Recombinant Xenopus tropicalis Coiled-coil domain-containing protein 90A, mitochondrial (ccdc90a)

What is Recombinant Xenopus tropicalis CCDC90A and what is its significance in research?

Recombinant Xenopus tropicalis CCDC90A (Coiled-coil domain-containing protein 90A, mitochondrial) is a protein expressed in E. coli systems that corresponds to the native protein found in the Western clawed frog (Silurana tropicalis). The protein spans amino acids 67-262 of the mature protein and has the UniProt ID Q0P4J6 . It is significant in research because it serves as a tool for studying mitochondrial calcium regulation and bioenergetics. CCDC90A is also known as MCUR1 (Mitochondrial calcium uniporter regulator 1) in some literature, although there is debate regarding its precise function . Xenopus tropicalis has emerged as an important vertebrate model for cellular and developmental biology research, making proteins from this organism valuable for comparative studies across species .

How does Xenopus tropicalis CCDC90A compare structurally and functionally to homologs in other species?

Xenopus tropicalis CCDC90A shares significant sequence homology with its mammalian counterparts, particularly in the coiled-coil domains and mitochondrial targeting sequences. While the specific conservation percentages are not provided in the available search results, the protein's function appears to be conserved across species based on similar localization patterns and proposed functions.

What are the optimal storage and handling conditions for Recombinant Xenopus tropicalis CCDC90A?

For optimal storage and handling of Recombinant Xenopus tropicalis CCDC90A, researchers should follow these evidence-based protocols:

Storage Conditions:

• Store the lyophilized protein at -20°C to -80°C upon receipt

• For extended storage, conserve at -20°C or -80°C

• Aliquoting is necessary for multiple use to prevent protein degradation

Reconstitution Protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended)

• Prepare working aliquots and store at 4°C for up to one week

Critical Considerations:

• Repeated freezing and thawing is not recommended as it may lead to protein denaturation and loss of activity

• The protein is typically supplied in Tris-based buffer with either 50% glycerol or 6% Trehalose at pH 8.0, optimized for stability

• Working aliquots should be maintained at 4°C and used within one week to ensure optimal protein activity

What experimental approaches can be used to study the function of CCDC90A in mitochondrial calcium regulation?

To investigate CCDC90A's role in mitochondrial calcium regulation, several complementary experimental approaches can be employed:

Calcium Imaging Techniques:

• Use calcium-sensitive fluorescent dyes (e.g., Rhod-2 AM) to measure mitochondrial calcium levels in cells expressing or depleted of CCDC90A

• Employ genetically encoded calcium indicators targeted to mitochondria to monitor real-time changes in calcium flux

• Apply calcium ionophores or physiological stimuli to trigger calcium release and measure the effects of CCDC90A manipulation on mitochondrial calcium uptake

Protein Interaction Studies:

• Conduct co-immunoprecipitation experiments to identify binding partners of CCDC90A, particularly components of the MCU complex

• Perform proximity ligation assays to visualize CCDC90A interactions with MCU or other mitochondrial proteins in situ

• Use yeast two-hybrid or split-luciferase assays to map specific interaction domains

Functional Assays:

• Measure oxygen consumption rates using a Seahorse XF analyzer to assess the impact of CCDC90A on mitochondrial bioenergetics

• Evaluate mitochondrial membrane potential using JC-1 or TMRM dyes in the presence or absence of CCDC90A

• Assess cytochrome c oxidase activity to test the hypothesis that CCDC90A functions as an assembly factor

These approaches should be combined with genetic manipulation techniques such as CRISPR/Cas9-mediated knockout or overexpression systems to establish causality between CCDC90A and observed functional changes.

How can I validate the purity and activity of commercially obtained Recombinant Xenopus tropicalis CCDC90A?

