Recombinant Danio rerio Coiled-coil domain-containing protein 47 (ccdc47)

What is the biological function of Danio rerio ccdc47?

Danio rerio ccdc47 serves multiple critical cellular functions primarily associated with calcium homeostasis and endoplasmic reticulum (ER) processes. The protein is predicted to enable calcium ion binding activity, function as a protein folding chaperone, and participate in ribosome binding . Research indicates that CCDC47 is involved in multi-pass transmembrane protein insertion into the ER membrane and plays a crucial role in endoplasmic reticulum calcium ion homeostasis .

The functional importance of ccdc47 is further demonstrated by studies in mammalian systems, where bi-allelic loss-of-function variants in CCDC47 have been linked to multisystem disorders . In zebrafish (Danio rerio), ccdc47 acts as part of the multi-pass translocon complex in the ER membrane, suggesting it has a conserved role in protein processing and cellular calcium regulation .

Methodologically, researchers investigating ccdc47 function should consider calcium imaging techniques, protein-protein interaction studies focusing on ER membrane complexes, and targeted gene manipulation approaches to elucidate its specific roles in calcium signaling pathways.

What is the structure and organization of the ccdc47 gene in zebrafish?

The zebrafish ccdc47 gene is located on chromosome 3 of the Danio rerio genome . It encodes a protein-coding gene officially named "coiled-coil domain containing 47" with the synonym zgc:92099 . The gene structure likely mirrors some aspects of its human ortholog, which contains 13 exon splice sites and 14 distinct introns spanning 3445 base pairs (after removal of exons) .

The encoded protein contains several key structural domains that are likely conserved across species, including:

• Coiled-coil domains (as the name suggests)

• A transmembrane domain

• Domains associated with calcium binding

• Potential signal peptides

While specific zebrafish ccdc47 structural information is limited in the current literature, comparative genomics approaches suggest high conservation with the human CCDC47, which is characterized by an acidic isoelectric point due to high content of negatively charged amino acids .

For researchers conducting structural analyses, techniques such as protein homology modeling, domain prediction algorithms, and evolutionary conservation mapping would be valuable approaches to predict functional domains in the zebrafish ortholog.

What are the expression patterns of ccdc47 during zebrafish development?

Zebrafish ccdc47 demonstrates specific temporal and spatial expression patterns during development, although comprehensive characterization is still emerging. According to the Zebrafish Information Network (ZFIN), expression data for ccdc47 includes at least one figure from Thisse et al. (2004), suggesting developmental regulation of expression .

While detailed expression information specifically for zebrafish is limited in the provided search results, insights from other model systems indicate that CCDC47 plays an essential role in early development. In mouse models, Ccdc47 knockout leads to embryonic lethality featuring delayed development, atrophic neural tubes, heart abnormalities, and deficient blood cell production in the dorsal aorta .

To effectively study ccdc47 expression patterns in zebrafish development, researchers should consider:

• In situ hybridization to visualize tissue-specific expression

• Quantitative PCR at different developmental stages

• Single-cell transcriptomics utilizing databases like FishSCT that contain zebrafish single-cell transcriptomic data covering various tissues/organs across 36 different developmental time points

• Transgenic reporter lines expressing fluorescent proteins under the ccdc47 promoter

Understanding these expression patterns can provide valuable insights into the protein's developmental roles and tissue-specific functions.

How conserved is ccdc47 between zebrafish and human models?

The ccdc47 gene demonstrates significant evolutionary conservation between zebrafish and humans, reflecting its fundamental importance in cellular function. The zebrafish ccdc47 is explicitly identified as orthologous to human CCDC47 , indicating a shared evolutionary origin and suggesting functional conservation.

Several features support this orthologous relationship:

• Both genes encode proteins with similar structural features including coiled-coil domains and transmembrane segments

• Both are predicted to function in calcium homeostasis and ER-related processes

• Both likely participate in the multi-pass translocon complex

The high degree of conservation suggests that insights gained from studying ccdc47 in zebrafish models can provide valuable understanding of human CCDC47 function. This conservation makes zebrafish an appropriate model organism for studying ccdc47-related pathologies observed in humans, such as the multisystem disorder associated with bi-allelic CCDC47 variants characterized by woolly hair, liver dysfunction, and developmental delays .

For researchers leveraging this conservation, comparative sequence analysis, functional complementation studies, and cross-species rescue experiments provide powerful approaches to translate findings between model systems.

