Recombinant Macaca fascicularis Coiled-coil domain-containing protein 47 (CCDC47)

What is CCDC47 and what are its structural characteristics in Macaca fascicularis?

CCDC47, also known as calumin, is a calcium-binding endoplasmic reticulum (ER) transmembrane protein found in Macaca fascicularis (cynomolgus macaque). Structurally, CCDC47 contains a globular N-terminal domain and a long, flexible C-terminal coiled-coil extension . The protein's architecture includes a conserved and positively charged coiled-coil that positions between Sec61 and rRNA H24 in the ribosomal complex, with the coiled-coil terminating near the mouth of the exit tunnel . The C-terminal region of CCDC47 forms significant contacts with ribosomal proteins including eL6 and rRNA H25, suggesting its important role in ribosome association during protein synthesis .

What is the primary function of CCDC47 in Macaca fascicularis cells?

CCDC47 functions as a critical component of an ER translocon complex that facilitates multi-pass membrane protein biogenesis . In this capacity, CCDC47 works alongside other proteins including TMCO1 and the Nicalin-TMEM147-NOMO complex to form a specialized machinery for membrane protein synthesis and integration . Additionally, CCDC47 plays a significant role in calcium signaling and homeostasis within the ER, binding calcium with low affinity but high capacity . This function is essential for various cellular processes including development, as evidenced by the embryonic lethality observed in CCDC47 knockout mouse models .

How conserved is CCDC47 across primate species, particularly between Macaca fascicularis and humans?

CCDC47 shows significant conservation across primate species, including between Macaca fascicularis and humans. While specific sequence homology percentages are not explicitly stated in the provided search results, the functional implications of CCDC47 mutations in humans and the protein's structural characterization in both species suggest high conservation . The C-terminal coiled-coil domain appears particularly conserved, as truncating this conserved motif causes developmental disorders in humans, indicating functional importance across primate species . Genomic studies across Macaca species have identified various genes with copy number variations, but CCDC47 conservation likely reflects its essential cellular functions in calcium homeostasis and protein biogenesis .

What expression patterns does CCDC47 exhibit in different tissues of Macaca fascicularis?

While the search results don't provide specific tissue expression profiles for CCDC47 in Macaca fascicularis, the protein's functional implications suggest widespread expression in tissues dependent on calcium signaling and active protein synthesis. Based on human data and other macaque studies, CCDC47 would likely be expressed in developing neural tissues, cardiac tissues, and other organs with high secretory activity . The embryonic lethality observed in mouse knockout models suggests essential expression during early development stages . For precise tissue-specific expression patterns in Macaca fascicularis, researchers would need to conduct targeted studies using immunohistochemistry, RNAscope, or tissue-specific transcriptomics.

How does the structural configuration of CCDC47 in Macaca fascicularis influence its role in the ER translocon complex?

The structural configuration of CCDC47 directly impacts its functionality within the ER translocon complex. Cryo-EM analysis reveals that CCDC47's globular domain and elongated C-terminal coiled-coil form specific interactions with both the ribosome and other translocon components . The globular domain of CCDC47 makes contact with ribosomal protein eL6 and rRNA H25, while the conserved positively charged coiled-coil wedges between Sec61 and rRNA H24 . This positioning allows CCDC47 to extend approximately 90 x 120 x 140 Å alongside Sec61 and its accessory factors near the ribosome exit tunnel .

These structural features enable CCDC47 to facilitate nascent chain emergence from the ribosome exit tunnel, as its cytosolic domains are positioned to interact with the newly synthesized proteins . The luminal Nicalin domain is positioned near translocated segments of the nascent chain, further supporting CCDC47's role in coordinating multi-pass membrane protein integration . This structural arrangement creates a specialized environment for the synthesis and folding of complex membrane proteins, distinguishing the CCDC47-containing translocon from other ER translocation machinery.

What methodologies are recommended for studying CCDC47 calcium-binding properties in Macaca fascicularis cell models?

To investigate the calcium-binding properties of CCDC47 in Macaca fascicularis cell models, researchers should employ a combination of complementary approaches:

• Calcium Imaging Techniques: Utilize fluorescent calcium indicators (e.g., Fura-2, Fluo-4) in CCDC47-manipulated cell lines to measure real-time changes in intracellular calcium concentrations following various stimuli .

• ER Calcium Storage Assays: Implement specific assays to measure total ER calcium storage capacity, which has been shown to decrease in cells with damaging CCDC47 alleles .

