCDC47 structure-function relationships?

Understanding CCDC47 structure-function relationships requires sophisticated analytical approaches spanning multiple scales of resolution. The most effective techniques include:

Structural Analysis Techniques:

• X-ray Crystallography: Provides atomic-level resolution of protein structure, especially valuable for the coiled-coil domains. Challenges include obtaining diffraction-quality crystals of membrane-associated proteins like CCDC47.

• Cryo-Electron Microscopy (Cryo-EM): Particularly suitable for visualizing CCDC47 in complex with other MPT components, offering insights into the native conformational states without crystallization.

• Nuclear Magnetic Resonance (NMR): Useful for analyzing dynamic regions and calcium-binding domains of CCDC47, though typically limited to smaller domains rather than the full-length protein.

Functional Mapping Approaches:

• Site-Directed Mutagenesis: Systematic alteration of key residues (particularly in the coiled-coil regions) followed by functional assays to establish structure-function correlations.

• Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS): Provides insights into protein dynamics and conformational changes upon binding to partners or calcium.

• Crosslinking Mass Spectrometry: Identifies interaction interfaces between CCDC47 and other components of the translocon complex.

Integration of Data:
Computational approaches, including molecular dynamics simulations and integrative modeling, are essential for combining data from multiple experimental techniques into coherent structural models that explain functional observations.

These complementary approaches collectively provide a comprehensive understanding of how CCDC47's structure relates to its multiple functions in membrane protein insertion, calcium homeostasis, and ER organization.

How can CCDC47 research contribute to understanding neurodevelopmental disorders?

CCDC47 research offers significant potential for advancing our understanding of neurodevelopmental disorders through multiple pathways. The protein's involvement in crucial cellular processes, particularly in the context of the developing nervous system, establishes it as a key player in neuronal function and development.

The identification of CCDC47 mutations in patients with Trichohepatoneurodevelopmental Syndrome provides a direct link between this protein and neurological manifestations . Research investigating CCDC47's function in neural cells reveals several mechanisms through which its dysfunction may contribute to neurodevelopmental disorders:

• Disruption of membrane protein trafficking: CCDC47's role in the multi-pass translocon complex is crucial for the correct insertion of neuronal membrane proteins, including ion channels and receptors essential for synaptic function.

• Altered calcium signaling: As a regulator of ER calcium homeostasis, CCDC47 dysfunction can disrupt calcium-dependent processes critical for neuronal development, axon guidance, and synapse formation.

• ER stress responses: Impaired CCDC47 function can trigger chronic ER stress, activating pathways that compromise neuronal viability and connectivity during critical developmental windows.

• Protein quality control defects: Through its role in the ERAD pathway, CCDC47 helps prevent accumulation of misfolded proteins that can be neurotoxic when allowed to aggregate.

Future research directions should include development of neural organoid models derived from patient cells with CCDC47 mutations, detailed electrophysiological characterization of neurons with altered CCDC47 function, and exploration of potential therapeutic strategies targeting ER stress pathways affected by CCDC47 dysfunction.

What are the emerging techniques for studying CCDC47 in cellular models?

Cutting-edge techniques are revolutionizing the study of CCDC47 in cellular contexts, providing unprecedented insights into its localization, dynamics, and function. These emerging approaches include:

Advanced Imaging Technologies:

• Super-resolution microscopy (STORM, PALM): Enables visualization of CCDC47 distribution within the ER at nanometer resolution, revealing microdomain organization not visible with conventional microscopy.

• Live-cell FRET sensors: Custom-designed sensors to monitor CCDC47 interactions with binding partners and conformational changes in real-time within living cells.

• Correlative light and electron microscopy (CLEM): Combines fluorescence imaging of tagged CCDC47 with ultrastructural analysis, providing context for its localization relative to ER subdomains.

Genome Engineering Approaches:

• CRISPR-Cas9 knock-in models: Generation of endogenously tagged CCDC47 allows visualization and analysis of the protein at physiological expression levels.

• Degron systems: Rapid, inducible degradation of CCDC47 to study acute loss-of-function effects without compensatory adaptations.

• Base editing technologies: Precise introduction of patient-specific point mutations to study pathogenic variants in isogenic cellular backgrounds.

Functional Genomics and Proteomics:

• Proximity labeling methods (BioID, APEX): Identification of the CCDC47 interactome in different cellular compartments and physiological states.

• Single-cell transcriptomics: Analysis of cell-type-specific responses to CCDC47 perturbation in complex tissues or organoids.

• Quantitative interaction proteomics: Mass spectrometry-based approaches to map dynamic changes in CCDC47 interactions during different cellular processes.

These technologies, particularly when used in combination, are transforming our ability to study CCDC47's roles in membrane protein insertion, calcium homeostasis, and ER organization in physiologically relevant cellular models.

What are the therapeutic implications of targeting CCDC47-related pathways?

The emerging understanding of CCDC47's functions opens several promising avenues for therapeutic intervention in disorders stemming from ER dysfunction and altered calcium homeostasis. These therapeutic strategies could target various aspects of CCDC47-related pathways:

Modulating ER Stress Responses:

• Chemical chaperones that mitigate ER stress in the absence of fully functional CCDC47

• Small molecules targeting specific branches of the unfolded protein response (UPR)

• Compounds that enhance remaining CCDC47 function in patients with partial loss-of-function mutations

Targeting Calcium Homeostasis:

• Calcium channel modulators that compensate for dysregulated ER calcium signaling

• Designer peptides that mimic CCDC47's calcium-binding domains

• Regulators of store-operated calcium entry (SOCE) to normalize cytosolic calcium levels

Enhancing Protein Quality Control:

• Activators of alternative ERAD pathways to compensate for CCDC47 dysfunction

• Proteostasis regulators that reduce the burden of misfolded proteins

• Targeted protein degradation approaches for aggregation-prone substrates

Gene Therapy Approaches:

• AAV-mediated delivery of functional CCDC47 for complete loss-of-function cases

• RNA therapeutics to modulate splicing or increase expression of remaining functional alleles

• CRISPR-based correction of specific pathogenic variants

These therapeutic directions are particularly relevant for neurodevelopmental disorders and other conditions associated with CCDC47 mutations. Preliminary research in cell and animal models suggests that early intervention targeting these pathways may prevent or mitigate the developmental consequences of CCDC47 dysfunction. As our understanding of CCDC47's regulation and interaction network continues to grow, additional therapeutic targets within these pathways are likely to emerge.