What cellular localization patterns does ccdc47 exhibit in zebrafish cells?

In zebrafish cells, ccdc47 is predicted to be predominantly localized to the endoplasmic reticulum (ER) membrane . This localization pattern is consistent with findings from mammalian systems, where CCDC47 functions as an ER transmembrane protein involved in calcium homeostasis .

Specifically, zebrafish ccdc47 is:

• Predicted to be located in the endoplasmic reticulum membrane

• Expected to be part of the multi-pass translocon complex

• Predicted to be active in the endoplasmic reticulum

This localization is functionally significant as it positions ccdc47 to participate in ER-associated calcium regulation, protein folding, and membrane protein insertion processes . The protein's transmembrane domain is likely crucial for its proper integration into the ER membrane.

To study ccdc47 localization in zebrafish cells, researchers should consider:

• Immunofluorescence with anti-ccdc47 antibodies along with ER markers

• Fluorescent protein fusion constructs for live-cell imaging

• Subcellular fractionation followed by Western blotting

• Electron microscopy with immunogold labeling for high-resolution localization

These approaches can help elucidate the precise distribution of ccdc47 within cellular compartments and provide insights into its functional interactions.

What experimental approaches are most effective for studying ccdc47 function in calcium homeostasis?

To investigate ccdc47's role in calcium homeostasis in zebrafish models, researchers should implement multifaceted experimental approaches that capture both cellular and organism-level effects:

Cellular Calcium Imaging Techniques:

• Fluorescent calcium indicators (Fluo-4, Fura-2) to measure intracellular calcium dynamics in isolated cells or tissues

• Genetically encoded calcium indicators (GCaMP variants) for in vivo calcium visualization

• Ionomycin-induced calcium release and reuptake measurements, as demonstrated in studies where CCDC47 overexpression affected these parameters

• Store-operated calcium entry (SOCE) assays to assess ER calcium storage capacity, which has been shown to be impaired in cells with CCDC47 dysfunction

Genetic Manipulation Approaches:

• CRISPR/Cas9-mediated gene editing to generate ccdc47 knockout or knock-in zebrafish lines

• Morpholino-based transient knockdown for rapid assessment of developmental phenotypes

• Conditional expression systems to study stage-specific functions

Functional Assessment Methods:

• IP3R calcium release channel activity measurements to assess impacts on downstream signaling

• ER calcium storage capacity assays, given findings that CCDC47 dysfunction leads to decreased total ER Ca2+ storage

• Protein-protein interaction studies focusing on ccdc47's association with other calcium handling proteins

Studies in mammalian systems have shown that CCDC47 affects ionomycin-induced calcium release and reuptake , suggesting similar experimental paradigms would be productive in zebrafish models. Additionally, measuring ER calcium refilling via store-operated calcium entry would be particularly informative given prior observations of its impairment in CCDC47-deficient cells .

How does dysregulation of ccdc47 affect zebrafish cardiac development and function?

The relationship between ccdc47 dysregulation and cardiac development in zebrafish represents an important research frontier, particularly given evidence from mammalian models linking CCDC47 to cardiac pathologies:

Developmental Impact:
Studies in mouse models demonstrate that Ccdc47 knockout results in heart abnormalities and embryonic lethality , suggesting that similar developmental cardiac defects might occur in zebrafish with ccdc47 disruption. While zebrafish-specific cardiac phenotypes await comprehensive characterization, the evolutionary conservation of ccdc47 suggests potential developmental cardiac roles.

Functional Cardiac Effects:
Evidence from mammalian systems indicates that CCDC47 dysregulation is associated with diabetic cardiomyopathy. In a diet-induced obese rat model, CCDC47 mRNA expression increased in both atrium and ventricle, while protein expression significantly increased specifically in the atrium . This differential expression pattern suggests tissue-specific regulatory mechanisms that might also exist in zebrafish hearts.

Research Methodology:
Researchers investigating ccdc47's cardiac effects in zebrafish should consider:

• High-resolution cardiac imaging in transparent zebrafish embryos

• Heart-specific conditional knockout models

• Calcium transient measurements in isolated zebrafish cardiomyocytes

• Functional assessments including heart rate, contractility, and response to stress

• Molecular profiling of heart chambers to detect differential expression patterns similar to those observed in mammalian models

Intriguingly, in mammalian models, no changes in established cardiac stress markers (ANP, BNP, β-MHC) were observed despite CCDC47 dysregulation , suggesting that ccdc47 might represent an early or alternative pathway in cardiac pathophysiology—a hypothesis that could be efficiently tested in zebrafish models.

