Recombinant Rat Coiled-coil domain-containing protein 47 (Ccdc47)

What is Recombinant Rat Coiled-coil Domain-containing Protein 47 (Ccdc47)?

Recombinant Rat Coiled-coil Domain-containing Protein 47 (Ccdc47), also known as calumin, is an endoplasmic reticulum (ER) transmembrane protein that functions as a calcium-binding protein. This recombinant protein represents the expression region 21-483 of the full-length protein and is derived from Rattus norvegicus . The protein has critical roles in calcium signaling and embryonic development. CCDC47 contains specific structural motifs including coiled-coil domains which facilitate protein-protein interactions and contribute to its functional properties in calcium homeostasis. The recombinant form is typically produced through heterologous expression systems to enable detailed laboratory investigation of its properties and functions .

The amino acid sequence of Rat CCDC47 includes multiple domains responsible for its calcium binding capabilities and interaction with other proteins in the ER calcium regulation system. The full sequence, as identified in the UniProt database (Q5U2X6), demonstrates conservation of key functional regions across mammalian species .

How does CCDC47 function in cellular calcium signaling pathways?

CCDC47 serves as a critical component in the regulation of calcium (Ca²⁺) homeostasis within the endoplasmic reticulum, which represents the largest intracellular Ca²⁺ store in most cells . The protein binds calcium with low affinity but high capacity, suggesting its role as a calcium buffer or sensor within the ER lumen . CCDC47 participates in multiple calcium-dependent cellular processes including synaptic vesicle exocytosis, muscle contraction, secretion regulation, gene transcription, and cellular proliferation .

Mechanistically, CCDC47 influences calcium signaling through interaction with store-operated calcium entry (SOCE) pathways, a process critical for ER calcium refilling following depletion . Experimental evidence from knockout models demonstrates that cells with decreased CCDC47 expression show impaired calcium signaling mediated by the IP₃R calcium release channel and reduced ER calcium refilling via SOCE . This indicates that CCDC47 may function as a regulatory component in maintaining proper calcium flux between cellular compartments.

What is the tissue expression profile of CCDC47 in rats?

CCDC47 demonstrates a broad tissue distribution pattern in rats, consistent with its fundamental role in calcium homeostasis. The protein is present in multiple tissues including brain, lung, heart, stomach, liver, spleen, kidney, muscle, and testis . This widespread expression pattern suggests that CCDC47 serves essential cellular functions across diverse tissue types rather than having tissue-specific roles.

The expression levels vary across different tissues, with notable expression in tissues with high secretory activity and calcium-dependent signaling requirements. While comprehensive quantitative tissue expression data specifically for rat CCDC47 is limited in the current literature, studies in other mammalian models suggest correlation between CCDC47 expression levels and tissue-specific calcium signaling demands.

What are the optimal storage and handling conditions for Recombinant Rat CCDC47?

Recombinant Rat CCDC47 requires specific storage conditions to maintain protein stability and functional activity. The recommended storage buffer is a Tris-based buffer containing 50% glycerol that has been optimized for this specific protein . For short-term storage, working aliquots can be maintained at 4°C for up to one week . For longer-term storage, the protein should be kept at -20°C, while extended storage periods require conservation at -20°C or -80°C .

Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of activity . To mitigate this risk, researchers should prepare small single-use aliquots upon receipt of the protein. When handling the protein, standard precautions for working with purified proteins should be followed, including maintaining sterile conditions and using appropriate personal protective equipment to prevent contamination.

What experimental approaches are most effective for studying CCDC47 function?

Multiple experimental approaches have proven effective for investigating CCDC47 function, with selection depending on the specific research question:

• Calcium Imaging Techniques: Fluorescent calcium indicators (Fura-2, Fluo-4) can be used to measure intracellular calcium dynamics in real-time. This approach enables direct observation of calcium flux in response to various stimuli in cells with altered CCDC47 expression .

• Store-Operated Calcium Entry (SOCE) Assays: These assays typically involve depleting ER calcium stores (using thapsigargin or other SERCA inhibitors) followed by reintroduction of extracellular calcium to measure SOCE activity. This methodology has successfully demonstrated impaired SOCE in cells with decreased CCDC47 levels .

• IP₃-Mediated Calcium Release Measurement: Stimulation with IP₃-generating agonists followed by calcium measurement can assess the role of CCDC47 in IP₃R-mediated calcium release .

• Cell-Based Functional Assays: These include assessing cellular processes that depend on proper calcium signaling, such as secretion, transcription, or proliferation in cells with modulated CCDC47 expression .

• Gene Expression Analysis: Quantitative PCR and RNA sequencing can measure CCDC47 mRNA expression levels in different experimental conditions or disease models .

How can researchers effectively quantify CCDC47 protein levels in experimental samples?