Validation of recombinant CCDC90A purity and activity is essential before proceeding with experiments. A comprehensive validation approach should include:

Purity Assessment:

• SDS-PAGE analysis - Commercial preparations typically have >90% purity as determined by SDS-PAGE

• Western blotting - Use anti-CCDC90A or anti-His tag antibodies (for His-tagged proteins) to confirm identity

• Mass spectrometry - For definitive identification and detection of potential contaminants or truncations

Functional Validation:

• Binding assays with known interaction partners (e.g., components of the MCU complex)

• Circular dichroism spectroscopy to confirm proper protein folding, particularly important for coiled-coil domains

• Size exclusion chromatography to assess oligomerization state, as CCDC90A may form complexes

Activity Assessment:

• Reconstitution into liposomes or permeabilized mitochondria to test effects on calcium transport

• In vitro interaction studies with purified mitochondrial calcium uniporter components

• Cytochrome c oxidase assembly assays to test the alternative proposed function

A typical validation workflow should begin with purity assessment before proceeding to functional and activity tests. Researchers should also perform side-by-side comparisons with positive controls when possible.

How can Xenopus tropicalis CCDC90A be used in studies of mitochondrial dysfunction in disease models?

Xenopus tropicalis CCDC90A represents a valuable tool for investigating mitochondrial dysfunction in various disease models. The protein can be employed in several sophisticated research applications:

Transgenic Xenopus Models:
Researchers can leverage the advantages of Xenopus tropicalis as a genetic model system to create transgenic animals with modified CCDC90A expression. These models can provide insights into how alterations in CCDC90A affect development and physiological processes related to mitochondrial function. The diploid genome and shorter generation time of X. tropicalis compared to X. laevis make it particularly suitable for genetic studies .

Calcium Homeostasis Dysregulation Studies:
CCDC90A/MCUR1 has been implicated in the regulation of mitochondrial calcium, which plays crucial roles in:

• Metabolic regulation and bioenergetics

• Cell death pathways including apoptosis and necrosis

• Reactive oxygen species (ROS) generation and signaling

Recombinant CCDC90A can be used in reconstitution experiments to determine how alterations in this protein contribute to calcium dysregulation observed in diseases such as neurodegeneration and cardiac pathologies .

Tissue-Specific Function Analysis:
By combining the recombinant protein with Xenopus tissue explants or by creating tissue-specific transgenic lines, researchers can investigate how CCDC90A functions differently across tissues. This approach takes advantage of the ease with which tissue chimeras can be created in Xenopus models .

What is the current understanding of the controversy regarding CCDC90A's function as MCUR1 versus a cytochrome c oxidase assembly factor?

The functional characterization of CCDC90A (also known as MCUR1) has generated significant scientific debate. The current understanding of this controversy can be summarized as follows:

Evidence Supporting MCUR1 Function:
Studies have characterized CCDC90A as MCUR1 (Mitochondrial Calcium Uniporter Regulator 1), suggesting it acts as a scaffold factor for the MCU complex. Research by Tomar et al. (2016) indicated that "MCUR1 is a scaffold factor for the MCU complex function and promotes mitochondrial bioenergetics" . This role would position CCDC90A as a critical regulator of mitochondrial calcium uptake and, consequently, cellular metabolism and energy production.

Evidence Supporting Cytochrome c Oxidase Assembly Function:
Contradicting this view, Paupe et al. (2015) presented evidence that "CCDC90A (MCUR1) is a cytochrome c oxidase assembly factor and not a regulator of the mitochondrial calcium uniporter" . This study suggested that the effects observed on calcium regulation might be indirect, resulting from altered mitochondrial function due to impaired cytochrome c oxidase assembly.

Methodological Considerations Contributing to the Controversy:
The discrepancy may stem from:

• Different experimental systems and conditions used across studies

• Varied methods for measuring mitochondrial calcium uptake

• Different approaches to CCDC90A knockout or knockdown

• Potential multifunctional nature of CCDC90A, which might perform different roles depending on cellular context

This controversy highlights the need for comprehensive studies using multiple experimental approaches and model systems, such as Xenopus tropicalis, to definitively resolve CCDC90A's function.

What techniques can be used to study CCDC90A's interaction with the mitochondrial calcium uniporter complex?

