Recombinant Taeniopygia guttata Coiled-coil domain-containing protein 58 (CCDC58)

What is CCDC58 and what is its primary function in Taeniopygia guttata?

CCDC58, also known as Mix23 in some contexts, is a coiled-coil domain-containing protein that functions primarily as a mitochondrial matrix import factor. In zebra finch, as in other species, CCDC58 likely acts as a regulator or stabilizer involved in the mitochondrial protein import machinery . Specifically, it functions in the inner mitochondrial membrane and intermembrane space to facilitate the effective import of proteins into the mitochondrial matrix . Given the high metabolic demands of avian species, particularly in flight muscles and song-related neural circuits of zebra finch, CCDC58 may play a crucial role in maintaining mitochondrial function under varying physiological conditions.

How does zebra finch CCDC58 compare structurally to its mammalian counterparts?

While specific structural comparisons aren't directly addressed in the current literature, comparative analysis suggests that CCDC58 maintains its core functional domains across vertebrate species. The defining coiled-coil domain, which facilitates protein-protein interactions, is likely conserved in the zebra finch ortholog. Researchers should perform detailed sequence alignments and structural predictions to identify zebra finch-specific features that might relate to avian physiology, such as adaptations to higher body temperature (40-42°C) compared to mammals. Specific attention should be paid to mitochondrial targeting sequences and interaction surfaces with other components of the mitochondrial import machinery.

What are the expression patterns of CCDC58 in different zebra finch tissues?

Expression analysis of CCDC58 in zebra finch would be expected to show highest levels in tissues with high mitochondrial content and energy demands. Based on the function of CCDC58 in mitochondrial processes, researchers should focus on examining expression in:

• Flight muscles (high oxidative metabolism)

• Heart tissue (continuous energetic demands)

• Brain regions involved in song learning and production (high neuronal activity)

• Liver (central metabolic regulation)

Quantitative PCR, in situ hybridization, and immunohistochemistry would be appropriate techniques for mapping tissue-specific expression patterns. Particular attention should be paid to song-control nuclei given the zebra finch's importance as a model for vocal learning .

What are the optimal expression systems for producing recombinant zebra finch CCDC58?

For recombinant expression of zebra finch CCDC58, researchers should consider multiple expression systems, each with distinct advantages:

For mitochondrial proteins like CCDC58, E. coli expression often results in inclusion bodies due to improper folding of the coiled-coil domain. A recommended approach is to use a combination of solubility-enhancing tags (such as MBP or SUMO) with low-temperature induction to maximize proper folding.

What purification strategies are most effective for recombinant zebra finch CCDC58?

Purification of recombinant zebra finch CCDC58 requires a multi-step approach to ensure high purity and native conformation:

• Initial capture: Affinity chromatography using His-tag or GST-tag depending on the expression construct

• Intermediate purification: Ion exchange chromatography (typically anion exchange at pH 8.0)

• Polishing: Size exclusion chromatography to ensure homogeneity

For CCDC58, which functions in mitochondrial membranes, buffer optimization is critical. Recommended buffers should include:

• Mild detergents (0.1% DDM or 0.5% CHAPS) to maintain solubility

• Reducing agents (1-5 mM DTT or TCEP) to prevent oxidation of cysteine residues

• Stabilizing agents like glycerol (10%) for long-term storage

Quality control should include mass spectrometry verification, circular dichroism to confirm secondary structure (expecting high alpha-helical content for the coiled-coil domain), and thermal shift assays to assess stability.

How can researchers verify the functional activity of recombinant zebra finch CCDC58?

Functional verification of recombinant CCDC58 should address its role in mitochondrial protein import. Several complementary approaches are recommended:

• In vitro reconstitution assays:

  • Isolated mitochondria depleted of endogenous CCDC58

  • Addition of recombinant protein

  • Measurement of import efficiency using fluorescently labeled reporter proteins

• Interaction studies:

  • Pull-down assays with known components of the mitochondrial import machinery

  • Surface plasmon resonance to determine binding kinetics

  • Crosslinking mass spectrometry to identify specific interaction interfaces

• Thermal stability assays:

  • Differential scanning fluorimetry to assess stability at varying temperatures

  • Particularly relevant given CCDC58's reported role in temperature-sensitive protein import

Researchers should compare results with mammalian CCDC58 to identify any zebra finch-specific functional characteristics.

How is the CCDC58 gene organized in the zebra finch genome?

While specific details of zebra finch CCDC58 genomic organization aren't directly addressed in the literature, researchers can analyze this based on the zebra finch genome data . Key considerations should include:

• Chromosomal location and local recombination landscape

• Exon-intron structure and potential alternative splicing events

• Regulatory elements in promoter and enhancer regions

The zebra finch genome shows distinctive patterns of recombination, with pronounced effects near telomeres and correlation with GC content . Researchers should determine if CCDC58 is located in a high or low recombination region, as this would influence its evolutionary dynamics. Analysis should include identification of GC-rich motifs like CCTCCCT that are associated with recombination hotspots in the zebra finch genome .

What evolutionary insights can be gained from studying CCDC58 in zebra finch?

Evolutionary analysis of CCDC58 across species can reveal selective pressures on mitochondrial function in avian lineages. A comprehensive approach would include:

• Phylogenetic analysis of CCDC58 sequences from diverse bird species

• Calculation of dN/dS ratios to identify signs of purifying or positive selection

• Correlation of sequence changes with ecological factors (flight capability, metabolic rate)

The zebra finch genome shows specific patterns of base composition and recombination that may influence the evolution of genes like CCDC58. Researchers should examine whether CCDC58 shows evidence of GC-biased gene conversion, which has been documented in avian genomes and is related to recombination rates .

