Recombinant Danio rerio Coiled-coil domain-containing protein 56 (ccdc56)

What is CCDC56 and what is its function in Danio rerio?

CCDC56 (Coiled-coil domain-containing protein 56) is a small protein of 96 amino acids expressed in zebrafish (Danio rerio). Based on comparative studies with homologs in other species, CCDC56 functions as a cytochrome c oxidase (COX) assembly factor, playing a crucial role in the proper assembly and function of Complex IV of the mitochondrial respiratory chain. The protein contains characteristic coiled-coil domains that facilitate protein-protein interactions essential for its assembly function. Research in Drosophila has demonstrated that CCDC56 is necessary for COX function and organism viability, suggesting similar critical roles in zebrafish development and physiology .

What is the molecular structure of Danio rerio CCDC56?

Danio rerio CCDC56 is a small protein with the following amino acid sequence: MSSQGEPKPEAQFAKRIDPTKEALTKEQLQFIRQVEMAQWKKKTDKLRGRNVATGLAIGAVVLGIYGYTFYSVSQEKIMDEIDEEAKVRVPKTGAN . The protein possesses coiled-coil structural motifs, which are characterized by heptad repeats that form alpha-helical structures. These domains typically mediate protein-protein interactions and molecular recognition. The protein has a UniProt accession number of A8KB87 and is encoded by the gene ccdc56 (also known by its ORF name zgc:171846) .

Why use zebrafish as a model organism for CCDC56 research?

Zebrafish present several advantages for studying CCDC56 function:

• Rapid development and high fecundity (300-600 eggs per female) allow for large-scale experiments with short timeframes .

• Optical transparency of embryos enables real-time imaging of developmental processes without invasive procedures .

• External fertilization provides easy access to embryos without maternal compartment influences .

• Zebrafish embryos can be precisely staged, allowing for consistent developmental exposure windows in experimental studies .

• Behavioral studies can be conducted on very early stages as swimming begins at hatching (48-72 hpf) .

• The zebrafish genome has been fully sequenced, facilitating genetic manipulation and comparative studies across species.

Importantly, CCDC56 appears to be evolutionarily conserved across species, with research suggesting functional homology between zebrafish, Drosophila, and human versions of the protein .

How does CCDC56 affect mitochondrial function in Danio rerio compared to other model organisms?

Based on studies in Drosophila, CCDC56 knockout results in significant reduction of fully assembled cytochrome c oxidase (COX) and its activity, while other oxidative phosphorylation complexes remain either unaffected or show increased activity . In zebrafish, we would expect similar mitochondrial dysfunction patterns, though species-specific differences may exist.

The comparative analysis between zebrafish and Drosophila CCDC56 function reveals important evolutionary conservation:

The differences in protein length and sequence between species suggest potential functional adaptations that warrant investigation in zebrafish-specific studies .

What are the optimal expression systems for producing recombinant Danio rerio CCDC56?

For efficient expression of functional recombinant Danio rerio CCDC56, several expression systems can be considered:

• E. coli expression systems: Suitable for basic structural studies but may lack post-translational modifications. Using BL21(DE3) strains with pET vectors under T7 promoter control typically yields high protein quantities. Optimization of induction conditions (IPTG concentration, temperature, duration) is critical for preventing inclusion body formation of this mitochondrial protein.

• Insect cell systems: Baculovirus-infected Sf9 or Hi5 cells offer eukaryotic post-translational modifications. This system is particularly advantageous when studying CCDC56 interactions with other mitochondrial proteins.

• Mammalian expression systems: HEK293 or CHO cells transfected with plasmids containing strong promoters (CMV) provide mammalian-specific modifications and folding environments, valuable for functional studies.

The recombinant CCDC56 protein should be produced with appropriate tags for purification (His, GST, or FLAG) that can be removed by protease cleavage if necessary for functional assays. Storage in a Tris-based buffer with 50% glycerol at -20°C (or -80°C for extended storage) helps maintain protein stability, with caution against repeated freeze-thaw cycles .

