Recombinant Human Coiled-coil domain-containing protein 47 (CCDC47)

What is the structural and functional characterization of CCDC47?

CCDC47 is a calcium-binding endoplasmic reticulum (ER) transmembrane protein that belongs to the CCDC47 family. Structurally, it contains multiple coiled-coil domains that facilitate protein-protein interactions within cellular compartments. The full-length human CCDC47 protein sequence begins with the amino acid sequence MKAFHTFCVVLLVFGSVSEAKFDDFEDEEDIVEYDDNDFAEFEDT and continues through its functional domains . Functionally, CCDC47 serves as a component of the multi-pass translocon (MPT) complex that mediates the insertion of multi-pass membrane proteins into lipid bilayers of membranes . The protein specifically occludes the lateral gate of the SEC61 complex within the PAT subcomplex, which is essential for proper protein folding and insertion . Additionally, CCDC47 plays significant roles in calcium ion homeostasis in the ER and is required for proper protein degradation via the ER-associated degradation (ERAD) pathway .

What expression systems are recommended for producing recombinant CCDC47?

For optimal expression of recombinant CCDC47, HEK293 cells have been demonstrated as an effective mammalian expression system, particularly for producing the protein with high purity (>95%) . This system allows for proper post-translational modifications essential for maintaining the protein's native conformation and functionality. When expressing recombinant CCDC47, researchers should consider including affinity tags such as His-tags to facilitate purification, while ensuring these modifications do not interfere with the protein's biological activity . For fragment expression, the amino acid region 1-135 has been successfully produced with high yield and purity . Expression conditions should be optimized to minimize endotoxin levels (<1 EU/μg) to ensure compatibility with downstream cellular and biochemical assays . Alternative expression systems such as bacterial or insect cell-based systems may be considered, but validation against the HEK293-expressed protein is recommended to confirm structural and functional integrity.

How can researchers detect and quantify CCDC47 in experimental samples?

CCDC47 detection and quantification can be achieved through multiple complementary approaches. For protein level detection, Western blotting using specific anti-CCDC47 antibodies provides qualitative and semi-quantitative assessment in cellular and tissue samples. For precise quantification, sandwich ELISA assays with a detection wavelength of 450 nm offer high sensitivity for CCDC47 in various sample types including serum, plasma, urine, tissue homogenates, and cell culture supernatants . When performing ELISA, sample volumes between 50-100 μl are typically required, and researchers should maintain strict temperature control during storage of reagents (2-8°C) . For mRNA expression analysis, quantitative real-time PCR can be employed using validated primer sets targeting conserved regions of the CCDC47 transcript. Immunohistochemistry and immunofluorescence techniques provide valuable information on subcellular localization, particularly for confirming ER membrane localization. Mass spectrometry approaches can be used for more detailed protein characterization, interaction studies, and post-translational modification analysis.

What are the established functions of CCDC47 in cellular physiology?

CCDC47 has several well-established functions in cellular physiology. Primarily, it serves as a critical component of the multi-pass translocon (MPT) complex that facilitates the insertion of multi-pass membrane proteins into lipid bilayers . The MPT complex takes over from the SEC61 complex after the initial membrane insertion of the first few transmembrane segments, with CCDC47 specifically occluding the lateral gate of SEC61 to promote insertion of subsequent transmembrane regions . CCDC47 plays a crucial role in calcium homeostasis within the ER, binding Ca²⁺ with low affinity but high capacity, which is essential for proper cell signaling . It also contributes to proper protein degradation through the ERAD pathway, highlighting its importance in protein quality control mechanisms . Developmental studies reveal that CCDC47 is essential for maintaining ER organization during embryogenesis, with knockout models demonstrating embryonic lethality, delayed development, atrophic neural tubes, and heart abnormalities . These functional aspects collectively underscore CCDC47's importance in fundamental cellular processes including protein folding, calcium signaling, and developmental progression.

How does CCDC47 contribute to calcium signaling and what methodologies are optimal for studying these dynamics?