How does genetic variation in CCDC58 correlate with zebra finch population differences?

Population genetic analysis of CCDC58 in different zebra finch populations would provide insights into natural variation and potential adaptive significance. Researchers should:

• Sequence CCDC58 from multiple populations (both wild and domesticated)

• Analyze nucleotide diversity and haplotype structure

• Perform association studies with phenotypic traits (if available)

Particular attention should be paid to variations in coding regions that might affect protein function versus regulatory regions that might influence expression levels. The zebra finch genome exhibits substantial variation in recombination rates , which would influence the pattern of linkage disequilibrium around the CCDC58 locus and should be considered in population genetic analyses.

How does CCDC58 contribute to mitochondrial function in zebra finch neurons?

Given the zebra finch's importance as a model for vocal learning , the role of CCDC58 in neuronal mitochondria is particularly relevant. Research approaches should include:

• High-resolution imaging of CCDC58 localization in neurons from song-control nuclei

• Analysis of mitochondrial morphology and function in neurons with CCDC58 manipulation

• Electrophysiological measurements to correlate mitochondrial function with neuronal activity

Neurons in song-control regions have high energy demands during periods of song learning and production. Researchers should investigate whether CCDC58 expression or function is regulated during these critical periods, potentially contributing to the metabolic support of neuroplasticity.

What is the role of CCDC58 in zebra finch development, particularly during critical periods for song learning?

Developmental analysis of CCDC58 expression and function would provide insights into its potential role during critical periods of zebra finch development:

• Quantitative expression analysis across developmental timepoints

• Correlation with markers of mitochondrial biogenesis

• Functional manipulation during specific developmental windows

If CCDC58 expression changes during the sensory acquisition phase of song learning (approximately 20-60 days post-hatch), this might suggest a role in supporting the metabolic demands of neural circuit formation. Researchers should examine both mRNA and protein levels, as post-transcriptional regulation might be important for fine-tuning CCDC58 function during development.

How does zebra finch CCDC58 respond to various physiological stressors?

Given CCDC58's role in mitochondrial function, its response to physiological stressors is an important area of investigation:

CCDC58 has been implicated in temperature-sensitive protein import processes , making thermal stress particularly relevant for zebra finch, which may experience significant temperature variations in their natural habitat.

How can zebra finch CCDC58 research contribute to understanding human mitochondrial diseases?

Comparative studies between zebra finch and human CCDC58 can provide insights relevant to human mitochondrial diseases:

• Identification of conserved functional domains that may be affected in human disorders

• Analysis of species-specific adaptations that might suggest therapeutic approaches

• Use of zebra finch as a model system for testing interventions targeting mitochondrial import

Recent research has shown that CCDC58 may function as a biomarker in various human cancers, including hepatocellular carcinoma . Researchers could investigate whether similar associations exist in avian tumor models, potentially revealing conserved mechanisms linking mitochondrial function to cellular proliferation.

What is the relationship between CCDC58 and mitochondrial genomic heterogeneity in zebra finch?

Research on CCDC58's relationship with mitochondrial genomic heterogeneity would explore several parameters:

• Correlation with mitochondrial DNA copy number variation

• Potential role in mitochondrial DNA maintenance or expression

• Relationship with nuclear-encoded mitochondrial genes

This research direction is supported by findings that CCDC58 expression in human cancers correlates with genomic heterogeneity indicators , including tumor mutation burden, microsatellite instability, and homologous recombination deficiency. Similar analyses in zebra finch would provide evolutionary context for these associations.

How does CCDC58 interact with the zebra finch mitochondrial proteome?

A comprehensive analysis of CCDC58's interaction network would provide insights into its functional role:

• Affinity purification-mass spectrometry to identify binding partners

• Proximity labeling approaches to map the local protein environment

• Structural studies of key protein-protein interactions

These approaches would build on findings from other species regarding CCDC58's role in mitochondrial protein import machinery . Particular attention should be paid to interactions with components identified in protein-protein interaction (PPI) networks from human studies , which could reveal both conserved and species-specific aspects of CCDC58 function.

What CRISPR-based approaches are most effective for studying CCDC58 function in zebra finch models?

While CRISPR techniques are still developing for avian models, several approaches can be considered:

• Ex vivo editing of primary zebra finch cells

  • Isolation of relevant primary cells (neurons, muscle)

  • Delivery of CRISPR components via nucleofection or viral vectors

  • Analysis of mitochondrial phenotypes following CCDC58 editing

• In vivo approaches

  • Viral delivery to specific tissues or brain regions

  • Primordial germ cell manipulation for germline editing

  • Inducible systems for temporal control of CCDC58 manipulation

The zebra finch genome has been well-characterized , facilitating guide RNA design and off-target prediction. Researchers should consider the high GC content in some regions of the zebra finch genome when designing efficient guide RNAs.

What are the best approaches for studying CCDC58 protein complexes in zebra finch mitochondria?

Mitochondrial protein complexes involving CCDC58 can be studied using multiple complementary techniques:

• Blue native PAGE to preserve native protein complexes

• Crosslinking mass spectrometry to identify interaction interfaces

• Cryo-electron microscopy for structural characterization of complexes

Researchers should isolate intact mitochondria from relevant zebra finch tissues, particularly those with high energy demands. Sample preparation requires careful optimization to maintain native interactions while allowing efficient solubilization of membrane-associated complexes.

How can multi-omics approaches be integrated to comprehensively study CCDC58 function in zebra finch?

An integrated multi-omics approach would provide the most comprehensive understanding:

Data integration using computational approaches like weighted gene co-expression network analysis (WGCNA) can reveal functional modules associated with CCDC58, providing context for its role in zebra finch physiology .