How can I overcome solubility issues when working with recombinant CCDC47?

Recombinant CCDC47 can present solubility challenges due to its membrane association and multiple domains. Researchers can implement several strategies to improve solubility:

Buffer Optimization:

• Include 6% Trehalose in storage buffer to enhance stability and prevent aggregation

• Maintain pH at 8.0 using Tris/PBS-based buffers

• Add low concentrations of non-ionic detergents (0.01-0.05% Triton X-100 or NP-40) to reduce hydrophobic interactions

• Include calcium (1-2 mM) to stabilize native conformation

Protein Engineering Approaches:

• Express soluble domains separately for domain-specific studies

• Create fusion constructs with solubility-enhancing partners (MBP, SUMO, Thioredoxin)

• Optimize the position and linker length of affinity tags

• Remove aggregation-prone regions identified through computational prediction

Expression Conditions:

• Lower induction temperature (16-18°C) to slow folding and improve solubility

• Co-express with molecular chaperones (GroEL/ES, DnaK system) in bacterial systems

• Consider mammalian expression systems for complex domains requiring specific modifications

Purification Strategies:

• Implement staged purification with gradually decreasing salt concentrations

• Use size exclusion chromatography as a final step to remove aggregates

• Consider on-column refolding for proteins recovered from inclusion bodies

• Add glycerol (up to 50%) to final storage buffer to prevent aggregation during storage

If persistent solubility issues occur, researchers should consider structural analysis of the problematic regions and possibly redesign constructs based on predicted domain boundaries and secondary structure elements.

What controls should be included in CCDC47 functional assays?

Robust experimental design for CCDC47 functional assays requires comprehensive controls to ensure valid and interpretable results. Essential controls include:

Positive Controls:

• Wild-type CCDC47 protein with confirmed activity

• Known interaction partners (SEC61 complex components) for binding assays

• Established CCDC47 substrates for membrane insertion assays

• Calcium ionophores (like ionomycin) for calcium homeostasis studies

Negative Controls:

• Heat-denatured CCDC47 protein to confirm specificity of observed activities

• CCDC47 with mutations in key functional domains (calcium-binding sites, coiled-coil regions)

• Empty vector or irrelevant protein controls for expression studies

• CCDC47-depleted systems (knockout or knockdown) to establish baseline signals

Specificity Controls:

• Competitive inhibition assays with known binding partners

• Dose-response relationships to demonstrate specificity

• Rescue experiments with wild-type CCDC47 in depleted systems

• Isoform-specific controls if multiple splice variants are present

Technical Controls:

• Loading controls for Western blots (housekeeping proteins)

• Calcium concentration controls for calcium-dependent assays

• Multiple independent biological replicates

• Validation with orthogonal methods for key findings

The specific combination of controls should be tailored to the particular assay being performed, with special attention to potential confounding factors like protein aggregation, tag interference with function, or endogenous CCDC47 expression in the experimental system.

How can I distinguish between direct and indirect effects of CCDC47 manipulation in cellular studies?

Distinguishing direct from indirect effects of CCDC47 manipulation represents a significant challenge in cellular studies due to the protein's involvement in multiple pathways. Researchers can employ several complementary strategies to address this challenge:

Temporal Analysis Approaches:

• Acute vs. chronic manipulations: Use inducible systems (Tet-On/Off, optogenetics, or chemical degrons) to achieve rapid CCDC47 depletion or activation, minimizing compensatory adaptations.

• Time-course experiments: Map the sequence of events following CCDC47 perturbation to identify primary (early) versus secondary (later) effects.

• Pulse-chase analysis: Track specific substrates through the secretory pathway to identify immediate consequences of CCDC47 dysfunction.

Rescue Experiments:

• Structure-function analysis: Rescue with wild-type versus domain-specific mutants of CCDC47 to link particular functions to observed phenotypes.

• Isoform-specific complementation: Determine which CCDC47 isoforms can rescue specific phenotypes.

• Heterologous protein rescue: Test whether orthologs from other species can substitute for specific functions.

Pathway Dissection:

• Epistasis analysis: Combine CCDC47 manipulation with perturbation of suspected downstream effectors to establish pathway relationships.

• Pharmacological interventions: Use specific inhibitors of calcium signaling, ER stress, or ERAD pathways to block potential indirect effects.

• Pathway reporters: Employ fluorescent or luminescent reporters for key cellular pathways to monitor their activation following CCDC47 manipulation.

Direct Interaction Validation:

• In vitro reconstitution: Recapitulate key biochemical activities with purified components.

• Proximity labeling in live cells: Use BioID or APEX2 fusions to identify proteins in direct physical proximity to CCDC47.

• FRET/BRET analysis: Monitor real-time interactions between CCDC47 and suspected direct partners.

By combining these approaches, researchers can build a comprehensive understanding of which cellular effects stem directly from CCDC47 function versus those arising from downstream signaling cascades or compensatory mechanisms.