What techniques yield highest purity recombinant ccdc47 for structural studies?

Obtaining high-purity recombinant zebrafish ccdc47 for structural and functional studies presents several technical challenges due to its transmembrane domain and calcium-binding properties. Based on approaches used for similar proteins, the following optimized methodology is recommended:

Expression Systems:

• Bacterial systems (E. coli): May be suitable for expressing soluble domains but challenging for full-length protein due to transmembrane regions

• Insect cell systems (Sf9, Hi5): Offer improved folding and post-translational modifications

• Mammalian expression systems: Provide native-like folding environment, particularly important for calcium-binding properties

Construct Design Considerations:

• Truncation constructs excluding the transmembrane domain for soluble expression

• Fusion tags that enhance solubility (MBP, SUMO, GST)

• Addition of purification tags (His6, FLAG, Strep-II) positioned to avoid interference with functional domains

• Codon optimization for the expression system of choice

Purification Strategy:

• Initial capture using affinity chromatography (IMAC for His-tagged constructs)

• Tag removal using site-specific proteases

• Ion exchange chromatography to separate charged species (particularly important given ccdc47's predicted acidic isoelectric point)

• Size exclusion chromatography as a final polishing step

• Consider calcium-free and calcium-bound states during purification to control protein conformation

Quality Control Assessments:

• Dynamic light scattering to assess monodispersity

• Circular dichroism to confirm secondary structure

• Thermal shift assays to evaluate stability

• Functional calcium-binding assays to confirm activity

How can researchers effectively study ccdc47's role in ER stress response?

CCDC47's involvement in the endoplasmic reticulum overload response makes it an interesting target for studying ER stress mechanisms in zebrafish. A comprehensive experimental approach would include:

ER Stress Induction Protocols:

• Pharmacological inducers: tunicamycin (N-glycosylation inhibitor), thapsigargin (SERCA inhibitor), DTT (disulfide bond reducer)

• Physiological stressors: glucose deprivation, hypoxia, calcium depletion

• Genetic models: overexpression of misfolding-prone proteins

Molecular Readouts of ER Stress:

• Unfolded Protein Response (UPR) activation markers:

  • Phosphorylation of PERK, IRE1α

  • XBP1 splicing assays

  • ATF6 cleavage and nuclear translocation

  • Upregulation of chaperones (BiP/GRP78, GRP94)

• Calcium homeostasis measurements:

  • ER calcium store depletion

  • Cytosolic calcium fluctuations

  • Store-operated calcium entry dynamics

Genetic Manipulation Approaches:

• Tissue-specific or inducible ccdc47 knockdown/knockout

• Rescue experiments with wild-type vs. mutant ccdc47

• Structure-function studies using domain-specific mutations

Experimental Systems:

• Zebrafish embryos: allowing visualization of whole-organism effects

• Primary cultures of zebrafish cells: for detailed cellular analysis

• Organ explants: for tissue-specific responses

Data Integration Framework:

• Correlate ccdc47 expression levels with ER stress markers

• Assess whether ccdc47 manipulation affects threshold for ER stress induction

• Determine if ccdc47 acts upstream or downstream of canonical UPR pathways

• Evaluate whether calcium perturbations modify ccdc47-dependent phenotypes

Given findings that CCDC47-deficient cells show impaired calcium signaling and reduced ER calcium storage , researchers should particularly focus on whether ccdc47 represents a link between calcium homeostasis and canonical ER stress pathways in zebrafish models.

What are the methodological challenges in analyzing ccdc47 expression in single-cell transcriptomic data from zebrafish?