CCDC47 plays a sophisticated role in calcium homeostasis within the endoplasmic reticulum, serving as a calcium-binding protein with low affinity but high capacity characteristics . Research methodologies for investigating CCDC47's contribution to calcium signaling should employ multiple approaches. Calcium imaging using fluorescent indicators such as Fura-2, Fluo-4, or genetically encoded calcium indicators (GECIs) allows real-time visualization of calcium flux in live cells with CCDC47 manipulations. Store-operated calcium entry (SOCE) assays, which can be conducted by depleting ER calcium stores with thapsigargin followed by calcium re-addition, reveal how CCDC47 affects ER calcium refilling mechanisms . Patch-clamp electrophysiology provides direct measurement of calcium currents across membranes. Researchers investigating CCDC47's calcium binding properties should use purified recombinant protein for isothermal titration calorimetry (ITC) or microscale thermophoresis (MST) to determine binding affinities and stoichiometry. In vitro studies have demonstrated that cells with CCDC47 deficiency exhibit impaired calcium signaling, specifically showing decreased total ER calcium storage, impaired calcium signaling through the IP3R release channel, and reduced ER calcium refilling via store-operated calcium entry . These methodological approaches provide complementary data to establish CCDC47's precise role in the complex calcium signaling network.

What protein-protein interactions does CCDC47 participate in, and how can these be experimentally characterized?

CCDC47 engages in several critical protein-protein interactions within the cellular environment, particularly as a component of the multi-pass translocon (MPT) complex and the PAT subcomplex . To comprehensively characterize these interactions, researchers should employ multiple complementary approaches. Proximity-based labeling techniques such as BioID or APEX2 can identify proximal protein interactors in living cells by tagging CCDC47 with a biotin ligase or peroxidase. Co-immunoprecipitation followed by mass spectrometry analysis provides a direct means to identify stable protein complexes containing CCDC47. For targeted validation of specific interactions, techniques such as fluorescence resonance energy transfer (FRET), bimolecular fluorescence complementation (BiFC), or split-luciferase assays can provide quantitative assessment of interaction dynamics in living cells. Crosslinking mass spectrometry (XL-MS) offers structural insights into the interacting domains and amino acid residues involved in these interactions. Surface plasmon resonance (SPR) or biolayer interferometry (BLI) with purified recombinant proteins enables determination of binding kinetics and affinities. Known interactions with components of the SEC61 complex should be carefully examined, as CCDC47 occludes the lateral gate of this complex during membrane protein insertion . Additionally, interactions within the PAT subcomplex that facilitate sequestration of polar transmembrane domains deserve particular attention for their functional significance .

What experimental approaches are recommended for investigating CCDC47's role in disease pathogenesis?

To investigate CCDC47's role in disease pathogenesis, particularly the multisystem disorder characterized by woolly hair, liver dysfunction, pruritus, dysmorphic features, hypotonia, and global developmental delay, several sophisticated experimental approaches are recommended . Patient-derived cell models including fibroblasts, lymphoblasts, or induced pluripotent stem cells (iPSCs) provide platforms for studying cellular phenotypes associated with CCDC47 variants. These can be analyzed for alterations in calcium homeostasis, protein folding, and ER stress responses. CRISPR/Cas9 gene editing enables creation of isogenic cell lines with specific CCDC47 mutations identified in patients, allowing direct comparison with wild-type cells under controlled conditions. Animal models carrying equivalent mutations can be generated to study systemic effects, though complete knockout in mice results in embryonic lethality, necessitating conditional or hypomorphic approaches . High-throughput drug screening using disease-relevant cellular phenotypes may identify compounds that rescue functional defects. Transcriptomic and proteomic profiling of patient-derived samples can reveal dysregulated pathways downstream of CCDC47 dysfunction. For clinical correlation, genotype-phenotype analyses across multiple patients with different CCDC47 variants should be conducted to identify mutation-specific effects. These approaches collectively provide a comprehensive framework for understanding how CCDC47 dysfunction contributes to disease and may reveal potential therapeutic targets .

What methodological considerations are important when analyzing calcium dysregulation in CCDC47-deficient cellular models?

