Recombinant Rat Coiled-coil domain-containing protein 90B, mitochondrial (Ccdc90b)

What is the basic structure of rat Ccdc90b protein?

Rat Ccdc90b belongs to the CCDC90 family and is characterized by the presence of a domain of unknown function, DUF1640. This domain is characteristic of the entire protein excluding the first 23 amino acid residues, which function as a mitochondrial targeting signal. The cleaved Ccdc90b is a mitochondrial protein without transmembrane spans or segments . Based on human CCDC90B, which shares high homology with rat Ccdc90b, the protein is expected to be a non-glycosylated polypeptide chain in its recombinant form with a molecular mass of approximately 24 kDa .

What are the known post-translational modifications of rat Ccdc90b?

Rat Ccdc90b, similar to its human ortholog, is predicted to contain at least three specific phosphorylation sites . These phosphorylation events may play crucial roles in regulating the protein's function within mitochondria. While glycosylation has not been reported for rat Ccdc90b, the recombinant form produced in E. coli would not contain post-translational modifications that typically occur in mammalian cells, which is an important consideration when using recombinant protein for functional studies .

How is rat Ccdc90b protein localized within cells?

Rat Ccdc90b primarily localizes to the mitochondria, directed by its N-terminal mitochondrial targeting sequence (first 23 amino acids) . Immunofluorescence studies of CCDC90B in human cells show co-localization with mitochondrial markers, including MitoTracker and COX1, confirming its mitochondrial localization . When designing experiments using recombinant rat Ccdc90b, researchers should consider whether the recombinant protein includes this targeting sequence, as this will affect its cellular localization in experimental systems.

What expression systems are optimal for producing recombinant rat Ccdc90b?

Based on protocols used for human CCDC90B, E. coli represents an effective expression system for recombinant rat Ccdc90b production . For optimal expression, the protein can be fused to a His-tag at the N-terminus to facilitate purification. The expression construct should be designed to either include or exclude the mitochondrial targeting sequence (first 23 amino acids) depending on the experimental requirements. If studying interactions within mitochondria, excluding this sequence may provide a protein form that better represents the mature, processed mitochondrial protein .

What purification strategies are most effective for recombinant rat Ccdc90b?

Recombinant rat Ccdc90b with an N-terminal His-tag can be effectively purified using proprietary chromatographic techniques, particularly immobilized metal affinity chromatography (IMAC) . A typical purification protocol would include:

• Cell lysis in a buffer containing 20mM Tris-HCl (pH 8.0), 0.15M NaCl, and protease inhibitors

• IMAC purification using nickel or cobalt resin

• Size exclusion chromatography for further purification

• Quality assessment using SDS-PAGE (purity >90% is typically achievable)

• Formulation in a stabilizing buffer containing 20mM Tris-HCl (pH 8.0), 0.15M NaCl, 10% glycerol, and 1mM DTT

What are the optimal storage conditions for maintaining rat Ccdc90b stability?

For short-term storage (2-4 weeks), recombinant rat Ccdc90b can be stored at 4°C. For longer periods, storage at -20°C is recommended. To enhance stability during long-term storage, the addition of a carrier protein (0.1% HSA or BSA) is advisable. Multiple freeze-thaw cycles should be avoided as they can compromise protein integrity and function . For experimental work, the protein concentration should be standardized (e.g., 0.25mg/ml) to ensure reproducibility across experiments.

How can mitochondrial localization of rat Ccdc90b be experimentally verified?

To verify the mitochondrial localization of rat Ccdc90b, researchers can employ dual-labeling immunofluorescence techniques. This involves:

• Transfecting cells with a rat Ccdc90b expression construct

• Staining cells with MitoTracker dye (for mitochondrial compartment)

• Immunostaining for Ccdc90b using specific antibodies

• Analyzing co-localization using confocal microscopy

• Calculating co-localization coefficients (e.g., Pearson's correlation coefficient)

This approach has been successfully used to demonstrate mitochondrial localization of human CCDC90B, showing distinct co-localization between CCDC90B protein staining and mitochondrial activity .

What methods can assess the impact of rat Ccdc90b on mitochondrial function?

Based on studies with human CCDC90B, several approaches can assess the impact of rat Ccdc90b on mitochondrial function:

• Mitochondrial membrane potential measurement: Using fluorescent dyes like MitoTracker Orange to quantify changes in mitochondrial membrane potential when Ccdc90b is overexpressed or knocked down .

• Mitochondrial compartment size analysis: Using MitoTracker Green to measure total mitochondrial compartment size, as studies have shown that T1-CCDC90B overexpression leads to decreased mitochondrial compartment size .

• Calculation of mitochondrial potential to compartment size ratio (MP/MC): This provides insight into mitochondrial function efficiency. In human cells, T1-CCDC90B overexpression resulted in approximately twice the MP/MC ratio compared to control cells .

• Analysis of respiratory chain components: Measuring levels of proteins like COX1 (a component of Complex IV of the electron transport chain) can reveal functional effects of Ccdc90b. Human studies showed that T1-CCDC90B overexpression increased COX1 protein levels .

How does rat Ccdc90b affect mitochondrial matrix volume regulation?

Evidence from human CCDC90B studies suggests that this protein may play a crucial role in regulating mitochondrial matrix volume through ion channel activity . To investigate this function in rat Ccdc90b, researchers should consider:

• Generating stable cell lines with controlled Ccdc90b expression (overexpression and knockdown)

• Measuring changes in mitochondrial compartment size using MitoTracker Green

• Assessing mitochondrial ion flux using ion-specific fluorescent probes

• Evaluating matrix volume changes in response to osmotic challenges

• Analyzing the effects of ion channel inhibitors on Ccdc90b-mediated changes

Research with human CCDC90B has shown that overexpression leads to decreased mitochondrial compartment size, while knockdown results in increased compartment size, suggesting a role in ion channel regulation affecting matrix volume .

