Recombinant Xenopus laevis Coiled-coil domain-containing protein 58 (ccdc58)

What is CCDC58 and what is its function in Xenopus laevis?

CCDC58 (Coiled-coil domain-containing protein 58) is a protein that functions primarily within mitochondria. In Xenopus laevis, as in other organisms, CCDC58 acts as a regulator or stabilizer involved in mitochondrial protein import machinery . The protein contains a coiled-coil domain, which is a highly conserved supercoiled protein motif with several alpha-helices, typically ranging from two to six . Approximately 10% of all proteins contain such coiled-coil domains .

Functionally, CCDC58 (also known as Mix23) is an intermembrane space protein that plays a critical role in the effective import of proteins into the mitochondrial matrix . The recombinant form of Xenopus laevis CCDC58 consists of 144 amino acids and has been successfully expressed in yeast expression systems for research purposes .

Why is Xenopus laevis a suitable model system for studying CCDC58?

Xenopus laevis serves as an excellent model system for studying proteins like CCDC58 for several compelling reasons:

• Well-characterized developmental stages and sequenced genome, allowing for gene expression modifications through exogenous molecules .

• Cells are easily obtained and maintained, not requiring special culture conditions, making them ideal for long periods of live imaging .

• The model has contributed significantly to understanding multiple cytoskeletal components, including actin, microtubules, and neurofilaments .

• Xenopus laevis has been used as a model organism for more than 60 years to study various aspects of early vertebrate development .

• The relatively large growth cones compared to other vertebrates provide a suitable system for imaging cytoskeletal components and associated proteins .

These characteristics make Xenopus laevis particularly valuable for studying mitochondrial proteins like CCDC58, especially when investigating their role in development, cellular function, and potential involvement in disease mechanisms.

What expression systems are optimal for producing recombinant Xenopus laevis CCDC58?

For recombinant Xenopus laevis CCDC58 production, researchers have several expression system options, each with distinct advantages:

The yeast expression system has been successfully used to produce recombinant Xenopus laevis CCDC58 with a His-tag, achieving purity levels exceeding 90% as determined by ELISA . This system offers an optimal balance between proper eukaryotic protein processing and cost-effectiveness for most research applications.

How should recombinant Xenopus laevis CCDC58 be stored and handled for optimal stability?

Proper storage and handling of recombinant Xenopus laevis CCDC58 is critical for maintaining its stability and functionality:

Storage recommendations:

• Store at -80°C for long-term preservation

• Thaw on ice when needed for experiments

• Aliquot into individual single-use tubes before refreezing to avoid repeated freeze-thaw cycles

• Limit freeze-thaw cycles to 2-3 maximum to preserve protein integrity

Buffer conditions:
For recombinant CCDC58 proteins, optimal buffer conditions typically include:

• 25 mM Tris.HCl, pH 7.3

• 100 mM glycine

• 10% glycerol

These storage and handling protocols ensure the recombinant protein maintains its structural integrity and biological activity for experimental use.

What purification methods yield the highest purity recombinant Xenopus laevis CCDC58?

Obtaining high-purity recombinant Xenopus laevis CCDC58 requires careful selection of purification methods:

• Affinity chromatography: The most common method uses the His-tag present on recombinant CCDC58, allowing purification via nickel or cobalt affinity columns. This typically achieves >90% purity when expressed in yeast systems .

• Size exclusion chromatography: Often used as a secondary purification step to remove aggregates and contaminating proteins of different molecular weights.

• Ion exchange chromatography: Can be employed to further improve purity based on CCDC58's charge properties.

Quality control assessment methods include:

• SDS-PAGE with Coomassie blue staining to verify purity (>80-90%)

• Western blotting to confirm identity

• ELISA to verify functionality of the purified protein

The combination of affinity chromatography followed by size exclusion chromatography typically yields recombinant CCDC58 of sufficient purity (>90%) for most research applications.

How can recombinant Xenopus laevis CCDC58 be used to study mitochondrial protein import mechanisms?

Recombinant Xenopus laevis CCDC58 provides a valuable tool for investigating mitochondrial protein import mechanisms through several methodological approaches:

• In vitro import assays: Researchers can use purified recombinant CCDC58 with isolated mitochondria to study:

  • The kinetics of CCDC58 import into mitochondrial compartments

  • Requirements for membrane potential and ATP

  • Interactions with components of the import machinery

• Structure-function analysis: Site-directed mutagenesis of the recombinant CCDC58 can help identify:

  • Critical residues required for mitochondrial targeting

  • Domains involved in interactions with import machinery components

  • Regions necessary for its function as a regulator or stabilizer of import processes

• Comparative studies: Xenopus laevis CCDC58 can be compared with orthologs from other species to identify:

  • Conserved functional domains

  • Evolutionary adaptations in mitochondrial import mechanisms

  • Species-specific regulatory elements

Research has shown that CCDC58 (also known as Mix23) acts specifically as a regulator or stabilizer in the process of effective protein import into the mitochondrial matrix, particularly in temperature-sensitive Tim17 mutants where the scarcity of Mix23 results in synthetic growth defects .