Quantification of CCDC47 protein levels in experimental samples can be accomplished through several validated techniques:

• Western Blotting: This remains the standard approach for semi-quantitative assessment of CCDC47 protein levels. Sample preparation should include appropriate cell lysis buffers containing protease inhibitors to prevent protein degradation. Detection using specific anti-CCDC47 antibodies allows visualization and relative quantification when compared to appropriate housekeeping proteins .

• ELISA-Based Quantification: Commercial ELISA kits or custom assay development using the recombinant protein as a standard can provide more precise quantification of CCDC47 in biological samples .

• Mass Spectrometry-Based Proteomics: For more comprehensive analysis, liquid chromatography-tandem mass spectrometry (LC-MS/MS) approaches can be used to quantify CCDC47 and identify post-translational modifications that may affect function.

• Immunofluorescence Microscopy: This technique allows visualization of CCDC47 subcellular localization and can provide qualitative assessment of expression levels in intact cells or tissues.

The choice of quantification method should be determined by the specific research question, with consideration for sensitivity requirements and the need for absolute versus relative quantification.

How can CCDC47 knockout or knockdown models be established and validated?

Establishing effective CCDC47 knockout or knockdown models requires strategic approaches due to the protein's essential developmental role. Complete knockout in mice causes embryonic lethality, suggesting similar concerns may apply to rats . Therefore, researchers should consider the following approaches:

• Conditional Knockout Systems: Cre-loxP or similar inducible systems allow temporal control of gene deletion, enabling study of CCDC47 function in adult animals while avoiding developmental lethality. Tissue-specific promoters can restrict deletion to tissues of interest.

• RNA Interference (RNAi): siRNA or shRNA approaches can achieve partial knockdown of CCDC47 expression, which may avoid complete loss of function while still revealing phenotypic effects. Multiple target sequences should be tested to identify those with optimal knockdown efficiency and specificity .

• CRISPR/Cas9 Gene Editing: This can be used to introduce specific mutations that mimic disease-associated variants rather than complete gene knockout. Careful guide RNA design is essential to minimize off-target effects.

Model validation requires comprehensive analysis including:

• Confirmation of successful genetic modification using genomic PCR and sequencing

• Verification of reduced mRNA levels via qRT-PCR

• Demonstration of reduced protein expression using Western blot or immunostaining

• Functional validation through calcium signaling assays to confirm altered ER calcium dynamics

What methodologies effectively analyze CCDC47's role in ER calcium homeostasis?

Investigating CCDC47's specific role in ER calcium homeostasis requires specialized techniques that directly measure ER calcium dynamics:

• ER-Targeted Calcium Indicators: Genetically encoded calcium indicators (GECIs) targeted to the ER lumen (e.g., ER-GCaMP, D1ER) provide direct measurement of ER calcium content and dynamics. These sensors can be expressed in cells with modified CCDC47 expression to observe real-time changes in ER calcium levels .

• Calcium Depletion-Refilling Protocols: These involve pharmacological manipulation of ER calcium stores using:

  • Thapsigargin (SERCA inhibitor) to deplete ER calcium

  • Ionomycin to assess total calcium store content

  • EGTA/calcium re-addition protocols to measure SOCE activity

• Patch-Clamp Electrophysiology: This technique can measure calcium-release activated calcium (CRAC) currents that represent SOCE at the electrophysiological level, providing detailed kinetic information about calcium flux alterations in cells with modified CCDC47 expression.

• Proximity Ligation Assays: These can identify protein-protein interactions between CCDC47 and other components of calcium handling machinery, revealing functional relationships within the ER calcium regulation system.

The data from these techniques can be presented in the following format to facilitate interpretation:

How do mutations in CCDC47 affect calcium signaling and cellular function?

Mutations in CCDC47 have significant impacts on calcium signaling and broader cellular functions, as evidenced by studies of disease-associated variants. Bi-allelic loss-of-function variants in CCDC47 are associated with a complex multisystem disorder characterized by woolly hair, liver dysfunction, pruritus, dysmorphic features, hypotonia, and global developmental delay .

At the cellular level, these mutations result in:

• Decreased Total ER Calcium Storage: Cells from affected individuals show reduced ER calcium content, which affects the cell's ability to respond to calcium-mobilizing stimuli .

• Impaired IP₃R-Mediated Calcium Release: The release of calcium through inositol trisphosphate receptors (IP₃Rs) is compromised, affecting downstream signaling pathways that depend on this mechanism .

• Reduced Store-Operated Calcium Entry: The refilling of ER calcium stores through SOCE is significantly impaired, creating a chronic state of ER calcium depletion .

These alterations in calcium homeostasis have cascading effects on multiple cellular processes:

• Disrupted protein folding in the ER, potentially leading to ER stress

• Altered gene expression due to calcium-dependent transcription factor dysregulation

• Compromised secretory pathway function, affecting protein processing and secretion

• Potential impact on cell proliferation and differentiation during development

The severity of these effects varies with the specific mutation type, with nonsense and frameshift mutations typically causing more severe phenotypes due to complete loss of protein function or nonsense-mediated mRNA decay .

How can CCDC47 research inform understanding of calcium-related disorders?