What phenotypes are observed in zebrafish CCDC56 knockout models?

While specific zebrafish CCDC56 knockout phenotypes are not explicitly detailed in the provided search results, we can make informed predictions based on Drosophila studies. In Drosophila, CCDC56 knockout resulted in:

• Developmental delay

• 100% lethality by arrest of larval development at the third instar

• Significant decrease in fully assembled cytochrome c oxidase (COX) levels

• Reduced COX activity while other oxidative phosphorylation complexes remained unaffected or showed increased activity

In zebrafish, we would expect CCDC56 knockouts to exhibit similar mitochondrial dysfunction, particularly affecting tissues with high energy demands such as the nervous system, cardiac tissue, and skeletal muscle. Possible observable phenotypes might include:

• Developmental arrest or delay

• Cardiac dysfunction (reduced heart rate, arrhythmias)

• Reduced swimming capacity

• Neurological abnormalities

• Increased susceptibility to oxidative stress

• Metabolic abnormalities reflecting mitochondrial dysfunction

These predictions are based on the evolutionary conservation of CCDC56 function and common consequences of COX dysfunction across species .

What are the recommended protocols for CCDC56 knockout generation in zebrafish?

To generate CCDC56 knockout models in zebrafish, researchers should consider the following methodological approaches:

• CRISPR/Cas9 genome editing:

  • Design sgRNAs targeting early exons of the ccdc56 gene

  • Inject Cas9 protein and sgRNAs into one-cell stage embryos

  • Screen F0 mosaic embryos for mutations using T7 endonuclease assay

  • Raise potential founders to adulthood and outcross to identify germline transmission

  • Establish stable homozygous lines through F1 and F2 generations

• Morpholino knockdown (for transient loss of function):

  • Design splice-blocking or translation-blocking morpholinos

  • Inject into one-cell stage embryos

  • Include appropriate controls (mismatch morpholinos)

  • Validate knockdown efficiency by RT-PCR or Western blotting

• P-element excision-based methods (adapted from Drosophila studies):

  • While less common in zebrafish, similar transposon-based approaches could be used

  • This approach was successfully employed in Drosophila to create ccdc56 knockout lines (ccdc56 D6 and ccdc56 D11)

Validation of knockout models should include:

• DNA sequencing to confirm mutations

• RT-PCR and Western blotting to verify absence of CCDC56 expression

• Functional assays for COX activity (spectrophotometric or histochemical)

• Phenotypic characterization at multiple developmental stages

How can recombinant CCDC56 be used to rescue knockout phenotypes?

Rescue experiments provide crucial evidence for the specificity of knockout phenotypes and the function of CCDC56. The following methodological approach is recommended:

• Generation of rescue constructs:

  • Clone the wild-type zebrafish ccdc56 cDNA into expression vectors

  • Create vectors with tissue-specific promoters for targeted rescue

  • Consider including epitope tags (FLAG, HA) for detection if not interfering with function

• Delivery methods:

  • Microinjection of mRNA into one-cell stage embryos for immediate but transient expression

  • Tol2 transposase-mediated transgenesis for stable integration

  • Use of the UAS-GAL4 system for conditional expression (similar to the UAS-ccdc56 transgene approach that partially rescued lethality and COX deficiency in Drosophila)

• Assessment of rescue efficiency:

  • Quantify survival rates and developmental progression

  • Measure COX activity restoration using enzymatic assays

  • Analyze restoration of normal mitochondrial morphology by electron microscopy

  • Conduct behavioral assays to assess functional recovery

For controlled temporal expression, consider using heat shock-inducible or chemical-inducible (e.g., Cd2+-inducible metallothionein promoter) expression systems similar to those used in mammalian Cre recombinase studies .

What techniques are most effective for analyzing CCDC56 interactions with the COX assembly pathway?

