Recombinant Danio rerio Coiled-coil domain-containing protein 25 (ccdc25)

What is the molecular structure and basic characterization of zebrafish ccdc25?

Zebrafish ccdc25 (also known as zgc:64173) is a protein-coding gene that encodes coiled-coil domain containing protein 25 . While detailed structural analysis of zebrafish ccdc25 remains limited, the protein belongs to the coiled-coil domain family characterized by alpha-helical structures that form supercoiled regions facilitating protein-protein interactions.

Current genomic data indicates that zebrafish ccdc25 contains conserved coiled-coil motifs that likely mediate its biological functions through interaction with other proteins. The predicted protein structure shares homology with human CCDC25, which is a 208-amino acid protein with a molecular weight of approximately 25 kDa .

Comparative analysis methodology:

• Sequence analysis using MUSCLE or CLUSTAL alignments to identify conserved regions

• Secondary structure prediction using PSIPRED or similar tools

• Domain identification using InterProScan or SMART databases

• Homology modeling based on related coiled-coil domain proteins

How is ccdc25 temporally and spatially expressed during zebrafish development?

While the search results don't provide specific temporal expression data for ccdc25 in zebrafish, insights can be gained from expression patterns of other genes in zebrafish. For developmental gene expression profiling, techniques similar to those used for analyzing slc39a14 (which is expressed during embryonic and early larval development between 3-120 hpf ) would be applicable.

For determining spatial expression patterns, methodologies include:

• Whole-mount in situ hybridization (WISH) with RNA probes specific to ccdc25 at key developmental stages (similar to techniques used for slc39a14 )

• Fluorescent in situ hybridization for co-localization studies

• Transgenic reporter lines with the ccdc25 promoter driving fluorescent protein expression

• RT-qPCR on dissected tissues to quantify expression levels

Previous studies of coiled-coil domain proteins in zebrafish have revealed diverse expression patterns, ranging from ubiquitous to highly tissue-specific. For example, studies of CCDC56 demonstrated its essential role in mitochondrial function, particularly in cytochrome c oxidase (COX) activity .

What are the optimal approaches for generating ccdc25 knockout models in zebrafish?

Based on successful strategies used for other zebrafish genes, CRISPR/Cas9 genome editing provides the most efficient approach for generating ccdc25 knockout models.

Recommended CRISPR/Cas9 protocol for ccdc25 knockout:

• gRNA design:

  • Target early exons to ensure complete loss of function

  • Use algorithms like CHOPCHOP or CRISPRscan to design multiple gRNAs with high specificity

  • Validate gRNA efficiency using in vitro assays

• Microinjection procedure:

  • Inject 2-3 nl of solution containing:

    • 300 ng/μl Cas9 mRNA or 500 ng/μl Cas9 protein

    • 25-50 ng/μl gRNA

    • 0.05% phenol red as injection tracer

  • Target one-cell stage embryos

• Mutation screening:

  • High-resolution melt analysis (HRMA) for rapid screening (as used for slc39a14 )

  • T7 endonuclease I assay as an alternative screening method

  • DNA sequencing to confirm exact mutations

• Establishing mutant lines:

  • Raise F0 injected fish to adulthood

  • Outcross to wild-type fish to identify germline transmission

  • Incross F1 heterozygotes to generate homozygous mutants for phenotypic analysis

For complete loss of function studies, additional strategies may be needed to address maternal contribution, such as generating maternal-zygotic mutants through germline replacement.

How can protein-protein interactions of zebrafish ccdc25 be effectively characterized?

Characterizing protein-protein interactions is crucial for understanding ccdc25 function. Based on approaches used for other zebrafish proteins, the following methodological strategy is recommended:

Step-by-step interaction analysis protocol:

• Bait protein preparation:

  • Generate epitope-tagged ccdc25 constructs (FLAG, HA, or GFP tags)

  • Express recombinant protein using bacterial or cell-free systems

  • Alternative: Create transgenic zebrafish expressing tagged ccdc25

• Primary interaction screening:

  • Co-immunoprecipitation (Co-IP) followed by mass spectrometry

  • Yeast two-hybrid screening

  • BioID or proximity labeling approaches

• Validation methods:

  • Reciprocal Co-IP experiments

  • GST pulldown assays with recombinant proteins

  • BiFC (Bimolecular Fluorescence Complementation) in zebrafish cells

• In vivo confirmation:

  • Co-localization studies using confocal microscopy

  • FRET (Förster Resonance Energy Transfer) analysis

  • Genetic interaction studies through combined knockdown/knockout approaches

Given the coiled-coil structure of ccdc25, interactions are likely mediated through these domains, which should be considered when designing truncation constructs for domain-specific interaction mapping.