To investigate CCDC90A's interaction with the mitochondrial calcium uniporter (MCU) complex, researchers can employ several sophisticated techniques:

Structural Biology Approaches:

• Cryo-electron microscopy to visualize the MCU complex with and without CCDC90A

• X-ray crystallography of purified components or subcomplexes

• Nuclear magnetic resonance (NMR) spectroscopy for mapping interaction interfaces of smaller domains

Advanced Imaging Techniques:

• Super-resolution microscopy (STORM, PALM) to visualize co-localization beyond the diffraction limit

• Förster resonance energy transfer (FRET) to detect direct interactions between labeled proteins

• Fluorescence lifetime imaging microscopy (FLIM) to quantify protein-protein interactions in living cells

Biochemical and Proteomic Methods:

• Chemical crosslinking followed by mass spectrometry (XL-MS) to map protein interaction networks

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions involved in binding

• Thermal shift assays to assess complex stability under various conditions

Genetic and Functional Approaches:

• Site-directed mutagenesis of specific CCDC90A domains followed by interaction studies

• Domain swapping experiments between species to identify critical interaction motifs

• Reconstitution of purified components in liposomes to recreate functional complexes in vitro

A comprehensive approach would integrate multiple techniques to build a complete picture of CCDC90A's role in the MCU complex, addressing the controversy regarding its function .

How is CCDC90A expression regulated during Xenopus tropicalis development?

While the specific expression patterns of CCDC90A during Xenopus tropicalis development are not directly addressed in the provided search results, we can infer several aspects of its regulation based on available information about Xenopus as a model organism:

Temporal Expression Patterns:
The expression of mitochondrial proteins, including CCDC90A, likely varies across developmental stages as energy demands change. Researchers can investigate this using:

• Quantitative PCR to measure mRNA levels across developmental stages

• Western blotting to track protein expression

• In situ hybridization to visualize spatial expression patterns

Transcriptional Regulation:
Xenopus tropicalis offers excellent opportunities for studying the transcriptional regulation of CCDC90A due to:

• The availability of full-length cDNA libraries and EST databases

• The ability to perform promoter analysis using BAC libraries containing the CCDC90A gene region

• The capacity to create transgenic reporter lines to monitor CCDC90A expression in vivo

Developmental Significance:
Understanding CCDC90A expression during development is particularly relevant given:

• The critical role of mitochondria in energy production during embryogenesis

• The potential involvement of calcium signaling in developmental processes

• The possibility that CCDC90A functions in cytochrome c oxidase assembly, which would impact cellular respiration during development

Xenopus tropicalis, with its external development and transparent embryos, provides an excellent system for tracking CCDC90A expression dynamics during development using transgenic approaches with fluorescent reporters.

What approaches can be used to study the role of CCDC90A in mitochondrial biogenesis during Xenopus development?

Investigating CCDC90A's role in mitochondrial biogenesis during Xenopus development can be accomplished through several sophisticated experimental approaches:

Genetic Manipulation Strategies:

• CRISPR/Cas9-mediated knockout of CCDC90A in Xenopus tropicalis embryos

• Morpholino-based knockdown for stage-specific inhibition of CCDC90A expression

• Creation of transgenic lines with inducible or tissue-specific CCDC90A expression

Mitochondrial Analysis Techniques:

• Live imaging of mitochondrial dynamics using fluorescent reporters in developing embryos

• Transmission electron microscopy to visualize mitochondrial ultrastructure across developmental stages

• Measurement of mitochondrial DNA copy number as an indicator of mitochondrial biogenesis

Functional Assays:

• Respirometry to assess oxidative phosphorylation capacity in tissues from normal versus CCDC90A-modified embryos

• ATP production assays to evaluate energetic consequences of CCDC90A manipulation

• Calcium imaging to monitor mitochondrial calcium handling during development

Molecular Pathway Analysis:

• Investigation of interactions between CCDC90A and known regulators of mitochondrial biogenesis such as PGC-1α

• Analysis of SIRT3 and AMPK signaling, which have been linked to mitochondrial function

• Examination of the relationship between CCDC90A and cytochrome c oxidase assembly

The experimental toolkit available for Xenopus tropicalis, including transgenic technologies and the ability to perform tissue chimeras , makes it particularly suitable for these studies. Additionally, the relatively rapid development of Xenopus tropicalis allows for efficient analysis across multiple developmental stages.

What are common challenges when working with Recombinant Xenopus tropicalis CCDC90A and how can they be addressed?