When analyzing calcium dysregulation in CCDC47-deficient cellular models, several methodological considerations are critical for obtaining reliable and interpretable results. Researchers should first establish appropriate cellular models, including patient-derived cells, CRISPR/Cas9-edited cell lines with specific CCDC47 mutations, or siRNA/shRNA knockdown systems, with careful validation of CCDC47 depletion at both mRNA and protein levels . For calcium imaging experiments, selection of appropriate calcium indicators is crucial—ratiometric dyes like Fura-2 are preferred for quantitative measurements as they control for variations in dye loading and cell thickness. Genetically encoded calcium indicators offer advantages for long-term or compartment-specific measurements. When measuring store-operated calcium entry, standardized protocols should include controlled ER calcium depletion (typically using thapsigargin or ionomycin) followed by external calcium re-addition, with precise timing between steps . Temperature control is essential as calcium dynamics are temperature-sensitive. Single-cell analysis is preferred over population measurements to account for cellular heterogeneity. Complementary electrophysiological measurements of calcium currents provide direct functional assessment. Researchers should include appropriate controls, such as rescue experiments with wild-type CCDC47 expression to confirm phenotype specificity. Previous studies have demonstrated that CCDC47-deficient cells exhibit specific defects in ER calcium storage, IP3R-mediated signaling, and store-operated calcium entry that contribute to cellular dysfunction . These methodological considerations ensure robust and reproducible assessment of calcium dysregulation in CCDC47 research.

How can findings from CCDC47 research be applied to understanding human genetic disorders?

Research on CCDC47 has direct translational implications for understanding human genetic disorders, particularly the multisystem disorder associated with bi-allelic CCDC47 variants. Studies have identified that patients with pathogenic CCDC47 variants present with a constellation of symptoms including woolly hair, liver dysfunction, pruritus, dysmorphic features, hypotonia, and global developmental delay . When investigating potential connections between CCDC47 variants and human disease, researchers should employ whole-exome or whole-genome sequencing to identify novel variants, followed by rigorous variant interpretation using ACMG-AMP guidelines and segregation analysis within families . Functional validation of identified variants is essential, using patient-derived cells to assess mRNA expression, protein levels, and calcium signaling defects . The documented reduction in CCDC47 mRNA and protein levels in cells from affected individuals provides a potential biomarker for assessing variant pathogenicity . Brain MRI findings in affected individuals, including ventricular enlargement, white matter paucity, and thin corpus callosum, suggest specific neurological manifestations that should be evaluated in suspected cases . These research approaches enable the development of diagnostic criteria for CCDC47-related disorders and potentially inform genetic counseling for affected families. As additional cases are identified and characterized, genotype-phenotype correlations may emerge to predict disease severity and progression based on specific variant types.

What experimental designs are most effective for screening potential therapeutic interventions targeting CCDC47-associated disorders?

Developing effective screening approaches for therapeutic interventions targeting CCDC47-associated disorders requires careful experimental design. High-throughput drug screening platforms using patient-derived cells or CCDC47-deficient cellular models should focus on phenotypic endpoints that reflect the fundamental cellular dysfunctions, particularly calcium homeostasis defects, ER stress responses, and protein trafficking abnormalities . Primary screening assays should include calcium imaging to identify compounds that restore normal calcium signaling patterns, with secondary validation using store-operated calcium entry measurements and IP3R-mediated calcium release assessments . Cell viability and proliferation assays under stress conditions can identify compounds that protect against CCDC47 deficiency-induced cellular dysfunction. For in vivo validation, conditional knockout animal models that survive beyond embryonic stages are necessary, as complete knockout causes embryonic lethality . Therapeutic approaches might include small molecules that stabilize residual CCDC47 protein, chaperone therapies to improve protein folding, calcium channel modulators to correct signaling defects, or gene therapy approaches for severe loss-of-function cases. Drug repurposing strategies focusing on approved compounds that modulate calcium signaling or ER stress responses offer accelerated development pathways. Given the clinical presentation that includes liver dysfunction and neurological impairments, cell type-specific effects should be evaluated in hepatocytes and neurons derived from patient iPSCs to account for tissue-specific manifestations of CCDC47 dysfunction . These comprehensive screening approaches maximize the potential for identifying effective therapeutic interventions.

What methodological approaches should be used to correlate CCDC47 function with developmental processes in model organisms?

