Recombinant Human Coiled-coil domain-containing protein 90B, mitochondrial (CCDC90B)

What is the basic structure and characterization of CCDC90B protein?

CCDC90B (Coiled-coil domain containing 90B) is a mitochondrial protein characterized by the presence of a domain of unknown function (DUF1640). This domain constitutes most of the protein structure except for the first twenty-three amino acid residues (MNSRQAWRLFLSQGRGDRWVSRP), which function as a mitochondrial targeting site and are cleaved in the mature protein . The protein contains seven predicted alpha helices, which is typical of coiled-coil proteins . CCDC90B has a molecular weight of approximately 26.72 kDa and an isoelectric point of 7.5, with no transmembrane helices . It is a member of the CCDC90 protein family and is expected to be localized to the mitochondria without any transmembrane spans or segments .

Where is the CCDC90B gene located in the human genome and what are its neighboring genes?

The CCDC90B gene is located on chromosome 11 in humans . Its genomic neighborhood includes several important genes: PCF11 (a mammalian pre-mRNA cleavage complex 2 protein), ANKRD42 (an ankyrin repeat protein involved with calcium ion bonding), BC070093, and DLG2 (a member of the membrane-associated guanylate kinase family) . This genomic context may provide insights into potential functional relationships or co-regulation patterns among these genes.

What post-translational modifications are known to occur in CCDC90B?

CCDC90B undergoes several post-translational modifications that are critical to its function. The most significant modification is the cleavage of its N-terminal mitochondrial targeting sequence (first 23 amino acids) . Additionally, CCDC90B is predicted to contain at least three specific phosphorylation sites: Protein Kinase C phosphorylation sites, Casein Kinase II phosphorylation sites, and cAMP/cGMP-dependent phosphorylation sites . The protein does not appear to undergo other common modifications such as chloroplast transit peptide processing, signal peptide cleavage, C-mannosylation, or N-glycosylation .

What is the functional role of CCDC90B in the mitochondrial calcium uniporter (MCU) complex?

CCDC90B interacts with components of the Mitochondrial Calcium Uniporter (MCU) complex, which is the primary mechanism for increasing matrix Ca²⁺ in most cell types . Research has demonstrated that CCDC90B physically interacts with MCU (the channel-forming component), MCUR1 (MCU Regulator 1), and EMRE (Essential MCU Regulator) . Despite these interactions, silencing of CCDC90B has been shown to have only a nominal effect on MCU-dependent mitochondrial Ca²⁺ uptake, suggesting its role may be regulatory rather than essential for channel function . This is in contrast to its paralog MCUR1, whose downregulation causes a significant decrease in agonist-induced mitochondrial calcium transients .

How does CCDC90B interact with other proteins in the MCU complex?

Coimmunoprecipitation studies have revealed that CCDC90B forms specific interactions with several components of the MCU complex. When HA-tagged CCDC90B was used as bait in coimmunoprecipitation experiments, it strongly pulled down MCU, MCUR1, and EMRE proteins . Similarly, when MCU-GFP was used as bait, it successfully coimmunoprecipitated with CCDC90B along with MCUR1, EMRE, and MICU1 . Notably, CCDC90B does not appear to interact with MICU1 when MCUR1 is used as bait, nor does it interact with LETM1 (Leucine zipper-EF-hand containing transmembrane protein 1) . These interaction patterns suggest that CCDC90B may be part of a specific sub-complex within the larger MCU complex.

What is the evolutionary conservation of CCDC90B compared to other MCU complex components?

An interesting aspect of CCDC90B is its evolutionary conservation. Unlike MCU, MICU1, MCUR1, and EMRE, which are absent in yeast, CCDC90B is present in yeast species . This evolutionary divergence suggests that CCDC90B may serve functions independent of the MCU complex, which evolved later in higher organisms. This unique evolutionary profile makes CCDC90B an interesting target for comparative studies between different species to understand the evolution of mitochondrial calcium regulation mechanisms.

How is recombinant human CCDC90B typically produced for research applications?

Recombinant human CCDC90B can be produced in E. coli expression systems as a single, non-glycosylated polypeptide chain . A typical construct contains 211 amino acids (corresponding to residues 43-230 of the native protein) and has a molecular mass of approximately 24.1 kDa . For purification and detection purposes, the protein is often fused to an affinity tag, such as a 23-amino acid His-tag at the N-terminus . The recombinant protein is typically purified using proprietary chromatographic techniques to achieve greater than 90% purity as determined by SDS-PAGE .

What are the optimal storage conditions for recombinant CCDC90B?

Optimal storage of recombinant CCDC90B is critical for maintaining its stability and functionality . For short-term use (within 2-4 weeks), the protein solution can be stored at 4°C . For longer-term storage, it is recommended to keep the protein frozen at -20°C . To enhance stability during extended storage periods, the addition of a carrier protein (0.1% HSA or BSA) is recommended . Multiple freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of activity . These storage guidelines ensure that the recombinant protein maintains its structural integrity and functional properties for experimental applications.