Research on CCDC47 provides valuable insights into the pathogenesis of calcium-related disorders through several mechanisms:

• Direct CCDC47-Associated Disorders: Bi-allelic variants in CCDC47 cause a distinct multisystem disorder characterized by woolly hair, liver dysfunction, pruritus, dysmorphic features, hypotonia, and global developmental delay . Studying this condition provides direct evidence of how calcium dysregulation manifests clinically.

• Model for ER Calcium Regulation: CCDC47 research offers a model system for understanding fundamental mechanisms of ER calcium homeostasis relevant to numerous diseases including:

  • Neurodegenerative conditions such as Alzheimer's and Parkinson's diseases

  • Metabolic disorders like diabetes mellitus

  • Cancer biology, where calcium signaling affects proliferation and apoptosis

• Developmental Implications: The embryonic lethality observed in Ccdc47-knockout mice highlights the critical role of proper calcium signaling in development . This provides insight into developmental disorders associated with calcium dysregulation.

Researchers investigating calcium-related disorders should consider incorporating CCDC47 functional analysis in their experimental design, particularly when evaluating disorders with unexplained alterations in ER calcium handling or store-operated calcium entry pathways.

What considerations apply when using Recombinant Rat CCDC47 for in vitro functional studies?

When utilizing Recombinant Rat CCDC47 for in vitro functional studies, researchers should consider several important factors:

• Protein Preparation and Quality Control:

  • Verify protein integrity through SDS-PAGE analysis before experimental use

  • Confirm functional activity using calcium-binding assays when appropriate

  • Avoid repeated freeze-thaw cycles which may affect protein structure and function

• Experimental Design Considerations:

  • Control for the presence of tags that may affect protein function

  • Account for the expression region (21-483) when interpreting results, as this may not represent the complete native protein functionality

  • Consider the buffer composition's effect on calcium binding properties

• Cell-Based Assay Adaptations:

  • When introducing recombinant protein into cells, optimize delivery methods (microinjection, cell-penetrating peptides, or permeabilization)

  • Account for potential competition with endogenous CCDC47

  • Include appropriate controls with mutated or heat-inactivated protein

• Quantitative Analysis:

  • Establish dose-response relationships to determine optimal protein concentrations

  • Account for potential time-dependent effects in longer experiments

  • Use appropriate statistical methods for data analysis and interpretation

Careful attention to these considerations will enhance experimental reproducibility and validity when working with recombinant CCDC47 protein.

What emerging technologies could advance understanding of CCDC47 function?

Several cutting-edge technologies hold promise for deepening our understanding of CCDC47 function:

• Cryo-Electron Microscopy: High-resolution structural analysis of CCDC47 alone and in complex with binding partners could reveal detailed molecular mechanisms of calcium binding and protein-protein interactions.

• Super-Resolution Microscopy: Techniques such as STORM, PALM, or STED microscopy can visualize CCDC47 localization within the ER with unprecedented precision, potentially revealing functional microdomains.

• Single-Cell Transcriptomics and Proteomics: These approaches can reveal cell-type specific expression patterns and correlations with other calcium handling proteins across diverse tissues.

• Optogenetic Calcium Manipulation: Light-activated calcium channels or pumps could be used in conjunction with CCDC47 studies to precisely manipulate calcium dynamics and observe effects on CCDC47 function.

• In Situ Structural Analysis: Techniques like proximity labeling combined with mass spectrometry (BioID, APEX) can map the protein's interaction landscape within its native cellular environment.

• CRISPR-Based Screening: Genome-wide or targeted CRISPR screens can identify genetic modifiers of CCDC47 function and reveal novel pathway connections.

These technologies will enable more precise dissection of CCDC47's molecular function and its integration into cellular calcium signaling networks.

What are the critical gaps in current CCDC47 research knowledge?

Despite progress in understanding CCDC47 function, several critical knowledge gaps remain that represent important opportunities for research advancement:

• Detailed Structural Information: The three-dimensional structure of CCDC47 has not been fully elucidated, limiting our understanding of how it physically interacts with calcium and binding partners .

• Precise Calcium Binding Mechanisms: While CCDC47 is known to bind calcium with low affinity and high capacity, the specific binding sites and calcium coordination mechanisms remain uncharacterized at the molecular level .

• Tissue-Specific Functions: Despite its widespread expression, potential tissue-specific roles of CCDC47 beyond basic calcium handling remain poorly understood, particularly in neuronal and muscle tissues .

• Regulatory Mechanisms: The factors controlling CCDC47 expression, post-translational modifications, and activity regulation in different physiological and pathological states require further investigation.

• Therapeutic Potential: The possibility of targeting CCDC47 or its pathways for therapeutic intervention in calcium-related disorders remains largely unexplored.

Addressing these knowledge gaps will require interdisciplinary approaches combining structural biology, cell physiology, and in vivo models to fully elucidate CCDC47's complex roles in calcium homeostasis and related pathologies.