• IP3R-mediated Calcium Release Measurements: Given that CCDC47 mutations impair calcium signaling mediated by the IP3R calcium release channel, researchers should conduct specific measurements of IP3R activity using selective agonists .

• Store-operated Calcium Entry (SOCE) Assessment: Examine SOCE mechanisms, as CCDC47 deficiency has been linked to reduced ER calcium refilling via this pathway .

• Purified Protein Binding Studies: For direct measurement of calcium-binding properties, express and purify recombinant Macaca fascicularis CCDC47 for in vitro calcium binding assays, including isothermal titration calorimetry or calcium overlay assays.

These methodologies should be applied in both wild-type cells and those with CCDC47 knockdown or overexpression to comprehensively characterize the protein's role in calcium homeostasis.

What are the challenges in expressing functional recombinant CCDC47 from Macaca fascicularis in heterologous systems?

Expressing functional recombinant Macaca fascicularis CCDC47 in heterologous systems presents several significant challenges:

• Structural Complexity: The protein's structure includes both membrane-spanning domains and an extended coiled-coil region, making complete and correctly folded expression difficult in bacterial systems .

• Post-translational Modifications: Any species-specific post-translational modifications necessary for CCDC47 function may be absent in heterologous systems, particularly bacterial expression platforms.

• Protein-Protein Interactions: CCDC47 functions as part of a multi-protein complex with TMCO1 and the Nicalin-TMEM147-NOMO complex . These interaction partners may be necessary for proper folding and function but absent in heterologous systems.

• Membrane Integration: As an ER transmembrane protein, CCDC47 requires proper membrane targeting and integration machinery, which may differ between expression systems and Macaca fascicularis cells.

• Calcium Binding Properties: Maintaining the calcium-binding properties of CCDC47 in recombinant systems requires appropriate buffer conditions and calcium concentrations that mimic the ER environment .

To address these challenges, researchers might consider mammalian expression systems closely related to primates, the use of microsomal preparations to provide appropriate membrane environments, or co-expression with known interaction partners to facilitate proper complex formation.

How does CCDC47's role differ between developing and adult tissues in Macaca fascicularis?

While the search results don't provide direct comparative data between developing and adult tissues in Macaca fascicularis, evidence from knockout studies and human disorders suggests significant developmental roles for CCDC47:

• Developmental Requirement: The embryonic lethality observed in Ccdc47-knockout mice indicates an essential role during early development that cannot be compensated by other proteins . This suggests particularly critical functions in developing tissues.

• Neurodevelopmental Implications: Human patients with bi-allelic CCDC47 variants display global developmental delay and hypotonia, suggesting important roles in neural development . By extension, CCDC47 likely serves similar neurodevelopmental functions in Macaca fascicularis.

• Tissue Differentiation: The developmental disorders associated with CCDC47 mutations affect multiple systems (hair, liver, muscle tone), indicating roles in tissue differentiation across various lineages .

• Adult Maintenance Functions: In adult tissues, CCDC47 likely transitions to maintenance roles in ER calcium homeostasis and continued production of multi-pass membrane proteins necessary for tissue function .

• Stress Response: Adult tissues may require CCDC47 for appropriate ER stress responses and calcium signaling during environmental challenges, whereas developmental roles may be more focused on tissue formation and differentiation.

Further research specifically tracking CCDC47 expression and function throughout Macaca fascicularis development would be necessary to fully characterize these differences.

##.3. Experimental Methodology FAQs

What are the optimal conditions for extracting and purifying native CCDC47 from Macaca fascicularis tissues?

For optimal extraction and purification of native CCDC47 from Macaca fascicularis tissues, researchers should follow this methodological approach:

• Tissue Selection: Choose tissues with high ER content such as liver, pancreas, or cultured fibroblasts from Macaca fascicularis. Fresh tissue samples should be immediately processed or flash-frozen in liquid nitrogen.

• Homogenization Buffer: Use a buffer containing:

  • 50 mM Tris-HCl (pH 7.4)

  • 150 mM NaCl

  • 1 mM EDTA

  • 1% Digitonin or 1% DDM (n-Dodecyl-β-D-maltoside)

  • Protease inhibitor cocktail

  • 1 mM CaCl₂ (to maintain calcium-binding properties)

• Membrane Fraction Isolation: Perform differential centrifugation to isolate ER-enriched membrane fractions before detergent solubilization.