Researchers working with Recombinant Xenopus tropicalis CCDC90A may encounter several technical challenges. The following methodological considerations can help address these issues:

Protein Solubility and Stability Issues:

Experimental Design Challenges:

Technical Implementation Tips:

• Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• When using antibodies against CCDC90A, validate specificity with recombinant protein

• For functional studies, consider the physiological calcium concentrations relevant to mitochondria

• When designing expression constructs, include appropriate mitochondrial targeting sequences

How can contradictory results regarding CCDC90A function be reconciled experimentally?

The conflicting reports regarding CCDC90A's function as either an MCU regulator (MCUR1) or a cytochrome c oxidase assembly factor present a significant challenge. A systematic experimental approach can help reconcile these contradictions:

Comprehensive Functional Assessment Protocol:

• Simultaneous Measurement of Multiple Functions:

  • Design experiments that simultaneously assess both proposed functions

  • Monitor calcium uptake and cytochrome c oxidase assembly in the same experimental system

  • Determine whether these functions are independent or interdependent

• Temporal Analysis:

  • Investigate whether CCDC90A performs different functions at different times or under different conditions

  • Use inducible expression systems to track the immediate versus delayed effects of CCDC90A expression

  • Examine function during different stages of mitochondrial biogenesis

• Domain-Function Relationship Analysis:

  • Create domain deletion or mutation constructs to map regions responsible for different functions

  • Identify domains required for interaction with MCU complex versus cytochrome c oxidase components

  • Develop separation-of-function mutants that affect one process but not the other

• Parallel Model Systems:

  • Compare CCDC90A function across multiple model systems (mammalian cells, Xenopus, etc.)

  • Use the advantages of Xenopus tropicalis for developmental studies

  • Examine whether observed differences are due to cellular context or experimental conditions

• Integrated Multi-omics Approach:

  • Combine proteomics, transcriptomics, and metabolomics data

  • Use systems biology approaches to model CCDC90A's role in mitochondrial networks

  • Identify contextual factors that might influence CCDC90A's primary function

This systematic approach can help determine whether CCDC90A has dual functions, context-dependent roles, or if methodological differences account for the contradictory reports in the literature .

What are the specific considerations for studying Xenopus tropicalis proteins in heterologous expression systems?

When studying Xenopus tropicalis CCDC90A in heterologous expression systems, researchers should consider several important factors to ensure meaningful results:

Expression System Selection:

Protein Tag Considerations:

• His-tags (commonly used for Xenopus tropicalis CCDC90A ) may affect protein folding or function

• Consider tag position (N- versus C-terminal) based on protein topology and function

• Include tag cleavage sites for removal after purification

• Validate that tagged protein retains native activity

Codon Optimization:

• Xenopus codon usage differs from mammalian and bacterial systems

• Consider codon optimization when expressing in heterologous systems

• Balance optimization with maintaining critical regulatory elements

Mitochondrial Targeting:

• Ensure proper mitochondrial targeting sequences are included for in vivo studies

• Verify mitochondrial localization using fractionation or imaging studies

• Consider species differences in mitochondrial import machinery

Functional Context:

• Reconstitute with appropriate binding partners from the same species when possible

• Consider the temperature sensitivity of Xenopus proteins (adapted to lower temperatures)

• Account for species-specific regulatory mechanisms

These considerations are particularly important given the debates about CCDC90A's function and will help ensure that results from heterologous expression studies accurately reflect the protein's native function in Xenopus tropicalis.

What are emerging techniques that could advance our understanding of CCDC90A's role in mitochondrial function?

Several cutting-edge technologies show promise for elucidating CCDC90A's precise role in mitochondrial function:

Advanced Imaging Technologies:

• Correlative light and electron microscopy (CLEM) to visualize CCDC90A localization with nanometer precision

• Live-cell super-resolution microscopy to track CCDC90A dynamics in real-time

• Expansion microscopy to physically enlarge subcellular structures for improved visualization of protein complexes

Novel Genetic Engineering Approaches:

• Mitochondria-targeted CRISPR systems for precise organelle-specific genome editing

• Optogenetic control of CCDC90A to manipulate its activity with spatiotemporal precision

• Prime editing techniques for introducing specific mutations without double-strand breaks