What role does CCDC58 play in mitochondrial function in Xenopus laevis development?

The role of CCDC58 in mitochondrial function during Xenopus laevis development represents an important research area:

• Developmental expression patterns:

  • CCDC58 expression can be studied across different developmental stages of Xenopus laevis

  • Temporal and spatial expression patterns may correlate with specific developmental events requiring mitochondrial remodeling

• Loss-of-function experiments:

  • Morpholino-based knockdown approaches in Xenopus embryos

  • CRISPR/Cas9-mediated gene editing to generate CCDC58-deficient models

  • Analysis of subsequent developmental defects related to mitochondrial dysfunction

• Rescue experiments:

  • Complementation studies using recombinant CCDC58 to rescue knockdown phenotypes

  • Structure-function analysis to identify critical domains required for developmental functions

The established Xenopus model system is particularly advantageous for these studies as its developmental stages have been widely characterized and its genome has been sequenced, allowing gene expression modifications through exogenous molecules . The ability to easily obtain and maintain Xenopus cell cultures makes it an ideal system for studying CCDC58's role in mitochondrial function during development.

How can researchers use recombinant CCDC58 to investigate its potential role as a biomarker in disease models?

Recent research has identified CCDC58 as a potential biomarker for various diseases, particularly cancer. Researchers can utilize recombinant Xenopus laevis CCDC58 to investigate its biomarker potential through the following approaches:

• Antibody production and validation:

  • Generate specific antibodies against Xenopus CCDC58 using the recombinant protein

  • Validate antibodies for applications including immunohistochemistry, Western blotting, and ELISA

  • Use these tools to study CCDC58 expression patterns in normal versus disease states

• Comparative proteomics:

  • Employ recombinant CCDC58 as a standard in proteomic analyses

  • Identify interacting partners through pull-down assays and mass spectrometry

  • Compare interaction networks in normal versus disease conditions

• Disease modeling in Xenopus:

  • Studies have shown that CCDC58 is upregulated in hepatocellular carcinoma (HCC) and correlates with poor prognosis

  • Researchers can model similar disease states in Xenopus to investigate whether CCDC58 shows conserved functions

Research data indicates that CCDC58 has been associated with:

• 28 GO terms related to mitochondria

• 5 KEGG pathways including oxidative phosphorylation

• Poor prognosis in HCC patients based on various survival metrics (OS, DFS, DSS, RFS, and PFS)

• Potential influence on tumor immune microenvironment

This makes recombinant CCDC58 a valuable tool for investigating the protein's role as a biomarker in both human diseases and comparative animal models.

What are the best methods to detect and quantify recombinant Xenopus laevis CCDC58 in experimental samples?

Researchers have several methods at their disposal for detecting and quantifying recombinant Xenopus laevis CCDC58:

For recombinant Xenopus laevis CCDC58 specifically, ELISA has been validated as an effective detection method with high sensitivity when the protein is expressed in yeast systems . The His-tag present on the recombinant protein also facilitates detection and quantification using anti-His antibodies.

What are the common challenges and solutions when working with recombinant Xenopus laevis CCDC58?

Researchers working with recombinant Xenopus laevis CCDC58 may encounter several challenges:

• Protein solubility issues:

  • Challenge: CCDC58 may form aggregates or insoluble fractions during expression or storage

  • Solution: Optimize buffer conditions with appropriate salt concentration, pH, and additives like glycerol (10%)

• Maintaining protein stability:

  • Challenge: Loss of activity during freeze-thaw cycles

  • Solution: Store at -80°C, thaw on ice, aliquot before refreezing, and limit freeze-thaw cycles to 2-3

• Species-specific differences:

  • Challenge: Applying findings from Xenopus CCDC58 to other species

  • Solution: Conduct comparative analyses with human, mouse, or other model systems to identify conserved and divergent features

• Expression system selection:

  • Challenge: Balancing yield, purity, and functionality

  • Solution: Yeast expression systems provide a good compromise between proper folding and cost-effectiveness for CCDC58 , while mammalian systems may be preferred for functional studies despite higher costs

• Antibody cross-reactivity:

  • Challenge: Limited availability of Xenopus-specific antibodies

  • Solution: Use the recombinant protein to produce and validate custom antibodies, or identify conserved epitopes for cross-species antibody selection

Addressing these challenges through careful experimental design and optimization will improve the reliability and reproducibility of research involving recombinant Xenopus laevis CCDC58.