To investigate CCDC56 interactions within the COX assembly pathway, researchers should employ multiple complementary approaches:

• Co-immunoprecipitation (Co-IP):

  • Express tagged versions of CCDC56 in zebrafish embryos or cell lines

  • Perform Co-IP followed by mass spectrometry to identify interaction partners

  • Validate specific interactions with known COX assembly factors

• Proximity labeling techniques:

  • APEX2 or BioID fusion proteins can identify proximal proteins in the mitochondrial environment

  • These approaches are particularly valuable for transient interactions during assembly processes

• Mitochondrial isolation and blue native PAGE:

  • Isolate intact mitochondria from CCDC56 wild-type and knockout models

  • Analyze COX assembly intermediates using blue native PAGE

  • Western blotting with antibodies against COX subunits can identify specific assembly defects

• Live-cell imaging:

  • Leverage the optical transparency of zebrafish embryos for in vivo imaging

  • Use fluorescent protein fusions to track CCDC56 localization during development

  • Employ zebrafish embryo microfluidic culture systems for continuous observation

• Proteomic analysis:

  • Compare the mitochondrial proteome between wild-type and CCDC56-deficient samples

  • Identify accumulating assembly intermediates or altered abundance of other assembly factors

These techniques should be applied systematically to build a comprehensive model of CCDC56's role in COX assembly, similar to the approach used in Drosophila studies that established CCDC56 as a putative COX assembly factor .

How should researchers interpret conflicting data on CCDC56 function across different model systems?

When faced with conflicting data on CCDC56 function across model systems (e.g., zebrafish vs. Drosophila vs. mammalian systems), researchers should:

• Systematically compare experimental conditions:

  • Developmental stages examined (precisely staged embryos are crucial as emphasized in zebrafish studies)

  • Knockdown/knockout approaches used (complete vs. partial loss of function)

  • Methods used to assess mitochondrial function

• Consider evolutionary context:

  • Despite 42% amino acid identity between Drosophila and human CCDC56, functional conservation may be higher

  • Domain-specific functions may be preserved even with sequence divergence

  • Compare phenotypes in the context of species-specific developmental programs

• Evaluate tissue-specific effects:

  • CCDC56 function may vary across tissues with different energy demands

  • Use of tissue-specific promoters in rescue experiments can help resolve apparent conflicts

• Perform direct comparative studies:

  • Express zebrafish CCDC56 in CCDC56-deficient Drosophila (or vice versa) to test functional equivalence

  • Conduct parallel experiments in multiple systems under standardized conditions

A methodical approach to resolving conflicts should include:

What are the best quantitative methods for measuring CCDC56's impact on COX assembly and activity?

To rigorously quantify CCDC56's impact on COX assembly and activity, researchers should employ these methods:

• Spectrophotometric enzyme activity assays:

  • Measure COX activity in isolated mitochondria using reduced cytochrome c as substrate

  • Calculate enzyme kinetics parameters (Vmax, Km) to assess catalytic efficiency

  • Normalize to other mitochondrial enzymes (e.g., citrate synthase) as internal controls

• Blue Native PAGE coupled with quantitative Western blotting:

  • Separate intact respiratory complexes and supercomplexes

  • Quantify the proportion of fully assembled COX vs. assembly intermediates

  • Track specific subunits using antibodies against nuclear and mitochondrially encoded components

• Oxygraphy (respirometry):

  • Measure oxygen consumption rates in isolated mitochondria or intact cells

  • Determine COX-dependent respiration using specific substrates and inhibitors

  • Assess coupling efficiency and respiratory control ratios

• In-gel activity assays:

  • Perform enzymatic staining after blue native PAGE to directly visualize active COX

  • Quantify band intensity relative to controls

• Pulse-chase labeling:

  • Track assembly kinetics and stability of newly synthesized mitochondrially encoded COX subunits

  • Compare half-lives and incorporation rates between wild-type and CCDC56-deficient samples

Based on Drosophila studies, CCDC56 knockout resulted in significant decreases in fully assembled COX and its activity, while other oxidative phosphorylation complexes remained either unaffected or showed increased activity. This specific pattern provides a quantitative signature of CCDC56 deficiency that should be systematically measured in zebrafish models .