Single-cell transcriptomic analysis of ccdc47 expression in zebrafish presents several technical and analytical challenges that researchers should address:

Data Source Considerations:
The FishSCT database contains extensive single-cell transcriptomic data for zebrafish, covering 25 different tissue/organ types across 36 developmental time points . This resource provides a valuable starting point for analyzing ccdc47 expression at single-cell resolution, but several methodological challenges remain:

Technical Challenges:

• Low expression detection: As a regulatory protein, ccdc47 may be expressed at low levels in some cell types, potentially falling below detection thresholds in droplet-based single-cell RNA sequencing

• Transcript dropout: Single-cell data often suffers from technical dropouts where expressed genes appear absent

• Batch effects: Integration of data across multiple experiments/platforms requires normalization

• Isoform discrimination: Detecting potential alternative transcripts of ccdc47

Analytical Approaches:

• Cell type annotation refinement:

  • Use established marker genes to accurately identify cell populations

  • Implement machine learning algorithms for unsupervised clustering

  • Cross-reference with published zebrafish cell atlases

• Expression analysis strategies:

  • Implement imputation methods to address dropouts for lowly expressed genes

  • Use RNA velocity analysis to determine transcriptional dynamics

  • Perform pseudotime analysis to track ccdc47 expression changes during differentiation

  • Apply spatial transcriptomic methods to correlate expression with anatomical context

• Correlation analyses:

  • Identify gene modules co-expressed with ccdc47

  • Perform pathway enrichment among correlated genes

  • Compare expression patterns with other calcium-handling genes

• Validation approaches:

  • Confirm key findings using in situ hybridization

  • Validate cell-type specific expression with immunohistochemistry

  • Use reporter constructs to track expression in live embryos

When utilizing resources like FishSCT , researchers should carefully evaluate quality metrics, assess coverage depth, and consider complementary validation of important findings through alternative experimental methods.

How does the function of ccdc47 in calcium homeostasis differ between zebrafish and mammalian models?

Understanding the similarities and differences in ccdc47 function between zebrafish and mammalian models is essential for translational research. Both systems share core functional properties but demonstrate species-specific characteristics:

Conserved Functional Elements:

• Endoplasmic reticulum membrane localization

• Calcium ion binding capabilities

• Involvement in ER calcium homeostasis

• Essential role in embryonic development

Potential Species-Specific Differences:

Research Approaches to Address Differences:

• Comparative protein domain analysis to identify divergent regions

• Cross-species rescue experiments (e.g., can human CCDC47 rescue zebrafish ccdc47 knockout?)

• Targeted mutagenesis of species-specific residues

• Parallel calcium imaging studies in both zebrafish and mammalian cells

While zebrafish ccdc47 likely maintains core functional properties similar to its mammalian ortholog, species-specific adaptations may exist that influence its precise role in calcium signaling networks. Understanding these nuances is crucial for appropriate translation between model systems.

What methods best characterize post-translational modifications of ccdc47 in zebrafish?

Post-translational modifications (PTMs) likely play a crucial role in regulating ccdc47 function, particularly in calcium homeostasis and ER-related processes. While specific information about zebrafish ccdc47 PTMs is limited, the following methodological approaches would enable comprehensive characterization:

Mass Spectrometry-Based Approaches:

• Sample Preparation Strategies:

  • Immunoprecipitation of endogenous ccdc47 from zebrafish tissues

  • Expression of tagged recombinant ccdc47 followed by affinity purification

  • Subcellular fractionation to enrich ER membrane proteins

• MS Analysis Methods:

  • Bottom-up proteomics using tryptic digestion

  • Middle-down approaches for improved PTM site localization

  • Top-down proteomics for intact protein analysis

  • Targeted MS methods (PRM/MRM) for quantifying specific modifications

• Enrichment Strategies for Specific PTMs:

  • Phosphorylation: TiO₂, IMAC, phospho-specific antibodies

  • Glycosylation: Lectin affinity, hydrazide chemistry

  • Ubiquitination: K-ε-GG antibodies, TUBEs

  • Disulfide bonds: Differential alkylation strategies

Complementary Techniques:

• Phosphorylation-specific antibodies (if available for conserved sites)

• Mobility shift assays to detect modified forms

• Site-directed mutagenesis of putative modification sites

• In vitro kinase/phosphatase assays

• Chemical inhibitors of specific PTM-related enzymes

Bioinformatic Prediction and Analysis:

• Comparative analysis with known mammalian CCDC47 modifications

• PTM site prediction algorithms

• Conservation analysis of putative modification sites

• Structural modeling to assess potential functional impacts of modifications

Considering that human CCDC47 contains two predicted disulfide bonds , special attention should be given to characterizing the oxidation state of corresponding cysteine residues in zebrafish ccdc47. Additionally, given CCDC47's role in calcium handling, phosphorylation sites that might regulate its activity in response to calcium-dependent kinases would be particularly interesting targets for investigation.