How can researchers design structure-function studies using recombinant Xenopus laevis CCDC58?

Structure-function studies of recombinant Xenopus laevis CCDC58 require methodical approaches to elucidate the relationship between protein structure and biological activity:

• Domain mapping strategies:

  • Generate truncated versions of CCDC58 focusing on the coiled-coil domain

  • Create deletion mutants to identify regions essential for mitochondrial localization

  • Develop chimeric proteins with domains from other species to identify species-specific functions

• Site-directed mutagenesis approach:

  • Target conserved residues identified through sequence alignment across species

  • Focus on regions implicated in protein-protein interactions

  • Create point mutations to disrupt or enhance specific functional capabilities

  • Pay particular attention to the coiled-coil domain, which is critical for protein-protein interactions

• Functional readouts for assessment:

  • Mitochondrial import efficiency assays

  • Protein-protein interaction studies using co-immunoprecipitation

  • Localization studies using fluorescently tagged variants

  • Rescue experiments in knockdown or knockout models

• Structural biology techniques:

  • Circular dichroism (CD) spectroscopy to analyze secondary structure changes

  • Limited proteolysis to identify stable domains

  • If feasible, X-ray crystallography or cryo-EM for detailed structural information

The 144-amino acid sequence of Xenopus laevis CCDC58 provides an excellent template for these structure-function studies, with the coiled-coil domain serving as a primary focus for understanding how this protein contributes to mitochondrial function and potentially to disease processes when dysregulated.

How is CCDC58 being investigated as a potential biomarker or therapeutic target in cancer research?

Recent studies have identified CCDC58 as a promising biomarker with therapeutic potential across multiple cancer types:

These findings position CCDC58 as a potential therapeutic target that could improve patient prognosis in the future .

What comparative studies can be performed between Xenopus laevis CCDC58 and its orthologs in other species?

Comparative studies between Xenopus laevis CCDC58 and its orthologs offer valuable insights into evolutionary conservation and functional specialization:

• Cross-species sequence and structure analysis:

  • Compare Xenopus laevis CCDC58 with orthologs from various species, including:

    • Xenopus tropicalis

    • Human

    • Cow

    • Atlantic salmon

    • Zebra finch

  • Identify conserved domains, particularly the coiled-coil motifs

  • Map species-specific variations to potential functional differences

• Functional conservation assessment:

  • Complementation studies in various model systems

  • Cross-species protein-protein interaction analyses

  • Comparative subcellular localization studies

  • Functional rescue experiments using orthologs from different species

• Developmental role comparison:

  • Analyze expression patterns across developmental stages in different species

  • Compare phenotypes resulting from CCDC58 deficiency across model organisms

  • Investigate whether developmental functions are more conserved than adult functions

• Disease relevance across species:

  • Compare associations with pathological conditions across different model systems

  • Evaluate whether CCDC58's role in cancer biology is conserved in Xenopus and mammalian models

  • Assess potential for Xenopus as a model for CCDC58-related human diseases

These comparative studies would leverage the availability of recombinant CCDC58 proteins from multiple species, including Xenopus laevis, Xenopus tropicalis, human, cow, Atlantic salmon, and zebra finch, all of which have been produced as His-tagged recombinant proteins .

What is the relationship between CCDC58 and mitochondrial function in cellular stress responses?

The relationship between CCDC58 and mitochondrial function during cellular stress represents an emerging area of research:

• Stress response pathways:

  • CCDC58 functions as a mitochondrial matrix import factor and regulator/stabilizer of mitochondrial protein import machinery

  • This positioning makes it a potential sensor or mediator of mitochondrial stress responses

  • Research can examine how CCDC58 expression and activity change under various stress conditions:

    • Oxidative stress

    • Metabolic stress

    • Temperature variations

    • Hypoxia

• Mitochondrial quality control:

  • Investigate CCDC58's potential role in mitochondrial quality control mechanisms

  • Examine interactions with mitochondrial protein degradation pathways

  • Study potential involvement in mitophagy (selective degradation of mitochondria)

• Energy metabolism under stress:

  • CCDC58 is associated with oxidative phosphorylation pathways

  • Research can explore how CCDC58 influences energy production during stress

  • Examine potential roles in metabolic adaptation to changing environmental conditions

• Cell survival mechanisms:

  • Given CCDC58's role in cancer progression , investigate its contribution to cell survival under stress

  • Study potential interactions with apoptotic and anti-apoptotic pathways

  • Examine whether CCDC58 modulation affects cellular resilience to stress

The Xenopus laevis model system provides an excellent platform for these investigations due to its well-characterized developmental stages and ease of experimental manipulation , allowing researchers to study how CCDC58 mediates mitochondrial responses to various stressors.