Investigating CCDC47's role in developmental processes requires sophisticated methodological approaches across multiple model organisms. Temporal and spatial expression profiling of CCDC47 throughout embryonic development using RNA in situ hybridization and immunohistochemistry provides the foundation for understanding when and where this protein functions during development . Conditional knockout strategies using tissue-specific Cre-lox systems circumvent the embryonic lethality observed in complete Ccdc47 knockout mice, allowing investigation of CCDC47's role in specific tissues during defined developmental windows . For rapid preliminary studies, morpholino knockdown or CRISPR/Cas9 editing in zebrafish offers advantages for visualizing developmental phenotypes in a vertebrate system with optical transparency. Lineage tracing experiments in embryos with fluorescently labeled CCDC47-expressing cells track their developmental trajectory and fate. Live calcium imaging during development reveals how CCDC47-dependent calcium signaling coordinates developmental processes. Single-cell RNA sequencing of embryos at different developmental stages with CCDC47 manipulation identifies downstream molecular pathways affected by its dysfunction. Detailed phenotypic characterization should focus on the neural tube, heart, and blood cell development, as these were specifically affected in Ccdc47-knockout mouse embryos, showing delayed development, atrophic neural tubes, and heart abnormalities . These methodological approaches collectively provide a comprehensive understanding of CCDC47's developmental functions and potential therapeutic windows for intervention in CCDC47-associated developmental disorders.

What are the optimal storage and handling conditions for recombinant CCDC47 protein and related reagents?

Proper storage and handling of recombinant CCDC47 protein and related reagents are critical for maintaining their stability and functionality in research applications. Purified recombinant CCDC47 protein should be stored at -80°C for long-term preservation, with aliquoting recommended to minimize freeze-thaw cycles that can lead to protein denaturation . For working solutions, temporary storage at 4°C (up to 1 week) may be acceptable, but validation of protein activity should be performed regularly. ELISA kits for CCDC47 detection have specific storage requirements: 2-8°C for short-term (up to 6 months) and -20°C for long-term storage (up to 12 months) . When preparing working solutions, use of high-quality, nuclease-free water and appropriate buffers is essential, with pH verification before use. Handling should include standard protein biochemistry precautions such as working on ice when possible and avoiding repeated freeze-thaw cycles. Addition of protease inhibitors to solutions containing CCDC47 protein is recommended to prevent degradation. For applications requiring maintenance of native conformation, reducing agents should be used cautiously as they may disrupt structural disulfide bonds if present. Endotoxin testing is advisable for preparations intended for cell culture applications, with a target of <1 EU/μg . Antibodies against CCDC47 should be stored according to manufacturer recommendations, typically at -20°C with glycerol to prevent freezing damage. These handling and storage practices ensure optimal performance and reproducibility in CCDC47 research applications.

What controls and validation steps are essential for CCDC47 functional studies?

Robust experimental design for CCDC47 functional studies requires comprehensive controls and validation steps. For gene expression manipulation experiments, multiple siRNA or shRNA constructs targeting different regions of CCDC47 should be employed to control for off-target effects, with validation of knockdown efficiency by both qRT-PCR and Western blot . Rescue experiments involving re-expression of wild-type CCDC47 in knockdown or knockout cells provide crucial validation that observed phenotypes are specifically due to CCDC47 deficiency rather than off-target effects . When studying CCDC47 variants, both wild-type and mutant versions should be expressed at comparable levels, confirmed by Western blot, to ensure differences in function are not simply due to expression differences. For calcium signaling experiments, positive controls using established calcium modulators (thapsigargin, ionomycin) should be included to confirm assay functionality . Subcellular localization of CCDC47 should be confirmed by co-localization with established ER markers in immunofluorescence or fractionation experiments. Protein-protein interaction studies should include non-interacting protein controls and validation by at least two independent methods (e.g., co-IP plus FRET). When using recombinant CCDC47 protein, batch-to-batch consistency should be validated by SDS-PAGE, with functionality confirmed by calcium binding assays . These validation steps ensure that experimental results are specifically attributable to CCDC47 function and provide the foundation for reliable interpretation of experimental outcomes.