Innovative Biochemical Methods:

• Proximity-dependent biotin identification (BioID) or APEX2 labeling to identify transient interaction partners

• Nanobody-based detection systems for improved specificity in protein interaction studies

• Native mass spectrometry to analyze intact protein complexes containing CCDC90A

Integrative Multi-omics:

• Single-cell proteomics to reveal cell-to-cell variation in CCDC90A function

• Spatial transcriptomics to map CCDC90A expression across tissues with subcellular resolution

• Metabolomics profiling to determine the metabolic consequences of CCDC90A manipulation

These emerging technologies, when applied to the Xenopus tropicalis model system , could help resolve the controversy regarding CCDC90A's function as either an MCU regulator or cytochrome c oxidase assembly factor .

How might the study of CCDC90A in Xenopus tropicalis contribute to our understanding of mitochondrial evolution across species?

Studying CCDC90A in Xenopus tropicalis offers unique insights into mitochondrial evolution due to amphibians' evolutionary position:

Evolutionary Conservation Analysis:
Xenopus tropicalis represents an important evolutionary position between fish and mammals. Comparing CCDC90A structure and function across species can reveal:

• Conserved domains that likely serve fundamental mitochondrial functions

• Species-specific adaptations that may reflect environmental or metabolic specializations

• Evolutionary patterns in mitochondrial calcium regulation and respiratory complex assembly

Functional Divergence Assessment:
The dual proposed functions of CCDC90A - as MCU regulator and cytochrome c oxidase assembly factor - raise interesting evolutionary questions:

• Did these functions evolve separately or from a common ancestral function?

• Does the relative importance of each function vary across species?

• How do species-specific interaction partners influence CCDC90A function?

Genomic Context Exploration:
The genomic environment of CCDC90A in Xenopus tropicalis can provide evolutionary insights:

• Analysis of synteny relationships across species

• Identification of conserved regulatory elements using the Xenopus tropicalis genome sequence

• Examination of paralogous genes that may have evolved specialized functions

Developmental Program Comparison:
Xenopus tropicalis allows for detailed study of developmental regulation:

• Comparison of CCDC90A expression patterns during development across species

• Assessment of how mitochondrial calcium regulation changes through evolutionary history

• Examination of how mitochondrial biogenesis programs have evolved in different lineages

Leveraging the genetic and genomic resources available for Xenopus tropicalis can provide a comprehensive picture of CCDC90A evolution that bridges findings from other model organisms.

What are potential therapeutic applications that might emerge from a better understanding of CCDC90A function?

A comprehensive understanding of CCDC90A function could lead to several therapeutic applications, particularly in diseases involving mitochondrial dysfunction:

Potential Therapeutic Targets:

• Neurodegenerative Disorders: Mitochondrial calcium dysregulation and oxidative stress are implicated in conditions like Alzheimer's and Parkinson's disease. If CCDC90A regulates mitochondrial calcium , it could represent a novel therapeutic target.

• Cardiac Pathologies: Heart failure and ischemia-reperfusion injury involve mitochondrial calcium overload. As mentioned in the search results, "mCa2+ overload" is related to cardiac injury , suggesting CCDC90A modulation could be cardioprotective.

• Metabolic Disorders: If CCDC90A influences mitochondrial bioenergetics , it might be targeted to improve metabolic efficiency in conditions like diabetes and obesity.

Therapeutic Approaches Being Explored:

• Small Molecule Modulators: Compounds that enhance or inhibit CCDC90A function could regulate mitochondrial calcium uptake or cytochrome c oxidase assembly.

• Gene Therapy Approaches: Using the understanding gained from Xenopus tropicalis models , gene therapy strategies could be developed to correct CCDC90A dysfunction.

• Mitochondrial-Targeted Therapeutics: Drug delivery systems that specifically target mitochondria could be employed to modulate CCDC90A activity with minimal off-target effects.

Research Models for Therapeutic Development:
Xenopus tropicalis offers several advantages for developing and testing CCDC90A-targeted therapies:

• The ability to create transgenic lines with human disease mutations

• High-throughput screening of compounds in Xenopus embryos

• Evaluation of developmental toxicity of potential therapeutics