• Affinity Purification Strategy:

  • Generate antibodies specific to Macaca fascicularis CCDC47 for immunoprecipitation

  • Alternatively, use ribosome isolation techniques followed by detergent extraction, as CCDC47 associates with ribosomes

• Complex Preservation: Consider mild crosslinking with low concentrations of formaldehyde (0.1-0.5%) before extraction to preserve native protein complexes for subsequent analysis .

• Quality Control: Verify purified CCDC47 integrity using Western blotting, mass spectrometry, and functional calcium-binding assays.

This approach should yield relatively pure native CCDC47, potentially in complex with its natural binding partners from the translocon complex, suitable for subsequent structural and functional analyses.

What cellular assays can be used to evaluate CCDC47 function in Macaca fascicularis cell cultures?

Several specialized cellular assays can effectively evaluate CCDC47 function in Macaca fascicularis cell cultures:

• ER Calcium Homeostasis Assays:

  • Measure ER calcium content using genetically encoded ER-targeted calcium indicators (e.g., ER-GCaMP)

  • Assess store-operated calcium entry (SOCE) using paired thapsigargin stimulation and calcium readdition protocols

  • Quantify IP3R-mediated calcium release using specific agonists such as ATP or carbachol

• Membrane Protein Biogenesis Assays:

  • Monitor integration efficiency of reporter multi-pass membrane proteins

  • Assess membrane protein folding using protease protection assays

  • Measure the kinetics of membrane protein synthesis and maturation using pulse-chase experiments

• CCDC47 Knockdown/Knockout Studies:

  • Generate CRISPR/Cas9-mediated CCDC47 knockout or knockdown in Macaca fascicularis cells

  • Compare protein synthesis rates of multi-pass membrane proteins versus soluble or single-pass proteins

  • Evaluate ER stress markers (XBP1 splicing, ATF6 cleavage, BiP upregulation)

• Protein-Protein Interaction Studies:

  • Perform proximity ligation assays to detect interactions with known partners (TMCO1, Nicalin-TMEM147-NOMO complex)

  • Implement FRET-based approaches to quantify dynamic interactions in living cells

• Ribosome Association Assays:

  • Conduct polysome profiling with CCDC47 immunoblotting

  • Perform ribosome footprinting to assess translation effects

  • Use cryo-EM to visualize CCDC47-ribosome complexes in native context

These assays provide complementary information about CCDC47's dual roles in calcium homeostasis and membrane protein biogenesis, yielding comprehensive functional characterization.

What is the recommended protocol for generating recombinant Macaca fascicularis CCDC47 for structural studies?

For generating recombinant Macaca fascicularis CCDC47 suitable for structural studies, researchers should follow this optimized protocol:

• Construct Design:

  • Consider separate expression of domains: N-terminal globular domain (residues 1-X) and the C-terminal coiled-coil domain (residues X-C-terminus) based on structural predictions

  • Include a cleavable purification tag (His6, GST, or MBP) that improves solubility

  • Optimize codon usage for the chosen expression system

  • Include TEV protease cleavage site between tag and protein

• Expression System Selection:

  • For full-length protein: Insect cell expression (Sf9 or Hi5) using baculovirus

  • For soluble domains: E. coli (BL21(DE3) or Rosetta) at reduced temperature (18°C)

  • Alternative: HEK293 mammalian expression for complex post-translational modifications

• Membrane Protein Considerations:

  • Include stabilizing mutations if needed

  • Consider fluorescence-based thermostability assays to identify optimal detergent conditions

  • Test various detergents: DDM, LMNG, GDN for extraction

• Purification Strategy:

  • Two-step affinity chromatography using tag

  • Size-exclusion chromatography in appropriate detergent

  • Consider amphipol or nanodisc reconstitution for increased stability

• Structural Analysis Preparation:

  • For X-ray crystallography: Screen crystallization conditions with LCP (Lipidic Cubic Phase) approach

  • For Cryo-EM: Apply to freshly glow-discharged grids with optimized blotting conditions

  • For NMR studies: Isotope labeling (¹⁵N, ¹³C) in minimal media

• Quality Control Checkpoints:

  • SEC-MALS for oligomeric state assessment

  • Thermal shift assays for stability

  • Negative stain EM for sample homogeneity

  • Mass spectrometry for sequence verification and PTM identification

This systematic approach addresses the challenges of membrane protein production while maximizing the likelihood of obtaining correctly folded protein suitable for high-resolution structural studies.

How can researchers effectively study CCDC47 interactions with the ribosome in Macaca fascicularis?