What role might ccdc25 play in cell proliferation and embryonic development?

While specific information about ccdc25's role in zebrafish development is limited, insights can be drawn from research on other zebrafish proteins. Cell proliferation is compartmentalized at the posterior end of zebrafish embryos through regulated expression of mitotic factors like Cdc25a . Given that some coiled-coil domain proteins are involved in cell cycle regulation, ccdc25 might have similar functions.

Experimental approaches to investigate developmental roles:

• Cell proliferation analysis:

  • BrdU or EdU incorporation assays in wild-type vs. ccdc25 mutants

  • Phospho-histone H3 immunostaining to detect mitotic cells

  • Cell cycle analysis using FACS in dissociated embryonic cells

• Developmental phenotyping:

  • Detailed morphological analysis at key developmental stages

  • Time-lapse imaging of embryonic development

  • Lineage tracing using photoconvertible fluorescent proteins

• Molecular pathway analysis:

  • Gene expression profiling using RNA-seq

  • Assessment of cell cycle regulators (cyclins, CDKs)

  • Analysis of developmental signaling pathways (Wnt, Notch, FGF)

Stat3/Cdc25a-dependent cell proliferation promotes embryonic axis extension during zebrafish gastrulation , suggesting that proteins involved in cell cycle regulation can have significant impacts on developmental processes. If ccdc25 functions in similar pathways, it might influence axis formation, gastrulation movements, or tissue-specific development.

How can zebrafish ccdc25 studies inform understanding of human diseases?

Human CCDC25 has been implicated in several disease contexts, including hepatocellular carcinoma , cholangiocarcinoma , and metabolic syndrome . Zebrafish models can provide valuable insights into these conditions through comparative functional studies.

Table 1: Disease associations of CCDC25 and potential zebrafish model applications

For disease modeling, key approaches include:

• Generating ccdc25 mutations that mirror human disease variants

• Creating liver-specific or other tissue-specific manipulations of ccdc25 expression

• Combining ccdc25 manipulation with established disease models

• Conducting drug screens to identify compounds that modulate ccdc25-dependent phenotypes

How can cellular localization and trafficking of ccdc25 be accurately determined in zebrafish?

Understanding the subcellular localization of ccdc25 is crucial for elucidating its function. While CCDC25 has been reported on the cell surface of cancer cells in humans , its localization in zebrafish has not been specifically described.

Comprehensive localization analysis protocol:

• Fluorescent fusion proteins:

  • Generate N- and C-terminal fluorescent protein fusions (GFP, mCherry)

  • Create stable transgenic lines with tissue-specific promoters

  • Validate functionality of fusion proteins through rescue experiments

• Subcellular marker co-localization:

  • Co-expression with established organelle markers:

    • Nucleus: H2B-BFP

    • Mitochondria: MitoTracker or Tom20-RFP

    • ER: Sec61-mCherry

    • Golgi: GalT-RFP

    • Cell membrane: membrane-targeted fluorescent proteins

• Super-resolution microscopy:

  • STED or PALM/STORM imaging for nanoscale localization

  • Live cell imaging to capture dynamic trafficking events

  • FRAP (Fluorescence Recovery After Photobleaching) to assess protein mobility

• Biochemical fractionation:

  • Cell fractionation followed by Western blotting

  • Density gradient centrifugation for membrane subdomains

  • Protease protection assays for membrane topology

Similar approaches have been successfully used to determine subcellular localization of other zebrafish proteins, such as CCDC56 which was shown to localize to mitochondria .

What approaches can resolve contradictory findings in ccdc25 research?

Scientific research often produces seemingly contradictory results due to methodological differences or biological complexity. For ccdc25 research, systematic approaches can help reconcile discrepancies.