How can zebrafish CCDC56 research inform human mitochondrial disease studies?

Zebrafish CCDC56 research can significantly contribute to human mitochondrial disease understanding in several ways:

• Model for COX deficiency disorders:

  • CCDC56-deficient zebrafish can serve as in vivo models for human mitochondrial diseases involving COX deficiency

  • The optical transparency and external development of zebrafish embryos enable real-time visualization of disease progression

• Drug screening platform:

  • Zebrafish embryos provide a cost-effective system for high-throughput screening of compounds that might restore COX function

  • The rapid development of zebrafish allows for quick assessment of drug effects on development and COX activity

• Genetic modifier identification:

  • Forward genetic screens in CCDC56-deficient zebrafish can identify suppressors or enhancers of the phenotype

  • These genetic interactions may reveal novel therapeutic targets for human mitochondrial diseases

• Translational validation:

  • Human CCDC56 variants of uncertain significance can be functionally characterized through expression in zebrafish knockouts

  • This approach may help classify human variants as pathogenic or benign

The evolutionary conservation of CCDC56 (42% amino acid identity between Drosophila and human versions) suggests that findings in zebrafish will likely have relevance to human mitochondrial biology and disease . The bicistronic arrangement observed in Drosophila, where CCDC56 is co-transcribed with mitochondrial transcription factor B1, adds another layer of regulatory complexity that may have implications for human mitochondrial gene expression .

What emerging technologies could enhance CCDC56 functional studies in zebrafish?

Several cutting-edge technologies can advance CCDC56 research in zebrafish:

• CRISPR base editing and prime editing:

  • Generate precise point mutations to study specific domains without creating double-strand breaks

  • Model human CCDC56 variants of interest with nucleotide-level precision

• Single-cell transcriptomics and proteomics:

  • Profile cell-type-specific responses to CCDC56 deficiency

  • Identify compensatory pathways activated in response to COX dysfunction

• Microfluidic organ-on-chip technologies:

  • Culture zebrafish embryos in continuous flow-through systems (9 μL well volume with 2 μL/(well min) buffer flow)

  • Enable real-time imaging while precisely controlling the microenvironment

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize CCDC56 localization within mitochondrial subcompartments

  • Live imaging of mitochondrial function using genetically encoded sensors for ATP, calcium, or reactive oxygen species

• Cryo-EM structural analysis:

  • Determine the structure of CCDC56 in complex with COX assembly intermediates

  • Provide atomic-level insights into the mechanism of CCDC56 function

These technologies, combined with the inherent advantages of zebrafish as a model system (optical transparency, external development, high fecundity), create powerful approaches for elucidating CCDC56 function .

What are the most significant unanswered questions in CCDC56 research?

Despite progress in understanding CCDC56 function, several critical questions remain:

• Precise molecular mechanism:

  • How does CCDC56 facilitate COX assembly at the molecular level?

  • Which specific COX subunits or assembly factors directly interact with CCDC56?

  • Is CCDC56 function limited to assembly, or does it play roles in COX stability or activity regulation?

• Developmental regulation:

  • How is CCDC56 expression regulated during development?

  • Are there tissue-specific variations in CCDC56 function or requirement?

  • What compensatory mechanisms exist in response to CCDC56 deficiency?

• Evolutionary aspects:

  • Why does CCDC56 length vary across species (87 amino acids in Drosophila, 96 in zebrafish, 106 in humans)?

  • How do species-specific differences in CCDC56 sequence relate to functional adaptations?

  • What is the significance of the bicistronic arrangement with mitochondrial transcription factor B1 observed in Drosophila?

• Disease relevance:

  • Are there human diseases directly associated with CCDC56 mutations?

  • Could CCDC56 modulation be a therapeutic strategy for mitochondrial disorders?

Addressing these questions through systematic research in zebrafish and other model systems will provide valuable insights into fundamental aspects of mitochondrial biology and potential therapeutic approaches for mitochondrial diseases.