To effectively study CCDC47 interactions with the ribosome in Macaca fascicularis, researchers should implement the following methodological approaches:

• Ribosome Profiling and Associated Protein Analysis:

  • Isolate cytosolic ribosomes through differential centrifugation

  • Perform sucrose gradient fractionation to separate polysomes, monosomes, and ribosomal subunits

  • Analyze fractions by Western blotting for CCDC47 co-sedimentation

  • Implement mass spectrometry to identify additional proteins in CCDC47-positive fractions

• Cross-linking Mass Spectrometry (XL-MS):

  • Apply chemical cross-linkers (DSS, BS3, or formaldehyde) to intact cells or isolated ribosomes

  • Isolate CCDC47-ribosome complexes via immunoprecipitation

  • Perform LC-MS/MS analysis of cross-linked peptides

  • Use software such as pLink, XlinkX, or MeroX for cross-link identification

  • Apply the linear support vector machine (SVM) model to classify cross-link spectral matches as described in the literature

• Cryo-Electron Microscopy:

  • Isolate native CCDC47-ribosome complexes from Macaca fascicularis cells

  • Apply to cryo-EM grids with optimization of blotting conditions

  • Collect high-resolution data and perform 3D reconstruction

  • Use molecular modeling and flexible fitting to interpret densities

  • Validate structural models against cross-linking data

• Ribosome Binding Assays:

  • Express recombinant CCDC47 domains

  • Perform in vitro binding assays with purified ribosomes

  • Quantify binding using fluorescence polarization or surface plasmon resonance

  • Characterize binding kinetics and thermodynamics

• Mutational Analysis of Interaction Interface:

  • Generate mutations in key residues identified from structural studies

  • Assess effects on ribosome binding and cellular function

  • Focus especially on the conserved C-terminal coiled-coil that extends to the ribosome exit tunnel

These complementary approaches provide a comprehensive characterization of CCDC47-ribosome interactions, from biochemical binding properties to high-resolution structural details.

What bioinformatic approaches can be used to analyze CCDC47 conservation and variation across macaque species?

To analyze CCDC47 conservation and variation across macaque species, researchers should implement these bioinformatic approaches:

• Multiple Sequence Alignment (MSA) Analysis:

  • Collect CCDC47 sequences from multiple Macaca species, including M. fascicularis, M. mulatta, M. nemestrina, and others mentioned in the literature

  • Perform MSA using tools like MUSCLE, MAFFT, or T-Coffee

  • Calculate conservation scores for each amino acid position

  • Generate conservation plots highlighting functional domains

  • Identify species-specific insertions or deletions

• Phylogenetic Analysis:

  • Construct phylogenetic trees using maximum likelihood (RAxML, IQ-TREE) or Bayesian methods (MrBayes)

  • Compare CCDC47 phylogeny with established species phylogeny to identify potential selective pressures

  • Calculate dN/dS ratios to assess selective constraints on different regions of the protein

• Structural Variation Analysis:

  • Apply methods like those used to identify copy number variations (CNVs) across Macaca species

  • Integrate reads from whole genome sequencing to detect structural variants

  • Use read depth-based methods to identify duplications or deletions

  • Employ paired-end mapping strategies to identify inversions or translocations

• Domain and Motif Analysis:

  • Apply RaptorX-Contact and other structural prediction tools to compare domain organization across species

  • Identify conserved motifs using MEME, GLAM2, or similar tools

  • Compare coiled-coil predictions across species using COILS or Paircoil2

  • Analyze conservation of known functional regions (calcium-binding motifs, ribosome interaction domains)

• Integrated Comparative Genomics:

  • Compare genomic context across species (synteny analysis)

  • Analyze promoter regions for conserved transcription factor binding sites

  • Examine intronic conservation that might indicate regulatory functions

  • Integrate with population-level transcriptome data similar to that used in macaque CNV studies

These approaches provide a comprehensive framework for understanding CCDC47 evolution across macaque species, revealing functional constraints and potential adaptive changes.

How can researchers differentiate between the functions of CCDC47 in calcium homeostasis versus protein biogenesis?