How might CRISPR/Cas9 gene editing be used to study CCDC58 function in Xenopus laevis?

CRISPR/Cas9 gene editing offers powerful approaches for investigating CCDC58 function in Xenopus laevis:

• Knockout model generation:

  • Design guide RNAs targeting the CCDC58 gene in Xenopus laevis

  • Implement tissue-specific or inducible knockout strategies

  • Analyze resulting phenotypes, particularly focusing on:

    • Mitochondrial morphology and function

    • Developmental abnormalities

    • Cellular stress responses

    • Energy metabolism alterations

• Knock-in strategies:

  • Generate fluorescent protein fusions for live imaging of CCDC58 localization and dynamics

  • Create specific point mutations to test structure-function hypotheses

  • Introduce human disease-associated variants to create disease models

• Regulatory element analysis:

  • Target CCDC58 promoter or enhancer regions to understand transcriptional regulation

  • Identify key regulatory elements controlling CCDC58 expression during development

  • Map tissue-specific regulatory networks

• Multiplexed gene editing:

  • Simultaneously target CCDC58 and interacting partners identified through protein-protein interaction studies

  • Study genetic interactions and potential compensatory mechanisms

  • Create complex disease models involving multiple genes

The well-characterized genome of Xenopus laevis makes it amenable to CRISPR/Cas9 approaches , though its pseudotetraploid nature presents some challenges that require careful guide RNA design to ensure target specificity.

What protein-protein interaction networks involve CCDC58 in Xenopus laevis cells?

Understanding the protein-protein interaction (PPI) networks involving CCDC58 in Xenopus laevis cells will provide critical insights into its functional roles:

• Interactome mapping approaches:

  • Immunoprecipitation coupled with mass spectrometry using recombinant His-tagged CCDC58

  • Proximity labeling techniques (BioID, APEX) to identify proteins in close proximity to CCDC58 in living cells

  • Yeast two-hybrid screening to identify direct binding partners

• Mitochondrial import machinery interactions:

  • Previous research indicates CCDC58 (Mix23) functions as a regulator or stabilizer in mitochondrial protein import

  • Focus on interactions with components of the mitochondrial import machinery, particularly Tim17

  • Investigate dynamic changes in these interactions under different cellular conditions

• Comparative interactome analysis:

  • Compare CCDC58 interaction networks in Xenopus laevis with those identified in human cells

  • Identify conserved core interactions versus species-specific interactions

  • Link interaction differences to functional specialization

• Protein interaction network visualization:

  • Generate comprehensive interaction maps that integrate experimental data

  • Identify key hub proteins and functional modules within the network

  • Map interactions to specific cellular compartments and functions

Research has already identified that CCDC58 interacts with at least 10 proteins that are constituent components of mitochondria , providing a starting point for more detailed interactome studies in Xenopus laevis.

How can multi-omics approaches enhance our understanding of CCDC58 function in Xenopus laevis?

Integrating multiple omics approaches offers a comprehensive strategy for understanding CCDC58 function:

• Transcriptomics:

  • RNA-seq analysis following CCDC58 knockout or overexpression

  • Identification of genes co-regulated with CCDC58 during development

  • Single-cell RNA-seq to map CCDC58 expression patterns across cell types

• Proteomics:

  • Quantitative proteomics to identify protein expression changes in response to CCDC58 modulation

  • Phosphoproteomics to map CCDC58-dependent signaling networks

  • Spatial proteomics to track mitochondrial protein composition changes

• Metabolomics:

  • Targeted analysis of mitochondrial metabolites

  • Changes in energy metabolism pathways following CCDC58 perturbation

  • Identification of metabolic signatures associated with CCDC58 function

• Integration of multi-omics data:

  • Network analysis to connect transcriptomic, proteomic, and metabolomic changes

  • Machine learning approaches to identify patterns across multiple data types

  • Pathway enrichment analyses to highlight biological processes affected by CCDC58

• Comparative multi-omics:

  • Cross-species analysis comparing Xenopus laevis data with human or other model systems

  • Evolutionary conservation of CCDC58-dependent networks and pathways

This integrative approach would build upon existing findings that CCDC58 is associated with 28 GO terms related to mitochondria and 5 KEGG pathways including oxidative phosphorylation , providing a systems-level understanding of CCDC58 function in normal development and disease states.