Strategies for resolving contradictory findings:

• Methodological standardization:

  • Document complete experimental conditions including:

    • Zebrafish strain and genetic background

    • Developmental stage and temperature

    • Detailed protocol parameters

  • Use multiple detection methods (e.g., different antibodies, tagged constructs)

  • Implement blinded analysis where possible

• Genetic and biological factors:

  • Test for genetic modifiers by using different zebrafish backgrounds

  • Consider maternal vs. zygotic contributions

  • Evaluate tissue-specific effects through conditional approaches

  • Assess dosage sensitivity through heterozygote analysis

• Integration of multiple approaches:

  • Combine knockdown, knockout, and overexpression studies

  • Utilize both in vivo and in vitro systems

  • Perform time-course experiments to capture dynamic processes

  • Apply systems biology approaches to model complex interactions

• Collaborative validation:

  • Independent replication in different laboratories

  • Data sharing through repositories

  • Multi-lab consortium approaches for complex phenotypes

Systematic documentation and reporting of negative results can be particularly valuable for resolving contradictions in emerging research areas.

How can high-throughput approaches advance understanding of ccdc25 function?

Modern high-throughput technologies offer powerful approaches for rapidly advancing understanding of ccdc25 biology in zebrafish.

High-throughput methodologies applicable to ccdc25 research:

• Transcriptomic approaches:

  • Single-cell RNA-seq to identify cell populations expressing ccdc25

  • Spatial transcriptomics to map expression in intact tissues

  • Time-series RNA-seq to capture dynamic expression changes

  • Differential expression analysis in ccdc25 mutants

• Proteomic strategies:

  • Proximity labeling coupled with mass spectrometry

  • Phosphoproteomics to identify post-translational modifications

  • Thermal proteome profiling to identify protein interactions

  • CETSA (Cellular Thermal Shift Assay) for drug target engagement

• Functional genomics:

  • CRISPR screens to identify genetic interactors

  • Chemical-genetic interaction screens

  • Enhancer/suppressor screens in sensitized backgrounds

  • Synthetic lethality approaches

• High-content imaging:

  • Automated phenotyping of embryonic development

  • Machine learning-based image analysis

  • 4D live imaging with computational tracking

  • High-throughput behavioral analysis

Table 2: High-throughput technologies and their applications to ccdc25 research

What are the promising therapeutic applications of targeting ccdc25 in disease models?

Based on human studies showing CCDC25's potential as a biomarker for multiple conditions , zebrafish models could be valuable for therapeutic development.

Therapeutic development framework:

• Target validation in zebrafish:

  • Phenotypic rescue experiments with chemical compounds

  • Structure-function studies to identify critical domains

  • Conditional expression systems to define therapeutic windows

• Drug discovery approaches:

  • Small molecule screens using embryo-based phenotypic assays

  • In silico docking to identified binding pockets

  • Peptide inhibitors targeting protein-protein interactions

  • Antisense approaches for targeted knockdown

• Delivery system development:

  • Nanoparticle-based delivery testing in zebrafish

  • Tissue-specific targeting strategies

  • Evaluation of off-target effects

  • PK/PD studies in larval and adult zebrafish

• Translational considerations:

  • Conservation of drug targets between zebrafish and humans

  • Biomarker development for treatment response

  • Combination therapies targeting multiple pathway components

  • Repurposing of approved drugs that modulate ccdc25 pathways

Given the association of CCDC25 with metabolic syndrome , liver cancer , and cholangiocarcinoma , therapeutic approaches targeting this protein could potentially address multiple pathological conditions.

What are the most promising future research directions for zebrafish ccdc25 studies?

Based on current knowledge and technological capabilities, several promising research directions emerge:

• Developmental biology:

  • Characterize spatial and temporal expression patterns throughout development

  • Determine cell type-specific functions in key developmental processes

  • Investigate potential roles in tissue patterning and morphogenesis

• Molecular mechanisms:

  • Define the ccdc25 interactome in different cellular contexts

  • Identify post-translational modifications and their functional significance

  • Determine structure-function relationships through domain analysis

• Disease modeling:

  • Develop zebrafish models for CCDC25-associated human diseases

  • Investigate cancer-related functions, particularly in liver cancers

  • Explore potential roles in metabolic regulation

• Therapeutic applications:

  • Screen for compounds that modulate ccdc25 function

  • Develop targeted approaches for disease intervention

  • Explore ccdc25 as a biomarker for disease progression or treatment response

The emerging role of human CCDC25 as a potential marker for conditions like metabolic syndrome suggests that zebrafish models could provide valuable insights into both basic biology and translational applications.