To differentiate between the dual functions of CCDC47 in calcium homeostasis versus protein biogenesis, researchers should implement these methodological approaches:

• Domain-specific Mutation Studies:

  Domain	Mutation Strategy	Expected Effect on Calcium Homeostasis	Expected Effect on Protein Biogenesis
  Ca²⁺-binding	Mutate putative calcium binding residues	Impaired calcium handling	Minimal effect if separate from structural role
  Coiled-coil	Truncate or disrupt C-terminal coiled-coil	Potentially unaffected	Severely compromised ribosome interaction
  N-terminal	Target residues at ribosome interface	Minimal effect	Disrupted translocon assembly

• Temporal Separation Experiments:

  • Use rapid calcium chelation (BAPTA-AM) to acutely disrupt calcium homeostasis without affecting existing protein complexes

  • Compare with long-term CCDC47 depletion affecting both functions

  • Measure immediate versus delayed effects on cellular phenotypes

• Rescue Experiments with Specialized Constructs:

  • Design chimeric proteins with calcium-binding domains from other proteins

  • Create constructs lacking ribosome-binding regions but retaining calcium functions

  • Assess which cellular defects can be rescued by each specialized construct

• High-resolution Imaging Techniques:

  • Implement live FRET sensors to monitor protein-protein interactions in the translocon

  • Use calcium sensors targeted to specific subcellular compartments

  • Perform correlative light and electron microscopy to connect calcium handling with physical translocon structure

• Multi-parameter Analysis:

  • Design experiments measuring both functions simultaneously

  • Plot correlation matrices between calcium homeostasis metrics and membrane protein synthesis rates

  • Use principal component analysis to identify independent versus linked functional parameters

These approaches allow researchers to dissect the independent contributions of CCDC47 to these interconnected cellular processes, providing mechanistic insight into how disruption of one function might impact the other.

What are the implications of CCDC47 research in Macaca fascicularis for understanding human developmental disorders?

Research on CCDC47 in Macaca fascicularis has significant implications for understanding human developmental disorders, particularly through these translational connections:

• Modeling Human Disease Mutations:

  • The high conservation of CCDC47 allows recreation of human disease-associated variants in macaque models

  • Bi-allelic CCDC47 variants in humans cause developmental disorders with woolly hair, liver dysfunction, pruritus, dysmorphic features, hypotonia, and global developmental delay

  • Macaque models can reveal tissue-specific and developmental stage-specific effects of these mutations

• Developmental Pathway Insights:

  • CCDC47 knockout in mice causes embryonic lethality, suggesting critical developmental functions

  • Macaque studies can bridge the evolutionary gap between mouse and human models

  • Investigation in primates allows for closer examination of neurodevelopmental processes affected by CCDC47 dysfunction

• Calcium Signaling in Development:

  • CCDC47's role in calcium homeostasis affects numerous developmental pathways

  • Macaque studies can reveal how altered calcium signaling contributes to specific developmental abnormalities seen in patients

  • The connection between CCDC47 dysfunction and decreased ER Ca²⁺ storage in patient cells can be further characterized in macaque models

• Tissue-specific Effects:

  • Patients with CCDC47 mutations display multi-organ involvement (hair, liver, brain)

  • Macaque studies can determine why certain tissues are particularly sensitive to CCDC47 dysfunction

  • Understanding tissue-specific expression patterns in macaques provides insight into human developmental vulnerability

• Therapeutic Development Platform:

  • Macaca fascicularis provides a physiologically relevant system for testing potential therapeutics

  • Compounds addressing calcium homeostasis or protein biogenesis defects can be evaluated in macaque cells

  • The close evolutionary relationship to humans increases translational potential for therapeutic discoveries

These connections highlight how CCDC47 research in Macaca fascicularis serves as a critical translational bridge between basic science and clinical applications for human developmental disorders.

How can multi-omics approaches enhance our understanding of CCDC47 function in Macaca fascicularis?

Multi-omics approaches offer powerful strategies to comprehensively characterize CCDC47 function in Macaca fascicularis through integrated data analysis:

• Integrated Transcriptomics and Proteomics:

  • Perform RNA-Seq and quantitative proteomics in CCDC47-depleted versus control cells

  • Identify discordant mRNA-protein pairs suggesting post-transcriptional regulation

  • Focus on membrane proteins potentially affected by translocon dysfunction

  • This approach mirrors population-level transcriptome analysis used in macaque CNV studies , but with specific focus on CCDC47's impact

• Structural Omics Integration:

  • Combine cryo-EM structural data of CCDC47 in the translocon complex with cross-linking mass spectrometry

  • Map interaction networks using proximity labeling (BioID, APEX) followed by mass spectrometry

  • Integrate with predicted protein structures using AlphaFold or RaptorX-Contact

  • Create structure-function networks highlighting domains critical for different activities

• Calcium Signaling Interactome Analysis:

  • Implement calcium-dependent proximity labeling to identify CCDC47 interaction partners

  • Perform phosphoproteomics to identify calcium-dependent signaling changes

  • Conduct metabolomics to detect downstream effects of altered calcium homeostasis

  • Link to clinical phenotypes observed in patients with CCDC47 mutations

• Developmental Time-course Multi-omics:

  • Sample multiple developmental timepoints in various tissues

  • Perform parallel RNA-Seq, proteomics, metabolomics, and calcium imaging

  • Create temporal maps of CCDC47 function during development

  • Compare with human developmental trajectories to identify conserved pathways

• Comparison with Human Disease Profiles:

  Data Type	Control Macaque	CCDC47-depleted Macaque	Human Patient Samples
  Transcriptome	Baseline expression	Altered ER stress response	Similar dysregulation patterns
  Proteome	Normal membrane protein profiles	Defects in specific multi-pass proteins	Concordant protein changes
  Calcium dynamics	Normal ER Ca²⁺ handling	Impaired store refilling	Matched calcium defects
  Metabolome	Normal ER-dependent metabolism	Altered lipid composition	Parallel metabolic shifts

This multi-dimensional data integration enables researchers to connect molecular mechanisms to cellular phenotypes and ultimately to organismal development and disease manifestations, providing a comprehensive understanding of CCDC47 biology.

What emerging technologies show promise for advancing CCDC47 research in Macaca fascicularis?

Several cutting-edge technologies show particular promise for advancing CCDC47 research in Macaca fascicularis:

• Cryo-Electron Tomography (Cryo-ET):

  • Enables visualization of CCDC47 in its native cellular environment

  • Allows direct observation of CCDC47-containing translocon complexes in situ

  • Can reveal structural variations that might not be captured in purified samples

  • Building on the existing cryo-EM findings that identified CCDC47's position in the translocon complex

• AlphaFold2 and RoseTTAFold Integration:

  • Provides accurate structural predictions to complement experimental approaches

  • Particularly valuable for predicting species-specific variations in structure

  • Can model complex formation with interaction partners

  • Extends the modeling approaches already applied using RaptorX-Contact

• Single-Cell Multi-omics:

  • Reveals cell-type specific functions of CCDC47

  • Identifies distinct roles in different cellular populations

  • Captures rare cell populations potentially most sensitive to CCDC47 dysfunction

  • Complements population-level transcriptome analyses previously employed in macaque research

• Spatial Transcriptomics and Proteomics:

  • Maps CCDC47 expression and function with spatial resolution

  • Reveals microenvironmental influences on CCDC47 activity

  • Particularly valuable for developmental studies and tissue-specific effects

  • Helps interpret the multi-system disorder phenotypes seen in human patients

• CRISPR Base Editing and Prime Editing:

  • Enables precise engineering of specific CCDC47 variants

  • Creates isogenic cell lines differing only in CCDC47 sequence

  • Facilitates modeling of human disease mutations

  • Allows testing of variant effects on calcium homeostasis and protein biogenesis functions

These emerging technologies, especially when used in combination, promise to reveal new dimensions of CCDC47 biology and transform our understanding of its roles in cellular physiology and development in primates.

What are the most significant unanswered questions about CCDC47 function in Macaca fascicularis?

Despite recent advances, several critical questions about CCDC47 in Macaca fascicularis remain unanswered:

• Functional Specificity: How does CCDC47 specifically contribute to multi-pass membrane protein biogenesis beyond other translocon components? The structural data shows CCDC47's position in the translocon , but its precise mechanistic contribution remains unclear.

• Calcium Sensing Mechanism: What is the molecular mechanism by which CCDC47 senses and regulates calcium levels? While we know CCDC47 binds calcium with low affinity and high capacity , the structural basis and regulatory mechanisms remain undefined.

• Evolutionary Adaptation: How has CCDC47 function evolved specifically in Macaca fascicularis compared to other primates, and does this reflect environmental adaptations? Macaque species show CNVs in genes related to metabolism and immune function , but CCDC47's evolutionary trajectory is unexplored.

• Developmental Regulation: What regulatory mechanisms control CCDC47 expression and function during development in Macaca fascicularis? The embryonic lethality in knockout mice suggests essential developmental roles, but the temporal and spatial regulation remains unknown.

• Disease Relevance: Beyond known human disorders , what role might CCDC47 play in more common diseases affecting both macaques and humans? The connection between CCDC47 dysfunction and pathologies beyond rare developmental disorders remains to be established.