What is the relationship between rat Ccdc90b and respiratory chain complex assembly?

While the exact relationship between rat Ccdc90b and respiratory chain complex assembly remains to be fully elucidated, studies with human CCDC90B provide valuable insights. Overexpression of T1-CCDC90B led to increased levels of COX1, a critical protein in Complex IV of the electron transport chain . To investigate this relationship in rat Ccdc90b, researchers should:

• Create cells with controlled Ccdc90b expression levels

• Measure the expression and activity of respiratory chain complexes (I-V)

• Assess mitochondrial oxygen consumption rates

• Evaluate the assembly of respiratory supercomplexes using blue native PAGE

• Analyze ATP production in relation to Ccdc90b expression levels

These approaches will help determine whether rat Ccdc90b directly influences respiratory chain complex assembly and function, potentially revealing its role in mitochondrial energy production.

How can researchers effectively design knockdown/knockout models to study rat Ccdc90b function?

To effectively design knockdown/knockout models for studying rat Ccdc90b function:

• Stable shRNA knockdown: Design multiple shRNA sequences targeting different regions of rat Ccdc90b mRNA. Validate knockdown efficiency by qRT-PCR and Western blot. Human studies have successfully used stable CCDC90B knockdown cells to demonstrate reversal of the effects seen with protein overexpression .

• CRISPR/Cas9 knockout: Design guide RNAs targeting exonic regions of rat Ccdc90b. Validate knockout by sequencing and Western blot. Consider generating conditional knockout models if complete knockout affects cell viability.

• Isoform-specific targeting: Design strategies to target specific isoforms (like T1-CCDC90B) to dissect isoform-specific functions, as different transcripts may have distinct effects on mitochondrial function .

• Rescue experiments: Include restoration of Ccdc90b expression in knockdown/knockout models to confirm specificity of observed phenotypes.

• Phenotypic characterization: Comprehensively assess mitochondrial morphology, membrane potential, compartment size, respiratory function, and cell viability in models with altered Ccdc90b expression.

What are the known protein-protein interactions of rat Ccdc90b?

While specific protein-protein interactions of rat Ccdc90b have not been extensively characterized, its mitochondrial localization suggests potential interactions with proteins involved in mitochondrial function, structure, and dynamics. Based on its effects on mitochondrial compartment size and COX1 levels observed in human studies , researchers can investigate potential interactions with:

• Respiratory chain components, particularly Complex IV proteins

• Mitochondrial ion channels and transporters

• Proteins involved in mitochondrial matrix volume regulation

• Mitochondrial structural proteins

Methodologies to identify these interactions include:

• Co-immunoprecipitation with tagged recombinant rat Ccdc90b

• Proximity labeling approaches (BioID or APEX)

• Yeast two-hybrid screening

• Mass spectrometry-based interactome analysis

How can phosphorylation sites in rat Ccdc90b be identified and characterized?

Based on human CCDC90B, rat Ccdc90b is predicted to contain at least three specific phosphorylation sites . To identify and characterize these sites:

• Computational prediction: Use phosphorylation site prediction tools to identify potential sites based on sequence analysis.

• Mass spectrometry analysis: Perform phosphoproteomic analysis of purified rat Ccdc90b using:

  • Phosphopeptide enrichment (TiO₂ or IMAC)

  • LC-MS/MS analysis

  • Site-specific identification using fragmentation patterns

• Phosphorylation-specific antibodies: Develop antibodies against predicted phosphorylation sites to monitor site-specific phosphorylation under different conditions.

• Mutagenesis studies: Create phosphomimetic (S/T to D/E) and phosphodeficient (S/T to A) mutants to assess the functional significance of identified phosphorylation sites.

• Kinase identification: Perform in vitro kinase assays with candidate kinases to identify enzymes responsible for Ccdc90b phosphorylation.

What is the role of rat Ccdc90b in mitochondrial dysfunction models?

Given the effects of CCDC90B on mitochondrial potential and compartment size observed in human cells , rat Ccdc90b likely plays important roles in mitochondrial dysfunction models. To investigate these roles, researchers should consider:

• Oxidative stress models: Assess how Ccdc90b expression affects cellular responses to oxidative stress inducers (e.g., H₂O₂, paraquat)

• Mitochondrial toxicity models: Evaluate the impact of Ccdc90b on cellular sensitivity to mitochondrial toxins (e.g., rotenone, antimycin A)

• Neurodegenerative disease models: Analyze Ccdc90b expression and function in rat models of diseases with known mitochondrial dysfunction components (e.g., Parkinson's disease)

• Metabolic stress models: Investigate how Ccdc90b affects mitochondrial adaptation to metabolic challenges (e.g., glucose deprivation, fatty acid overload)

How can rat Ccdc90b research be translated to understand human pathologies?

To translate rat Ccdc90b research to human pathologies, researchers should:

• Comparative analyses: Perform sequence and functional comparisons between rat and human CCDC90B to identify conserved features and species-specific differences.

• Clinical sample studies: Analyze CCDC90B expression in human patient samples from diseases with mitochondrial dysfunction components, correlating with findings from rat models.

• Cell-based validation: Validate key findings from rat studies in human cell lines, comparing effects of rat Ccdc90b and human CCDC90B overexpression or knockdown.

• Therapeutic target assessment: Evaluate whether modulation of CCDC90B function could have therapeutic potential in mitochondrial diseases based on findings from rat models.

The high conservation of mitochondrial proteins between species suggests that insights from rat Ccdc90b research will likely have relevance to human mitochondrial biology and pathology.