What methodologies are effective for studying CCDC90B interactions with the MCU complex?

Several methodological approaches have proven effective for studying CCDC90B interactions with the MCU complex components. Coimmunoprecipitation using epitope-tagged proteins (such as Flag, V5, HA, or GFP tags) has successfully demonstrated physical interactions between CCDC90B and other MCU complex proteins . Both forward and reverse coimmunoprecipitation approaches are recommended to confirm interactions, as shown in studies where CCDC90B-HA pulled down MCU, MCUR1, and EMRE, and reciprocally, MCU-GFP pulled down CCDC90B . Proper controls, including single-transfection samples and non-interacting proteins (like LETM1), should be included to verify antibody specificity and interaction authenticity .

How can CRISPR/Cas9 technology be utilized to generate CCDC90B knockout models?

CRISPR/Cas9 lentiviral gene-knockout technology has been successfully employed to generate CCDC90B knockout models for functional studies . The process typically involves designing guide RNAs targeting specific regions of the CCDC90B gene, followed by lentiviral delivery of the CRISPR/Cas9 system to target cells . Western blot analysis can confirm successful knockout by demonstrating the absence of CCDC90B protein expression in generated clones . These knockout models are valuable tools for investigating the physiological roles of CCDC90B through loss-of-function approaches, allowing researchers to assess the consequences of CCDC90B deficiency on mitochondrial calcium handling and related cellular processes.

What is the relationship between CCDC90B and MCUR1 (CCDC90A), and how do their functions differ?

CCDC90B and MCUR1 (formerly known as CCDC90A) are paralogs with distinct functional characteristics . Both proteins are integral components of the inner mitochondrial membrane and are broadly expressed across tissues, with CCDC90B mRNA generally present at higher levels than MCUR1 . Functionally, while silencing of either protein leads to decreased mitochondrial calcium influx, MCUR1 appears to play a more critical role in this process . MCUR1 downregulation causes a significant decrease in agonist-induced mitochondrial calcium transients and can bind MCU but not MICU1, suggesting MCU may exist in two different complexes: one with MCUR1 and another with MICU1 . In contrast, CCDC90B knockdown has a more modest effect on mitochondrial calcium uptake . The table below summarizes key comparative aspects:

How does the calcium signaling role of CCDC90B relate to broader mitochondrial functions?

The role of CCDC90B in calcium signaling has implications for broader mitochondrial functions, as mitochondrial calcium homeostasis impacts various aspects of cellular metabolism and physiology . Mitochondrial calcium uptake through the MCU complex influences ATP production, mitochondrial reactive oxygen species generation, and can trigger apoptosis under certain conditions . Given CCDC90B's interaction with the MCU complex components, it may participate in fine-tuning these processes, potentially serving as a regulatory factor that modulates mitochondrial calcium uptake under specific physiological conditions. Future research exploring the effects of CCDC90B modulation on mitochondrial bioenergetics, redox status, and cell survival pathways would provide valuable insights into its broader functional significance.

What are common challenges in detecting endogenous CCDC90B in experimental samples?

Detection of endogenous CCDC90B presents several challenges that researchers should consider. The relatively small size of mature CCDC90B (after cleavage of the mitochondrial targeting sequence) may require optimized gel conditions for proper resolution in Western blots. Additionally, antibody selection is critical, as antibodies targeting the N-terminal region might not recognize the mature processed protein. Furthermore, the mitochondrial localization of CCDC90B necessitates effective mitochondrial isolation procedures to enrich for the protein in experimental samples. To overcome these challenges, researchers might consider using multiple antibodies targeting different epitopes and employing appropriate subcellular fractionation techniques.

What controls are essential when studying CCDC90B-protein interactions?

When studying CCDC90B-protein interactions, several controls are essential to ensure result reliability. First, single-transfection controls should be used to verify antibody specificity, as demonstrated in studies where V5-tagged MCUR1 was immunoprecipitated while Flag-tagged candidate proteins did not pull down independently . Second, inclusion of known non-interacting proteins (such as LETM1 for CCDC90B) serves as negative controls . Third, reciprocal coimmunoprecipitations using differently tagged versions of the proteins should be performed to confirm interactions from multiple angles, as shown in studies using both CCDC90B-HA and MCU-GFP as baits . These controls help establish the specificity and authenticity of observed protein-protein interactions.

How can researchers distinguish between the roles of CCDC90B and MCUR1 in experimental settings?

Distinguishing between the roles of CCDC90B and MCUR1 requires careful experimental design. One approach is selective knockdown or knockout of each protein individually and in combination, followed by assessment of mitochondrial calcium uptake and related functions . Another strategy involves overexpression studies with wild-type and mutant versions of each protein to identify functional domains and potential redundancies. Detailed interaction mapping through truncation mutants or domain swapping between the two proteins can further illuminate their distinct binding properties and functional contributions. Finally, tissue-specific analyses comparing expression patterns and knockout phenotypes across different cell types may reveal context-dependent roles, given that CCDC90B mRNA is generally expressed at higher levels than MCUR1 in many tissues .